1 INTRODUCTION

Symbiosis is an association between two or more different species of organisms. The association
may be permanent, the organisms never being separated, or it may be long lasting. This definition
excludes populations, which are associations between individuals of the same species. Organisms
that are involved in a symbiosis may benefit from, be harmed by, or not be affected by the
association. Symbiotic associations are common in nature, from bacteria and fungi that form close
alliances with the roots of terrestrial plants to those between giant tube worms and sulfur-oxidizing
bacteria that live together in the deepest depths of the oceans. No organism is an island--each one
has a relationship to other organisms, directly or indirectly. Even humans bear a reminder of an
ancient symbiosis--their cells contain mitochondria, organelles which once were symbiotic bacteria.
In addition, each of us harbors several types of viruses and bacteria in our skin and intestinal tract.
Similarly, chloroplasts in plant cells are organelles which have evolved from ancient symbiotic
photosynthetic bacteria. Bacteria which form symbioses with higher forms of life are themselves
hosts to symbiotic viruses. Satellite viruses depend on other viruses for their expression. It is
difficult to imagine life and its evolutionary history without symbioses ( Khakhina, 1992; Sapp,
1994a,b).

This book focuses on interspecific relationships, their importance in the evolution of living
organisms, and their significance in the earth's economy. We examine in detail symbiotic
associations that represent the different kingdoms of the living world ( Rennie, 1992).

1.1 SYMBIOSIS AND ITS SIGNIFICANCE IN MODERN

BIOLOGY
Many biologists study organisms and their natural history. Organisms have been collected, hunted,
stuffed, preserved, classified, and dissected, and their life histories have been subjected to
microscopic details, yet we are hard pressed to define an organism. After decades of cellular and
molecular emphasis in biology, the organismal concept is once again drawing attention due to
renewed interest in biodiversity and conservation biology. Organisms do not occur axenically in
nature. Does the physical boundary contain and define an organism? At the genetic level we learn
that an organism's genes are "selfish," genomic conflicts abound, and transposons and viruses play
critical roles in genetic variability. All symbioses involve interspecific genetic interactions ( Dyer,
1989).

Endocytosis is central to all cellular symbioses. The process involves the uptake of extracellular
material into a cell in a membrane bound vacuole. Endocytosis also plays a significant role in: (1)
antigen presentation, (2) nutrient acquisition, (3) clearance of apoptic cells, (4) receptor
regulation, (5) hypertension, and (6) synaptic transmission. The process involves several distinct
morphological and biochemical mechanisms and understanding it through a high-resolution three-
dimensional view of the clathrin coat has opened a new vista ( Marsh and McMahon, 1999).

Symbiosis and Coevolution

Modern evolutionary biologists are deeply entrenched in conceptual frameworks of group selection,
genetic kinship, inclusive fitness, and gradual versus punctuated equilibrium. What has been
missing from their inquiries is the role of symbiosis in the evolutionary explanations. Lynn Margulis
( 1992a) expressed her frustration by wondering how one can talk about the evolution of the cow
without mentioning its cellulose-digesting microbes. Most biologists have accepted the concept of a
serial endosymbiosis origin of eukaryotes, but other coevolutionary themes have yet to receive the
attention they deserve. The evolutionary impact of biological interactions is only now beginning to
be appreciated in terms of its influence on speciation and biodiversity, microbial pathogenesis, and
the evolutionary arms race between a host and its symbiont ( Maynard- Smith, 1991).

Generating Novelty by Symbiosis

Plants and animals have acquired new metabolic capabilities through symbioses with bacteria and
fungi ( Olff and Ritchie, 1998). Mammalian herbivores and termites digest cellulose with the help of
microbial symbionts. Marine bioluminescence in some fishes and squids is produced by luminescent
bacteria contained in specialized light organs. Diverse animal life around the deep-sea vents is
based on symbiosis with bacteria that oxidize hydrogen sulfide and chemosynthetically fix carbon
dioxide into carbohydrates. Associations between fungi and algae have resulted in unique
morphological structures called lichen thalli ( Margulis and Fester, 1991).

Early Life and Origin of the Eukaryotic Cell

A major event in which symbiosis played a crucial role is the evolution of cell organelles such as
mitochondria and chloroplasts through associations involving prokaryotic organisms. In this
manner, eukaryotes acquired the metabolic machinery of cell respiration and photosynthesis from
endosymbionts. Another ancient symbiotic union may have produced the microtubule-based
motility of the modern cell ( Margulis and McMenamin , 1990).

Colonization of Land by Plants

All living things need phosphorous to make nucleic acids (DNA and RNA) and ATP. On land, early
plants were faced with the difficulty of absorbing phosphorous. They formed associations with
mycorrhizal fungi, which greatly facilitated their phosphorous uptake. This crucial event occurred
some 400 million years ago and may have played a significant role in the plant's ability to colonize
terrestrial habitats ( Atsatt, 1991; Newsham et al., 1995).

Horizontal Gene Transfer: Symbioses that Affect

Gene Pools
Evolutionary changes in organisms and their gene pools are not restricted to nuclear events and
sexual mechanisms. Horizontal gene transfer between species has been documented in all forms of
life. Bacterial cells possess plasmids and prophages that transfer new genetic properties from one
cell to another ( Dubnau, 1999). Many virulence factors in pathogenic bacteria are expressed
through plasmid-borne genes. Similarly, bacteria become resistant to antibiotics when they take up
plasmids with genes for antibiotic resistance (fig. 1.1). Horizontal gene transfer has been
suggested in the evolution of flowers, fruits, and storage structures from gall-forming insects and
fungi. The role of viruses as genetic engineers is gaining importance in evolutionary biology (
Amabile- Cuevas and Chicurel, 1993).

Bacterial Symbionts that Behave as Cell Organelles

The Rhizobium--legume symbiosis is an example of how host cells and bacterial symbionts within
root nodules undergo transformations which allow the bacterial cells to fix nitrogen, which then is
transferred to the host plant. Newly formed root nodules continuously replace old nodules
throughout the host plant's life span. Rhizobia as bacteroids within the host cortical cells behave as
temporary cell organelles that fix nitrogen.

In the rice weevil--bacteria endosymbiosis, bacteria are permanent "organelles" of female germ
line cells. The host insect benefits by receiving vitamins, and through rapid development and
increased fertility. The bacterial symbionts cannot live independently and occur temporarily in
larvae from where the future egg cells become infected.

The Evolution of Sex

Intragenomic conflict as an evolutionary force is providing new insights into the history of
 Fig. 1.1 Plasmid-mediated horizontal gene transfer of antibiotic resistance among gram-
negative bacteria. In a similar way, genes encoding for virulence factors in bacterial
pathogenesis can be transferred among bacterial species. Adapted from Atlas (1997).

life on earth. The evolution of sex is a form of genomic conflict management ( Lively, 1996).
Uniparental inheritance of cytoplasmic genes, mating types, and many features of sexual
behavior may have evolved as a result of evolutionary conflict ( Partridge and Hurst, 1998).
The two-sex model which is widespread throughout eukaryotic organisms may have been the
result of ancient intracellular symbioses ( Hutson and Law, 1993; Vollrath, 1998; Moller et al.,
1999).

The Red Queen hypothesis suggests that harmful parasites and virulent pathogens exert
selection pressure on their hosts so that sexual reproduction is maintained, while asexual
reproduction becomes an unstable strategy. So how do we account for asexual reproduction
among organisms that form symbioses? According to this hypothesis, ancient asexual
reproduction was a viable strategy only when asexual hosts did not have a long generation
time compared to their parasitic symbionts ( Ladle et al., 1993).

In 1930 Ronald Fisher proposed that the development of male characteristics and female
preference for certain characteristics speeds up the evolutionary process. In birds, female
choice is responsible for bigger tails, brighter colors, and exaggerated displays; matings with
males with these traits will produce sons with the preferred characteristics. During the 1980s,
Maynard Smith and Hamilton and Zuk examined this issue in light of parasitic load, host
fitness, and the degree of host sexual dimorphism. They argued that parasites and pathogens
and their hosts are involved in a microevolutionary arms race and that in time the symbiont's
offense and host defense produce cycles of coadaptation. An accurate measure of female
choice is for healthy males that are free of harmful symbionts, and displaying that character in
males communicates viability ( Hamilton, 1982; Hamilton and Zuk, 1982; Maynard- Smith,
1989; McLennan and Brooks, 1991; Zuk, 1996).

Symbiotic interactions occur even at the molecular level, with the concept of "selfish DNA"
being the ultimate parasite, spreading around as transposons without causing too much harm (
Kunze et al., 1997). Computer models have been designed to study the influence of symbiotic
interactions on ecosystems

( O'Callaghan and Conrad, 1992). Looking into the future, some have envisioned a type of
"cybersymbiosis" or the interactions between human and manufactured body parts in new life
forms ( Margulis and Sagan, 1986b).

1.2 SUBDIVISIONS OF SYMBIOSIS

In this book, we use the term "symbiosis" in a broad sense, as originally intended by Anton de Bary
in 1879 (appendix 1) to refer to different organisms living together. Proposals to change this
definition and redefine symbiosis, such as equating it with mutualism, have led to confusion (
Saffo, 1992a; Lewin, 1995). Various types of symbioses, whether beneficial or harmful, are
described by the terms commensalism, mutualism, and parasitism. An association in which one
symbiont benefits and the other is neither harmed nor benefited is a commensalistic symbiosis. An
association in which both symbionts benefit is a mutualistic symbiosis. A relationship in which a
symbiont receives nutrients at the expense of a host organism is a parasitic symbiosis.

Commensalism
The term commensal was first used by P.J. van Beneden in 1876 for associations in which one
animal shared food caught by another animal. The two animals were considered to be "messmates"
that ate from the same table. An example of a commensalistic relationship is that between
silverfish and army ants. The silverfish live with the army ants, participate in their raids, and share
their prey. They neither harm nor benefit the ants. We use the term commensalism in its broadest
sense, where the benefit to one of the symbionts may be nutritional or protective.

Mutualism
In a mutualistic symbiosis, both partners benefit from the relationship. The extent to which each
symbiont benefits, however, may vary and generally is difficult to assess ( Cushman and Beattie,
1991; Bronstein, 1994a). The benefit a symbiont receives from an association should be considered
in terms of its costs. As de Bary ( 1879) suggested, there probably is not an example of mutualism
in which both partners benefit equally. In most symbioses, despite numerous experimental studies,
we do not fully understand the complex interactions that take place between the symbionts (
Bronstein, 1994b; Knowleton, 1998). In many associations there is a reciprocal exchange of
nutrients. For example, in the symbioses of algae and invertebrates, the algae provide the animals
with organic compounds, which are products of photosynthesis, while the animals provide the algae
with waste products such as nitrogenous compounds and carbon dioxide, which the algae use in
photosynthesis. Such a close complementarity between partners increases the success and
evolution of the mutualistic association. Marine animals such as corals grow well in nutrient-poor
tropical oceans because their symbiotic algae provide them with food and oxygen. Another benefit
that may be obtained in a mutualistic association is protection. Some bacteria and unicellular algae
are protected from a hostile environment by residing within a host cell. Such protection has
resulted in adaptations of the symbiont such as a greatly reduced or absent cell wall and loss of
sexual reproduction.

Mutualism, symbiosis, and cooperation

Charles Darwin's theory of natural selection, with its emphasis on competition, struggle for
existence, and survival of the fittest, left out the role of cooperation as a factor in biological
evolution. Darwin was keenly aware of biological interactions in nature, as is evident from his
publications: The Different Forms of Flowers on Plants of the Same Species ( 1877), The Effects of
Cross and Self Fertilization in Vegetable Kingdom ( 1876), The Various Contrivances by which
Orchids are Fertilized by Insects ( 1862), Insectivorous Plants ( 1875), and Earthworms ( 1881).

Unfortunately, in many academic circles, the terms symbiosis, mutualism, and cooperation have
similar meanings and are often used interchangeably. Mutualism also has been used widely to
describe intraspecies cooperative behavior in various animal species. The study of cooperation has
enjoyed a resurgence during the past three decades ( table 1.1 ). Cooperation is defined as a
beneficial outcome relative to cost that an individual or a group requires for a collective action (
Gadagkar, 1997). Cooperation in an evolutionary perspective can be divided into four categories:
(1) cooperation via kin selection, (2) cooperation via group selection, (3) cooperation via
reciprocity, and (4) cooperation Table 1.1 Significant publications on the evolutionary study
of cooperation
Author Publication Year
A. V. Espinas Des Societes Animales 1878
Peter Kropotkin Mutual Aid. A Factor in Evolution 1902
W. M. Wheeler "The ant-colony as an organism" 1911
H. Reinheimer Evolution by Cooperation 1913
W. C. Allee Animal Aggregations 1931
W. C. Allee The Social Life of Animals 1938
A. E. Emerson Social Coordination and the Superorganism 1939
A. E. Emerson The Biological Basis of Social Cooperation 1946
A. Montagu Darwin: Competition and Cooperation 1952
W. D. Hamilton "The evolution of altruistic behavior" 1963
R. Trivers "The evolution of reciprocal altruism" 1971
E. O. Wilson The Insect Societies 1971
E. O. Wilson Sociobiology. The New Synthesis 1975
 Author Publication Year
R. Axelrod The Evolution of Cooperation 1984
D. S. Wilson Reviving the Superorganism 1989
E. O. Wilson Consilience: The Unity of Knowledge 1998

via byproduct mutualism ( Dugatkin, 1997). Although the first three categories have received
sufficient attention from modern evolutionary biologists, the case for byproduct mutualism has yet
to be established. Byproduct mutualism is generally found in the context of interspecific
associations and involves pseudo-reciprocity ( J. Brown, 1983; Connor, 1986, 1995).

Parasitism
Parasitism is a symbiosis in which one of the symbionts benefits at the expense of the other. As in
mutualism, the primary factor in parasitism is nutrition: the parasite obtains its food from the host.
Parasitic symbioses affect the host in different ways. Some parasites are so pathogenic that they
produce disease in the host shortly after the parasitism begins. In other associations the symbionts
have coevolved into a controlled parasitism where death of the host cells is highly regulated ( Ebert
and Herre, 1996).

Parasitologists often describe a host in relation to the role it plays in the life cycle of a parasitic
symbiont ( Toft and Karter, 1990). A definitive or final host is one in which a symbiont reaches
sexual maturity. An intermediate host may also be necessary for the completion of a symbiont's life
cycle. For example, in the life cycle of the sheep liver fluke, Fasciola hepatica, sheep are the
definitive hosts and snails are the intermediate hosts; the fluke's larval stages develop in the snail.

Some parasites need more than one intermediate host. Reservoir hosts are species that harbor
potential pathogenic symbionts often without showing signs of disease. Wild animals are frequently
the source of infection for humans and domesticated animals. Vectors are carriers of transmittable
pathogenic parasites. Many blood-sucking arthropods, such as horseflies, tsetse flies, mosquitoes,
and fleas, are well-known vectors of viral, bacterial, and protozoan pathogens.

Mutualism, parasitism, and commensalism are the only categories of symbiosis considered in this
book. Some scientists recognize other categories, such as phoresis and inquilinism, which are
based on the transport or shelter of one of the symbionts. However, we consider such associations
to be commensalistic. In phoretic relationships, for example, some insects deposit their eggs on
the bodies of other animals, which transport them to new habitats. Some marine hydroids and sea
anemones attach themselves to the shells of molluscs and crabs and are carried about by their
hosts. In inquilinism, two or more animals of different species share a dwelling place. The predation
of one animal by another has been considered to be a type of symbiosis by some investigators.

Terms such as mutualism, parasitism, and commensalism are used to conveniently categorize
associations. But many relationships are not static, and there may be frequent transitions from one
type to another. Symbiotic associations may change because of environ- mental factors or internal
influences caused by the development of the symbionts. A parasitic association could evolve into
one of mutualism or commensalism. Indeed, it is difficult to conceive of two organisms starting out
in a mutualistic association. Most mutualistic symbioses probably began as parasitic ones, with one
organism attempting to exploit another one. If we consider parasitism as an antagonistic
relationship, then mutualism can be regarded as a standoff or a draw between two antagonists. For
example, during the course of a parasitic relationship between two organisms, the host's defenses
may be strong enough to slow or stop the growth of the parasite. Two extreme examples of such a
mutualistic evolution are mitochondria and chloroplasts in eukaryotic cells. These organelles are
transformed bacteria that may have begun as parasitic symbionts in ancient prokaryotic cells.
Conversely, a mutualistic association may degenerate into a pathogenic one if the defenses of the
host weaken. For example, the common intestinal symbiont of humans, Escherichia coli, can
become a pathogen when it acquires plasmid-borne virulence factors from other bacteria.

Pathogens and Disease

Because early workers equated the term symbiosis with mutualism, parasitism is generally
considered to be separate from symbiosis. Terms such as "parasitology," "parasites," and
"parasitism" continue to be used without considering their position in the wider picture of
symbiosis. Parasites have a poor public image because scientists and laymen tend to link disease
and parasitism ( Windsor, 1997). Many parasites, however, do not cause disease; they do not
disrupt or seriously diminish the performance of their host even though they take nutrients from
the host. Parasites and parasitism should be viewed in the broader context of symbiosis and
coevolution ( Ewald, 1994). Pathogens are defined as entities that produce disease conditions in
their host ( Ewald, 1993; Read, 1994).

Common themes in microbial pathogenesis

Recent advances have shown that many bacterial pathogens follow similar strategies to infect their
host and cause disease ( Nee and Smith, 1990). We are ignorant of how diverse microorganisms
are and the concepts of microbial disease causation need to be reassessed ( Relman, 1999).
Common virulence factors include toxins, adhesion mechanisms, how pathogens enter the host
cells, and how they survive once inside the host cell (e.g., remaining in the vacuole or escaping
into the cytoplasm). Avoiding the host immune response is the key to the success of pathogens,
and methods of doing this include antigenic variation, camouflage by host, molecular mimicry, and
enzymatic disabling of the host immune system. Virulence factors are maintained on plasmids and
pathogenicity islands that are easily acquired. Therefore, new strains of pathogens are constantly
evolving ( Finlay and Falkow, 1997) ( fig. 1.1 ).

1.3 SYMBIOSIS IN ALL FORMS OF LIFE

One of the earliest systems of classification, developed by Linnaeus, consisted of two kingdoms,
plants and animals. Although this classification lasted a long time, it was not satisfactory because
some organisms such as Euglena, bacteria, and slime molds did not fit well in either kingdom.
These odd groups were arbitrarily placed into either the plant or animal kingdom depending on the
personal biases of scientists. As techniques in cell biology improved and details of the cell were
better understood, it became clear that the two-kingdom system of classification was too restrictive
and would have to be expanded. In 1969, R.H. Whittaker proposed a five-kingdom classification,
which met with initial resistance but later gained general acceptance. The five kingdoms proposed
by Whittaker were the Monera, Protista, Fungi, Plantae, and Amimalia. In recent times,
phylogenetic lineages based on molecular homologies of more than 60 ancient proteins and rRNA
sequences have provided a new perspective on the evolutionary tree of life ( fig. 1.2 ) ( Corliss,
1994; Brown and Doolittle, 1997).

Viral Symbioses
Symbiotic associations are common in all biological kingdoms, as well as among viruses. We
consider viruses as noncellular entities that have some basic characteristics of living organisms.
Viruses contain genetic material that is transmittable. Their level of symbiotic interaction is
molecular rather than organismal, and they generally are more integrated than other symbioses.
Viruses can form long- lasting associations with their hosts; for example, prophage viruses insert
part of their DNA into a chromosome of the host cell and replicate along with the host. The genetic
colonization of host cells by viruses is a subtle type of symbiosis that has far-reaching implications
for people. This natural form of genetic engineering is mirrored by human attempts to create
artificial symbioses by manipulating the genetic material of different organisms. Recombinant DNA
technology strives to produce symbiotic hybrids that will be of use in medicine and agriculture.

Based on molecular information, the modern domains of life include: (1) Archaea (archaebacteria),
(2) Bacteria (eubacteria), and (3) Eukarya (eukaryotes) ( Doolittle, 1999) ( fig. 1.2 ). Genetic
incongruity between Archaea and Eukarya suggests a sisterhood between these domains. One
horizontal gene exchange might have involved the gram-positive bacteria and Archaea, while
another might have taken place between proteobacteria and eukaryotes through endosymbiosis (
Brown and Doolittle, 1997).
Archaea
Archaebacteria are a major part of the global biomass ( Forterre, 1997) and are useful for studying
basic questions in biology ( Jarrell et al., 1999). All archaebacteria possess ether linkages in the
lipids of their cell membranes. These linkages are absent in eukaryotes and most bacteria. Most
archaebacteria that have been cultured came from extreme environments, hence their popular
name of extremophiles ( Horikoshi and Grant, 1998). Some archaebacteria can grow at
temperatures > 100°C. Methanogens form symbioses with other microorganisms in sediments of
lakes, rivers, and bogs, and they metabolize hydrogen produced by anaerobic bacteria.
Methanogens are also endosymbionts of

Fig. 1.2 Phylogenetic tree of life based on universal rRNA sequences. Six kingdoms of eukarya are
recognized. (Compiled from various sources cited in the text.)

anaerobic ciliates that live in ruminants. Thermoplasma, a wall-less archaebacterium, is often

considered to be a progenitor host cell of eukaryotes that may have acquired ancient bacteria
through endosymbiosis in the evolution of mitochondria and chloroplasts ( Aravalli et al., 1998).

Bacteria as Multicellular Organisms

Bacteria in nature exist as films, mats, colonies, aggregates, and chains and rarely as isolated
cells. The concept of bacteria as unicellular organisms, a very successful strategy that began with
the research successes of Robert Koch, is about to undergo a major paradigm shift in microbiology.
Koch invented the pure (axenic) culture method in which monomorphic traits remain constant, but
studies on mixed cultures proved difficult and frustrating because they often produced
pleomorphism; that is, any microbe could become any other. Pure culture methodology was soon
adopted by mycologists to study fungi for essentially the same reasons. The legacy of Koch's
method was that it eliminated as many variables as possible from the tangled web that normally
exists in nature and opened the way to a reductionist approach to studying the causes of infectious
diseases. After more than 100 years, an alternative view of microbiology is gathering momentum.
Now it has been shown that microorganisms rarely exist alone under natural conditions but rather
interact and form a range of associations with other organisms. Some microbial interactions involve
competition, and successful competitors excrete inhibitors. Microbe-microbe interactions form
biofilms and are very complex, and tools of molecular biology are now being applied to
understanding the dynamics of microbial communities ( Cirillo, 1999). Every day we learn how
versatile the bacteria are, and appreciate their impact on global ecology and geochemical
processes. Bacteria make efficient use of other organisms for their benefit. Bacteria are sensitive,
they communicate and integrate information from their neighbors and their environment, and they
reproduce and survive as multicellular populations. This view takes us far from the old notion that
bacteria are simple, small, and primitive and respond automatically to their changing environment
( Margulis et al., 1986; Sonea, 1988; Shapiro and Dworkin, 1997).

The Aquifex and hydrogenobacter lineage is believed to be the oldest branch of the bacterial
domain. Aquifex pyrophilus is an extreme thermophile that occurs in hydrothermal vents and grows
well at 85°C. Proteobacteria (purple bacteria) are a diverse group of bacteria, and several
members of alpha proteobacteria form significant symbioses with plants and animals. They include
species of Agrobacterium, Phyllobacterium, Rhizobium, Rickettsia, and Wolbachia. The
cyanobacteria are another phylogenetically related group that form symbioses with host organisms.
Cyanelles are cyanobacteria that are endosymbionts. In the case of one cyanelle, that within
Cyanophora paradoxa, because some functional genes have been transferred to the host nucleus, it
has lost the ability to live independently and thus has become equivalent to a cell organelle like the
chloroplast (cyanoplast).

Kingdoms of Eukarya: Archezoa, Protozoa,

Chromista, Fungi, Animalia, and Plantae
Eukarya is a vast assemblage of organisms that includes protozoans, fungi, animals, and plants
grouped into six kingdoms. Although the kingdom Protista is no longer recognized, the term protist
is still used for all protozoa, eukaryotic algae, and lower fungi ( Corliss, 1994). Most of the
primitive lineages of eukarya have yet to be discovered, but some present-day primitive
eukaryotes live anaerobically in thermophilic environments.

Archezoa
Archezoa are the earliest eukaryotes that evolved before the acquisition of mitochondria. They
include Enterocytozoon, Giardia, Hexamita, and Trichomonas, which are traditionally considered to
be protozoans. These amitochondrial organisms have evolved nuclei, endoplasmic reticulum,
primitive cytoskleton, and a 9+2 arrangement of microtubules in the flagella. Giardia lamblia, a
human pathogen, has 70s ribosomes like those of bacteria and also lacks the Golgi apparatus.
Sexual reproduction in Giardia has not been observed.

Protozoa

Protozoa represent lineages of eukarya that evolved after the acquisition of mitochondria and
chloroplasts through endosymbiosis. Most protozoans have a phagotrophic mode of nutrition, and
many are involved in symbiotic associations. The kinetoplastid protozoans such as Euglena,
Leishmania, and Trypanosoma represent early lineages among protozoans. This group produced
two branches, one leading to the cellular slime molds, such as Dictyostelium discoideum, and the
other to apicomplexans such as the malarial parasite, Plasmodium falciparum. Most of these
protists have mitochondria with tubular cristae. In an evolutionary sense, dinoflagellates such as
Symbiodinium are latecomers as protists. They are closely related to ciliate protozoans. By the time
ciliates emerged as an evolutionary group, meiosis and fertilization were regular eukaryotic
features. Phylogenetic lineage based on rRNA homology indicates that at about the time ciliates
appeared, animals, fungi, plants, chlorophytic algae, and chromophytic algae began to evolve.

Chromista

Phylogenetic analysis indicates that there were several evolutionary lineages of photosynthetic
eukaryotes. Chromista includes diatoms and brown algae whose chloroplasts are surrounded by a
unique periplasmic membrane. Diatoms and brown algae evolved in a two-step symbiosis. It began
when a phagocytic protozoan engulfed a photosynthetic protist, which entered the host
endoplasmic reticulum by fusion of phagosome and host nuclear membranes. Most chromists
possess another unifying feature, the retronemes, which are rigid structures that may have
facilitated the endosymbiotic process.

Fungi

The kingdom Fungi includes organisms that are heterotrophic and obtain nutrients by absorption
from living or nonliving organic sources. Phylogenetically, fungi are more closely related to animals
than to plants. They evolved from protozoa some 400 million years ago by acquiring rigid chitinous
cell walls. Fungi excrete enzymes that break down organic matter into simpler, usable compounds
and, like bacteria, are important decomposers in nature. Most fungi grow by means of filamentous
hyphae, although some, such as yeasts, are unicellular. Fungi have diverse reproductive strategies.
Many fungi reproduce sexually, but the resulting diploid zygote undergoes meiosis immediately or
after a resting period to form haploid spores or offspring. Fungi produce many spores and grow
practically everywhere. Fungi possess mechanisms to resist desiccation. Fungi form numerous
wide-ranging associations with cyanobacteria, algae, plants, and animals, and some species are
important pathogens (fig. 7.1 ). Fungi even parasitize other fungi. These mycoparasites are
common and include different groups of fungi. Marine fungi form close associations, called
mycophycobioses, with marine algae. Such associations probably have enabled ancient marine
organisms to colonize the terrestrial environment. Presumably, the fungi protected the more
sensitive algae from drying and helped them obtain water and minerals from the ground. Other
highly successful lines of evolution for many fungi have been associations with algae and
cyanobacteria to form lichens.

Animalia
The kingdom Animalia consists of multicellular organisms that ingest their food. Reproduction
consists of fertilization of an egg by a sperm, and the resulting zygote forms a blastula from which
the animal develops. Animal cells lack walls and most are diploid, the haploid condition occurring
only in the sperm and eggs. Animals, like plants, are hosts to many symbionts and have developed
sophisticated defense mechanisms. The host immune response to symbionts in some cases is such
that mutualistic relationships have evolved ( Clay and Kover, 1996). Insects and flowering plants
have evolved many close relationships during a long period of coevolution. We consider pollination
to be a type of symbiosis, as did de Bary. The contacts between insects and plants during
pollination are transitory, but the repetitive nature of the contacts qualifies these associations as
symbiotic. Insects also use plants as a place to deposit eggs or to hide from prey. Seed plants form
symbioses with birds to achieve seed dispersal, and some birds such as hummingbirds are efficient
pollinators. Numerous symbioses between animals exist, Some birds commonly associate with
large land mammals and not only remove insects from the animal's skin but also provide early
warning of an impending threat from a predator. In the oceans, small fish and shrimp maintain
cleaning stations and remove surface parasites from larger fish that periodically visit the stations.

Plantae

The kingdom Plantae consists mostly of photosynthetic, multicellular organisms, such as green and
red algae, mosses, ferns, and seed plants. The green and red algae and higher plants evolved from
a protozoan ancestor which acquired chloroplasts through endosymbiosis of photosynthetic
cyanobacteria. The red algae have two or more membranes around their plastids and store starch
in the cytoplasm.

Some plants parasitize other plants. Most parasitic plants lack chlorophyll and obtain food from
their hosts, but some, such as mistletoe, can manufacture food and also obtain nutrients from their
hosts. Plant cells have walls that contain cellulose and undergo sexual reproduction. They develop
from embryos which are surrounded by a jacket of sterile tissues. Plants show alternation of
generations in which a haploid generation (gametophyte) alternates with a diploid generation
(sporophyte). In mosses and liverworts the haploid generation is dominant; in more advanced
plants, such as gymnosperms and angiosperms, the diploid generation is dominant. Most terrestrial
plants form symbioses with mycorrhizal fungi. These root -- fungus associations are vital for the
well-being of plants and are far more complex than originally thought. Many of the symbiotic
associations that involve plants are parasitic, as plants serve as hosts to fungi, bacteria, protozoa,
and animals.

1.4 CLASSIFICATION OF SYMBIOSES

Mortimer P. Starr ( 1975) developed a classification of symbiotic associations to standardize the
many conflicting terms that have been used to describe different symbioses. Following is a
modification of Starr's system.

Location of the Symbionts

A symbiont may be inside or outside another symbiont. An endosymbiont is one that resides inside,
whereas an ectosymbiont resides outside a host organism. The distinction between inside and
outside may not be clear. We restrict the term "endosymbiont" to those organisms that reside
within the cells of other organisms. Thus, the extracellular microbes of the digestive tracts of
animals are ectosymbionts.

Technically, few symbionts lie within the host cytoplasm. Most endosymbionts are surrounded by
the host plasma membrane, which separates them from the cytoplasm. Therefore, in a cellular
sense, the symbionts are not within the host cell. The structure with the symbiont enclosed has
been termed a symbiosome and is considered to be an organelle ( Roth et al., 1988).

Persistence of the Symbiosis

Most symbioses are persistent; that is, the symbionts remain together for a long time or their
contacts are frequent. Associations in which one partner is an endosymbiont are usually the most
persistent type. The symbionts remain together through all stages of their life cycles. Many
endosymbionts have an arrested life cycle and generally remain in the vegetative stage. In some
symbioses, the contacts between the partners are intermittent, as in the cleaning symbiosis of
marine fishes or the pollination of flowers by insects and birds. In cases of parasitism, the
symbiosis persists throughout the host's life, the duration of which depends on the virulence of the
parasite.

Dependence on the Symbiosis

Obligate symbionts are so highly adapted to a symbiotic existence that they cannot live outside
it. Facultative symbionts can also live in the free-living condition. Sometimes, it is hard to tell
whether a symbiont is obligate or facultative. For example, a symbiont may exist free-living but in
specialized niches or in small populations that are difficult to identify ( Douglas, 1996). Algae such
as Trebouxia are highly adapted to the lichen symbiosis, but scattered colonies of these algae have
been found living separately. Are such colonies truly nonsymbiotic, or are they established by
individuals that escape from the symbiosis? Obligate symbionts depend on the symbiosis for
nutrients. A symbiosis that involves two organisms, when at least one obtains nutrients from the
other, is called a biotrophic symbiosis. If one of the symbionts dies and the other uses it as a
source of nutrients, the association is called a necrotrophic symbiosis. Organisms may obtain
physical protection from a symbiotic association or receive some other benefit, such as the light
produced by symbiotic bacteria in marine fishes.

Specificity of the Symbionts

Symbionts may be highly specific to one organism, such as the bacterial symbionts of
Paramecium aurelia, or they may associate with different organisms. How specific an organism
is may relate to the evolutionary stage of the symbiosis. Presumably, the more highly evolved
a symbiosis is, the longer the symbionts have had to adapt to each other, and the more
specific is the association ( Douglas, 1995). An extreme example of specificity is seen in
mitochondria, which are semi-independent, transformed organisms that cannot exist outside a
eukaryotic cell.

Symbiotic Products
In some associations interaction of the symbionts results in the formation of new structures or
chemical compounds. For example, a lichen thallus and many of its chemical compounds
develop only as a result of the symbiosis. Similarly, legume root nodules and the red pigment
leghemoglobin are products that develop from symbiotic associations of Rhizobium bacteria and
legumes. Cooperation as a byproduct of mutualism occurs in the context of interspecific
associations.

1.5 SUMMARY
Symbioses are interspecific associations that have played a significant role in the evolution of
plants and animals and in shaping the earth's physical features. There are three main types of
symbiosis: mutualism, parasitism, and commensalism. The boundary lines between these
categories are not clear, and there are frequent transitions between them. The most common
association among living organisms is that of parasitism, and it appears, from an evolutionary
point of view, that mutualism and commensalism arose from parasitism.

Parasites that cause disease are pathogens, and they may be localized or spread throughout a
host. There are several types of host: intermediate host, final host, and reservoir host.

The most widely accepted classification of living organisms recognizes three domains; Archaea,
Bacteria, and Eukarya, and six kingdoms of eukaryotes, Archezoa, Protozoa, Chromista, Fungi,
 Animalia, and Plantae. All forms of life contain symbiotic associations. Viruses are subcellular
symbionts because they have basic properties of living organisms and are commonly found in
many life forms.

Because of the wide diversity of symbioses and the growing number of terms used to describe
them, a classification system for symbiotic associations has been developed. This classification
is based on several features: location of the symbionts, whether ectosymbionts or
endosymbionts; persistence of the symbiosis; dependence on the symbiosis, whether obligate
or facultative symbionts; specificity of the symbionts and symbiotic products. Biotrophic and
necrotrophic symbionts are distinguished on the basis of whether nutrients are obtained from a
living or dead partner.

Research on symbiosis is occurring in many disciplines of biology. There is a growing

awareness of the fundamental importance of symbiosis as a unifying theme in biology, an
awareness that organisms function only in relation to other organisms.

2 VIRAL SYMBIOTIC ASSOCIATIONS

Viruses fascinate us not only because they cause disease, but also because they challenge the way
we define life ( Radetsky, 1994; Dimmock and Primrose, 1994). Viruses are intracellular, obligate
parasites which have evolved numerous methods to survive and reproduce. Ability to reproduce is
a central feature of life, and in the living world the only systems that can reproduce are the ones
that contain nucleic acids ( Lederberg, 1993). Viruses consist of nucleic acids that replicate inside
living cells and cause the synthesis of virions that can transfer the genome to other cells ( Luria et
al., 1978).

Viruses lack metabolic activity and therefore depend on host cells for many of their functions. Like
most living organisms, viruses replicate, mutate, and adapt to their host cells. Many viruses are
pathogenic, but, in some cases, viruses and hosts have evolved together. Today, we know of about
3000 different viruses, but only a few of these have been studied intensively. New viral host
relationships are constantly being discovered. Viruses have been found in all major categories of
life forms. Despite their diversity, viruses have many features in common ( Voyles, 1993).

2.1 VIRUS STRUCTURE

Viral Genome
Viral genes may be clustered together on a single DNA or RNA molecule, or a virus may have a
segmented genome consisting of several nucleic acid molecules. Influenza virus has a segmented
genome of eight unique strands of RNA, whereas measles and rabies viruses each have a single
RNA molecule ( Webster et al., 1992).

The simplest virus has a single strand of RNA made up of several thousand nucleotides. If this RNA
is a plus strand, its genetic message can be translated directly by the host cell's ribosomes.
Examples of plusstranded RNA viruses are the bacteriophage QB, which infects Escherichia coli, and
the polio virus in humans. Minus-strand RNA must first be transcribed into a complementary plus
strand before viral replication can begin. Influenza A virus in humans is an example of this type.

Retroviruses contain single-stranded RNA which, after entering a host cell, changes into a double-
stranded DNA with the help of a viral enzyme, reverse transcriptase. The newly synthesized DNA
can then integrate into the host's own DNA. Examples of retroviruses include human
immunodeficiency virus (HIV1), human T-cell leukemia virus (HTLV), and Rous sarcoma virus
(RSV) ( Varmus, 1988).

Viral capsid
A capsid is the protein coat that surrounds the viral genome. It may be composed of only one type
of protein or of several different types. Capsids are made up of capsomers, which are arranged in
either isometric or helical symmetry. Tobacco mosaic virus (TMV) is a helical virus that forms a
spiral around its nucleic acid. The capsid of an isometric virus, such as polio virus, forms an
icosahedron of 20 facets ( Guo, 1994) ( fig. 2.1 ).

Fig.2.1 Types of viral particles and their structural features. Adapted from Matthews ( 1991a).

Viral Envelope
Some animal viruses have a host-derived membrane or envelope around the capsid coat. The
envelope is made up of phosopholipids and proteins. The envelope proteins are specified by viral
genes and may have free ends consisting of carbohydrates. Together, they form spikes on the
outer surface of the virus ( Fields et al., 1996). Some envelope proteins help a virus attach itself to
the host cell; others cause the host cell to lyse. The envelope is acquired during the maturation
process after the virions have been assembled. An envelope is a common feature of animal viruses
but is absent in plant viruses. In herpes virus, the envelope is derived from the nuclear membrane,
whereas in vaccinia virus, it comes from the Golgi apparatus. Nonenveloped or naked viruses lack
lipids.

Structure of the Human Immunodeficiency Virus

Human immunodeficiency virus is a complex virus which contains two identical copies of a positive-
sense RNA (similar to mRNA) of about 9500 nucleotides. The two RNA strands are linked to form a
genomic RNA dimer, which associates with a nucleocapsid (NC) protein called p9/6. The
ribonucleoprotein, in turn, is enclosed by the capsid protein (CA), p24. The capsid thus formed also
contains other viral proteins such as integrase and reverse transcriptase along with
macromolecules derived from the host cell cytoplasm. The capsid has an icosahedral structure and
is enclosed by a layer of matrix protein (MA), p17, that is associated with an envelope. The HIV-1
envelope is acquired when the virus exits through the host cell membrane. A viral envelope may
carry viral proteins that form spikes. In HIV-1, the most significant of these spike-forming proteins
is gp120/41, which attaches the virus to a host cell. Gp41 is a transmembrane protein; gp120 is a
surface protein that is attached to gp41. Both proteins are manufactured in the host cell's
endoplasmic reticulum and are transported to the cell surface by means of the Golgi complex ( Nye
and Parkin, 1994) ( fig. 2.2 ).

2.2 VIRUS TAXONOMY

The classification of viruses appears confusing because the system is a hybrid of several traditional
approaches. Animal virologists have used a system modeled after Linnaeus's zoological
classification, with families, genera, and species, while plant virologists have classified viruses
according to their hosts.

Today all viruses are ranked into species, genera, and families. The important taxonomic
criteria include host organism(s), virus morphology, and genome type. In 1966, the
International Committee on Taxonomy of Viruses developed a system of virus classification and
nomenclature, and an outline of its latest edition ( 1995) is presented in an appendix to this
chapter.Viruses may be investigated by examining infected host cells, observing the effect of
viral growth on a cell culture, or measuring the antibody response that a virus evokes in a host
animal. Much has been learned about viruses from their cytopathic effects on hosts and from
electron microscopic observations of viral particles in host cells. In many naturally occurring
virus -- host symbioses, there are no recognizable cytotopathic effects, and such infections are
called persistent ( Murphy et al., 1995).According to the "Baltimore Classification" there are
seven different groups of viruses:
1. Double-stranded DNA viruses (dsDNA) (adeno-, herpes, and pox viruses) have DNA which
may be linear or circular. Adenoviruses replicate in the nucleus using cellular proteins,
whereas others such as poxviruses replicate in the cytoplasm and make their own
enzymes for viral DNA synthesis.
2. Circular, single-stranded DNA viruses (ssDNA) (parvoviruses) replicate in the nucleus by
forming a minus-sense strand, which serves as a template for plus-strand RNA and DNA
 synthesis.
3. dsRNA viruses (reoviruses, birnaviruses) have segmented genomes. Each segment
produces a distinctive monocistronic mRNA.
4. ssRNA viruses with a plus-sense genome (picornaviruses, togaviruses). Picornaviruses
(hepatitis A) have polycistronic MRNA. The naked RNA is infectious and when translated
forms a polypeptide chain from which mature protein is cleaved. Togaviruses require two
or more rounds of translation to produce viral RNA.
5. ssRNA viruses with minus-sense genome (orthomyxoviruses, rhabdoviruses) have viral
RNAdependent RNA polymerase.
6. "Diploid" ssRNA viruses (retroviruses) have a plus-sense genome that does not serve as
mRNA but as a template for reverse transcription.

a HIV-1 virus

b The attachment of HIV-1 to CD4 T cell

c HIV-1 replicative cycle in the infected host cell Fig. 2.2 Human immunodeficiency virus, HIV-
1: (a) principal structural features, (b) interactions between viral proteins gp 41 and gp 120
with host cell surface receptor, (c) steps involved in HIV-1 entry into and release from host
cell. (Compiled from various sources cited in the text.)
1. dsDNA viruses (hepadnaviruses) also need reverse transcription, which occurs inside the
mature virus particle.

2.3 VIRAL REPLICATION: VIRUSES AND

HOST CELL INTERACTIONS
All viruses penetrate the host cell, replicate, and then exit the infected cell. Six generalized stages
are recognized in a typical viral replication ( fig. 2.3 ).
1. Adsorption involves the attachment of the virus to a host cell. Specific regions on the host
cell surface serve as virus receptors. Viral adsorption involves weak forces such as ionic or
hydrogen bonds. In addition, there may be a complex interplay at the receptor sites.
2. Penetration and uncoating introduce the viral genome into the host cell. Viral structural
identity is lost during this stage. Some viruses inject their nucleic acid into a host cell and
leave their protein coats behind as empty shells. Other viruses enter a cell with their protein
coats intact. After a whole virus has entered a cell, the viral nucleic acid must be freed from
its protein coat before the virus can replicate and initiate viral protein synthesis.
3. Synthesis of viral proteins and viral genome depends on the activity of a specific enzyme
replicase. Viral mRNA directs the synthesis of enzymatic proteins by mobilizing the host cell's
resources. The normal host metabolism is altered after the infection process so that it
produces new viral particles.
4. Synthesis of viral structural proteins may occur at the same time as the viral genome is
being replicated, but it usually lags behind. New viral genome and structural proteins
accumulate within the host cell.
5. Maturation is the phase during which newly synthesized nucleic acid molecules and protein
subunits assemble into virions (virus particles). Symmetry is important in viral assembly.
Some viruses such as tobacco mosaic virus can selfassemble, while others such as polio virus
require the participation of cellular chaperones in the viral assembly ( Fields et al., 1996).
Herpes viruses assemble in the host cell nucleus and acquire an envelope as they pass
through the inner nuclear membrane during the budding process. Virions accumulate as
inclusions in either the cytoplasm or nucleus.
6. Release is the exit of mature virions from the infected host cell and can occur suddenly or
gradually. The host cell often disintegrates because viral replication damages the normal
cellular functions. Some viruses remain in a resting stage until the cell dies.
2.4 VIRAL PATHOGENESIS
Viruses produce disease in the host in different ways. Capsid proteins are often toxic to host cells
and cause cell death and the release of new viral particles. Viruses show a high degree of host
tissue specificity because of surface receptor molecules that are used for virus attachment.
Adenoviruses, orthomyxoviruses, and paramyxoviruses have spikes on their outer surfaces that
attach to the host cell. Viruses such as polio, herpes simplex, togavirus, and pox inhibit protein
synthesis of the host cells. Herpes simplex virus can also inhibit and degrade cellular DNA ( Cann,
1997).

Virus-infected cells often show distinctive structural and physiological abnormalities which are
collectively termed cytopathic effects. The most common of these effects are the inclusion bodies,
which are usually the sites of viral assembly or cellular damage. Virus-infected cells may detach
from the substrate, lyse, or form a syncytium, which is a multinuclear giant cell that results from a
mass of fused cells. Syncytia are characteristic of cytomegalovirus, measles, herpes simplex,
paramyxoviruses, and HIV-1 ( T. E. Shenk, 1993).

Some viruses depress host cell activities in general but stimulate biochemical processes that
involve viral replication. The viral genomes of herpes viruses, hepatitis B virus, and HIV-1 contain
enhancer regions that increase the efficiency of viral transcription in specific host tissue cells. For
example, the papilloma virus has an enhancer region in its genome that becomes activated only in
skin cells ( Bernard, 1994).

Virulence of the influenza virus is controlled by hemaglutinin and neuraminidase, proteins that form
HA and N spikes in the envelope of the virus particles. Hemaglutinin needs to be cleaved at a
specific amino acid site in order to activate viral virulence. A mutation with a different amino acid
at the cleavage site renders the virus avirulent ( Webster et al., 1992; Scholtissek, 1996).

After a virus replicates in one type of tissue, it can spread to other host tissues that have the
appropriate cell surface receptors. Polio virus replicates first in intestinal cells and then spreads to
cells of the central nervous system, where it causes paralysis of the limbs by destroying motor
neurons in the spinal cord ( White and Fenner, 1994). Respiratory viruses such as influenza
replicate first in the epithelial cells of the lungs, where they cause lesions and block air passages.
The virus then spreads to cells lining the trachea ( Atlas, 1997).

Viral infections that produce diarrhea often interfere with absorption of fluids and loss of lactose-
degrading enzymes in the intestine. As lactose accumulates in the digestive system, it lowers the
sodium -- potassium ATPase activities and leads to acidosis, which changes the rate of potassium
ion exchange and causes a buildup of water in the intestine ( Atlas, 1997).

In some human viral infections, the disease symptoms are the result of the body's own immune
response, such as the skin rashes associated with measles ( G. L. Smith, 1994). Cytomegalovirus
and HIV-1 cause host cells to fuse and thereby allow the viruses to infect new cells without
encountering host antibodies. Glycoproteins in the envelope of HIV-1 inhibit T-cell proliferation (
Nye and Parkin, 1994). Hepatitis B virus produces large amounts of antigens that are not
associated with the fully assembled virions. These antigens counteract antibodies in circulation so
that when the virus particles are released, there are few antibodies present to interact with them (
Tiollais and Buenidia, 1991; Bradley, 1993).

Some viruses, such as HIV-1, herpes simplex virus 2, hepatitis B virus, human papilloma virus, and
Epstein-Barr virus, have oncogenes. When the oncogenes are transferred to new host cells, the
cells become malignant. Cancer results from either activation of oncogenes or repression of tumor-
suppressor genes. Oncogenic viruses have v-onc genes that are similar to the host's normal
developmental genes (proto-oncogenes). An oncogene may encode for a growth factor, a signal
transducer, or a transcription factor ( Hausen, 1991).

Virokines (also called viral cytokines) are viral proteins which are not needed for replication but
are necessary for host cell infection. Virokines suppress the host's immune system and often mimic
host cell molecules that are active in the immune response. Virokines are produced by retroviruses,
pox viruses, herpes viruses, and adenoviruses. Pox viruses produce virokines that mimic receptors
for tumor necrosis factor; HIV-1 produces a virokine that inhibits protein kinase C, thereby blocking
signal transduction ( Atlas , 1997). The herpes simplex virus produces a protein (UL6) similar to
one in the cornea, causing a human autoimmune disease, herpes stromal keratitis, which leads to
blindness. Tcells with UL6 protein from virus-infected animals produced a severe corneal
autoimmune disease in healthy animals. This study suggests that molecular mimicry by viral
proteins may be a significant component of virus-induced autoimmune disease ( Zhao et al.,
1998).

Case Study: HIV-1 Pathogenesis

The success of HIV-1 infection is largely due to (1) a "Trojan horse" mechanism which allows the
virus to escape detection by replicating in lymphocytes; (2) latency, which allows the virus to
control its own expression by means of virus-encoded regulatory proteins (tat and rev); and (3)
antigenic variation, which allows new strains to emerge.

The depletion of CD4+ cells of the immune system is a characteristic feature of AIDS ( Levy, 1993).
HIV-1 can replicate in blood, brain, intestinal, and skin tissues. With the help of the envelope
proteins gp 120 and gp41, an HIV-infected cell can fuse with other infected CD4+ cells. This leads
to multinucleate syncytia, which die from altered permeability of their cell membranes. Cell death
also occurs when viral DNA accumulates in the host cell cytoplasm, as has been observed with
brain cells. Gp41 envelope protein may be cytotoxic to the infected host cell. HIV-infected cells
produce large amounts of gp120, some of which is stablized in the membrane by gp41, while the
rest is released from the infected cell. The freed gp120 binds to the CD4+ receptors of healthy T-
cells and brings about their programmed death or apoptosis ( Johnson son et al., 1992). Gp120
also can bind with immature T lymphocytes in the thymus and cause their early death.

Decline of CD4+ T-cell populations in an HIV-infected person alters the immune system. HIV-1-
infected T-cells produce viral cytokines, which may cause Kaposi's sarcoma, B-cell lymphoma, and
anal carcinoma. As HIV-infected individuals survive longer, other cancers such as cervical
carcinoma and Hodgkin's lymphoma may appear, and other viruses may become involved ( Wei,
1995). Cytomegalovirus, human papillomavirus, and hepatitis B virus are all associated with
Kaposi's sarcoma. Similarly, the Epstein-Barr virus is correlated with the development of Bcell
lymphomas ( Levy, 1993; Weiss, 1993; Masucci and Ernberg, 1994).

2.5 PERSISTENT VIRAL INFECTIONS

Not all viruses kill their host cells or trigger an immune response. Some viruses form longlasting
symbioses with their host by altering a specialized function of the infected cells such as hormone
production. Viruses may be responsible for human illnesses such as growth retardation, diabetes,
neuropsychiatric diseases, autoimmune diseases, and heart disease ( Oldstone, 1989).

Viruses are genetic parasites that have evolved different strategies for survival and reproduction.
They must infect a host cell and exploit its cellular machinery in order to replicate. Both polio and
common cold viruses cause acute infection and rapid cell death, whereas herpes simplex virus can
remain dormant in a cell for long periods before being activated. Oldstone ( 1989) has investigated
the lymphocytic choriomeningitis virus (LCMV), which is endemic in some wild mice populations.
Virus-infected neuroblastoma cells in tissue culture cannot synthesize and degrade acetylcholine.
The infected cells continue to grow and metabolize normally. Microscopically, virus-infected cells
are indistinguishable from noninfected cells. In laboratory studies, the pituitary glands of LCMV-
infected mice produced 50% less growth hormone than normal glands ( Old- stone , 1989).

Most persistent viral infections are asymptomatic, but a few cause cancers or severe organ
dysfunction. When human cytomegala virus infects early embryonic cells, a few viral genes are
expressed in the host cells, and the infection becomes persistent. In an immunocompromised host,
however, the virus is reactivated and causes a life-threatening disease. Herpes simplex virus
normally kills the epithelial cells it infects, but in neurons the virus becomes latent ( White and
Fenner, 1994).

The introduction of myxoma virus to control the expanding rabbit population in Australia is an
excellent example of virus-host coevolution. European rabbits were introduced into Australia in the
mid-1850s as a source of meat, and they multiplied uncontrollably in the absence of natural
enemies. Myxoma virus is endemic in its natural host, the South American rabbit, in which the
virus produces wartlike, benign skin tumors. The virus is spread in nature by mosquitoes or rabbit
fleas, but it does not multiply in its vector. In European rabbits the virus causes the disease
myxomatosis, which is characterized by fatal skin lesions. When the myxoma virus was first
introduced into Australia in 1950, it killed more than 99% of the infected rabbits, but it did this so
rapidly that the most virulent strains of the virus died with the rabbits. Virus-resistant rabbits
multiplied, and 7 years after the virus was introduced, only 25% of infected rabbits died. The virus
is now established within the Australian rabbit population as a persistent, nonlethal infection (
Fenner , 1983).

2.6 INTERFERONS
Interferons are protein molecules that are secreted by animal cells in response to infection with
viruses. Although all types of viruses can stimulate interferon production, the most significant
response is obtained with doublestranded RNA viruses. Interferon (INF) does not affect viral
metabolism in the host cell, but when neighboring healthy cells are treated with interferon, they
become resistant to viral infection. There are three types of interferons, a, b, and v, of which INF V
is the most effective cell regulator. The interferon molecules bind to the cell membrane and inhibit
synthesis of viral nucleic acid and protein by inducing the host cell to produce two enzymes, 2,5-
oligoA synthetase and an RNAactivated protein kinase, to create an "antiviral state." Interferons
are powerful drugs against virus infection but produce severe side effects; therefore, their use as
antiviral therapy agents has been limited to life-threatening infections. Interferon produced by an
infected cell may constitute its primary defensive response until the host can produce antibodies.
Several biotechnology companies are attempting to clone human interferon genes in order to
mass-produce interferons. Large quantities of specific interferons will allow studies to be made on
their effectiveness against human cancers ( Cann, 1997).

2.7 RETROELEMENTS: RETROVIRUSES

AND SELFISH DNA
Retroelements are genetic sequences that occur as DNA and RNA and can move through an RNA
molecule and make DNA by reverse transcription. The discovery of the enzyme reverse
transcriptase in the 1970s revolutionized ideas about genomic evolution. Retroelements are
grouped into three major categories:
1. Viral retroelements (e.g., retroviruses, hepadnaviruses, and caulimovirus).
2. Eukaryotic, nonviral retroelements: transposable retroelements, (e.g., copia and gypsy
elements in Drosophila; copialike elements in plants); retroposons (e.g., long interspersed
nucleic acid elements [LINEs] in mammals; mitochondrial introns and plasmids); and
retrosequences (e.g., short interspersed repetitive elements [SINEs]).
3. Bacterial retroelements (retrons). All retroelements are made up of a gag-pol replication core,
to which are added other genes that adapt the retroelements to its host.

Retroelements, in many copies, are widely dispersed throughout the genomes of animals, plants,
fungi, protozoa, and bacteria.

The discovery of retrons in the archaebacterium Myxococcus xanthus and the myxobacterium
Stigmatella aurantiaca in 1981 established the reverse transcriptase genes as the oldest known
genetic elements. Retron genes may have served as a genetic link between prokaryotes and
eukaryotes and thus have been the possible ancestor of all retroelements ( Patience et al., 1997).

Retroviruses are widespread throughout vertebrate species. Retroviruses copy their own RNA
genome into DNA with the help of the virally encoded enzyme reverse transcriptase. The newly
synthesized DNA (provirus) then becomes inserted into the host chromosomal DNA with the help of
another viral enzyme, integrase. The integration site of the provirus is random and often triggers
disruptions of adjacent genes. Oncoviruses, for example, involve insertion activation of cellular
oncogenes that closely resemble the host cell's regulatory genes. In 1989, Howard Temin proposed
that all viral DNA or RNA, which share similarities, were once transposons or retrotransposons that
arose from the host cellular genome by acquiring the ability to replicate autonomously.
Accordingly, some ancient transposable elements, by possessing additional genes that allowed
them to be transmitted horizontally, evolved into infectious particles. The infectious particles may
infect the same cell type or a new type of host cell, making it possible for them to spread across
the species barrier ( Varmus, 1988; Temin, 1989).

Reverse transcriptase associated with eukaryotic nonviral retroelements generates extra gene
copies by using mature mRNAs as a template. In addition, reverse transcriptase produces DNA
from all types of nonmessenger RNAs, which upon insertion in the host genome become long
repeated sequences of LINEs/SINEs. Novel genes may emerge when such retrogenes move into
new genomic neighborhoods and thus become a powerful driving force of evolutionary change (
Brosius and Tiedge, 1996).

Retroelements can be regarded as molecular parasites with selfish nucleic acid, which may enhance
their own survival and also that of the host ( Leib-Mosch and Seifarth, 1996). Thus, there has been
a kind of mutualism between the selfish genes and the host genome that gains genetic variability
as a consequence of retroelement translocation. The coevolution of retroelements and their host
DNA has been going on, perhaps, throughout the history of life on earth ( Hull and Covey, 1996;
Patience et al., 1997).

2.8 VIRUSES IN BACTERIA

Bacterial viruses (bacteriophages or phages) are distributed widely throughout the world of
bacteria and cyanobacteria. They are excellent models of virus-host interplay and have played a
significant role in bacterial evolution. Bacteriophages serve as vectors in the genetic engineering of
bacteria, being used to transfer genes of interest to areas near particular promotor sites.

There are about 12 groups of bacteriophages, including the most widely studied phage system, the
T-series (T1 to T7) phages, which infect Escherichia coli. T-even phages (T2, T4, and T6) have
elaborate capsid proteins and contractile tails, whereas T-odd phages (T3, T7) lack contractile tails.
T-even phages have terminal redundancies; that is, some base pairs are repeated at both ends of a
linear DNA molecule. After adsorption to a specific receptor in the bacterial cell wall, the phage
DNA enters the cell, while capsid proteins remain outside the infected cell. When an infected
bacterial cell undergoes lysis, it releases 70-300 new phage particles. The entire infection process
from initial contact to the release of new bacteriophages (latent period) takes less than 30 min (
fig. 2.3 ).

Virulent phages such as T-even phages do not integrate their DNA into the host cell chromosome
and usually cause the host cell to lyse. Temperate phages can integrate into the host DNA, causing
lysogeny. This condition of the bacteriophage is the prophage state. As a prophage, the viral DNA
replicates along with the bacterial DNA and, through such an integration, transforms the genetic
properties of the bacterium that determines virulence. This phenomenon is called viral conversion.
Some environmental stresses such as UV radiation, X-rays, or chemicals may activate the
temperate phage and cause it to replicate. Lysis of the host cell results, and a new generation of
viral particles is ready to infect other host cells. Bacterial cells containing prophages are lysogenic
and immune to superinfection by the same phage, due to repression of transcription by the
resident prophage. Some temperate phages, such as Mu, act as transposons and move from site to
site in the bacterial chromosome.

The λ phage establishes a stable lysogenic relationship as a prophage. This phage can be made to
alternate between the lysogenic and lytic growth cycles and is one of the best understood genetic
control systems. When phage λ injects its DNA into the host cell, the bacterial cell has neither
repressor nor Cro proteins. The balance between the λ repressor and Cro protein determines
whether a lysogenic or lyric pathway is followed. The C1 repressor gene of the λ phage encodes for
a wellcharacterized repressor protein that binds to two operator regions called OL and OR. When
repressor protein is bound to these two operators, RNA polymerase is unable to bind and initiate
transcription of λ lytic genes. Thus, the lytic genes are repressed and the dormant prophage is
passed from one generation to another. The lysogenic state is maintained as long as the lyric
pathway genes are tightly repressed. In addition, the presence of lambda

Fig. 2.3 Schematic life cycle of a T-even bacteriophage on its host Escherichia coli. The
bacteriophage enters the lytic or lysogenic cycle depending on the synthesis of repressor cl protein.

repressor stimulates the synthesis of more represser in a self-regulation process. Exposure of

lysogenic cells to UV light activates a protease which cleaves the λ repressor protein and renders it
nonfunctional.
In the absence of λ repressor protein, two new proteins, N and Cro are synthesized. Mutations in
the operator regions, OL and OR, will prevent the repressor protein to bind and thus end any
possibility for lysogeny. Similarly, mutation of Cro or N will abolish any potential for host cell lysis.

Lysogenic conversions, which result in new genetic properties for the host, have been observed in
Clostridium botulinum, Corynebacterium diphtheriae, Salmonella sp., and Vibrio cholerae ( Atlas,
1997; Voyles, 1993). In Salmonella, ε phages were the first viruses known to alter the antigenic
properties of host bacteria. The cholera pathogen, Vibrio cholerae, though rare in Western
countries, threatens the rest of the world with sporadic outbreaks. These sudden outbreaks of the
disease have been linked to the conversions of surface antigens induced by phages. Significant
discoveries have been made on the nature of proteinaceous toxins of C. botulinum and C.
diphtheriae. It has been confirmed that these toxins are the products of prophages residing on the
host chromosome. Currently, scientists think that several toxins previously believed to be of
bacterial origin may in fact be the products of phage genes.

Some intron-containing bacteriophages have been described as parasites of bacteriophages (

Belfort, 1989). The bacteriophage introns contain a gene for endonuclease that allows the introns
to splice themselves and thus behave as mobile genetic elements within the phage DNA. These
self-splicing introns confer no particular selective advantage to a T4 phage. In a strict sense, the
introns act like genetic commensals. Endonuclease genes may be viewed as symbionts of introns,
as introns are genes within genes, with their own transcriptional and translational signals. The
bacteriophage introns containing genes for endonuclease can thus be viewed as mutualists because
introns get propagated, and a gene for endonuclease is provided with a safe haven by the intron.
Together, they form a highly efficient mobile element ( Derbyshire et al., 1995).

Cyanobacteria contain viruses that are similar to T-phages in morphology and life cycle but are not
genetically related. Cyanophages inhibit growth of the infected cyanobacteria. In this respect,
cyanophages may have a useful role, as "blooms," or population explosions of some cyanobacteria,
can kill aquatic organisms and diminish water quality in ponds, lakes, and reservoirs.

Viruses have been reported in several actinobacteria such as Actinomyces bovis and Nocardia
farcinia which are abundant in soil. These viruses resemble a T-phage but differ in the structure of
their tail pieces and in the absence of base plates and contractile fibers.

2.9 VIRUSES IN INSECTS

Under natural conditions, most viral infections of insects are subclinical or latent and are often
difficult to detect. Insects may have evolved an efficient mechanism that prevents viral infections
from reaching pathogenic levels. Under ecological stresses, however, these viral infections may be
expressed, a situation that often results in host death. Below are some examples of well-
established virus-insect symbioses.

Wasp Polydnavirus Symbiosis

One of the most complex symbiotic associations in nature involves a wasp, a caterpillar, and a
virus ( Beckage, 1997b). The wasp is an endoparasite of the caterpillar, and when it deposits its
eggs it transmits a virus that disables the caterpillar's immune system. The association is so close
that if the caterpillar dies before the wasp larvae have matured, the wasp also dies. One of the
polydnavirus-encoded proteins called early protein 1 (EP1) is produced within minutes by the virus-
infected hemocytes. As long as EP1 protein is produced, the wasp eggs will continue to develop
within the caterpillar. Additional studies have indicated that immediate, short-term protection
against the host's immune system is conferred by wasp ovarian protein molecules which are
injected during oviposition ( Lavine and Beckage, 1995).

The virus also delays caterpillar development by stimulating its production of juvenile hormone,
which suppresses the caterpillar's pupation. Each polydnavirus particle is composed of up to 28
separate, circular DNAs, which are scattered throughout the chromosomes of both male and female
wasps. Virusfree wasps have not been found. The integration of polydnavirus into wasp
chromosomes raises some interesting questions about the
origin of this virus. Researchers have noted a similarity between the viral protein EP1 and wasp
venom. Antibodies against wasp venom can also recognize the viral protein ( Flemming , 1992). In
an evolutionary sense, polydnavirus may have picked up the wasp venom genes, or the wasp may
have found an efficient way to use its venom protein by copying these genes and packaging them
in the caterpillar's hemocytes. Both wasp and virus seem to share a genetic evolutionary history (
fig. 2.4 ).

Baculoviruses as Viral Pesticides

Baculoviruses are one of the largest groups of insect pathogenic viruses. They can be isolated from
vegetation and soil, where they remain viable for several years. Field studies have demonstrated
the usefulness of baculoviruses as pesticides. Two subgroups of baculoviruses are the nuclear
polyhedrosis viruses and granulosis viruses. These viruses have a narrow host range, and their
replication is limited to insects. Therefore, these viruses are considered safe and ecologically
acceptable as pesticidal agents.

Nuclear polyhedrosis viruses (NPVs) form polyhedral bodies in cells of insects such as the
caterpillars of butterflies and moths and larvae of bees and flies. The rod-shaped virus, which
contains DNA, multiplies within the host cell nucleus, which enlarges, and this ultimately causes the
cell to burst. Infected larvae often stop feeding and move, en masse, to different areas of the
plant. For example, infected nun moth larvae gather on tree tops. There they change color,
become fragile, and die when their epidermis ruptures and releases virus inclusion bodies. The
polyhedral inclusion bodies contain virus particles, which are enclosed either singly or in bundles

Fig. 2.4 An example of a three-way symbiosis among polydnavirus, a parasitoid wasp, and its host
the caterpillar. Adapted from Beckage ( 1997b).

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within a crystalline protein. Because of their size (up to 15 µm in diameter) and their ability to
refract light, inclusion bodies are easily seen with a light microscope. Upon liberation, the virus is
spread to neighboring plants by rain and wind. When ingested by larvae, the virus migrates from
the intestine to susceptible tissues in the hemocoel. Nuclear polyhedrosis viruses may also be
transmitted by eggs of adult insects ( Wood and Granados, 1991).The United States and Canada
have registered seven baculoviruses for use against forest and agricultural pests. Gypchek, for the
control of Gypsy moth, and Tm-BioControl-1, for the control of Douglas-fir tussock moth, are two
successful viral pesticides used by the U.S. Forest Service ( Federici and Maddox, 1996).
Baculoviruses are also being used as viral pesiticides in Europe, Russia, and China. The most
successful commercial use of a baculovirus has been to control the velvet bean caterpillar
(Anticarsia gemmatalis), which is a serious pest of soybeans in Brazil ( Wood and Granados, 1991).

Iridescent Viruses
One of the best known viral infections of insects involves the larvae of the crane fly. These larvae
have a blue-green iridescence from the crystalline arrays of viral particles in their tissues. The virus
also occurs in beetles, mosquitoes, and midges. Most insects acquire the virus during feeding. The
virus, in addition to DNA, contains lipids and proteins, resides in the host cytoplasm, and in the
advanced stage of infection may account for one-quarter of the insect's weight. This is the highest
weight proportion known for an animal viral infection. Iridescent viruses have been used as a
biological control for mosquitoes.
2.10 MYCOVIRUSES: VIRAL INHABITANTS OF FUNGI
Mycoviruses share the following features: (1) they are endogenous to the host cell not only during
replication but also during transmission; (2) their genomes consist of dsRNA; and (3) many cause
asymptomatic infections and can be described as latent and persistent. Mycoviruses occur in all of
the major groups of filamentous fungi. Some examples of mycoviruses include:
1. Agaricus bisporus viruses that cause degenerative diseases of cultivated mushrooms. These
viruses are transmitted through hyphal fusions and are also carried by spores of the fungus.
2. Viruses of Penicillium, including species that produce the antibiotic penicillin, of
Gaeumannomyces graminis, a causal agent of take-all disease in cereals, of Helminthosporium
 maydis, a causal agent of corn blight, and of Helminthosporium victoriae, which causes blight
of oats.
3. Killer system viruses in the yeast Saccharomyces cerevisiae and in the corn smut fungus
Ustilago maydis.

Agaricus bisporus viruses have been studied for their virulence and pathogenicity ( Lemke, 1979).
Disease severity is correlated with the concentration of viral particles. The virus accumulates in the
vegetative mycelium as well as in the fruiting body and spores. In spores, the virus particles occur
in the cytoplasm; in the vegetative mycelium they accumulate in the vacuoles in crystalline arrays.
Disease symptoms include deformities of the mushrooms because of degeneration of infected
mycelium.

Saccharomyces cerevisiae consists of several strains. The killer strain produces a toxin that kills the
cells of sensitive strains. The killer system in this yeast was earlier thought to have been a genetic
phenomenon with three phenotypic expressions: killer, sensitive, and neutral. It is now believed
that a virus is closely integrated with the yeast cells. The infected host cells contain segments of
dsRNA, which represent the viral genome and contain the determinants for toxins and immunity in
the killer system. In the yeast, more than 20 nuclear genes may be required to regulate the dsRNA
segment, which is maintained as a wild-type allele. The viral genome is frequently lost through
mutation, and the daughter cells then become sensitive to the killer strains.

A killer system also exists in Ustilago maydis, where extra dsRNA genomes have been noted in
killer and immune strains of the fungus. The killer strain secretes a protein toxin that is lethal to
sensitive strains. The killer toxins are encoded by satellite dsRNAs, which are dependent on a
helper virus.

Many fungal viruses are associated with their hosts as latent infections. Several viruses

that infect plants are transmitted by fungi. It has been suggested that fungi may be alternate hosts
for plant viruses. Tobacco mosaic viruslike particles have been found in fungi such as rusts and
powdery mildews. Virulence of plant pathogenic fungi may be altered because of their mycoviral
symbionts. The chestnut blight fungus, Cryphonectaria (Endothia) parasitica, causes a devastating
disease of the American chestnut. The tree now survives as stump sprouts, whose shoots become
infected with the fungus and die. The strain of C. parasitica commonly found in Europe shows a
reduced level of virulence along with a few fungal spores that are depigmented. This hypovirulent
strain contains a dsRNA virus. In 1992, virus-infected strains of C. parasitica were isolated from
recovering chestnut trees in New Jersey ( Ghabrial, 1994). Another mycoviral infection reduces the
pathogenicity of Gaeumannomyces graminis, which causes take-all disease in cereals. Mycoviruses
have been considered as a possible means of biological control of pathogenic fungi.

Viruslike structures called concentric bodies have been found in more than 100 species of
lichenized fungi as well as in some common plant pathogenic fungi. Concentric bodies occur in all
parts of a lichen thallus and do not appear to harm the mycobiont ( Ahmadjian , 1993).

Mycoviruses offer an excellent model for understanding the molecular basis of the evolution of
virus--host relationships. Mycoviruses produce stable associations with their host. Viral latency
benefits host survival, whereas viral persistence benefits the virus. In this mutualistic symbiosis,
the virus gains a more efficient means of transmission when the integrity of a host system is
maintained. There is a close interaction between host genes and fungal viruses ( Ghabrial, 1994).
Viral replication in fungi is controlled in such a way that its rate does not exceed the rate of host
cell division. Host-mediated transmission of mycoviruses may sometimes lead to viral
degeneration. For example, many viral particles in mushrooms lack nucleic acids.

2.11 VIRUSES IN ALGAE

Viruses or viruslike particles (VLP) have been reported in a number of algae ( Van Etten 1999). A
virus capable of degrading the nuclei of a green alga, Uronema gigas, was first described in 1972 (
Mattox et al., 1972). The virus consists of a large polyhedron capsid bounded by an outer
membrane and dsDNA; some viral particles have a prominent tail. Algae in the two- to four-cell
stage of division are the most susceptible to viral infection.
Virus or VLPs have been observed in vegetative cells of the red alga Sirodotia tenuissima, in
germlings of the green alga Oedogonium sp., and in spores of the brown alga, Chorda tomentosa.
Algal viruses or VLPs are difficult to study because (1) of low concentrations of viral particles, (2)
usually few algal cells contain particles, (3) particles are found only in certain stages of the algal
life cycle, (4) algal cells containing particles seldom lyse, and (5) in most cases, the particles are
not infectious. The virus-infected filamentous brown alga Feldmannia showed deformed sporangia
and significantly reduced photosynthesis ( Robledo et al., 1994). Viruslike particles have been
observed in Chlorella symbionts of Hydra viridis and Paramecium bursaria, suggesting that they
may play some role in their symbiosis. Chlorella viruses are similar to iridescent viruses that infect
insects, and their infection process resembles that of bacteriophages. Chlorella viruses are
excellent subjects for studying gene regulation in photosynthetic eukaryotes. The viral genomes
are thought to be excellent sources of control elements such as origins of replication or promoters
for plant genetic engineering ( Van Etten et al., 1991).

Algal viruses are a major factor in marine ecology. They have been linked to rapid changes in algal
populations and are thought to have a significant role in genetic exchange among algal species (
Suttle et al., 1990). Algae may serve as reservoirs or vectors for pathogenic viruses that infect
animals ( Van Etten et al., 1991).

2.12 VIRUSES IN PLANTS

The "breaking of colors" in tulips, a condition that is produced by a virus, was beautifully illustrated
in the still-life oil paintings by Dutch artists in the early seventeenth century. The streaking effect
produced on infected flowers was much prized in Holland. In the heyday of "tulip mania," farmers
traded cattle, grain, and cheese for the much desired bulbs. Infected tulips have been known for
more than 400 years. It was not until 1892, however, that a Russian scientist, Dimitri Iwanovski,
showed in the tobacco plant the existence of a disease agent that could pass through a filter
capable of holding back the smallest known bacteria ( Matthews, 1991). In 1898, a Dutch biologist,
Martinus Beijerinick, confirmed Iwanovski's observation and described the infectious agent as
contagium vivum fluidum, meaning "contagious living fluid." Since 1900, scientists have described
more than 600 plant disorders in which the causal agent is suspected to be a virus.

Viral infections in plants vary from clearing of leaf veins and chlorotic mottling to severe necrosis.
Symptoms of a viral disease may be generalized throughout the plant or localized. A common sign
of a virus infection is retarded growth, which results in stunted plants, lowered productivity, and
leaf deformation. Tumors are characteristic of several viral infections, notably, the wound tumor of
clover and swollen shoot of cacao. The mechanism of tumor formation resulting from wound tumor
virus infection is unknown. Viral plant infections vary widely. At one end of the spectrum are
virulent infections that result in necrotic lesions--the cells die so rapidly that they are unable to
pass on viral particles to neighboring cells. At the other end are asymptomatic or latent infections,
in which the cells are not seriously damaged.

Most plant viruses are spread by fungi, nematodes, and arthropods such as leaf hoppers and
aphids. Leaf hoppers, because they feed on phloem tissue, are vectors of viruses that become
systemic throughout the plant. This type of viral infection is usually called yellows. Aphids feed on
superficial parenchyma cells and are vectors of mosaic viruses. Most plant viruses produce
inclusion bodies, which may be amorphous or crystalline and reside either in the cytoplasm or the
nucleus. These inclusions, in most instances, consist of aggregates of viral particles. Plant inclusion
bodies, which are similar to those of insect viruses, are large structures that are visible under a
light microscope. Plant cells infected with tobacco mosaic virus contain two kinds of inclusions,
hexagonal crystals and irregular masses called X bodies.

More than 95% of all plant viruses possess ssRNA. Tobacco mosaic virus (TMV) is one of the best
studied of the plant viruses. In 1946 Wendell Stanley was awarded a Nobel Prize for crystallizing
TMV particles, and in 1957 Heinz Fraenkel-Conrat showed that the RNA of TMV was infectious (
Atlas, 1997; Fraenkel- Conrat and Williams, 1955). When inoculated into plants, TMV RNA initiates
synthesis of more viral RNA and also of viral protein subunits, which, together with the RNA,
assemble into typical rods of TMV ( fig. 2.5 ). An example of a dsRNA plant virus is the wound
tumor virus. Studies have confirmed the existence of DNA plant viruses such as the cauliflower
mosaic virus (dsDNA) and the bean mosaic virus (ssDNA).
Many plant viruses carry more than one

Fig. 2.5 Morphology and detailed organization of the protein subunits and nucleic acid of tobacco
mosaic virus. Adapted from Fraenkel-Conrat and Kimball ( 1982).

RNA strand in each viral particle. There are four strands of RNA in alfalfa mosaic and cucumber
mosaic viruses. Viral infection and the production of new viruses require the genetic information of
three of the four RNA strands. Cowpea mosaic, tobacco ringspot, and tobacco mosaic viruses have
two RNA strands, and both are necessary for viral reproduction.

Gemini viruses and cauliflower mosaic viruses are the only plant viruses that contain DNA
genomes. Gemini viruses, such as bean mosaic virus and corn streak virus, occur chiefly in the
tropics and form viral aggregates in the nuclei of plant cells. The use of DNA viruses as vectors of
beneficial genes into plants is being studied ( Matthews, 1991).

Associated with the tobacco necrosis virus (TNV) is a smaller, unrelated virus, the satellite virus.
The satellite virus is an obligate symbiont and requires the presence of TNV, the host virus, for its
replication. The nature of the dependency is not known. The satellite virus is not needed by the
TNV, but when present it modifies the infectious nature of the virus. Some virologists predict that
similar satellite viruses will be discovered among bacteriophages and animal viruses.

Although virus-infected plants often recover and new growth may be free of symptoms, the virus
still remains in the plant. Such a phenomenon is called acquired immunity. Some viruses can
develop intracellular symbiotic associations in both plant and animal cells. These viruses have
become adapted to a "double life" of molecular biosynthesis in two distant life forms. Many plant
viruses multiply in the tissues of their insect vectors. Leaf hoppers, for example, are well-known
hosts of many systemic plant viruses. Development of the wound tumor virus in a leaf hopper's
intestinal filter chamber and in its hemolymph has been well documented. The ability of insects to
transmit a plant virus is genetically controlled.

2.13 VIROIDS AND SATELLITE RNA

VIRUSES
Viroids are the smallest and simplest naked RNA plant pathogens. They are noncoding genomes
that require host enzymes for their replication and transmission. About 20-25 different viroids have
been identified, including potato spindle tuber viroid, coconut cadang-cadang viroid, and
chrysanthemum stunt viroid. The viroid RNA genome usually consists of a single strand of 246-375
nucleotides, whereas RNA viruses are 10-100 times larger. Viruses often have methylated
ribonucleotides in their genomes, whereas viroid RNA lacks modified bases. All virus RNAs are
subject to a host cell's enzymatic degradation; viroid genomes are resistant ( Voyles, 1993).
Viroids produce disease in their host plants either by viroid RNAs or by other RNAs generated
during the infection process. The coconut cadang-cadang disease, caused by the smallest of
viroids, is a virulent pathogen that has killed thousands of coconut palms in the Philippines. Other
viroids have little (e.g., hop latent viroid) to mild (e.g., apple scar skin viroid) pathogenic effects.
Viroids cause diseases mostly in agricultural crops and are believed to have originated from the
ancestral plants as a result of past plant breeding practices ( Diener, 1996).

Even viruses have their own subviral symbionts. Satellite RNAs consist of long, circular, single
strands of several hundred nucleotides and require a helper virus for their replication and
transmission.

Hepatitis δ virus (HDV) has some common features with satellite RNA and viroids. Hepatitis δ virus
was first described as a nuclear antigen (δ antigen) in the 1970s and later was found to cause a
virulent form of hepatitis (type D hepatitis) among indigenous inhabitants of South America. Delta
antigen association is necessary for the expression of the hepatitis B virus (HBV). Viral RNA of
HBV/HDV are packaged together in hepatitis B virion particles. Unlike viroids, the RNA of HDV
encodes for a nuclear phosphoprotein that is essential for viral replication as well as virion particle
formation. Viral hepatitis is a worldwide disease with increased mortality when HDV and HBV co-
infections are involved. The transmission of viral hepatitis is linked with drug abuse and transfusion
of blood products. Because HDV depends on HBV for its replication, vaccination against HBV
protects against the disease ( Tiollais and Buenidia, 1991).

2.14 PRIONS
Prions are infectious proteins that are not associated with nucleic acids ( Prusiner, 1994). Some
scientists believe that prions may be the cause of several diseases such as scrapie, a disease of
sheep and goats; transmissible mink encephalopathy (TME) and chronic wasting disease (CWD) of
mule deer and elk; Gerstmann Straussler and Scheinker syn-drome (GSS) (the new name of
CreutzfeldtJacob disease [CJDI); kuru, a disease of the New Guinea Fore tribe; and mad cow
disease.

During the 1960s, similarities between kuru and scrapie became evident, and later on kuru, CJD,
and GSS were shown to be similar in their infectiousness and familial transmission. All these
diseases are associated with neuronal cell death and spongiform changes in brain tissue, along with
proliferation of glial and astrocyte cells. Many regions of the brain were affected, and amyloid
plaques of insoluble protein aggregates, called PrP protein, were always observed. The function of
normal cellular PrP protein is not known, but mutant forms of PrP change shape and attach to other
prions and thus damage brain tissue. Stanley Prusiner of the University of California Medical School
at San Francisco received a Nobel Prize in 1997 for his research on prions, which he began in 1972
to resolve cases of degenerative brain disease. The prion hypothesis remains controversial, with
critics contending that a protein could not cause a disease without DNA or RNA to direct its
replication and synthesis. Some also believe that a virus co-factor may be involved. No one has
ever isolated pure prions and injected them into an animal to cause disease. Disease in
experimental animals occurs only after they are injected with ground brain tissue from sick
animals. PrP is a brain glycoprotein, and the disease is associated with the accumulation of an
abnormal form of PrP protein. Recent studies indicate that PrP protein can be produced in several
forms ( Prusiner and Scott, 1997). The normal cellular PrP protein, called PrPc, can be converted
into an abnormal form, termed PPrSc, and its accumulation in the brain leads to the GSS disease. In
mice, a PrP protein called PrPctm produces neurodegenerative symptoms that are similar to the
prion diseases. This significant research offers the first experimental model for the study of prions (
Hedge et al., 1998).

2.15 THEORIES ON THE ORIGIN OF

VIRUSES
Regressive theory postulates that viruses are degenerate forms of intracellular parasites, similar to
mitochondria and chloroplasts. Progressive theory suggests that viruses evolved from normal
cellular DNA and RNA that gained the ability to replicate autonomously. DNA viruses may have
evolved from plasmids or transposons, which later evolved protein coats and became infectious.
Retroviruses may have evolved from retrotransposons, while RNA viruses may have had their
origin as mRNA ( Becker, 1996a).

Coevolutionary theory argues that viruses and living organisms coevolved through ancient
biological interactions. Small DNA viruses (papilloma viruses and parvoviruses) are species specific
and genetically stable. They show a close link between virus evolution and host speciation.
Experimental observations strongly suggest that viral replicators are ancient systems with their
own origins of replication and replication proteins that have co-evolved with their host ( Shadan
and Villarreal , 1996).

2.16 SUMMARY
Viruses are the smallest of all symbionts, and they occur intracellularly in all forms of life. Some
viruses kill their host cells, whereas others may live for years in concert with their hosts, often
conferring new genetic properties. There are DNA viruses and RNA viruses, retroviruses, satellite
viruses, viroids, and viruslike particles such as prions. Bacterial viruses, or bacteriophages, carry
DNA from one host to another. Insect viruses have been studied more closely than viruses of other
animals.

Some scientists believe that invertebrates may be natural reservoirs of viruses that infect humans
and livestock. Viruses cause a variety of cytopathic effects in vertebrate hosts, including cancers.
Vertebrate host cells respond to viral infections by producing defensive compounds such as
interferons and antibodies. Mycoviruses usually form stable associations with their host, whereas
many plant viruses cause virulent infections of host plants. Concentric bodies are viruslike
structures that are common in lichen fungi. Retroelements are widely dispersed in the genomes of
living organisms.

Viruses are significant disease agents of plants and animals and are among the most difficult
parasites to control. Viruses may have a useful role in human affairs. Some viruses are used as
biological control agents for various types of pests; others such as λ phages are commonly used as
vectors in recombinant DNA research. Viruses in hosts that belong to different kingdoms have
common patterns of behavior in their life cycle. Viruses represent the ultimate stage in the
evolutionary development of host-parasite relationships. Their

extreme reduction in structure and their ability to integrate themselves into the genome of the
host cell have parallels with mitochondria and chloroplasts as well as bacterial symbionts, such as
K, which also have developed close relationships with the host genome. Satellite viruses modify the
infectious ability of the TNV virus, showing that even viruses are not immune from parasitic agents.
In a similar fashion, it is becoming increasingly recognized that the traits of some organisms are
the result of viruses that infect these organisms. For example, pathogenic virulence of certain
bacteria and protozoans has been correlated with the presence of viral genomes.Advances in
structural analysis of capsid protein and sequencing of the animal viral genome have provided new
insights into the basic nature of viral symbioses, enabling scientists to discover how a virus enters,
replicates, and assembles inside a host cell and to identify the molecular mechanisms of viral
pathogenesis. The presence of viruses in many different forms of life suggests that viruses may
have a much greater role in symbiotic associations than previously realized. There are several
theories on the origin of viruses, including the regressive theory which states that viruses are
degenerate forms of intracellular parasites.

APPENDIX: AN OUTLINE OF VIRUS

CLASSIFICATION
In 1966, the International Committee on Nomenclature of Viruses produced the first unified system
for virus classification. In 1973, this committee expanded its objectives and renamed itself the
International Committee on Taxonomy of Viruses (ICTV). The ICTV meets every 4 years. The sixth
report, published in 1995, recognized 3465 virus species belonging to a single order
(Mononegvirales), 50 families, 9 subfamilies, and 164 genera ( Murphy, F. A. et al., eds. Virus
Taxonomy: Classification and Nomenclature of Viruses: Sixth Report of the International
Committee on Taxonomy of Viruses. Archives of Virology, supplement 10, Springer-Verlag,
1995).Some of the significant rules for virus taxonomy include:
1. A virus name should be short and meaningful. Latin binomial names for viruses have been
abandoned.
2. A virus species is represented by a cluster of strains, all of which have some stable properties
that separate the cluster from clusters of other strains.
3. A genus is a group of virus species sharing common features.
4. A family is a group of genera with common characteristics. Groups of related viruses are
placed in families whose names are capitalized, italicized and end in the suffix -viridae.

Following are some examples of viruses discussed in this text which are arranged according to the
Baltimore Classification System.

Family Genus Species

Group 1. dsDNA Viruses

Adenoviridae Mastadenovirus Human adenovirus 2
Baculoviridae Nucleopolyhedrovirus Nucelopolyhedrovirus
Family Genus Species

Granulovirus Granulovirus
Herpesviridae Simplexvirus Human herpesvirus 1
Cytomegalovirus Human Herpesvirus 5
Iridoviridae Iridovirus Chilo iridescent virus
Myoviridae T4-like phages Coliphage T4
Papovaviridae Papillomavirus Cottontail rabbit papillomavirus
Phycodnaviridae Phycodnavirus Paramecium bursaria Chlorella virus 1
Polydnaviridae Ichnovirus Campoletis sonorensis virus
Poxviridae Orthopoxvirus Vaccinia virus
Leporipoxvirus Myxoma virus
Entomopoxvirus A Entomopoxvirus
Family Genus Species
Group 2. ssDNA Viruses
Geminiviridae Geminivirus Maize streak virus
Group 3. dsRNA Viruses
Partitiviridae Partitivirus Gaeumarmomyces graminis virus
Chrysovirus Penicillium chrysogenum virus
Reoviridae Botavirus Simian rotavirus SA 11
Phytoreovirus Plant wound tumor virus
Totiviridae Totivirus Saccharomyces cerevisiae virus
Giardiavirus Giardia lamblia virus
Leishmaniavirus Leishmania RNA virus
Group 4. Plus-sense RNA Viruses
Barnaviridae Barnavirus Mushroom bacilliform virus
Bromoviridae Alfamovirus Alfalfa mosaic virus
Ilarvirus Tobacco streak virus
Cucumovirus Cucumber mosaic virus
Flaviviridae Flavivirus Yellow fever virus
Pestivirus Bovine diarrhea virus
Hepatitis C-like virus Hepatitis C virus
Leviviridae Luteovirus Barley yellow dwarf virus
Necrovirus Tobacco necrosis virus
Picornaviridae Enterovirus Poliovirus 1
Rhinovirus Human rhinovirus
Hepatovirus Hepatitis A virus
Potexvirus Potato virus X
Tetraviridae Tobavirus Tobacco mosaic virus
Tobravirus Tobacco rattle virus
Tombusviridae Tombusvirus Tomato bushy stunt virus
Togaviridae Alphavirus Sindbis virus
Rubivirus Rubella virus
Group 5. Minus-sense RNA Viruses
Paramyxoviridae Paramyxovirus Human parainfluenza virus
Morbillivirus Measles virus
Rubulavirus Mumps virus
Rhabdoviridae Lyssavirus Rabies virus
Nucleorhabdovirus Potato yellow dwarf virus
Family Genus Species

Arenaviridae Arenavirus Lymphocytic choriomeningitis virus

Bunyaviridae Hantavirus Hantaan virus
Orthomyxoviridae InfLuenzavirus A, B Influenza A virus
Influenzavirus C Influenza C virus
Group 6. RNA Reverse-Transcribing Viruses
Retroviridae Mammalian type B retrovirus Mouse mammary tumor virus
Avian type C retrovirus Avian leukosis virus
Lentivirus Human immunodeficiency virus 1
Group 7. DNA Reverse-Transcribing Viruses
Caulimovirus Cauliflower mosaic virus
Hepadnaviridae Orthohepadnavirus Hepatitis B virus

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3 BACTERIAL ASSOCIATIONS OF
BACTERIA, PROTOZOA, AND ANIMALS
Prokaryotes are simple, primitive organisms that dominate life on earth in terms of numbers of
individuals. They also support life by recycling carbon, nitrogen, and sulfur in the biosphere.
Prokaryotes, in particular the bacteria, have adapted to almost every ecological niche, ranging from
plant surfaces to the skin and digestive system of animals. Bacteria are key players in the Gaia
hypothesis ("life regulates life"), and their controversial presence in a Martian meteorite has
spurred interest in the possibility of life on other planets ( Gibson et al., 1997). Bacteria are
important in human affairs. Some cause disease, but others are used to make vaccines and
antibiotics which cure disease. Bacteria are an integral part of the current biotechnology revolution.

The origin and evolution of prokaryotes has long been a mystery ( Woese, 1994). Today, such
mysteries are being addressed by scientists such as Carl Woese of the University of Illinois, whose
findings have raised new and perplexing questions of phylogeny ( Morell, 1997). Scientists classify
life into three domains, archaea, bacteria, and eukarya. The first two domains are prokaryotes; the
third one includes all the eukaryotes. Archaeans are represented by specialized microbes such as
methanogens, thermophiles, and species that live in high salt and highly acidic environments.

Prokaryotes provide significant insight into our rich and complex biochemical heritage that began
3.5 billion years ago ( Mathieu and Sonea, 1995). Some bacteria obtain energy from
photosynthesis, others by means of chemosynthesis or by fermentation. Many prokaryotes and
some eukaryotes grow without oxygen and may even be killed by its presence. Core samples
collected from a depth of 2.75 km in the earth's crust contained Bacillus infernus which was living
without oxygen at a temperature of 75°C. Anaerobic deep life accounts for about 0.1% of the
earth's total biosphere ( Kerr, 1997). Examples of anaerobic communities include those found in
ancient microbial mats, subsurface soils, freshwater and marine sediments, and the digestive
systems of animals. Anaerobic communities have been important participants in symbioses with
plants and animals ( Fenchel and Finlay, 1995). Interactions between bacteria and their hosts
involve a specialized protein secretion system that exports and translocates the bacterial protein to
the host cell and induces or interferes with host cell signal transduction pathways ( Galan and
Bliska, 1996; Yang He, 1998).

Prokaryotes differ from eukaryotes in several ways ( Watt, 1999). The prokaryotic cell divides by
binary fission or by budding, but never by mitosis. Each cell has only one chromosome that lacks
histone proteins. Sexuality occurs in some prokaryotes, but it is distinctive in that the cytoplasm of
the cells never fuse; only the chromosome, or part of it, passes from one cell to another.
Eukaryotic cells divide by mitosis and meiosis, the latter being part of a sexual process that
involves cell fusion and is well developed and regulated. The prokaryotic cell does not contain
membrane-bound organelles such as nuclei, mitochondria, and chloroplasts and on the average is
smaller than eukaryotic cells, usually < 10µm long. The ribosomes of prokaryotic cells have a
sedimentation coefficient of 70S, compared to 80S for the ribosomes of eukaryotic cells. Motile that
lack microtubules and consist of the protein flagellin. The cell walls of prokaryotes are made of
peptidoglycans and never cellulose or chitin. Motile structures of eukaryotic cells consist of
microtubules, which contain the protein tubulin, in a 9+2 paired arrangement. Prokaryotic cells
have a tubulinlike protein called FitsZ ( Erickson, 1997). In this chapter and in chapters 4 and 5 we
focus on prokaryotes as symbionts.

3.1 BACTERIAL SYMBIONTS OF BACTERIA

Bdellovibrio: Predatory Bacteria
Most of the known bacterial symbionts of bacteria have been placed in three genera: Bdellovibrio,
Microvibrio, and Vampirovibrio. In 1962, Heinz Stolp, while searching for bacterial viruses from soil,
observed plaques on cultures of the bacterium Pseudomonas phaseolicola ( Stolp, 1979). The
plaques continued to grow in size, a feature not found in plaques caused by bacteriophage
infection. Stolp examined the colonies with a phasecontrast microscope and observed small
bacteria attacking the pseudomonads. These unusual bacteria infect and kill other Gramnegative
bacteria.

Bdellovibrios are bacteria (1-2 µm long) whose natural habitat is the periplasmic space of other
bacteria. Bdellovibrios attack sensitive bacteria, often 10-20 times larger than themselves, by
penetrating their cell walls and growing and multiplying in the periplasmic space. The symbiont
breaks down the host's cellular constituents, and when the host cell dies, progeny of the symbiont
are liberated. When bdellovibrios are first introduced into a bacterial colony, they move quickly,
about 100 cell lengths per sec, and collide with a host bacterium. Just before colliding, the
symbiont spins rapidly, making contact with the surface of the host cell within seconds. The end of
the cell opposite the flagellum attaches to the host cell wall by means of tiny filaments. The
symbiont penetrates the cell wall by making a hole, but exactly how it does this is not fully
understood. Penetration is completed in 5-20 min. Young host cells are penetrated in less time
than older or metabolically inactive cells.

After infection, the bdellovibrio grows in size, elongates into a large, helical-shaped structure, and
divides to produce vibrioshaped, motile individuals. The life cycle of a bdellovibrio is completed in
1-3 h. The host cell, or part of it, swells into a globular body, the bdelloplast, because the cell wall
is weakened by enzymes produced by the symbiont. Infected bacteria stop moving within seconds
after a bdellovibrio attaches to their cells; their synthesis of nucleic acids and proteins stops after
penetration. Permeability of the host cell membrane changes, allowing degraded products to leak
into the periplasmic space. In the final stage of the infection, the host membrane breaks down, and
bdellovibrio progeny are liberated (fig. 3.1). The number of progeny per cell depends on the host
species: for example, 5 for Escherichia coli, 10 for Pseudomonas fluorescens, and 20 for Spirillum
serpens. The differences in the number of symbiont offspring per cell reflect the size and metabolic
rate of the host and its ability to provide nutrients to the symbionts. Bdellovibrios are not true
endosymbionts because they are outside the host cytoplasm. Some strains attack a wide range of
bacterial species, whereas others have a limited host range. Three species of Bdellovibrio are
recognized on the basis of nucleic acid homology and DNA base composition analysis: B.
bacteriovorus, B. starrii, and B. stolpii. The predator--prey relationship between Bdellovibrio and its
host cells is unique among the bacteria ( Guerrero, 1991).

Bdellovibrios possess exoenzymes that degrade nucleic acids, proteins, lipids, and peptidoglycan,
and they are noted for their high rate of respiration, which is seven times that of E. coli.
Bdellovibrio's life cycle includes a parasitic phase, during which it infects the host bacterium, and a
reproductive phase in which it grows and multiplies within the periplasmic space. Bdellovibrio can
be cultured axenically. Microvibrio is an ectosymbiont of Xanthomonas maltophilia, which kills its
host without penetrating it ( Atlas, 1997).

Bdellophages, or viruses that attack bdellovibrios, were first noted in 1970 ( Varon, 1974).
Bdellophages develop only in a sensitive bacterial host or in the presence of its extract. This is a
unique compound symbiotic association that involves a bacterial virus, a small prokaryote (the
bdellovibrio), and a Gram-negative prokaryote (the host bacterium).
Pseudomonads and Cyanobacteria
Studies have suggested that a mutualistic relationship exists between nitrogen-fixing,

prokaryotes have flagella A. Two Growth Patterns of Bdellovibrio B. Bdellovibrio penetrating E. coli
C. Bdellovibrio inside periplasmic space of E. coli Fig. 3.1 Bdellovibrio--Escherichia coli symbiosis.
(a) Schematic diagram of Bdellovibrio life cycle stages as symbiont of E. coli or growth under
axenic conditions. (b) penetration of Bdellovibrio into E. coli showing the invagination of host cell
membrane. (c) Bdellovibrio symbiont inside periplasmic space of host cell. (Drawn from several
sources cited in the text.)

blue-green bacteria and pseudomonads attached to the host filaments. Nitrogen compounds
excreted by the host are used by the attached bacteria, which in turn consume oxygen and liberate
carbon dioxide when they respire organic compounds. Growth and nitrogen fixation of the host are
stimulated by the reduced oxygen and increased CO 2 concentrations caused by the
pseudomonads.

3.2 BACTERIAL SYMBIONTS OF

PROTOZOA
Paramecium and Other Ciliates
In 1856, Johannes Muller observed rodshaped particles (bacteria) inside Paramecium caudatum,
and since then the literature on endosymbionts of ciliates and protozoa has grown considerably (
Lee et al., 1985). Bacteria have been observed in the micro- and macronuclei as well as in the
cytoplasm of protozoans. The bacterial symbionts of Paramecium aurelia, or κ particles, have been
studied the most thoroughly. In 1943, Tracy Sonneborn described these particles as cytoplasmic
factors that killed R. aurelia. The particles were considered to be an example of cytoplasmic
inheritance ( Sapp, 1994b).

All symbionts of Paramecium are rodshaped, gram-negative bacteria that cannot live outside their
host (fig. 3.2). Thousands of symbiont particles may exist in one host cell. Previously, the bacteria
were designated by Greek letters, but now they are assigned to the genera Caedibacter, Holospora,
Lyticum, Pseudocaedibacter, and Tectibacter ( Heckmann and Gortz, 1992). Some strains of the
bacteria, called killers, are toxic to sensitive paramecia. Killer strains develop only when the
bacterial symbionts contain phage or plasmids. Below is a list of some well-known symbionts of R.
aurelia. All symbionts, except α, occur in the cytoplasm of the host.

α (Holospora caryophila). These appear in the macronucleus as short rods, crescents, or spirals,
the shape depending on the growth rate of the host; a nonkiller. Infection of Paramecium bursaria
with Holospora acuminata is highly specific and complex ( Rautian et al., 1993).

δ (Tectibacter vulgaris). Generally nonmotile rods, which are often associated with other symbionts
such as κ and µ; a nonkiller.

γ (Pseudocaedibacter minuta). Frequently appear as small doublets; a strong killer; death of

sensitive paramecia results from vacuolization of the cell.

κ (Caedibacter taeniospiralis). Kappa symbionts contain unusual inclusions called R bodies which
form tightly coiled proteinaceous ribbons that are refractile and shine brightly. Kappas with R
bodies are toxic; those without R bodies are nontoxic. Sensitive paramecia, which includes most
strains, are killed by the R protein. Killing is highly specific, each killer being resistant to its own κ
symbiont but sensitive to other symbiont strains. R bodies are also found in other bacteria such as
Pseudomonas avenae (a plant pathogen), P. taeniospiralis (a hydrogen-oxidizing soil bacterium),
and Rhodospirillum centenum (a photosynthetic bacterium). R bodies are classified into five groups
based on their size, morphology, and unrolling behavior of the ribbons. Genes coding for R bodies
have been cloned in Escherichia coli. The genes are encoded in either bacteriophages or DNA
plasmids that are present in nonbright κ particles. Induction of the genes may occur spontaneously
or result from stimuli such as UV light. R bodies are unusually resistant to drying and are similar to
ejectosomes of Cryptomonas and Pyramimonas ( Pond et al., 1989).

λ (Lyticum flagellatum). Motile, large rods that are covered with flagella and cause rapid lysis of
sensitive paramecia.

µ (Pseudocaedibacter conjugatus). Mate killer; elongated rods whose killing action depends on the
cell-to-cell contact that occurs during mating. Particles of µ are transferred from killer to sensitive
mate.

σ (Lyticum sinuosum). The largest symbiont and a close relation of λ; a curved rod and a rapid
killer.

ο. The ciliate Euplotes aediculatus contains particles of the symbiont ο, which is essential for
survival of the host.

Amoeba: Evolution of a New Symbiosis

In 1966, Kwang W. Jeon noted a bacterial infection in a laboratory culture of Amoeba proteus (
Jeon, 1991). The bacteria initially killed most of the host amoebae, but some survived. By 1971,
the association had become so integrated that the amoebae could not survive without their
symbionts. New amoebae could be induced to establish symbioses after about 200 cell generations,
or 18 months. The bacteria were Gram-negative rods which resembled Escherichia coli. In the
amoebae, large numbers of symbionts became enclosed in membrane-bound vesicles of different
sizes (symbiosomes). The bacterial symbiont was named x-bacteria ( fig. 3.3 ). It infected only
amoebae and in doing so released large amounts of a protein into the host cytoplasm. This protein
appeared to prevent lysosomes from fusing with the symbiosomes, thus protecting the bacteria (
Jeon, 1992; Pak and Jeon, 1997). Attempts to grow x-bacteria apart from the host amoebae have
failed. The x-bacteria contain two types of plasmids. Newly infected amoebae grow faster and are
more sensitive to starvation than symbiont-free amoebae. The nuclei of amoebae with x-bacteria
are not only incompatible with the original amoebal strain but are now lethal to them. Jeon has
suggested that the symbiont-bearing amoebae represent either a new strain or species of Amoeba
( Jeon, 1991). The bacterial endosymbionts produce high levels of stress proteins, suggesting that
the symbiotic relationship may be less benign than supposed ( Choi et al., 1991). The amoeba-
bacteria association is an excellent model for studying the evolution of new symbiotic associations (
Jeon, 1995).

Amoeba--Bacterium Symbiosis in Entamoeba

histolytica
Pathogenicity of the intestinal protozoan Entamoeba histolytica (amoebic dysentery) is determined
in part by its association with specific bacteria. Trophozoites usually live as harmless commensals
in humans but can become virulent pathogens because of changes in the intestinal bacteria which
trigger development of the disease. Isozymes of cloned cultures of the bacteria can change from
one state to another ( Mirelman, 1987).

3.3 BACTERIAL SYMBIONTS OF ANIMALS

Bacterial Luminescence
Photobacterium, Photorhabdus, Vibrio, and Xenorhabdus contain luminous species that form
symbioses with animals. Photorhabdus and Xenorhabdus are pathogenic bacteria that associate
with soil nematodes and insects. Photobacterium and Vibrio occur in marine organisms such as
cephalopod molluscs, teleost fish, and tunicates ( Meighen and Dunlap, 1993; Forst and Nealson,
1996; Ruby, 1996).

Marine bioluminescence
More than 1500 marine animal species are bioluminescent, and they form two groups: those that
produce their own light and those that use bacteria to produce light ( Morin, 1999). Light that is
emitted by symbiotic bacteria, although apparently of no direct benefit to the bacteria, is important
for the host. Luminescent bacteria occur free-living, as plankton, and they also grow on the surface
of dead marine animals. The light produced by these bacteria attracts feeding organisms.
Luminescent bacteria that are ingested by other organisms resist digestion and are expelled into
the ocean and in this way are dispersed. Bioluminescence may have an evolutionary selection value
because it enhances a symbiont's survival and propagation ( T. Wilson and Hastings, 1998).

Photobacterium and Vibrio live as ectosymbionts on teleost fish and as endosymbionts in squids
and tunicates. They are heterotrophic on dead and decaying organisms, parasites of marine
crustaceans, and commensals in the digestive tract or surface of marine fishes and crustaceans.
Vibrio is the more cosmopolitan and nutritionally versatile genus. Luminescent bacteria are
important symbionts in the alimentary canals of marine animals. The bacteria produce extracellular
chitinase, which helps invertebrates such as mussels, scallops, and crabs digest chitin.

Some fish have light-emitting organs that contain large numbers of luminescent bacteria (
Haygood, 1993). The bacteria receive nutrients from the host and are in a protected environment.
Light produced by the bacteria frightens or diverts predators of the fish and also helps the fish
communicate with mates. Some teleost fish, such as the flashlight fish, harbor dense colonies of
bacteria in specialized light organs. The bacteria produce light continuously, but the fish controls
light emissions by means of shutterlike structures on the organs. The angler fish has a modified
dorsal-fin ray that contains a light organ at its tip. The organ contains luminescent bacteria and is
dangled in front of the fish, like bait, to lure prey ( Haygood, 1993) ( fig. 3.4 ). The gas bladder of
leiognathid fishes serves as a complex light organ system for camouflage, communication,
schooling behavior, and sexual displays. Purine crystals covering the gas bladder reflect light; other
structural modifications control the direction and timing of the emitted light ( Haygood, 1993). The
squid Loligo harbors bacteria in an ink sac near its anus and when alarmed discharges lumines-
cent secretions from this organ. The secretions distract or frighten other organisms and are
effective substitutes, under dark conditions, for the black ink that the squid usually secretes (
Montgomery and McFall-Ngai, 1995).

Biochemical pathways for bioluminescence are similar in most luminescent bacteria. One common
feature is regulation of the enzyme luciferase. This enzyme mediates a reaction in which reduced
flavin mononucleotide (FMNH) and a long-chain aldehyde are oxidized, and blue light (490 nm) is
emitted in the oxidation process ( Nealson and Hastings, 1992). Bacterial symbionts also produce
small amounts of an autoinducer molecule (N-acyl homoerine lacotone), which must be present in
high concentrations for the light-emitting system to function. Luminescence is controlled by a
number of environmental signals such as salinity, oxygen level, nutrients, and transcription of the
lux operon. Free-living bacteria produce low levels of autoinducers and therefore do not synthesize
luciferase. When bacteria are concentrated in a host, however, autoinducers accumulate in
sufficient quantity to generate the light-producing compounds. LuxA and LuxB encode for α and β
subunits of luciferase. Other genes located here are LuxC, LuxD, and LuxE which regulate synthesis
of the aldehyde. When a high cell density is detected (also called quorum sensing), the lux operon
is activated ( Meighen, 1991; Ruby, 1996) ( fig. 3.5 ).

Squid-Vibrio fischeri light organ symbiosis

The small Hawaiian squid Euprymna scolopes uses the light emitted by its bacterial symbiont,
Vibrio fischeri, to camouflage itself at night in a strategy called counter-illumination ( Ruby and
McFall-Ngai, 1992; McFall- Ngai , 1999). Light from the ventral organ simulates moonlight and
starlight and makes it difficult for predators to see the squid. In this association, both the squid
and the bacterial symbiont can be grown separately and re-

Fig. 3.5 The role of autoinducer in lux gene regulation and biochemical production of
bioluminescence in Vibrio fischeri. Autoinducer is synthesized from S-adenosyl-methionine (S-AM)
and fatty acyl-acyl carrier protein (FA-ACP). Adapted from Gray ( 1997

combined to form the symbiosis ( McFall-Ngai and Ruby, 1998). The light organ of the adult squid
is a bilobed structure with epithelial tissue within the mantle cavity that contains luminous bacterial
symbionts. Other accessory tissues include the ink sac, which controls the light intensity, a
reflector, and a lens that directs and diffuses light over the ventral surface of the squid. The host
can thus control and modify the light emitted by the bacteria. The squid embryo develops specific
tissues that become infected with V. fischeri cells, which are carried with the flow of water through
the mantle cavity. The surface of the light organ is covered with cilia which direct the sea water
toward a central opening. The porelike opening leads to a duct lined with cilia. The juvenile light
organ thus creates dead-end crypts that become populated with bacteria. Within 10 h after
colonization, the autoinducer is activated and luminescence begins. The bacteria cause death of the
ciliated epithelium and at the same time change the shape and size of the crypts. Each morning the
squid expels more than 90% of its symbionts from the light organ crypts, and by nightfall the
remaining bacteria have multiplied to maximum numbers again ( fig. 3.6 ). Genetic and
biochemical analyses of host and symbiont have provided insights into how a complex light-
emitting organ is formed through molecular mechanisms that involve recognition, remodeling of
host tissue, and a sustained symbiotic dialogue. Mutations that lead to loss of the symbiosis offer
exciting opportunities to explore how the host species and the bacteria have coevolved ( McFall-
Nagi and Ruby, 1998).

Xenorhabdus and Photorhabdus:

symbionts of nematodes
Species of Xenorhabdus and Photorhabdus form symbioses with soil nematodes that are virulent
pathogens of insects ( Nealson et al., 1988; Forst et al., 1997). The Gram-negative bacteria live
harmlessly in the gut of the nematodes and together with insects they form a three-way symbiosis.
The bacteria produce pigments, antibiotics, and intracellular protein crystals that are toxic when
released into the insect host. When nematodes in the infective stage are ingested by an insect,
they migrate into the hemocoel and release their bacterial symbionts to rapidly kill the host. While
the nematodes feed and reproduce in the dead host, the bacteria stop multiplying. Bacteria then
infect the new generation of nematodes.

Fig. 3.6 Squid--Vibrio fischeri symbiosis. Bacteria move in a specific manner as they pass from the
mantle cavity to the brush border of the crypt. (a) Scanning electron micrograph showing the
ciliated surface of a juvenile light organ. The arrows point in the direction the symbionts move
toward the pore (po) on the light organ surface. (b) The duct connecting the mantle cavity with the
crypt epithelium (ep). Bacterial symbionts enter the crypt spaces. (c) Bacterial association with the
microvilli (mv) of the crypt epithelium. (d) An electron micrograph of bacteria (b) associated with
the adult light organ. From McFall-Ngai and Ruby ( 1998), with permission.

-41-

Bioluminescence and red pigments produced by the bacterial symbionts in the dead insect attract
new insects in search of food. In addition to limiting the growth of other bacteria, antibiotics
produced by the bacterial symbiont retard decay of the host. Toxin genes from R. luminescens are
being considered for use in making insect-resistant (transgenic) plants ( Bowen et al., 1998).

Bioluminescence in this case is due to bacterial luciferase that resembles the luciferases of other
marine luminous bacteria. The lux operons from R. luminsecens have been cloned, sequenced, and
expressed in Escherichia coli. Nematodes may protect bacteria from the insect's immune system.
The bacteria are pathogenic to the insect host only in the presence of the nematodes. Similarly, the
nematode cannot complete its life cycle without the bacteria ( Nealson et al., 1988).

Bacterial Symbionts of Insects

There are more species of insects than any other group of animals; thus it is not surprising that
there are numerous examples of symbiotic associations between insects and bacteria (
Schwemmler, 1989, 1991, 1993; Moran and Telang, 1998). Insects that depend on a restricted
diet such as blood or plant sap contain bacterial symbionts that provide them with nutrient
supplements such as amino acids, vitamins, and nucleic acids. The bacteria may be extracellular or
intracellular within the insect tissues.

About 10% of the known species of insects, including cockroaches, leaf hoppers, and aphids, have
well-defined organs called mycetomes (bacteriomes). A mycetome consists of mycetocytes
(bacteriocytes), or highly specialized cells that contain symbionts. Mycetomes are usually in the
body cavity mixed with fat tissues, although in some insects they occur in the intestines and
Malpighian tubules. Several generalizations can be made about insect symbioses: (1) The primary
intracellular symbionts belong to the eubacterial class Proteobacteria and are round or oval. (2) In
most cases, the bacteria are enclosed within host-derived vesicles. (3) Some species have
secondary Gram-negative, rod-shaped symbionts located in cells that surround the mycetome.
Secondary endosymbionts coexist in the same host with primary endosymbionts. How the
secondary endosymbionts affect their hosts is not clear, but they are believed to be of more recent
origin than primary symbionts. (4) All symbionts reproduce by binary fission. (5) The symbiont's
ribosomes are about the same size as the ribosomes of free-living bacteria. (6) The bacterial
symbionts are cytoplasmically inherited when eggs become infected with bacteria, thus ensuring
continued host association from one generation to another. (7) The symbionts excrete vitamins and
nutrients that are used by the host ( Gutnick, 1992).

Paul Buchner ( 1965), an authority on insect symbioses, believed that the mycetocyte, or host cell,
controlled the rate of division of the primary symbionts. The number of dividing symbionts
increases significantly just before embryogenesis and transovarial infection. Mycetocytes destroy
some symbionts by lysosomal enzymes and in this way control the size of the bacterial population.
Lysosomal breakdown of secondary symbionts is more pronounced and is a way of removing
nonviable individuals, a means by which the host acquires nutrients from the symbionts, or both.
The insect hemocoel is a hostile environment for primary mycetocytal symbionts, and they are
rarely found there except during a brief period of transovarial infection. Secondary symbionts,
however, are often found in hemocytes, which may indicate their recent symbiotic association with
the host.

Buchnera aphidicola--aphid mutualism

Some 250 million years ago, a bacterium infected an ancestor of modern-day aphids, and it has
since been a constant companion of these insects. Like eukaryotic mitochondria and chloroplasts,
all aphids today carry Buchnera aphidicola in their cell cytoplasm. A mature aphid contains about
5.6 million bacterial symbionts, and the symbiosis is obligate for both partners ( Douglas, 1998).
Aphids die when treated with antibiotics. The amino acid tryptophan, which is rare in plant sap, is
provided by Buchnera to its host ( Douglas and Prosser, 1992). The tryptophan gene has been
isolated from a Buchnera chromosome and amplified on a plasmid. Similarly, the gene for leucine
production is plasmid amplified ( Bracho et al., 1995). Genetic analysis of Buchnera shows that it
has additional copies of genes that are involved in vitamin synthesis. Buchnera endosymbionts
possess high concentrations of GroEL proteins, a chaperonin that functions in the refolding of
polypeptides ( Baumann et al., 1995, 1997). Buchnera reproduction and host growth rates are
tightly coupled. For example, although most living bacteria have several copies of rRNA genes,
Buchnera contains only one gene, suggesting that its growth rate is at a minimum ( Baumann et
al., 1995). During embryogenesis, aphid mycetocytes release their bacteria, which then enter the
developing eggs. The infection process during embryogensis is complex.

Parthenogenesis is common among aphids. Some aphids face extinction because the Buchnera
they carry has a heavy mutation load and dies out. In sexual reproduction, genes for harmful
mutations are reshuffled during meiosis and mating, but in parthenogenetic reproduction there is
no gene recombination, and lethal mutations are expressed in each generation. Similarly,
mutations in mitochondria and chloroplasts over a long period of time can also cause extinction for
their host organisms. In modern eukaryotes, many mitochondrial and chloroplast genes have
become incorporated with the host nuclear genome ( Lynch, 1996).
Wolbachia--insect symbiosis and the
diversity of life
Wolbachia is an intracellular bacterium that infects a wide range of invertebrates, including insects,
crustaceans, and nematodes ( Leong, 1999; Rigaud, 1999). About 16% of all insect species carry
this bacterium, and, like mitochondria, it is inherited through the maternal cytoplasm ( Werren,
1997; Wilkinson, 1998). The Wolbachia--insect symbiosis is more recent than the Buchnera--aphid
association. It contributes little to its host, and the bacteria do not reside in specialized
bacteriocytes. Wolbachia infects several types of host cells and can live under different conditions (
Stevens and Fialho, 1999). It was first described in 1936 ( O'Neill, et al., 1997) as an unculturable
bacterial symbiont which infected ovaries of the mosquito Culex pipens. Its role as a source of
cytoplasmic incomptability in mosquitoes has been studied extensively ( Yen and Barr, 1973).
Recent DNA sequence analysis shows that Wolbachia is closely related to Rickettsia, and its two
major groups, A and B, diverged some 50 million years ago ( Werren et al., 1995). Wolbachia can
be transferred among different arthropod species by hemolymph injection.

Wolbachia affects the insect host in some interesting ways. In woodlice, Wolbachia infects the
salivary glands and converts embryonic males into females. In wasps, which can lay eggs with or
without mating, Wolbachiainfected females produce only daughters, while uninfected eggs develop
into males ( Hurst et al., 1993). In a most complex symbiosis, a Wolbachia strain in a male insect
modifies the sperm DNA so that when it fertilizes eggs, the sperm DNA is cut to pieces and the egg
dies. Insects carrying different strains of Wolbachia are mutually incompatible and cannot
interbreed. It has been suggested that Wolbachia may have been responsible for speciation among
insects ( Werren, 1997). Wolbachia-infected insects that reproduce parthenogenetically can be
"cured" with antibiotic treatment and produce sexually reproducing offspring ( Stouthamer et al.,
1990).

Wolbachia has attracted attention as an insect pest control because of its ability to cause
cytoplasmic incompatibility, thereby introducing sterility into wild populations of mosquitoes (
Bourtzis and O'Neill, 1998). Some of the most devastating diseases are transmitted by arthropod
vectors. Alternative strategies for vector control include introducing foreign genes into insects to
make them unable to transmit disease causing agents, genetic transformation of bacterial
symbionts, and foreign gene expression in symbiotic bacteria ( Beard et al., 1993; Federici and
Maddox, 1996).

Microbial Fermentation in Animal Digestive Systems

Cellulose is a major constituent of plant cells and accounts for approximately half of the global
carbon dioxide that is fixed by photosynthesis. Cellullose is degraded by microbial symbionts under
the anaerobic conditions that occur in digestive systems of animals where dense populations of
bacteria, protozoans, and fungi carry out a variety of metabolic pathways. The digestive tracts
have anatomical, biochemical, and behavioral modifications that allow for fermentation either in a
foregut or hindgut ( Troyer, 1984).

Most herbivorous mammals have postgastric fermentation in the colon or cecum in which ingested
fibers are degraded by microbial symbionts to release nutrients. Cecal fermentation occurs in small
mammals such as rodents and rabbits, where plant substrates are fermented to fatty acids that are
absorbed in the hindgut. The colon is the fermentation site in primates, horses, elephants, and
manatees.

Ruminants are specialized herbivores that retain ingested plant food in their pregastric
fermentation chambers. True ruminants such as sheep, cattle, goats, and deer have stomachs that
consist of four pregastric chambers (rumen, reticulum, omasum, and abomasum), whereas camels
and llamas have three-chambered stomachs. Anaerobic fermentation occurs in the rumen and
reticulum, which are usually considered as a single organ. The rumen is lined with fingerlike
projections called papillae that increase its absorptive surface. In sheep and cattle, the rumen
occupies more than 65% of the volume of the digestive system. Pregastric fermentation allows the
host animal to use volatile fatty acids and vitamins as well as microbial proteins. Secondary
metabolites that are ingested are detoxified by microoganisms in the pregastric chambers.
Nitrotoxins that occur in certain legumes are converted by rumen bacteria into harmless amines.
Acidaminococcus fermentans converts trans-aconitate, which is present in grasses, into harmless
acetate ( Van Soest, 1994; Douglas, 1994a).

There are more than 3.5 billion domesticated ruminants worldwide, and they are an important
resource to agriculture. Human domestication of cattle during the dawn of the agricultural
revolution some 10,000 years ago represents another example of behaviorial symbiosis between
humans and ruminants ( Rindos, 1984). Biotechnology firms are using transgenic sheep to make
pharmacologically active proteins. Even though obligate anaerobic organisms have been difficult to
study in culture, analysis of 16S rRNA sequences and polymerase chain reaction-based detection
and profiling techniques have revealed the complex nature of microbial ecosystems and new
species from the specialized niches have been described ( Amman et al., 1995; Flint, 1997).

Fermentation, whether in the rumen or the lower digestive tract, is a continuous system. According
to the feast-famine hypothesis, when an animal eats, pulses of potential substrates are sent down
the digestive tract and the microbial population dynamics shift because of death by lysis of those
who exhaust their substrate ( McBurney et al., 1987). Ruminants use lysozymes to digest bacteria
in the gastric chamber. The normal expression of the gene that codes for this enzyme is restricted
to cells in the tear glands. In ruminants, however, duplicate copies of this gene are expressed in
digestive functions.

Adhesion is a microbial strategy where bacteria attach to their substrates and avoid being washed
away by the digestive flow. Mechanisms for bacterial attachment include fimbria, lectins, and
glycocalyx. Many rumen microbes attach to surfaces of food particles. Competition for surface
attachment sites may result in old cells being replaced with new ones.

Cellulose digestion, although generally rare among insects, has been demonstrated in silverfish,
roaches, beetles, and wood wasps. Termites are the most efficient cellulose digesters. Recent
evidence indicates that in some cockroaches and in higher termites cellulose is not digested by
microbial symbionts. However, anaerobic microbial symbionts in the hindgut are fully exploited in
the lower termites Kalotermes flavicollis and Retculitermes flavipes and in the wood roach,
Cryptocerus punctulatus ( Breznak, 1999). Symbiont-mediated cellulose fermentation occurs
commonly among insect species that are scavengers and detritivores. It is not yet understood how
a symbiont-free cellulolytic system evolved in some insect species ( M. M. Martin, 1991). The
curious South American bird hoatzin has a diet of leaves which it ferments in a foregut similar to
that of ruminants ( Grajal and Strahl, 1991).

Rumen microbial ecosystem

Rumen microbiology began in 1947 when Robert Hungate developed the roll-tube technique to
study anaerobic organisms ( Hungate, 1950, 1966). Microorganisms can live in most parts of an
animal gut, but certain regions, such as the rumen, are more suitable. Bicarbonate ions from saliva
are continuously being added to the rumen to maintain a pH of about 6.5. Peristalsis occurs at 1-
to 2-min intervals and mixes and moves the rumen contents. The rumen is almost free of oxygen
and has a relatively constant temperature of about 39°C. Carbon dioxide and methane are the two
principal gases of the rumen. Methane is a direct product of fermentation; carbon dioxide is
produced by fermentation and also by neutralization of bicarbonate ions ( Leschine, 1995).

Cellulolytic bacteria in the rumen degrade the cellulose of plant material into its constituent
subunits, cellobiose and glucose. The sugar molecules are then fermented into volatile fatty acids,
such as acetic acid, bu- tyric acid, and propionic acid, which are then metabolized by the host. The
ruminant converts propionic acid into carbohydrates such as lactose and glycogen. Syntrophy is a
beneficial association where the metabolites of one organism serve as substrates for another
organism. The interdependence of several species in rumen fermentation results in beneficial
associations ( fig. 3.7a ). For example, carbohydrate-fermenting bacteria produce hydrogen,
carbon dioxide, and formate as metabolic end products, which methanogens use to produce
methane. Similarly, plant proteins that enter the rumen are digested and fermented by bacteria,
and ammonia is liberated. Nearly all of the bacterial species in the rumen can use ammonia as a
major source of nitrogen. Small amounts of amino acids are also produced and are used by the
bacteria in protein metabolism. Interactions between cellulose-degrading bacteria and the
nitrogenfixing bacterium Clostridium and a noncellulolytic anaerobe Klebsiella illustrate the
phenomenon of microbial interdependence. In this association, Clostridium fixes nitrogen and
provides Klebsiella with amino acids, while Klebsiella provides two vitamins, biotin and p-
aminobenzoate, which are needed by Clostridium. Both bacteria use the energy molecules obtained
from cellulolytic degradation by Clostridium ( Cavedon and Canale- Parola , 1992) ( fig. 3.7b ).

Bacteria and protozoa in the rumen benefit each other. Digestion of starch and proteins by the
protozoa releases sugars and amino acids, which the bacteria use to grow and reproduce. The
protozoa, in turn, ingest bacteria and use their protein as a source of amino acids. The protozoa in
the rumen stimulate bacterial growth, which in turn increases bacterial ingestion by the protozoa.
As a result, there is a greater turnover of bacterial carbon and nitrogen. The ruminant participates
in this symbiosis by providing nutrients for the rumen symbionts and by regulating the physical and
chemical conditions of its fermentation chamber. The ruminant meets its protein needs and also
obtains many vitamins by digesting some of its rumen residents.

Interdependent obligate anaerobic bacterial species make up about half of the total biomass of a
normal rumen. Species of Butryovibrio, Fibrobacter, and Ruminococcus are the prinicipal cellulose-
degrading bacterial symbionts. In addition, a chytrid, Neocallimastix frontalis, secretes cellulases
that are more active than those of other cellulolytic microorganisms ( Bauchop, 1989). Anaerobic
chytrids are new to rumen microbiology because they were first mistakenly identified as flagellates.
These anaerobic fungi have hydrogenosomes instead of mitochondria and occur only in the gut of
herbivorous animals. The cellulolytic bacteria and protozoans also compete with cellulose-
degrading ciliates. Anaerobic ciliated protozoa are uniquely adapted to the rumen environment and
make up about 40% of the rumen biomass ( Williams and Coleman, 1991; Tannock, 1995) ( fig.
3.7a ).

The role ciliates play in rumen fermentation is controversial. Their predation on rumen bacteria is
thought to cause rumen inefficency. Ciliates have slow turnovers. They feed on bacteria and can
ingest small plant fragments that enter the rumen. Digestion occurs in the food vacuoles and
produces volatile fatty acids, ammonia, and hydrogen, which in turn serve as a substrate for the
methanogens which are ectosymbionts of the ciliates. There are two major groups of ciliates in the
rumen, the holotrichs and the entodiniomorphs. The holotrichs have cilia all over their cell surface
and include the genera Dasytricha and Isotricha. Holotrichs convert sugar into a type of starch that
is chemically similar to plant starch. Entodiniomorphs have a firm pellicle that is often drawn out
posteriorly into spines and cilia and are represented by the genera Diplodinium, and Entodinium
(see fig. 9.1 ). Rumen protozoa do not survive in culture.

Clostridium and Peptostreptococcus produce ammonia, which is required by many rumen microbes.
Rumen fermentation of cellulose degradation products results in hydrogen, formate, succinate, and
lactate.

Methanogens are obligate anaerobic archaebacteria. They use hydrogen and carbon dioxide as
substrates for their energy-yielding metabolism and release methane into the environment. They
were first observed as ectosymbionts of rumen ciliates ( Krumholz et al., 1983). Methanogens as
endosymbionts of Dasytricha ruminantium and Entodinium sp. occur in membrane-bound vacuoles;
they escape digestion and grow and divide synchronously with their host cells ( Finlay and Fenchel,
1993). Methane is the most abundant hydrocarbon in the trophosphere, and its emission from
ruminants is second only to emission from rice cultivation. Termites are also major contributors of
global methane. The role of methane in global warming has attracted the attention of
climatologists.

The rumen system is an excellent model for studying symbiotic associations under stable
environmental conditions ( Duncan and Edberg, 1995). On a practical basis, rumen fermentation is
of special significance to humans. Cattle and other domesticated ruminants are efficient converters
of the bioenergy of green plants into animal protein, woolen fiber, and leather products.

Chemolithotrophy: Life without Oxygen

The finding of luxuriant biological communities around deep-sea hydrothermal vents and in other
sulfide-rich environments such as mudflats and subtidal sediments were important discoveries in
biology. An obvious question about these strange, new animals was the nature of their food source
( table 3.1 ). It was soon discovered that endosymbiotic sulfuroxidizing chemoautotrophic bacteria
lived within tissues of the organisms ( Cavanaugh 1994). Hydrogen sulfide in the bacterial
symbionts is oxidized in the process of oxidative phosphorylation to ATP ( Doeller, 1995). A more
common pattern occurs when the hydrogen sulfide that enters the host body is oxidized to
thiosulfate, which is then supplied to all the symbionts.

Methanotrophic bacteria also form endosymbioses with members of the vent animal community as
well as with protozoans that live in the anaerobic marine sediments. Such associations are found in
marine bivalves, which use methane as a source of carbon and energy ( Distel and Cavanaugh,
1994; Distel, 1998).

Anaerobic marine ciliates and their symbionts

Free-living ciliates are common inhabitants of marine sandy sediments and often form intimate
associations with bacteria. Sulfate-reducing bacteria are ectosymbionts of anaerobic ciliates such
as Caenomorpha levanderi, Metopus contortus, and Parablepharisma pellitum. The bacteria occupy
pits on the outer wall of P. pellitum, whereas in the other ciliates they form layers that attach to
host cell membranes. Ectosymbionts may add more than 10% to the biovolume of P. pellitum (
Fenchel and Finlay, 1995). Purple nonsulfur photosynthetic bacteria and methanogens live as
endosymbionts in their protozoan hosts ( fig. 3.7 ). In general, all anaerobic protozoans enhance
their energy metabolism by acquiring hydrogen-consuming symbionts. Methanogens live as
endosymbionts of anaerobic ciliates because they have a steady source of hydrogen that is
generated in the hydrogenosomes.

Strombidium purpureum is reddish brown because of the photosynthetic nonsulfur bacteria in its
cytoplasm. The ciliate can be cultured anaerobically with frequent exposure to light. Other closely
related species are aerobic, suggesting that S. purpureum evolved from an aerobic ancestor.
Strombidium purpureum produces large amounts of hydrogen, which then is consumed by the
bacterial symbionts. It is not known whether S. purpureum receives any products of the symbiont's
pho- tosynthesis. The bacterial symbiont is closely related to Rhodopseudomonas, which in the
dark and at low oxygen concentration can carry out oxidative phosphorylation by using hydrogen
and fatty acids as substrates. In the dark, the symbiont benefits from oxygen, and the host ciliate
reduces its threat of oxygen toxicity. Strombidium purpureum and its cytoplasmic symbionts
provide an evolutionary window into how an anaerobic host can convert into an aerobic ciliate (
Fenchel and Finlay, 1995) ( fig. 3.8 ). Modern-day mitochondria may be descendents of ancient
ancestors that are similar to present-day nonsulfur photosynthetic bacteria ( Woese, 1977).

Pelomyxa palustris is an interesting ciliate because, although lacking hydrogenosomes, it contains

three different rod-shaped bacterial endosymbionts, two of which are methanogens, a third type
which surrounds the nucleus. The endosymbiont associated with the nucleus is thought to perform
the hydrogenosome function. If true, the cytoplasm of Pelomyxa would demonstrate a three-step
food chain consisting of eukaryotic fermentation to volatile fatty acids, prokaryotic hydrogen
production, and methanogensis ( Fenchel and Finlay, 1995).

Psalteriomonas lanterna, an anaerobic amoeboflagellate, also contains hydrogenosome-like

organelles that form tight complexes with methanotrophic symbionts. Inactivation of methanogenic
symbionts in Metopus contortus and Plagiopyla frontata retarded the host's growth rate by 25-30%
( Fenchel and Finlay, 1995). Methanogen symbiosis in Plagiopyla frontata is the most complex. The
methanogenic symbionts and host hydrogenosomes form alternating layers. The host regulates the
number of methanogens so that when it divides both methanogens and hydrogensomes increase in
number, an example of co-evolution between this specific host and its symbionts.

A typical Metopus contortus has about 7000 methanogens as endosymbionts and about 4000
sulfate-reducing bacteria on its outer surfaces ( fig. 3.9 ). The ciliate breaks down its food into
pyruvate and NADH, which in turn are oxidized in hydrogenosomes to produce acetate and
hydrogen. Methanogens use hydrogen and carbon dioxide to liberate methane and water. The
ectosymbionts metabolize acetate, lactate, or alcohol that diffuse out of the host cell.

Bacterial symbionts of tubeworms and other animals

The deep-sea vents of the Pacific Ocean are unusual habitats of symbiotic associations ( Childress

et al., 1987; Fisher, 1990; Childress

 Fig. 3.9 Marine anaerobic ciliates such as Plagiopyle frontata and Metopus contortus possess
hydrogenosomes and release hydrogen as metabolic waste, which in turn is used by methanogens
that live as endosymbionts. Redrawn from Fenchel and Finlay (1994).

and Fisher, 1992; Van Dover, 1996). Hot water rises from these vents as a result of water seeping
through cracks in the ocean floor and being boiled by the underlying molten rock. The water has a
high concentration of hydrogen sulfide. These vents are identical to the hot springs found on land,
the only difference being that the marine vents are about 2 miles below the surface of the ocean.
Surprisingly, these remote and seemingly inhospitable habitats contain a rich and diverse group of
organisms ( Tunnicliffe, 1991, 1992; Childress , 1995). One of the strangest members of this group
is the red tube worm, Riftia pachyptila (phylum Pogonophora). Large colonies of this giant worm
are attached to rocks along the path of the water that flows from the hydrothermal vents ( Hessler
and Kaharl, 1995).

Pogonophorans live inside stiff, chitinous shells, which they secrete around themselves. Their body
consists of a long trunk, a short anterior piece, and a short posterior piece, which anchors the
animal to the tube and to the ground. Numerous long, cilia-bearing tentacles project from the
anterior end of the animal. Pogonophorans that live in regions such as the Galapagos rift may be
up to 3 m long. They glide partially in and out of their tubes and move with the currents of water
that flow from the vents. The animals lack a mouth and a digestive tract.

Part of the body cavity of a tube worm, the trophosome, is highly vascularized and contains dense
colonies of endosymbionts belonging to the γ group of Proteobacteria. The bacteria are
chemoautotrophs, which means that they obtain energy from the oxidation of sulfides and fix
carbon dioxide through the Calvin-Benson cycle. The symbionts are most likely acquired from the
environment, but they have not been detected outside of their host. The well-developed circulatory
system in the trophosome and the high oxygen-binding capacity of the blood hemoglobin are
adaptations that ensure anaerobic conditions for the bacterial symbiont. In the bivalves
Bathymodiolus thermophilus and Calyptogena magnifica and the gastropod Alvinconcha hessleri,
endosymbionts are located within the gill tissues. These vent molluscs have a digestive system
which allows them to feed on free-living bacterial mats. Filaments of Beggiatoa and Thiotrix form
thick mats through which the hydrothermal water percolates. The free-living sulfuroxiding species
Thiobacillus hydrothermalis and Thiomicrospira crunogena, as well as heterotrophic bacterial
species, have been described.

Shrimps and annelids have only ectosymbionts, whose role in the symbiosis is not clear. In the
annelids Alvinella caudata and A. pompejana, the ectosymbionts consist of complex bacterial
filaments of the E group of Proteobacteria, which are inserted into the host epidermis at specific
regions ( Nelson and Hagen, 1995).

Hydrothermal vent communities continue to be a source of new research challenges. The

mechanisms of symbiont transmission, genetic exchange between host and symbionts, and the
upper temperature limits to life are some of the unanswered questionsTable 3.1 Animals with
chemoautotrophic bacterial symbionts
Pogonophora
Riftia pachyptila, vestimentiferan tube worm
Molluscs (bivalves)
Bathymodiolus thermophilus, hydrothermal vent mussel
Calyptogena magnifica, hydrothermal vent vescomyid mussel
Lucina pectinata, lucinid mussel in mangrove sediments
Lucina floridana, warm sulfide-rich shallow sediment
Lucinoma aequizonata, Santa Barbara basin
Solemya togata, mudflats
Solemya borealis
Solemya velum
Thysaria, subtidal sediments
Annelids (alvinellid polychaetes)
Alvinella sp.
Paralvinella sulfinocola, sulfide worm in subtidal sediments
Nematodes
Stilbonematid, free-living marine nematode in subtidal sediments

3.4 SUMMARY
Prokaryotes form two domains, the archaea and the bacteria. Bacteria are prokaryotes whose cells
are fundamentally different in organization and chemistry from those of eukaryotes. Bacteria are
symbionts in diverse organisms, from one-celled paramecia to humans. Bdellovibrios are parasitic
bacteria that forceably enter other bacteria. Their mode of entry is being used as a model of how
ancestral prokaryotes may have become colonized by symbiotic organisms.

Paramecium aurelia is host to different types of rod-shaped, Gram-negative bacteria such as κ,

strains of which are killers. Why organisms such as P. aurelia are more predisposed to form
symbiotic unions than others is not clear.

Laboratory studies have shown that Amoeba proteus can establish artificial symbioses with bacteria
within a few years. The artificially induced symbiosis between the bacteria and A. proteus
illustrates how symbiotic associations may have evolved and suggests that even today symbiotic
unions are being formed and dissolved continually in natural situations. Within 5 years, bacteria
that began as pathogens of A. proteus assumed a role of organelles and became indispensable to
their hosts. Pathogenicity of Entamoeba histolytica is influenced by its bacterial symbiont.

Luminescent bacteria occur in different marine organisms and terrestrial insects, and the light they
produce may lure prey, attract feeding organisms, or frighten predators. The diversity of organisms
that house luminescent bacteria suggests that bioluminesence has a selective value in terms of the
evolution of organisms.

Bacteria are common in insects and are housed in specialized cells called mycetocytes, which form
unique organs called mycetomes. The life cycles of bacterial symbionts inside insects is complex
and not clearly understood. Aposymbiotic insects have been freed of mycetocytal symbionts,
usually by means of antibiotics fed to the insects.

The Buchnera--aphid mutualism is 250 million years old and very complex. An adult aphid may
contain more than 5 million bacterial symbionts. Wolbachia is an intracellular parasite that
determines the sex of its insect host. Its ability to cause cytoplasmic incompatibility in mosquitoes
has gained it attention as an insect pest control.

Ruminant animals contain large populations of cellulolytic bacteria in their stomachs along with
various types of protozoa, which also digest cellulose. Rumen bacteria, in addition to their
relationship with their host, also interact mutually with rumen protozoa. The hoatzin is a bird with
foregut fermentation that eats leaves. Anaerobic marine ciliates often form associations with
bacteria.

Sulfur-oxidizing bacteria are symbionts of tube worms, which live near deep-sea vents in the
Pacific Ocean. The bacteria synthesize carbon compounds, which are used by the host.
Methanotrophic bacteria occur in marine bivalves.

4 BACTERIAL PATHOGENESIS
Molecular Mechanisms
De Bary ( 1879) broad definition of symbiosis includes parasitism and disease, areas in which
significant discoveries are being made. This has been most evident in bacterial pathogenesis. In
this chapter we introduce some of the recent findings of molecular mechanisms of bacterial
pathogens. During the past decade, scientists have introduced innovative approaches and concepts
from disciplines such as bacteriology, cell biology, and immunology in an attempt to understand
the molecular basis of infectious diseases. Stanley Falkow calls such attempts the "Zen" of bacterial
pathogenesis, believing, in the Zen Buddhist tradition, that true enlightenment is achieved through
meditation and insight ( Falkow, 1988, 1990)."Virulence" and "pathogenicity" refer to the ability of
bacteria to cause disease. The traditional criteria for establishing that a bacterium is responsible for
a disease have been Koch's postulates, which were developed in 1882. Although serving well for
many years, these postulates have limitations: (1) not all bacteria can be cultured, (2) not all
members of a species are equally virulent, and (3) adequate animal hosts are not always available.
Host susceptibility is an important virulence factor for bacteria. Falkow has proposed the "molecular
Koch's postulates" to examine the potential role of genes and gene products in pathogenesis. The
new postulates are:
1. The gene or its product should be found in strains of bacteria that cause the disease and
not in bacteria that are avirulent.
2. A mutation of a virulent gene should reduce its virulence, and, conversely, introduction of a
virulent gene into an avirulent bacterial strain should make it virulent.
3. The gene should be expressed by the bacterium during the infection process.
4. Antibodies to a gene product should be protective, or the gene product should elicit an
immune response.

Molecular biology techniques now make it possible to detect and identify bacteria and their genes
even though they cannot be cultured ( Finlay and Falkow, 1989; Relman et al., 1992).

4.1 HOW BACTERIA COLONIZE A HOST

The first important step in bacterial pathogenesis is adherence to a host cell. This occurs by means
of pili, which consist of long rods that extend out from the bacterial surface. The tips of the pili
contain proteins that attach to host cell receptors. In some cases, pilin, the protein subunits of the
pilus shaft, attach to the host cell's receptors. In addition to pili, bacterial surface proteins called
adhesins attach firmly to the host cells. Sometimes the host makes antibodies against pili or
adhesin proteins, and this induces the bacteria to make different types of adhesins. Only
Gramnegative bacteria make adhesins; the mechanism of how Gram-positive bacteria attach to a
host cell is not known.

Bacterial surface proteins called invasins cause cytoskeletal changes in host cells that result in
pseudopodial formation and engulf- ment of bacteria in phagocytic vesicles. Some bacterial
pathogens escape from the vesicles and multiply in the host cell cytoplasm. Bacterial invasion can
become systemic after the pathogens cross the mucus membrane by moving through M cells, but
they must survive the attack of phagocytes in the lymphatic system ( Cundell and Tomanen,
1995). Once established in the host tissue, bacteria obtain nutrients from the host in different
ways. For example, iron, which is an essential mineral for living cells, occurs in complexes with
iron-binding proteins. Some bacteria concentrate the iron they need by producing a siderophore, a
compound which has a high affinity for iron. Other bacteria use surface proteins to remove iron
from host iron-binding proteins such as transferrin, lactoferrin, ferritin and hemin.Bacteria survive
by avoiding the host's immune system. Some bacteria have a capsule that consists of a loose
network of polysaccharides or a protein--carbohydrate mixture. Capsules protect the bacteria from
the host's inflammatory response after phagocytosis. Antibodies that bind to a capsule help a host
facilitate phagocytosis. Some bacteria evade antibodies by covering themselves with host proteins
such as fibronectin, changing their surface antigens, or mimicking host molecules such as
hyaluronic acid and sialic acid ( Finlay and Cossart, 1997).
4.2 HOW BACTERIA DAMAGE HOST CELLS
The virulence of many bacterial pathogens is due to the toxins they produce, which disrupt normal
cell functions and cause cell death. Exotoxins are proteins that are excreted by dividing bacteria.
Exotoxins that attack a variety of cell types are called cytotoxins; those that attack a particular cell
type or tissue have specific names, such as neurotoxin, leukotoxin, hepatotoxin, or cardiotoxin.
Exotoxins can be associated with a specific bacterial disease. For example, cholera toxin is
produced by Vibrio cholerae, diphtheria toxin by Corynebacterium diphtheriae, and tetanus toxin by
Clostridium tetani. Microbiologists place exotoxins into three groups:
1. A-B exotoxins consist of one enzymatically active portion (A) and another portion (B) that
binds to the host cell. The B portion is recognized by a host cell-surface receptor; the A
portion enters the cell cytoplasm and disrupts either protein synthesis or cAMP regulation.
2. Membrane-disrupting exotoxins make holes in the host's endosomal membrane by
hydrolyzing phospholipids, thereby enabling bacterial pathogens to escape from phagocytic
vacuoles into the cytoplasm. These toxins are also responsible for apoptosis of the host cell.
Two examples from this group of toxins are listeriolysin O produced by Listeria
monocytogenes and α-toxin produced by Corynebacterium perfringens.
3. Superantigens bind to major histocompatibility complex (MHC) receptors and to T-cells and
 cause them to produce protein cytokines such as interleukin-2 (Il-2). High levels of Il-2 in the
blood result in toxic shock syndrome, which is characterized by nausea, vomiting, malaise,
and fever. Examples of superantigen exotoxins are found in staphylococcal foodborne diseases
( Johnson et al., 1992).

An example of an endotoxin is the lipopolysaccharide (LPS) component of the Gram-negative

bacterial cell wall. The lipid A portion of this endotoxin is exposed only when the bacteria lyse. LPS
triggers the release of cytokines and activates the complement cascade in the host, which leads to
fluid leakage from blood vessels. This reaction, along with blood clotting in the peripheral
circulation, often leads to septic shock and death.

In addition to toxic proteins, pathogenic bacteria also produce hydrolytic enzymes that degrade
host tissues and disseminate bacteria within the host. Heat-shock proteins produced by bacteria
stimulate autoimmune responses so that host antibodies and T-cells attack healthy host cells (
Salyers and Whitt, 1994).

Role of Adhesins in Pathogenesis

Adhesins are surface proteins involved in protein--carbohydrate or protein--protein interactions
between bacteria and host cells. Bacteria can attach only when their adhesins match receptors of
the host cell membrane. This association of bacteria with a eukaryotic cell often brings about
changes in the host and pathogen. Bordetella pertussis (whooping cough), Hemophilus influenzae,
Psuedomonas aeruginosa, and Streptococcus pneumoniae (bacterial pneumonia) are pathogens
that destroy the ciliated epithelium and alveolar macrophages. Damage to the ciliated cells occurs
only when bacterial adhesins attach to the cilia and cause bacteria to release toxins. Two adhesin
proteins of B. pertussis, filamentous hemagglutinin (FHA) and pertussis toxin (Ptx), have been
characterized ( Cundell and Tomanen, 1995). Filamentous hemagglutinin is a 220-kDa protein with
several epitopes that recognize receptors called integrins on host cells. Attachment of pathogenic
bacteria to eukaryotic integrins involves three strategies: lectin binding, masking, and mimicry.
During lectin binding, as seen in E. coli and group B Streptococci, bacterial proteins recognize the
carbohydrate moiety of integrins. Masking, as seen in Mycobacteria and Legionella sp., involves
adsorption of serum protein, such as C3bi, onto the bacterial surface, which then binds to a C3bi
binding site on the integrin. Mimicry occurs when the bacteria produce a protein containing an
epitope that binds directly with integrin. Mimicry is the binding mechanism for the three epitopes of
FHA of B. pertussis. Mutations in the genes for adhesin proteins result in loss of virulence in the
pathogen ( Isberg, 1991; Cundell and Tomanen, 1995).

Role of Iron in Pathogenesis

Bacteria need iron to grow, reproduce, and make toxins, but iron concentrations in nature are
usually low. Acquiring host iron is an important virulence factor. The concentration of free iron in
the human body is low because compounds such as ferritin, hemin, lactoferrin, and transferrin
strongly bind to most of the available iron. Increased iron levels in individuals have been
associated with susceptibility to bacterial infections. Overdose of iron tablets, excess red meat
consumption, alcoholism (resulting in accumulation of iron in the liver), and smoking all contribute
to iron overload. The ability of macrophages to phagocytize bacteria is compromised because high
iron concentrations reduce their adherence and endocytic capabilities. Patients with a high iron
overload have been given injections of siderophore desferrioxamine to reduce the levels of iron in
circulation.Pathogenic bacteria have developed several strategies for getting iron from the host: (1)
lysis of red blood cells, digestion of hemoglobin, and binding and assimilating the heme, (2) cell
surface binding of transferrin, (3) production of siderophores to gather iron from ferrated
transferrin, and (4) assimilation of intracellular iron from pools of low-molecular weight host iron-
binding compounds.Pathogenic strains of Neisseria gonorrhoeae can obtain iron from human
transferrin, whereas commensal strains of this bacterium cannot gather iron from the host
environment. A wide variety of bacteria synthesize siderophores to gather iron from transferrin or
lactoferrin. Catechols and hydroxamates are two classes of siderophores that form tightly chelated
complexes with iron. These then bind to siderophore receptors on the bacterial surfaces and are
internalized in the cytoplasm. The siderophore--iron complex is cleaved to free the iron molecule.
Some bacteria not only make their own siderophores and surface receptors, but they also have
siderophore receptors for other bacteria. Highly virulent strains of Escherichia coli produce
siderophores called aerobacterin. Pathogens such as Legionella pneumophila, Listeria
monocytogenes, and strains of Mycobacterium, Salmonella, and Shigella obtain iron intracellularly
and do not need siderophores ( Weinberg, 1997). Lactobacilli prevail over other bacteria in host
environments where iron is scarce because they use cobalt or manganese in their metabolism
instead of iron.
4.3 BACTERIAL PATHOGENS
The term parasite-specified endocytosis describes microbial entry into phagocytic cells such as
macrophages ( Moulder, 1985). Alternatively, the bacterial entry may be through a host-specified
endocytosis. Phagocytic cells have many lysosomes with hydrolytic enzymes that can digest
microbial pathogens. Once a pathogen is engulfed, it is contained in a vacuole called a phagosome.
The phagosome fuses with a lysosome to make a phagolysosome, and digestion of the engulfed
microbes then occurs by one of the following mechanisms:
1. Oxygen-dependent phagocytosis, which requires increased oxygen consumption by
phagocytic cells and conversion of oxygen into toxic
intermediate forms such as the superoxide anion, hydrogen peroxide (H 2 O 2 ), singlet
oxygen, and hydroxyl radicals.
2. Oxygen-independent mechanisms, which involve the activities of lysosomal enzymes such
as phospho-lipases, proteases, RNase, and DNase.
3. Nitrogen-dependent mechanisms, which use reactive forms of nitrogen intermediates such
as nitric oxide (NO), nitrite (

) and nitrate (

). Nitric oxide is the most effective killing agent of the three and is produced from arginine
when the phagocytes are stimulated by interferons or tumor necrosis factor ( Lancaster,
1992).

Lactic acid production, a result of altered metabolism in phagocytes, lowers pH and enhances the
activities of many lysosomal enzymes. Examples of intracellular pathogens are given below.

Yersinia Entry into Mammalian Cells

Yersinia pestis, the causal agent of plague, enters the host from the bite of an infected flea.
Yersinia enterocolitica and Y. pseudotuberculosis enter the host's digestive system from
contaminated food or water ( fig. 4.2b ). All yersiniae eventually end up in lymphatic tissue. The
invasin protein of Y. pseudotuberculosis was originally identified by its giving E. coli the ability to
enter cultured mammalian cells. Later, it was found that this membrane protein confers invasive
properties by binding to ß-1 integrin receptors on the host cell membrane. The gene (inv) for this
protein has been cloned. Another Yersinia gene located on the virulence-associated plasmid, YadA,
mediates another pathway into the host cell. In animal models mutations for inv and YadA have
resulted in loss of virulence ( Miller et al., 1994).

During the infection process, pathogenic Yersinia spp. export proteins known as the Yersinia outer
membrane proteins (YOPs). The YOPs are encoded by 70-75 kb virulence plasmids (pYV) that are
highly conserved among the three pathogenic species of Yersinia. Yersinia strains that lack pYV can
enter host cells but are unable to multiply. Yersinia strains with virulent pYV can overcome host
inflammatory responses and multiply extracellularly in host tissue ( Salyers and Whitt, 1994).

Pathogenicity Islands and How Salmonella Became a

Pathogen
Salmonella is usually acquired from contaminated food or water. Because there is about a 90%
DNA-DNA homology between Salmonella sp. and Escherichia coli, some microbiologists want to
reclassify them within a single genus. Another proposal is to place all pathogenic Salmonella in one
species, Salmonella enterica. The traditional approach is to include the following three species in
the genus Salmonella: (1) Salmonella typhi, which causes typhoid fever in humans. Bacteria enter
the bloodstream following their uptake in M cells of the Peyer's patches and are spread throughout
the body. Death results from septic shock. (2) Salmonella enterditis, which causes diarrhea in
humans and animals and has more than 2000 serotypes, including Salmonella typhimurium, whose
infection in humans is characterized by diarrhea, fever, and abdominal pain. The disease is usually
self-limiting. Young children and immunocompromised adults are most susceptible to it. Salmonella
typhimurium infections are usually acquired from poultry products; (3) Salmonella cholerasuis,
which causes systemic infections in humans, but is primarily a pathogen of pigs.

Recent studies have shown that at least 60 genes are involved in virulence of Salmonella, perhaps
because of the complexity of its life cycle. The bacteria must survive the acidic pH of the stomach
before they attach to intestinal epithelial cells, thereby bringing about their own phagocytosis and
replication in macrophages ( Groisman and Ochman, 1997).

Laboratory studies have shown that Salmonella strains adhere to cultured mammalian cells and
cause cytoskeletal changes. The cell's outer membrane is modified to produce pseudopodia which
engulf the bacteria and internalize them inside endocytic vesicles ( fig. 4.1b ). Bacteria multiply
inside the vesicles which may fuse together. How Salmonella triggers cytoskeletal changes of the
host cell is not known. One explanation is that a host cell signal-transduction pathway increases
intracellular calcium, and this activates actin depolymerization enzymes ( Pace et al., 1993).

Many Salmonella strains, with the excep- Fig. 4.1 Escherichia coli and Salmonella cause
cytoskeletal changes in the host cell membranes. (a) Infection with enteropathogenic strain of
Escherichia coli induces the host cell membrane to form dome-shaped pedestals on top of which
bacteria attach. (b) Salmonella strains cause the host cell membrane to form pseudopodia before
the bacteria are engulfed. Adapted from Salyers and Whitt ( 1994).

tion of S. typhi, contain a large virulence plasmid. Loss of this plasmid significantly reduces
virulence in mice. Ability to survive inside the phagocytes depends on the expression of many
genes. These include genes that help bacteria withstand exposure to reactive forms of oxygen, low
pH, and a number of host defenses. Only a few of these genes have been identified, such as the
PhoP/PhoQ operon, which regulates prg and pag genes.

Most of Salmonella's virulence genes occur in large clusters in several regions of the bacterial
chromosome called pathogenicity islands. The best known pathogenicity island of Salmonella is
SPI-1, a cluster of 25 genes. Experimental evidence has shown that this island was acquired
through horizontal gene transfer from Shigella ( Baumler, 1997). The size, order, and orientation of
the inv and spa genes in the SPI-1 island are similar to the invasion genes on Shigella's virulence
plasmid. Homologs of the inv and spa genes occur in a broad range of bacterial pathogens, such as
Erwinia, Pseudomonas, Xanthomonas, and Yersinia. Mutations in the SPI-1 gene cluster prevent
Salmonella from invading epithelial cells in vitro ( Groisman and Ochman, 1997).

Shigella: Subversion of Host Cellular

Cytoskeleton
Shigella spp. and certain strains of Escherichia coli cause dysentery in humans and produce blood
and mucus in diarrheal stools. The disease is self-limiting in healthy adults, but among infants and
children in developing countries it often causes death.

Shigella spp. adhere to mammalian cells in a culture medium and disrupt the host's cytoskeleton (
Theriot, 1995). The bacteria become engulfed within vacuoles. Once inside the host cell the
bacteria escape into the cytoplasm, where they multiply. Bacterial surface proteins, the invasion
plasmid antigens (IpaB-D), are essential for adherence and invasion. The release of Ipa proteins
from the bacteria is controlled by another set of proteins called the membrane excretion of Ipas
(Mxi). Bacteria move within the cell cytoplasm and from one cell to another by polymerizing the
host's actin filaments. Shigellae are unable to invade the mature cultured and normal intestinal
columnar epithelium of a host animal. The free surfaces of these host cells lack receptors called
integrins. However, integrins are present on the lateral surfaces of mucosal cells and free surfaces
of M cells of the Peyer's patches. The M cells are phagocytic and are positioned along the mucosal
lining to sample antigens from the lumen of the intestine. Phagocytic uptake of Shigella (bacterial)
protein by M cells are then presented to lymphatic tissue of the Peyer's patch. Shigella invades
other body tissues by escaping the M cell phagocytosis ( Jepson and Ann Clark, 1998) ( fig. 4.2a ).

Shigella invasion of macrophages in tissue culture causes apoptosis or programmed cell

Fig. 4.2 Shigella and Yersinia bacteria enter the host through M cells of Peyer's patches, which are
common throughout the intestinal mucosa. (a) Shigella is unable to invade the normal columnar
epithelial cells, and their phagocytosis through M cells, followed by escape from the infected
macrophages, allow the bacteria to spread to epithelial cells. The bacteria spread through epithelial
cells by propelling themselves by using the host cell's cytoskeletal proteins. (b) Yersinia invasion of
M cells depends on the production of Yersinia outer proteins. The bacteria then escape and multiply
extracellularly in host tissue. Adapted from Salyers and Whitt ( 1994).

death. Apoptosis is characterized by DNA breakdown and condensation of chromatin at the nuclear
boundary while the cell organelles remain structurally sound. On the other hand, in accidental cell
death (cell necrosis), the chromatin becomes granular and the cytoplasmic organelles disintegrate.
The ability to trigger apoptosis in macrophages explains how Shigella sp. survive a phagocytic
attack of macrophages ( Sansonetti , 1992).

Shigella dysenteriae produces an A-B exotoxin called shiga toxin. This toxin inactivates ribosomes
and stops protein synthesis in the infected host cells. Shigella strains possess large plasmids that
have virulence genes for invasion and intracellular movement ( Zychlinsky et al., 1994).

Listeria monocytogenes

Listeria monocytogenes is a foodborne pathogen that attacks the cells of the intestinal lining and
produces mild influenzalike symptoms in healthy adults. The disease is commonly associated with
unpasteurized milk or foods such as coleslaw and shrimp. The bacterium can pass through the
placental barrier in pregnant women and can cause stillbirth, premature birth, and systemic
infection in newborn infants. Up to 5-10% of the human population may be asymptomatic carriers
of this pathogen.

Listeria monocytogenes enters a variety of mammalian cells in situ and in culture. A surface
protein, internalin, mediates entry into intestinal epithelial cells in culture. The host cell surface
receptor of intemalin is a transmembrane adhesion protein called E-cadherin, which is bound to
actin filaments of the cellular cytoskeleton through a complex with catenines ( Ireton and Cossart,
1997). A mutation in the gene for intemalin, InlA, prevents the pathogen from invading host cells.
Internalin induces phagocytosis of L. monocytogenes and creates an endocytic membrane-bound
vacuole containing bacteria. The bacteria escape from the vacuole by producing a hemolysin,
listeriolysin O (LLO), which is a sulfhydral-activated, pore-forming cytotoxin. The gene for LLO
protein is a major virulence factor in L. monocytogenes. In addition, two other hemolysins called
phospholipases disrupt host cell membranes and contribute to the spread of bacteria to other cells
( Farber and Peterkin, 1991; Tilney et al., 1992).

The movement of Listeria monocytogenes in the host cell cytoplasm is the result of a bacterial
protein called ActA, which causes the polymerization of actin and the formation of long tails. ActA is
an essential determinant of pathogenicity in Listeria because bacteria use the actin tails to pass
from one host cell to another ( G. A. Smith and Portnoy, 1997; Dramsi and Cossart, 1998). Listeria
virulence genes are clustered together on the bacterial chromosome.

Mycobacterium tuberculosis

More than 2 billion people are infected with Mycobacterium tuberculosis, and about 8 million new
cases occur each year. Tuberculosis is the most common infectious disease of humankind,
accounting for more than 26% of the avoidable deaths in the developing world. Mycobacterium
tuberculosis grows in alveolar macrophages of the lungs. Macrophages that ingest M. tuberculosis
use the bacterial antigens to evoke T-helper cell response (CD+4) and cytotoxic T-cell response
(CD+8). The CD+4 cells stimulate antibody response in B-cells and release interferon, which
activates the macrophages. The CD+8 cells kill macrophages that contain bacteria. The cell wall of
Mycobacterium consists of a thick peptidoglycan layer containing arabinogalactan,
lipoarabinomannan (LAM), and mycolic acids that protect it from host cell phagocytosis. LAM helps
reduce the receptor-mediated phagocytosis of mycobacteria by suppressing T-cell proliferation. The
mylonic acid component of mycobacterial cell walls is toxic to the host cells and stimulates the
host's inflammatory response. The ability of M. tuberculosis to survive in macrophages is a major
virulence factor. Bacteria survive in phagocytic vacuoles by preventing the acidification of the
phagosome ( MacDonough et al., 1993; Salyers and Whitt, 1994). Examining the genetics of M.
tuberculosis is difficult because of its slow growth under laboratory conditions.

Legionella pneumophila: Phagocytosis

with a Twist
Legionnaire's disease is a form of pneumonia caused by Legionella pneumophila and is usually
spread by inhaling aerosols from contaminated water that undergoes rapid cooling, as found in air-
conditioning cooling towers. L. pneumophila occupies a unique intracellular niche inside a
macrophage. The macrophage takes up the bacterial pathogen by means of a novel mechanism
called "coiling phagocytosis." A long, thin pseudopod from the macrophage coils around the
bacterium and engulfs it. Once internalized, the host cell's mitochondria gather around the
phagosome. The phagosome does not acidify and thus avoids fusion with lysosomes. An outer
membrane protein of L. pneumophila, called MIP (macrophage invasion protein) aids in the
bacterial invasion. Bacterial genes designated as DOT (defect in organelle trafficking) encode
proteins involved in intracellular survival. Damage to the lungs is caused not only by accumulation
of macrophages but also by the production of several proteases by L. pneumophila. A zinc
metalloprotease, in particular, is very destructive to the host tissue.

Legionella spp. are common in soil and freshwater and parasitize Acanthamoeba and Naeglaria,
which may serve as reservoir hosts of the bacterial pathogen ( Barker et al., 1992).

Diphtheria Toxin and Temperate Bacteriophages

Diphtheria is primarily a disease of children and is caused by Corynebacterium diphtheriae. It can
be fatal if not treated, but an effective vaccine is available. Diphtheria toxin is an example of how a
single molecule can produce disease symptoms, and its study helped give rise to the concept of
virulence factor, which is defined as any product or strategy of a bacterial pathogen that enhances
infection of a host. The toxin suppresses the protein synthesis in the host cell. The use of
diphtheria toxin to kill tumor cells or HIV-infected cells has attracted attention from the scientific
community ( Salyers and Whitt, 1994). The toxin gene is carried by a temperate bacteriophage,
and only those strains of C. diphtheriae that have this phage can cause the disease. The toxin gene
is regulated by an irondependent repressor protein, DtxR. It is suggested that the role of diphtheria
toxin is to provide iron for the bacteria ( Roth et al., 1995).

Diphtheria toxin selectively binds to the host cell receptor protein, the heparin-binding epidermal
growth factor. Cardiac muscles and neurons have many more toxin receptors, and therefore heart
failure and brain damage are common symptoms of this disease ( Salyers and Whitt, 1994).

Vibrio cholerae: Cholera as a Paradigm

Vibrio cholerae is a motile, Gram-negative, curved rod with a single flagellum. The bacterium grows
in freshwater and seawater and can colonize the human intestine, where it produces an exotoxin.
The toxin disrupts ion transport across the epithelial lining and causes diarrhea and water loss.
Cholera is

most severe among children. Individuals who recover from cholera may become asymptomatic
carriers and spread the bacteria during a subsequent epidemic.

The principal virulence factors in V. cholerae are pili and cholera toxins. Filamentous bundles of pili
on the bacterial surface are used to colonize the intestinal mucosa. The pili are called toxin
coregulated pili (TCP) because the regulation of genes for pili formation is similar to the way
cholera toxin genes are transcribed. The TCP pili are important cholera adhesins. Mutants deficient
in TCP pili are avirulent. Vibrio cholerae also makes a hemoagglutinin, which may serve as a
cholera adhesin.
Cholera toxin, an A-B type of exotoxin, is the main virulence factor responsible for the disease. The
toxin has five B subunits and one A subunit, which in turn is made up of two parts, A1 and A2.
Genes ctxA and ctxB are organized into an operon and are responsible for transcription of the toxin
subunits. The excreted toxin binds to GM1 ganglioside on host mucosal cells. An unknown
mechanism translocates the Al subunit into the host cell cytoplasm, where it triggers adenylate
cyclase activity, resulting in high levels of cAMP. Sodium ion transport is blocked, creating an ion
imbalance that leads to diarrhea ( fig. 4.3 ). Mutants for cholera toxin genes fail to produce the
disease. Vibrio cholerae also produces Zot toxin, which disrupts the tight junctions binding the
mucosal cells together, and Ace toxin, which produces a milder form of diarrhea. The genes for Zot
and Ace toxins are located next to each other on the bacterial genome. Together they increase
virulence during infection ( Atlas, 1997).

Cholera and El Niño

Cholera is an ancient disease with historical records in Greek and Sanskrit tracing back 2000 years.
The disease has existed for centuries on the Indian subcontinent. Medical historians have noted
that since 1817, seven major pandemics have killed millions of humans worldwide. Six pandemics
believed to have started in the coastal waters of the Bay of Bengal were caused by the classic
Vibrio cholerae 01 serotype. The seventh and most recent outbreak of cholera in the 1990s was
caused by V. cholerae 0139, and the first outbreak of this new serotype occurred in the port city of
Chancay in Peru. Many Vibrio sp., including V. cholerae, occur in chitinaceous zooplankton
(copepods) and shellfish. The as-

Fig. 4.3 Cholera toxin, an A-B exotoxin which is encoded by the ctx gene, binds to the host cell
receptors. A portion of the toxin triggers adenylate cyclase activity which in turn results in a high
level of AMP. Sodium channels are blocked, causing a loss of electrolytes and the disease
symptoms. Redrawn from Atlas ( 1997).
sociation of V. cholerae and copepods is key to understanding the global nature of cholera
epidemics. It is now believed that cholera is carried away from its endemic area by means of ocean
currents that transport copepods and their bacterial symbionts to new coastal communities.The
Peruvian outbreak of cholera has been correlated with plankton blooms triggered by the climatic
phenomenon known as El Niño, which causes rainfall that washes nutrients from the land and
brings about warmer sea surface temperatures. A single copepod may contain 10,000 bacterial
cells, a dose sufficient to infect humans. The probability of ingesting several copepods with drinking
water increases with planktonic blooms ( Colwell, 1997).
The birth of Vibrio cholerae 0139
In 1993 a cholera outbreak by Vibrio cholerae serogroup 0139 in South Asia was a significant
turning point in the history of the disease. Evidence shows that this strain arose as a result of
genetic recombination and horizontal gene transfer. With the exception of V. cholerae 0139, all
known cholera epidemics have been caused by the classic serotype 01 of V. cholerae.The cause of
the sixth cholera epidemic, which originated in Indonesia in 1991, was designated as Vibrio
cholerae 01 E1 Tor. Vibrio cholerae non-01 serotypes do not cause epidemics of diarrhea. An
outbreak of a choleralike disease occurred in Madras, India, in October 1992 and was identified as
Vibrio cholerae non-01. As the epidemic spread along the coastal communities on the Bay of
Bengal, a new serotype, Vibrio cholerae 0139, was identified. The new serotype was identical to V.
cholerae 01 E1 Tor except that it had a capsule. Vibrio cholerae 0139 not only had the virulence
factors of V. cholerae 01 but could better survive external environmental conditions. The 1991V.
cholerae 01 El Tor strain has virtually disappeared from many areas. The V. cholerae 0139
lipopolysaccharide (LPS) virulence factor has an additional O-antigen. Somatic antigens or O
antigens are those associated with the surface of LPS of the cell wall. Genetic analysis of V.
cholerae 0139 revealed a deletion of about 22 kb DNA from chromosome 1 and an insertion of a
new 35-kb DNA fragment that encodes the 0139 LPS and capsule proteins. Molecular evidence
suggests that 01 E1 Tor is transformed into 0139 by acquiring new DNA through a horizontal gene
transfer ( Mooi and Bik, 1997).The modern study of cholera has engaged scientists from such
disparate disciplines as oceanography, marine biology, microbiology, molecular biology,
immunology, medicine, epidemiology, and space satellite imagery to produce a new conceptual
understanding of this ancient disease.
4.4 ESCHERICHIA COLI: A VERSATILE HUMAN PATHOGEN
Escherichia coli can cause many different types of diseases in humans. Most strains of E. coli are
avirulent because they lack virulence genes. E. coli is classified into the following five "virotypes"
on the basis of how the bacterium attaches to host cells, how host cells are affected following
attachment, and the kind of toxins produced:
1. Enterotoxigenic E. coli (ETEC). Strains of ETEC resemble Vibrio cholerae in that they adhere
to the mucosa of the small intestine and produce diarrhea in the host by secreting toxins.
They are unable to invade the host tissue. Infants and children are most susceptible to this
disease, the adult version of which is called traveler's diarrhea. A choleralike toxin, called
heat-labile toxin, and a diarrheal toxin, heat-stable toxin, are involved. Genes for these toxins
are carried on plasmids.
2. Enteroaggregative E. coli (EAggEC). The bacteria adhere to the mucosal cells in clumps and
produce a heatstable--like toxin and a hemolysinlike toxin. The EAggEC strains cause diarrhea
in children.
3. Enteropathogenic E. coli (EPEC). The EPEC strains produce dramatic changes in the host
mucosal cells. The epithelial cells to which the bacteria bind lack the normal microvilli and
instead have a cup-shaped pedestal under each bacterium. EPEC strains are the major cause
of an often fatal diarrhea in children and infants. The EPEC strain invades cultured host cells in
three stages. First, bacteria bind to the host cell surface via bundleforming pili (BFP). (The BFP
gene is carried on a plasmid.) Second, a signal
transduction pathway is triggered that activates host cell tryosine kinases and increases Ca2+
levels. Third, the host actin cytoskeleton is rearranged, and a pedestal where the bacterium
become firmly attached is formed ( Donnenberg et al., 1997) ( fig. 4.1a ).
4. Enterohemorrhagic E. coli (EBEC). The EHEC strain such as 0157:H7 causes dysentery and
hemolytic uremic syndrome, which can result in death from acute kidney failure. EBEC strains
produce a toxin similar to shiga toxin, the gene for which is located on a temperate phage.
5. Enteroinvasive E. coli (EIEC). The EIEC strains cause a disease identical to those caused by
Shigella sp. but do not produce shiga toxin. The genes for virulence are located on a large
plasmid and are regulated in a manner similar to those of Shigella species.

4.5 PSEUDOMONAS AERUGINOSA:

QUORUM SENSING AND VIRULENCE GENE
REGULATION
Pseudomonas aeruginosa is a metabolically versatile Gram-negative bacterium found in soil, fresh
water, plants, and animals. In humans, R. aeruginosa is an opportunistic pathogen which causes
infections in burn patients or immunocompromised patients and those with cystic fibrosis. The
bacteria produce pili and nonpilus adhesins. Pili adhesins bind to ganglioside receptors of the
epithelium, whereas the nonpilus adhesins bind to mucin associated with the epithelial cells. R.
aeruginosa also produces a thick coat of exopolysaccharides or alginate, which covers the bacterial
outer surfaces. In strains associated with cystic fibrosis, alginate is a mechanism for adherence and
also prevents phagocytosis of the bacterium. Alginate synthesis is energy consuming, and its gene
expression is tightly regulated ( Deretic et al., 1994).

R. aeruginosa secretes several cellular products into the surrounding environment which supply
nutrients and create conditions suitable for bacterial survival. Three extracellular proteases, LasB
elastase, LasA elastase, and alkaline protease, have been identified, and the genes responsible for
their expression have been cloned and characterized ( Passador et al., 1993). These proteases
cause lung damage in cystic fibrosis patients. Exotoxin A, transcribed by the toxa gene, is the most
toxic substance produced by R. aeruginosa. Recent experiments with R. aeruginosa show that gene
rega regulates toxa expression. Another gene, LasR, though not required for toxa, contributes
significantly to enhance toxin production. The amino acid sequence of LasR protein is similar to
another bacterial protein, LuxR, which controls the bioluminescence genes of the marine microbe
Vibrio fisheri. The LuxR protein binds DNA and controls a signal molecule known as the Vibrio
autoinducer. A Pseudomonas autoinducer has been isolated, purified, and identified as N-3-
oxododecanoyl homoserine lactone, which is similar to other autoinducers ( Gray, 1997; Pesci and
Iglewski, 1997).

Quorum sensing (a cell-density--dependent phenomenon) is mediated through autoinducer

molecules. Many organisms make autoinducer molecules to monitor their population size, which in
turn controls the expression of density-dependent genes. Classic examples of cell-to-cell
cooperation are fruiting body formation in the slime mold Dictyostelium discoideum and sporulation
in Bacillus. Quorum sensing also ensures the proper timing in producing virulence factors that
overwhelm the host defense response. Quorum sensing mechanisms have been identified in other
bacteria ( Strauss, 1999). In the plant pathogen Agrobacterium tumefaciens, the Ti plasmid directs
the production of opines, which serve as food for the bacterium. Erwinia carotovora, a plant
pathogen that causes bacterial soft-rot in vegetables, possesses a Lux-homologous system that
regulates the synthesis of carbapenum antibiotics and cell wall degrading enzymes. The
autoinducer of E. carotovora is similar in structure to that of Vibrio ( Passador et al., 1993; Pesci
and Iglewski, 1997) (fig. 4.4).

4.6 HELICOBACTER PYLORI: MOLECULAR MIMICRY

BETWEEN PATHOGEN AND HOST
Until 1991, medical microbiologists had been teaching that the stomach environment was too harsh
to support microbes. This changed following the discovery of Helicobacter pylori, which opened an
exciting new chapter in our understanding of bacterial life ( Rabeneck and Ranshoff, 1991).
Helicobacter pylori not only grows and thrives in the stomach, but it may also be responsible for
90% of gastric and (a)

Pseudomonas aeruginosa (b)

Erwinia carotovora Vibrio fischeri Yersinia enterocolitica (c)

Vibrio fischeri (d)

Agrobacterium tumefaciens (e)

Rhizobium leguminosarum Fig. 4.4 Structural comparison of N-acylhomoserine lactone autoinducer

molecules in five species of bacterial symbionts. Autoinducers play an important role in quorum
sensing, a cell-density-dependent phenomenon. Adapted from Gray ( 1997).

duodenal ulcers, though most infections are asymptomatic.

Helicobacterpylori produces large amounts of urease, which converts urea into ammonia and
carbon dioxide. Bacteria survive the stomach environment by being surrounded with ammonia
molecules, which neutralize stomach acids. Many bacteria invade the mucin layer, but only H.
pylori has the adhesins that bind to mucosal cells ( fig. 4.5 ). These adhesins include Lewis blood
group O antigens, phosophatidyl-ethanolamine, sialic acid, and laminin. H. pylori also produces
cytotoxins that cause the symptoms of peptic ulcers. The cytotoxins produce vacuoles within the
mucosal cells lining the stomach and small intestine. When the injected mucosal cells die, gastric
acids and digestive enzymes cause the formation of ulcers. H. pylori isolated from patients with
peptic ulcer disease and with gastric cancer contained a 38-kb fragment of DNA that was not
present in asymptomatic carriers. A cytotoxin-associated gene, the CagA gene, occurs in this
stretch of DNA called the pathogenicity island. Another virulent gene, the vacuolating toxin gene,
the VacA, is located 300 kb away from CagA. The genes are coexpressed in the most severe forms
of gastroduodenal diseases. Strains lacking the CagA gene also lack the vacuolating activity of
VacA. Mutations of CagA show that virulence in H. pylori has evolved through the inheritance of
one or more DNA insertions ( Covacci et al., 1997).

Molecular mimicry is observed between pathogen and the host when certain protein sequences are
compared. The LPS of H. pylori is unusual in that it expresses Lewis x and y blood group antigens.
Such antigens are also expressed on the host mucosal cell. Sequence similarity exits between the
vacuolating toxin of H. pylori and the host gastric H+K+-ATPase. Gastric H+K+-ATPase is the
principal target of the autoimmune response in pernicious anemia. Whether H. pylori plays a role in
triggering antibodies against host gastric proteins remains to be determined ( Appelmelk et al.,
1997).

The DNA sequence of Helicobacter pylori strain 26695 has also provided new insights into its
pathogenesis ( Zhongming and Taylor, 1999). The H. pylori genome contains the two best-known
virulence determinants, the vacuolating cytotoxin allele and the 38-kb cagA pathogenicity island.
Though H. pylori is a Gram-negative pathogen, many of its proteins correspond to proteins in
eukaryotes, archaea, and Gram-positive bacteria. This suggests that horizontal gene transfer from
disparate phylogenetic groups into H. pylori lineage occurred during evolution ( Berg et al., 1997).

(A)

(B)

(C)

Fig. 4.5 Steps in Helicobacter pylori invasions of mucus-producing cells of stomach epithelium
and the subsequent development of ulcers. Redrawn from Atlas (1997).

4.7 SUMMARY
Much progress has been made toward understanding the molecular basis of infectious diseases.
Koch's postulates, the standard test for determining if a bacterium causes disease, have
limitations. A new set of postulates, the molecular Koch's postulates, has been proposed and
focuses on the role of genes and gene products in disease. Adherence to a host cell is an
important step in bacterial pathogenesis. This is accomplished by means of pili and surface
proteins such as adhesions and invasions. Attachment of bacteria to eukaryotic integrins
involves three strategies: lectin binding, masking, and mimicry. Bacteria use siderophores to
obtain the iron they need from a host. Bacterial pathogens produce exotoxins that disrupt host
functions and cause cell death. Digestion of microbes in a phagosome occurs by several
methods including nitrogen- and oxygen-dependent phagocytosis.

Intracellular pathogens include Yersinia pestis (plague), which uses different ways to enter into
host cells. About 60 genes are in-

volved in virulence of Salmonella, and many of them occur in clusters called pathogenicity islands.
When Shigella invades macrophages, it causes apoptosis. Up to 5-10% of the human population
may be asymptomatic carriers of Listeria monocytogenes. Mycobacterium tuberculosis causes one
of the most common of human diseases. Legionella pneumophila occupies a unique niche inside a
macrophage, which engulfs the pathogen by means of a novel mechanism called coiling
phagocytosis. The gene which codes for diphtheria toxin is carried by temperate phages, and only
strains of Corynebacteria diptheria that carry the phage can cause the disease. The main virulence
factors in Vibrio cholerae are pili and cholera toxins. Outbreaks of cholera in Peru have been linked
to planktonic blooms (copepods) caused by El Niño. New strains of V. cholerae arise from genetic
recombination and horizontal gene transfer.

Escherichia coli is now classified into five virotypes. Pseudomonas aeruginosa is an opportunistic
pathogen in humans. Helicobacter pylori responsible for most human gastric and duodenal ulcers
shows molecular mimicry between pathogen and the host protein sequences, suggesting horizontal
gene transfer.

5 BACTERIAL ASSOCIATIONS OF PLANTS

Relationships between bacteria and plants are widespread and include mutualistic and parasitic
associations ( Preston et al., 1998). Bacteria constitute more than 90% of the total biomass of
agricultural soils, followed by fungi and protozoa. Bacteria obtain energy and nutrients through
decomposition of dead plant material and from exudates of living plants, and they may significantly
influence interactions that take place between plants, including competition ( Chanway et al.,
1991). Most plant bacteria are facultative saprobes and contain plasmids in addition to large,
circular chromosomes.
5.1 NITROGEN-FIXING SYMBIOSES
Plant growth is limited by how much nitrogen is in the soil. Nitrogen is an important element for
living organisms and is used to make amino acids, proteins, and nucleic acids. Nitrogen is common
in our atmosphere, but plants cannot use it in its elemental form and must absorb it from the soil
principally in the form of nitrates. The supply of nitrogenous compounds in the soil is being
continually replenished by bacteria that fix atmospheric nitrogen into ammonia and from
decomposing organic matter. Other soil bacteria then convert ammonia into nitrites and nitrates.
Nitrates absorbed by plant roots are converted back to ammonia, which is used to form the
complex molecules needed by plants. Reactions involving nitrogen are part of a nitrogen cycle,
which is an important part of every ecosystem.

Nitrogen-fixing bacteria commonly form mutualistic relationships with plants. The bacteria contain
the enzyme complex nitrogenase, which can catalyze complex reactions, involving N 2, hydrogen
ions, and free electrons, leading to the formation of ammonia. For these reactions to occur, the
bacteria need energy-rich compounds, such as ATP, and also electrons, both of which are obtained
from respiring sugars supplied by the plant. Bacteria need several factors to fix nitrogen: (1)
access to atmospheric nitrogen; (2) a nitrogenase enzyme complex; (3) large amounts of ATP (16
moles ATP per mole N2 reduced); (4) an anaerobic environment; (5) a supply of iron, magnesium,
and molybdenum, which are required for nitrogenase activity; (6) temperatures below 30°C; (7) a
means of regulating the amounts of ATP available for the system and for controlling fluctuating
ammonia production.

Because oxygen readily denatures nitrogenase, nitrogen fixation must take place under low
concentrations of oxygen. Symbiotic bacteria fix nitrogen in specialized structures such as nodules,
heterocysts, and vesicles in which low levels of oxygen can be maintained. Another important
constraint on bacterial nitrogen fixation is the high energy cost of the process. A symbiont's
capacity for nitrogen fixation is controlled largely by the host because it regulates how much
energy, in the form of sugar, the symbiont receives. In some cases as much as 30% of the
photosynthetic products of a host plant may be used to support nitrogen fixation and assimilation.

As the bacterial symbionts fix nitrogen, the host inhibits their ammonia-assimilating enzymes, and
they excrete much of the nitrogen they fix as ammonia. Host cells contain en- zymes to convert
the excreted ammonia into useful compounds. Such adaptations exist in the Rhizobium--legume
and Anabaena-Azolla symbioses.

Rhizobium--Legume Symbiosis
All rhizobia (Azorhizobium, Bradyrhizobium, Rhizobium, Sinorhizobium) belong to the a group of
the Proteobacteria(purple bacteria). Rhizobium is closely related to Agrobacterium, but not to
Bradyrhizobium ( Young and Johnston, 1989).

Rhizobium is a genus of rod-shaped, motile bacteria that live in the soil, usually in areas where
legumes grow. These bacteria form symbiotic associations with the roots of legumes such as
alfalfa, clover, peas, and soybeans. High levels of gene transfer might obscure the broad
coevolution of bacteria with plants ( Doyle, 1998). The bacteria stimulate the roots to form unique
structures called nodules, within which nitrogen fixation occurs. In soil that is deficient in nitrogen,
legumes that form nodules grow better than other plants. Root nodules can supply a plant with all
the nitrogen it needs. Plants without nodules must obtain their nitrogen from the soil, where it is
often in short supply ( Van Rhijn and Vanderleyden, 1995).

Signal molecules

Two main groups of signal molecules have been discovered: one for short-range interaction (e.g.,
cell-cell attachment), and the other for long-range signals (e.g., diffusible substances between
rhizobia and host cells). Flavonoid molecules released by legume roots induce the transcription of
rhizobial nodulation (nod) genes that code for enzymes needed for the synthesis of nodulation
factors. Nod factors are lipo-oligosaccharide molecules that have a chitinlike sugar backbone and
an N-linked long-chain fatty acid. These factors are released by rhizobia and act as long-range
signals that cause root-hair deformation and cortical cell division in suitable legumes ( Hirsch,
1992; Fisher and Long, 1992; Franssen et al., 1992; Verma, 1992; Denarie and Cullimore, 1993;
Kannenberg and Brewin, 1994; Spaink, 1995) (fig. 5.1 b).

Fig. 5.1 Rhizobium--legume symbiosis. (A) Stages in the infection of legume roots by Rhizobium
species. (B) Interaction between Rhizobium and legume roots. Host plant secretes flavonoids,
which along with Nod D proteins regulate the bacterial nodulation genes (nod). The expression of
nod factors is essential for root-hair curling, cortical cell multiplication, and other steps leading to
nodule formation. (Redrawn from Van Rhijn and Vanderleyden ( 1995).

Infection process
Infection of a legume begins when bacteria attach, by one end, to newly formed root hairs of
plant seedlings. Complementary chemical bonding may be involved. Proteins called lectins on the
surface of root hairs bind to polysaccharides on the bacterial cell wall. In some instances, the
binding is specific; that is, only certain lectins will bind to certain polysaccharides, which means
that some strains of rhizobia infect only certain types of legumes. For example, Bradyrhizobium
japonicum commonly infects soybeans; Rhizobium trifolii infects only white clover. Some strains
have a broad host range. In some cases plant lectins may enhance nodulation rather than
determine specificity ( Kannenberg and Brewin, 1994). Recent studies, however, have shown
that transferring a lectin gene from one plant to another can extend host range ( Diaz et al.,
1989; Van Rhijn et al., 1998).

When bacteria attach to a root hair, the hair deforms in response, some forming 360° curls called
"shepherd's crooks." The exact mechanism of root hair curling is unknown. The bacteria then
penetrate the cell wall and come in contact with the plasma membrane. Cell wall synthesis by the
root hair is redirected toward the site of infection. As a result of this redirected cell wall
synthesis, a tubular infection thread forms and grows inwardly toward the root cortex. The
nucleus of the root hair doubles in size and directs the growth of the infection thread. The
infection thread contains rhizobial cells, which are surrounded by a slimy substance
(polysaccharide) in which they multiply. The infection thread is surrounded by cell wall except at
its tip, where there is only naked plasma membrane. Specific antibodies and enzyme--gold-
labeled probes have been used to observe the development of infection threads ( Rae et al.,
1992).

Bacteroids

As the infection thread grows through the root cells, cortical cells are activated to divide some
distance away from the thread, possibly because of Nod factors released by the bacteria. These
dividing cortical cells form nodules whose cells become colonized by bacteria delivered by the
infection threads. After a thread penetrates a cortical cell, the bacteria become surrounded by a
peribacteroid membrane (PBM), which originates from the host plasma membrane. They occur
either singly or divide to form groups. Bacteria that are released from the infection thread are
called bacteroids. Interactions between the plant and the bacteroids are regulated by
transporters and channels on the PBM and the bacterial membrane ( Udvardi and Day, 1997).

The cells of many strains of Rhizobium undergo radical changes in morphology and physiology
when they become bacteroids. Some bacteroids are 40 times larger than the small rods they
develop from, and their numbers, up to several thousand, can fill a host cell. Each legume host
determines the size and shape of its enclosed bacteroids and also the number of bacteroids
contained within a peribacteroid membrane. One strain of Rhizobium may form different types of
bacteroids in different species of legumes. Bacteroids are nonmotile and assume a wide variety of
shapes. They have thin walls that allow for easy passage of nutrients from the plant to the
bacteroids and of nitrogen from the bacteroids to the plant. The transformation of rodshaped
rhizobia into bacteroids inside the legume cells initiates the process that leads to nitrogen
 fixation. Bacteroids behave as nitrogen-fixing organelles within the host cells. They cannot use
the nitrogen they fix, but rather depend on the plant for their nitrogen and carbon compounds.

Role of oxygen-binding proteins

Legume root nodules contain large amounts of a proteinaceous red pigment called
leghemoglobin. Leghemoglobin is located in the cytoplasm of the plant cell and forms only after
the symbiotic association has taken place ( O'Brian, 1996). The pigment binds oxygen to
maintain a low concentration of free oxygen in the nodule. Nitrogenase is inactivated at high
oxygen concentrations. Leghemoglobin is similar in structure and function to hemoglobin found in
the red blood cells of mammals.

Nodules

Legume root nodules are generally spherical or club shaped, but lupine nodules are collar shaped.
Nodules develop in a region behind the root tips in the emerging root hair zone. Nodules are
outgrowths of plant tissue, and they consist of uninfected tissue as well as tissue that contains
bacteroids ( Nap and Bisseling , 1990; Hirsch, 1992; Brewin, 1993; Long, 1996; Hirsch and
LaRue, 1997). The

vascular system of the plant extends into the nodules and transports nutrients in and out of the
nodule.

Some strains of Rhizobium that nodulate legumes can also form nodules with a nonleguminous
plant, Parasponia, a member of the elm family. Nevertheless, whatever unique trait legumes have
that enables them to fix nitrogen in association with rhizobia is so complex that it has rarely
evolved in other plants. An example of such a unique trait might be isoflavonoid synthesis, which is
rare in other plant families ( Young and Johnson, 1989). As indicated earlier, the fixation of
atmospheric nitrogen uses significant amounts of energy-rich compounds, which are supplied by
the host plant. Such an energy-dependent process may explain why most plants do not form
symbiotic associations with nitrogen-fixing bacteria.

Nodules have a limited life span. Only those that function at maximum capacity are maintained on
the plant. When nodules decay, bacteria that have remained in the infection threads and the
bacteroids present in the nodules are released into the soil. These bacteria most likely can reinfect
other root hairs.

Leaf and stem nodules. Leaf nodules have been observed in more than 400 species of the family
Rubiaceae, and leaf nodules of Psychotria and Pavetta have been studied in detail. The leaf nodules
become covered with multicellular hairs and contain rod-shaped bacteria. The bacteria isolated
from mature leaf nodules belong to the genus Phyllobacterium and do not fix nitrogen. Ardisisia,
Pavetta, and Psychotria do not develop beyond the seedling stage without the presence of leaf
nodule bacteria. It has been suggested that host plants are cytokinin-deficient mutants and depend
on leaf nodule bacteria to provide them with plant hormones ( Miller, 1990).

Azorhizobium produces nodules on stems of species in Aeschynomene, Neptunia, and Sesbania. In

addition, some cultivated varieties of Arachis hypogaea and Vicia faba form nodules on the lower
part of their systems ( Fyson and Sprent, 1980). The bacterioids, when isolated from stem nodules
and grown in culture, can fix nitrogen by using glucose, succinate, and lactate as energy sources (
Trinchant and Rigaud, 1987). Nodules on stems develop at specific sites called mamillae, and
bacteria occur both in the intercellular and intracellular spaces of the cortex. The bacteria multiply
and stimulate host cell division. In contrast to the Rhizobium-legume symbiosis, Sesbania seeds
contain large numbers of Azorhizobium caulinodans ( Werner, 1992).

Root nodules. Root nodules formed on actinorhizal plants in response to Frankia or those on
Parasponia roots following an infection with Bradyrhizobium are modified lateral roots. Though
legume nodules have been considered homologs of lateral roots by some, others have suggested
alternative developmental pathways. It has been hypothesized that nodules (1) originated as
highly modified stems ( Sprent, 1989), (2) evolved from carbon storage ( Caetano- Anolles et al.,
1993), (3) originated as a new organ formed from endomycorrhizae--root interaction ( LaRue and
Weeden, 1994), (4) derived from a defense mechanism used to protect the plant from pathogenic
bacteria ( Verma and Delauney, 1989), and (5) arose as an elaboration of a wound response (
Sprent, 1997). Some experimental and anatomical evidence suggests that a nodule develops
instead of a lateral root when meristematic cell divisions occur in the cortex instead of in the
pericycle ( Hirsch and LaRue, 1997).

Rhizobium genes
Legume mutualism is determined by many genes, among them genes called nod and fix, which
code for nodulation and nitrogen fixation, respectively. Among the fix genes the most significant
are the nif genes, which code for the synthesis of the enzymes of the nitrogenase complex. In most
of the fast-growing Rhizobium species that have been examined, these genes are located on a
large plasmid called psym (symbiotic). Scientists have demonstrated a close linkage between the
nitrogenase (nif) and nodulation (nod) genes on the psym plasmid ( Vlassak and Vanderleyden ,
1997). The formation of a nodule in the host tissue is a complex, multistep process involving
recognition (signals), binding receptors, infection, host cell multiplication, and cell enlargement (
Hirsch, 1992). Mutants have been discovered for a number of nodular stages. These mutants have
been used to discover how the process of nodulation is governed and controlled. Both bacterial and
plant genes are involved in the formation of nodules. More than 50 different nod genes have been
identified ( Downie, 1994). Rhizobium genes controlling infection and nodule for-mation fall into
two groups, one consisting of genes involved in the synthesis of exopolysaccharides (exogenes)
and the other of nod genes ( Verma et al., 1992; Gottfert, 1993; Van Rhijn and Vanderleyden,
1995). Rhizobium genetics has been advanced by transposon mutagenesis, recombinant cloning,
and plasmid transfer experiments ( Long, 1989, 1996).

Many aspects of the Rhizobium--legume symbiosis are not fully understood, and the information
that is available comes from only a few species, which may not represent the situation in nature (
Van Rhijn and Vanderleyden , 1995; Pueppke, 1996). Research on this symbiosis continues
because of its agricultural significance and fundamental importance to natural plant communities.
Efforts to increase food production to feed a growing world population require new supplies of
fertilizer, of which nitrogen is a key element. It is more economical, as well as environmentally
sound, to increase nitrogen production through biological systems, such as that of Rhizobium and
legumes, than to produce nitrogen fertilizers by expensive chemical processes ( Maier and Triplett,
1996).

Actinorhizal Symbiosis
Nitrogen-fixing nodules are also formed on nonleguminous plants by actinomycetes, which are
filamentous soil bacteria ( Pawlowski and Bisseling, 1996). Actinomycetes that form nitrogen-fixing
symbioses belong to the genus Frankia. These associations, involving about 200 species of
angiosperms ( HussDanell, 1997), are called actinorhizae, and they develop on the roots of woody
dicotyledonous plants. Symbiotic Frankia form numerous terminal swellings, called vesicles, within
which nitrogen fixation occurs. Nodules of some actinorhizae also contain hemoglobin, which is
similar to leghemoglobin in Rhizobium--legume root nodules. The stages of infection that lead to
actinorhizal nodules are similar to those of Rhizobium. The sequence of events is generally as
follows: root hair curling, infection through root hairs, growth of the bacterial filaments through the
plant cell, rapid division of cortical cells to form nodules, invasion of the cortical cells by bacteria,
and formation of hyphae, vesicles, and spores in the plant cells. Depending on the type of host
plant, one bacterial strain may form club-shaped or spherical vesicles. In Casuarina the bacteria do
not form vesicles, but nitrogen fixation still occurs. Strains of Frankia have been isolated from more
than 160 species of woody dicotyledonous plants, including species of Alnus (alders), Casuarina
(beefwood), Comptonia (sweet fern), and Myrica (bayberry). Plants with actinorhizae are important
sources of nitrogen in woods, fields, bogs, and roadsides, both from their leaf litter and decay of
dead plants. Frankia strains have been cultured on artificial media. Their growth is much slower
than that of most free-living bacteria, and they assume a variety of shapes. The molecular genetics
of Frankia is being studied to determine if the symbiotic genes of this bacterium are similar to
those of Rhizobium ( Simonet et al., 1990; Franches et al., 1998). So far, the nif genes seem well
conserved, but there is no evidence for other similarities in symbiotic genes.
Cyanobacteria and Plants
Cyanobacteria are another group of nitrogenfixing bacteria. In addition to their probable
contribution to the origin of plants, as chloroplasts, they form associations with a wide range of
organisms that include fungi, algae, bryophytes, the water fern Azolla, cycads, and the angiosperm
genus Gunnera. Cyanobacteria colonize a variey of plant organs such as roots, stems, and leaves (
Bergman et al., 1996), and they fix nitrogen within heterocysts, which contain nitrogenase ( Rai,
1990; Bergman et al., 1992b; Paerl, 1992). Recent advances in the molecular biology of
cyanobacteria have increased our understanding of these organisms ( Bryant, 1995).

Symbiotic associations between cyanobacteria and plants have a number of common features (
Meeks, 1998). Symbiotic cyanobacteria usually move in a slow, gliding manner by means of short
filaments called hormogonia and can penetrate cavities and spaces of plant organs and form
colonies. After a population of cyanobacteria is established in a plant, growth of the endosymbiont
either stops or slows down. The host plant may regulate the growth and population size of the
endosymbiont, but exactly how this is done is not known. Symbiotic cyanobacteria have more
heterocysts and fix nitrogen at higher rates than free-living cyanobacteria. The symbiotic forms
have larger vegetative cells, fewer nutrient reserves, do not photosynthesize, and cannot
assimilate the nitrogen they fix. The host plant inhibits the nitrogen-assimi- lating enzymes of the
endosymbiont. As a result, the ammonia produced by the symbiont is excreted and passed to the
host plant, which then converts it to other compounds. Symbiosis alters host plants. They produce
hairs and extensions of cells that increase the surface contact between host and cyanobacteria.

Azolla

Azolla is a genus of small, aquatic ferns that float on the surface of freshwater ponds and marshes
throughout the world ( Peters and Meeks, 1989). The plant has tiny roots and a short, branched
stem covered with small, overlapping leaves ( fig. 5.2 ). The ferns multiply rapidly by vegetative
reproduction. They can double their biomass in 2 days if growth conditions are optimal and quickly
cover large bodies of water with a dense and sometimes impenetrable growth. Each leaf of Azolla is
divided into a dorsal and ventral lobe. The ventral lobe floats on the water and contains filaments
of the cyanobacterium Anabaena azollae, which lives in symbiosis with the fern. The bacteria are
inside mucilage-filled cavities of the leaves. The cavities are sealed so that cyanobacterial filaments
cannot escape and other organisms cannot enter. Sexual structures that the fern uses to
overwinter contain Anabaena spores, so that when germination occurs in the spring the new
individuals of Azolla are infected by the bacteria. Thus, no stage in the life cycle of the fern is free
of cyanobacteria.

Anabaena fixes nitrogen in the leaf cavities of Azolla and supplies the fern with all the nitrogen it
needs. With its own built-in fertilizer generator, the fern can grow in waters that are deficient in
nitrogen. The fixed nitrogen, or ammonia, that is released by Anabaena is absorbed by specialized
hairs in the leaf cavity. Each cavity has about 20 randomly located simple hairs that transport
sucrose from the fern to the cyanobacteria, where it is used in nitrogen fixation. Each cavity also
has two branched hairs that are always near a vein. These hairs are thought to assimilate the
ammonia produced by the cyanobacteria, form amino acids, and transport them to the fern (
Plazinski, 1990).

The leaf cavity that contains cyanobacteria acts as a greenhouse. Inside this space, the bacterial
symbiont has a moist, protected environment, and its nutrient needs are provided by the fern. The
extent to which Anabaena azollae contributes to the total photosynthesis of the symbiosis is not
clear. The isolated Anabaena symbiont fixes carbon dioxide and undergoes normal photosynthesis,
but this process is inhibited when the bacterium is in the leaf cavity. The fern inhibits the glutamine
synthetase activity of the cyanobacterium. When this enzyme is inhibited, the Anabaena symbiont
excretes rather than assimilates the ammonia it produces by nitrogen fixation. Anabaena filaments
are attached to the growing tip, or meristem, of the Azolla plant. From this region the bacteria
become incorporated into each new leaf as it develops. If the Anabaena colony on the meristem is
sparse or absent, then bacteria-free leaves of Azolla will form. The bacteria on the meristem do not
fix nitrogen because they lack heterocysts. Such bacteria receive nitrogenous compounds from the
fern.
The development of the fern and Anabaena is synchronous. As each new fern leaf develops, the
bacteria that become trapped inside the leaf cavity multiply, form many heterocysts (25-30% of
total cell number), and

A. Azolla caroliniana in water

B. Section of leaf cavity showing filaments of Anabaena azollae

C. Filaments of Anabaena azollae Fig. 5.2 Azolla-Anabaena symbiosis

begin to fix nitrogen. Cells of the Anabaena symbiont enlarge and divide slowly when they are in
the leaf cavity.

Coryneform bacteria also grow in Azolla leaf cavities, along with Anabaena. Whether these
secondary bacteria have a role in the symbiosis is not known. They may produce some of the slime
that is found in the leaf cavities, which is important for the growth of the Anabaena filaments (
Braun-Howland and Nierzwicki-Bauer, 1990).

Experimental studies on the AzollaAnabaena symbiosis have been hindered by the inability of
bacteria-free (aposymbiotic) ferns to reestablish the symbiosis with isolated Anabaena symbionts.
The specific recognition factors that are necessary to bring about the symbiosis are not known.

There are six species of Azolla, but whether they all contain Anabaena azollae as a symbiont has
not been determined. There are no detailed studies to identify the cyanobacteria in the different
species of Azolla. Morphologically, the species of fern differ according to their reproductive
structures. Physiologically, the species differ only in their optimum growth temperature.

Much research is being conducted on the Azolla-Anabaena association. Azolla is used as a fertilizer
in place of manure in Asian countries such as China, Thailand, and Vietnam because it grows well
in the warm, stagnant waters of rice paddies and provides nitrogen for the rice plants. Farmers
grow Azolla alongside rice plants or mix the fern with the soil of the paddies. The use of natural
fertilizers such as Azolla has a long history in Asia. Natural fertilizers are an important and
inexpensive alternative to commercial fertilizers ( Nierzwicki-Bauer, 1990). Azolla is also used to
feed animals and purify water ( Wagner, 1997).

Bryophytes

Hornworts, such as Anthoceros punctatus, and thallose liverworts, such as Blasia pusilla, form
symbiotic associations with the cyanobacterium Nostoc. Filaments of Nostoc live in mucilage-filled
cavities on the undersurface of the bryophyte gametophytes. The bacteria fix nitrogen into
ammonia, which is excreted into the cavities and absorbed by the bryophytes. The bryophytes
provide the bacteria with carbon compounds such as sucrose and a protected place in which to live
( Meeks, 1990).

Both symbionts are altered after they enter the association. The Nostoc symbionts virtually stop
reproduction and do not photosynthesize. Their cells have less pigment and fewer storage granules
than free-living cyanobacteria, and they appear to be nitrogen-starved, which suggests that they
excrete most of the nitrogen they fix. As in the Azolla symbiosis, the host plant may inhibit the
cyanobacterial enzymes that assimilate ammonia. The heterocyst frequency of Nostoc in the
gametophyte cavities is much higher than that of the same symbiont growing outside the cavities
(30-43% versus 3-6%, respectively). When Blasia becomes infected with Nostoc, its cavities form
filamentous extensions of tissue that closely associate with the cyanobacteria. These extensions
increase the area of contact between the symbionts and facilitate the exchange of nutrients (
Kimura and Nakano, 1990).

Cycads
Cycads are primitive seed plants that were common and widespread during the Mesozoic era. Only
about 9 genera and 100 species of cycads still grow in tropical and subtropical regions of the earth.
About one-third of the known cycads contain symbiotic cyanobacteria in their roots. The bacteria
are located in specialized branches of the roots that, unlike normal roots, are negatively
geotrophic. Instead of growing down into the ground, these roots grow on and above the soil
surface. The roots develop nodules that contain cyanobacteria. Because of their warty appearance,
these specialized structures are called coralloid roots. The roots are loosely organized, and gases
circulate easily in and out of them ( Lindblad and Bergman, 1990).

The endosymbionts of cycads are strains of Nostoc. The cyanobacteria penetrate the coralloid roots
through cracks or openings and move between the middle lamellae of cells until they reach the
middle of the root cortex. Small host cells in this area degenerate and form mucilage-filled spaces
where the cyanobacteria divide. Other host cells in the cyanobacterial zone elongate, possibly
because of growth factors excreted by the cyanobacteria, and develop fingerlike extensions that
may facilitate the exchange of nutrients between the symbionts ( Caiola, 1992).

Cyanobacteria of cycad roots fix nitrogen, which they supply to the cycad in the form of ammonia.
Cycads that grow in the forests of the Southern Hemisphere are important for the nitrogen
economy of those forests.

Gunnera

Gunnera is an angiosperm genus with about 50 species, all of which are perennial herbs. The
plants grow commonly in wet areas of the tropics and Southern Hemisphere. Near the base of the
leaf petioles, there are secretory glands that contain the cyanobacterium Nostoc punctiforme.
Filaments of Nostoc multiply in the mucilage that fills the glands and then penetrate cells at the
base of the glands ( Bonnett and Silvester, 1981). This association is unusual because the
cyanobacterial symbionts are intracellular; that is, they are surrounded by the plasmalemma of the
host cell. Each gland cell forms fingerlike extensions that increase the area of contact with the
endosymbiont. Like other cyanobacterial symbionts, the Nostoc endosymbiont of Gunnera does not
photosynthesize; it has the highest frequency of heterocysts recorded in any cyanobacterial
population ( Bergman et al., 1992a) and fixes nitrogen at a high rate. The Nostoc symbiont
supplies Gunnera with all the nitrogen it needs and receives carbohydrates from the plant. Thus, in
symbiosis the Nostoc endosymbiont behaves as a heterotroph ( Bergman et al., 1996).

5.2 AGROBACTERIUM, GALLS, AND GENE

TRANSFER
Galls are produced on plant roots and stems by species of Agrobacterium, Corynebacterium, and
Pseudomonas and are composed of a disorganized mass of plant tissue. Agrobacterium
tumefaciens causes crown gall disease on many woody plants, A. rubi forms cane galls on
raspberries and blackberries, and A. rhizogenes induces development of hairy roots on apples. The
type of symptoms that develop on the host are a function of the kind of plasmids the bacteria
carry. An Agrobacterium with a tumor-inducing (Ti) plasmid produces crown gall symptoms; the
hairy roots of apple are caused by a root-inducing (Ri) plasmid found only in strains of A.
tumefaciens and A. rhizogenes. Crown gall disease is unique among plant diseases because it is
malignant. Host plant cells once stimulated by A. tumefaciens continue to divide and no longer
obey the host's hormonal commands.

Crown gall disease affects approximately 100 families of plants and causes millions of dollars worth
of damage each year to agricultural and ornamental plants. Agrobacterium tumefaciens, a rod-
shaped, Gram-negative bacterium is closely related to the Rhizobium bacteria that form nodules on
the roots of legumes. Agrobacterium tumefaciens lives in the soil and infects plants through
wounds that the plants suffer either during seed germination or from insects and nematodes.
Infection is usually near the crown of a plant--that is, at the junction between the stem and the
roots. The bacteria stimulate the plant cells around the wound to multiply and form a tumorlike
growth, or gall. The disease generally affects only wounded plants because it is the new cells
developing around the wound that are most susceptible to bacterial infection ( Baron and
Zambryski, 1995a). The wounded plant cells produce signal molecules such as acetosyringone and
a hydroxyacetosyringone that turn on the virulence systems (vir genes) of Agrobacterium. These
compounds are thought to develop from the wounding-induced degradation, or the repair, of cell
wall lignin ( Baron and Zambryski, 1995a). Galls generally appear several weeks after a plant is
infected and may be a millimeter or more in diameter and may weigh up to 100 lbs depending on
the plant host ( fig. 5.3 ). Plants rarely die from crown gall disease, but they become weakened,
grow more slowly, and produce fewer flowers and fruits. Infected plants are susceptible to other
disease-causing agents, such as fungi.

The infectious process of crown gall disease has been studied intensively ( Kado, 1991). Each cell
of A. tumefaciens contains one or more large plasmids. The plasmid responsible for tumorigenesis,
the Ti plasmid, contains about 100 genes. After the bacterium attaches to a plant cell, the plasmid
passes through the host cell wall and part of the plasmid, called tDNA (transfer DNA), becomes
integrated into the host chromosome ( Hooykass and Schilperoort, 1992). Genes on the tDNA are
expressed along with the plant's genes ( Gelvin, 2000). The integrated bacterial genes cause the
plant cell to multiply abnormally and to produce unusual chemical compounds. In effect, the plant
cell is partly controlled by genes from the bacterium. Crown gall disease is an example of genetic
parasitism in which the genes of A. tumefaciens are used to parasitize another organism. Once the

plasmid is incorporated into the host

Fig. 5.3 Genetic colonization of a flowering plant by Agrobacterium tumefaciens.

chromosome, it replicates along with the plant's chromosomes. Thus, all the progeny of a cell with
tDNA will have copies of the tDNA, and the bacterium no longer is necessary for the disease to
progress. Crown gall is the only known example of a disease that persists in the absence of the
inciting agent. The Ti plasmid, in part at least, may represent ancient plant genes that have
become incorporated into A. tumefaciens.

Genes on the tDNA code for the production of auxin and cytokinins, plant growth hormones that
stimulate growth of the tumor. Other genes on the tDNA segment code for the synthesis of opines,
which are derived from amino acids and produced only by plant cells infected with tDNA. Opines
are sources of carbon, nitrogen, and energy for Agrobacterium in the soil and they increase the
rate of transfer of Ti plasmids to avirulent strains of A. tumefaciens that are in the soil near the
crown galls. Opines also stimulate plasmid exchange between strains of Agrobacterium in the
crown gall tissue.

Six different strains of A. tumefaciens are recognized, based on their ability to metabolize specific
types of opines. For example, the octopine strain uses opines such as octopinederived from the
amino acid arginine; lysopine, derived from lysine; histopine, derived from histidine; and octopinic
acid, derived from ornithine. The nopaline strain uses nopaline, derived from arginine, and
nopalinic acid, derived from ornithine. Most strains have a wide host range, but a few are limited in
the types of plants they can infect.

That a segment of a plasmid of A. tumefaciens, a prokaryotic cell, can fuse with a chromosome of a
eukaryotic plant cell is surprising. It is equally intriguing that genes of a plasmid are expressed
within a eukaryotic cell. This occurs because tDNA includes genetic sequences that can be read by
enzymes of the eukaryotic cell. Agrobacterium is the only bacterium known to transfer DNA to
eukaryotes as part of its life cycle ( Winans, 1992).

The ability of A. tumefaciens to insert part of its plasmid into a plant chromosome has considerable
implications for the improvement of crops ( Vasil, 1994). Through the techniques of genetic
engineering, A. tumefaciens has been used to introduce desired traits into plant cells. In fact,
transformation brought about by Agrobacterium is the most popular and reliable method of
creating transgenic plants in different species that can grow in tissue culture and regenerate. Such
modified plants have been used to produce new products such as antibiotics, oral vaccines, and
biodegradable plastic ( Walden and Wingender , 1995).

An important event in the A. tumefaciens infection of plants is the binding of the bacterium to the
host cell wall, a process that may be genetically controlled by the A. tumefaciens chromosome. A
lipopolysaccharide constituent of the bacterial cell wall binds to a specific component of the plant
cell wall. Only young, actively dividing plant cells, such as those that form around a wound, seem
to have this component.
Agrobacterium tumefaciens infects a wide variety of plants. Dicotyledonous angiosperms and
gymnosperms are susceptible to infection, but monocotyledonous plants appear to be resistant.
The nature of this resistance is not understood. It may result from the inability of the bacterium to
bind to the cell walls of these plants or from a failure of the tDNA to become integrated in the host
DNA or even to notice such an integration. Suitable culture and regeneration techniques for these
groups of plants have yet to be discovered ( Walden and Wingender, 1995).

5.3 BACTERIA AS PLANT PATHOGENS

In 1878 an American plant pathologist, Thomas Burrill, discovered that bacteria (Erwinia
amylovora) caused fire blight disease that killed apple and pear trees ( Agrios, 1997). Although
many plants are susceptible to bacterial diseases, mosses, ferns, gymnosperms, and monocots are
generally resistant. Bacteria enter plants through wounds and natural openings and survive winter
in plant debris.

The first avirulence gene was isolated in 1984 from Pseudomonas syringae, a bacterial pathogen of
soybean, and since then more than 30 bacterial avr genes have been cloned. Most avr genes
encode for hydrophobic proteins that require additional bacterial genes called hypersensitive
reaction and pathogenicity (hrp). The gene products of avr and hrp confer resistance in the host,
which is characterized by rapid localized cell death. The bacterial avr protein is translocated into
the plant cell, where it affects nuclear gene expression. Cloned avr genes have been introduced
into host plants. The avr gene activity in plant cells with an R gene is measured by induction of
hypersensitive cell death ( Ackerveken and Bonas, 1997) ( fig. 5.4 ).

Several hrp proteins are homologous to proteins from human bacterial pathogens such as
Salmonella, Shigella, and Yersinia that make up the type III secretion system. Several Yersinia
proteins called YOP are translocated into the host cells. In Pseudomonas, type III protein called
hrpA is associated with the formation of piluslike structures which are thin enough to pass through
the host cell wall and allow the avr protein to enter the host cell cytoplasm. Hypersensitive cell
death in resistant plants occurs when avr protein acts within the host cell ( Lee, 1997). The
following are some well-known examples of bacterial symbionts of plants.

Vascular Wilts
Several species of Clavibacter, Erwinia, Pseudomonas and Xanthomonas cause vascular wilts in
cultivated plants. The pathogens multiply in the host's vascular tissues and destroy the cells using
enzymes that cause leaves to wilt and die. Growth regulators secreted by the bacteria may cause
hypertrophy and hyperplasia of xylem tissue. Fig. 5.4 Bacterial pathogen-mediated host cell
apoptosis in plants. Host resistance is characterized by sudden, rapid death of the infected cell
(hypersenstivity response). When a host resistance gene product (PTO) interacts with an
avirulence gene product (Avr Pto), the resulting phosphorylation cascade leads to cell death.
Adapted from Lamb (1996).

Soft Rots
Bacterial soft rots occur commonly in plants during storage, and the decomposing plants give off
volatile gasses. The bacteria produce enzymes that dissolve the middle lamella in plant tissues.
Erwinia carotovor and E. chrysanthemi cause soft rots and wilting and dwarfing of many fruits,
vegetables, and flowers. The bacteria produce different pectinolytic enzymes by means of several
gene regulatory systems ( Hugouvieux-Cotte-Pattat et al., 1996). Pseudomonas marginalis causes
pink eye disease in potatoes and slippery skin in onions.

Scabs
Streptomyces scabies causes the common scab of potato and is distributed worldwide. Bacteria
multiply in the intercellular spaces of parenchyma cells, causing healthy cells around the lesion to
divide and form layers of cork cells.

Cankers
Bacterial cankers destroy stems and twigs and cause economic losses. Pseudomonas syringae
infects stone fruits and ornamentals such as roses and lilacs. Most strains of P. syringae produce a
plasmid-encoded phytotoxin.

Citrus canker, caused by Xanthomonas compestris pv. citzi, is one of the most feared citrus
diseases in Florida because of the heavy economic losses it produces. The disease is endemic in
Asia and was accidently introduced into Florida in the early 1910s with the import of infected
nursery trees from Japan. It took more than 30 years of sustained eradication efforts to eliminate
the bacterial pathogen that resulted in destruction of millions of fruit-bearing trees ( Schumann,
1991).

Role of Phytotoxins in Bacterial Plant Diseases

Syringomycin is one of several types of phytotoxins that determine bacterial pathogene- sis in a
host plant. Purified toxin applied to young peach trees produces typical disease symptoms ( DeVay
et al., 1978). Recent studies on the chemical structure of syringomycin and other related toxins
have provided significant insights into their role in disease physiology. The toxin lipopeptide
molecule acts on host cell membranes, affecting electrical potential and pH gradients, thereby
causing leaf stomata to close ( Mott and Takemoto, 1989).

Rhizobitoxin is produced by Bradyrhizobium japonicum, Pseudomonas aeruginosa, and P.

andropogonis. The toxin inhibits biosynthesis of methionine, which is needed to produce ethylene.
Phaseolotoxin, a tripeptide toxin produced by Pseudomonas syringae pv. phaseolicola, causes
chlorosis of leaves. Tagetitoxin is a phytotoxin produced by P. syringae pv. tagetis that interferes
with chloroplast genes in developing tissues.

5.4 MYCOPLASMAS: BACTERIA WITHOUT

CELL WALLS
Mycoplasmas are regarded as the smallest and simplest cellular organisms. Their genomes are the
smallest of any organism, and the DNA sequences of Mycoplasma genitatium and M. pneumoniae
encode for 470500 proteins ( Rottem and Naot, 1998). These prokaryotes lack the characteristic
cell wall of other bacteria and were first seen in the phloem cells of plants with yellows disease.
Yellows disease was long suspected to be caused by a virus because it was transmitted by insects,
and the infectious agent passed through bacterial filters. Antibiotic treatment of sick plants,
however, restored them to good health. Mycoplasmas are resistant to penicillin and other
antibiotics that inhibit cell wall formation but are sensitive to antibiotics such as tetracycline.
Today, we know of several different kinds of mycoplasmas and mycoplasmalike organisms (MLOs).
Mycoplasmas are similar in many respects to Gram-positive bacteria ( Dybvig and Voelker, 1996).

Plants infected with phloem-specific mycoplasmas are often stunted and have small flowers and
fruits. Corn stunt and citrus stubborn are two diseases that are caused by the mycoplasma
Spiroplasma. Aster yellows, lethal yellowing of coconut palms, elm phloem necrosis, peach X
disease, and pear decline disease are other plant diseases suspected of being caused by
mycoplasmas. These obligate symbionts occur intracellularly in sieve-tube elements of the phloem.
Sieve-tube cells are a highly specialized environment. A large volume of nutrients passes through
these cells to areas of the plant where active growth is taking place or to storage areas of the
roots. A unique feature of these cells is their high sugar concentration and hence high turgor
pressure gradient. Additionally, sieve cells contain significant concentrations of K+, Mg+, Cl- and PO

ions, amino acids, proteins, and ATP molecules. It is this uniquely specialized cellular environment
to which the symbionts have become adapted.

Leaf hoppers and other insects feed on phloem sap, and the chemical constituents of their
hemolymph are strikingly similar to those of the sieve-tube cell. Insect hemolymph has high
concentrations of organic and inorganic nutrients, which result in a high osmotic pressure. Leaf
hoppers feeding on phloem cells acquire mycoplasmas that multiply rapidly in the alimentary canal,
hemolymph, salivary glands, and ovaries of the insects. Mycoplasmas are eventually transported to
the salivary glands, from which they are transferred to new phloem cells during a leaf hopper's
feeding. The same mycoplasmas can reproduce in different hosts, such as flowering plants and
insects, because the intracellular environments of the hosts are remarkably similar. The lack of cell
walls in mycoplasmas may be viewed as an evolutionary consequence of intracellular symbiosis.

In recent studies, mycoplasmas have been observed in the leachates of flowers, suggesting that
flowers may also be habitats for these prokaryotes. Possibly, they survive in nectar and are
transmitted by pollinating insects. Indeed, insects could be the principal hosts of these
prokaryotes, and the flowers may represent only transient habitats.

Animal-associated mycoplasmas first gained the attention of researchers when tissue culture cells
obtained from respiratory and urogenital tracts developed large populations of these organisms.
Scientists are beginning to realize that mycoplasmas may be a normal constituent of human and
animal mucous membranes. Mycoplasmas have been reported from cells of the oral and nasal
cavities, urogenital tracts, and lungs of cattle and humans. Some authors estimate that 20% of all
human pneumonia may be caused by Mycoplasma pneumoniae. The significance of mycoplasmas in
human and animal health should be better understood now that the complete nucleotide sequence
of the M. pneumoniae genome is known ( Krause, 1998).

5.5 SUMMARY
Some bacteria form mutualistic, nitrogen-fixing symbioses with plants. The nitrogen fixed by these
organisms is an important part of different ecosystems throughout the world. The best known
nitrogen-fixing symbiosis is that between Rhizobium and legumes. Intensive research is being
conducted on this symbiosis to understand the genetics of nitrogen fixation and nodulation. The
long-term goal of such research is to be able to introduce nodulating bacteria into crop plants and
thus eliminate the need for artificial nitrogen fertilizers. Unfortunately, nitrogen fixation involves
complex interactions between the host plant and its microbial symbionts, and therefore the
symbiosis must be studied as an integrated whole to understand it fully. Frankia is an actinomycete
that forms nitrogen-fixing nodules on the roots of many trees and shrubs and fixes nitrogen within
root nodules in specialized vesicles.

Cyanobacteria also fix nitrogen and form associations with plants such as Azolla, bryophytes, and
cycads as well as with lichenforming fungi. Nitrogen is fixed in specialized structures called
heterocysts, which are similar in function to vesicles of Frankia. In all nitrogen-fixing symbioses,
the host inhibits the nitrogen-assimilating enzymes of the bacteria and thereby causes ammonia to
be excreted from the symbiont to be used by the host.

The three principal features of the Agrobacterium symbiosis are tumor production, opine
production, and Ti plasmids. Tumors occur when a plant cell with a tDNA segment derived from a
bacterial Ti plasmid divides in an uncontrolled fashion and produces opines. Synthesis of these
molecules is controlled by tDNA. Opines permit the symbiont to exploit the host's carbon and
nitrogen energy sources. Ti plasmids contain genes that allow the bacteria to use opines as energy
molecules, and they possess DNA that is transferred to the host genome. The infection process of
this disease is a subject of much study because TDNA is an important vector for carrying genes into
plant cells using techniques of genetic enginering. The A. tumefaciens symbiosis may serve as a
model for studying whether genetic information passes between symbionts. In other associations
as well, including those of rhizobia and legumes, lichens, and mycorrhizas, there are morphological
transformations of the symbionts and new chemical compounds produced, which future studies
may show are caused by genetic colonization of a host by its symbiotic partner. At present, little is
known about gene flow between the partners of a symbiosis, but considering that genetic exchange
occurs between A. tumefaciens and plant cells as well as between the organelles of a eukaryotic
cell, it appears likely that a similar situation may exist between partners of other symbioses,
especially highly integrated ones. Gene flow would be one form of signal exchange.

Many species of bacteria cause plant diseases such as vascular wilts, soft rots, scabs, and cankers.
Bacterial pathogens produce different phytotoxins that cause pathogenesis in plants. The gene-for-
gene hypothesis explains the genetic basis of resistance and virulence in plants and pathogens.
Mycoplasmas are the smallest known cellular organisms. They cause many plant diseases and also
have been found in different animal tissues.

6
SYMBIOSIS AND THE ORIGIN OF THE
EUKARYOTIC CELL
The cell theory, long a basic tenet of biology, states that all organisms are made up of cells and
that cells are the basic units of living organisms. This traditional view of an indivisible cell has been
challenged by the serial endosymbiosis theory (SET), which claims that the eukaryotic cell is not a
single entity but rather a multiple of the prokaryotic cell ( Margulis, 1992a). This controversial
theory has slowly gained acceptance by biologists until now, according to Doolittle and Brown (
1994), "the endosymbiont hypothesis for the origin of mitochondria and chloroplasts is as firmly
established as any fact in biology" (p. 6721). Given the widespread acceptance of the SET, a
revision or rejection of the cell theory is inevitable. Even the boundaries of an organism can be
clouded over by symbiotic associations ( Dyer and Obar, 1985).

Another firmly held biological tenet is the distinction between prokaryotes and eukaryotes, and this
too is under attack. Critics contend that the definition of a prokaryotic cell is a negative one (it
lacks what the eukaryotic cell has--namely, a nucleus and organelles) and therefore has no
phylogenetic value (i.e., the absence of a particular feature does not mean that all prokaryotes are
related) ( Lake, 1991; Lake and Rivera, 1996). Prokaryotes should be defined on shared molecular
traits, such as the structure of the small subunit rRNA. This challenge comes from Carl Woese and
colleagues ( Woese and Fox, 1977; Woese et al., 1990; Woese, 1993; Olsen et al., 1994), who
from their phylogenetic analyses based on RNA structure and sequences, have found that
archaebacteria are more closely related to eukaryotes than to the true bacteria. Margulis ( 1996)
and others ( Mayr, 1990), however, argue that despite their molecular differences, archaebacteria
and eubacteria are prokaryotes and should be kept in one inclusive taxon, the Prokaryota
(Prokarya). Nucleated organisms that evolved from symbiotic integration of several prokaryotes
are placed in the Eukaryota (Eukarya).

6.1 KINGDOMS AND DOMAINS

The jump from a two-kingdom classification (Plants and Animals) to one that involves five
kingdoms (Plantae, Animalia, Fungi, Monera, Protista) was a radical step a few years ago ( Margulis
and Schwartz, 1997). Despite the general acceptance of this new classification, it is now being
challenged by new molecular findings ( Woese et al., 1990; Sogin et al., 1996). Critics of the five-
kingdom classification feel that it does not reflect the true evolutionary history of life on earth.
Supporters of the five kingdoms ( Margulis and Guerrero, 1991; Margulis, 1992a) are not
persuaded.

The use of molecular information to determine evolutionary relationships has revolutionized

taxonomy, especially among the bacteria, which do not display conspicuous morphological diversity
as do large organisms, and for associations whose symbionts cannot be cultured independently of
each other. Woese and colleagues ( Woese et al., 1990) divide living organisms into three
domains: Archaea, Bacteria, and Eukarya. Domains are taxonomic categories above the kingdom (
Williams and Embley, 1996). Figure 1.2 shows the three domains with the Archaea being distantly
related to the Eukarya ( Keeling, 1998). Some molecular features shared by these two domains
include N-linked glycoproteins, absence of formylmethionine, shared resistance or sensitivity to
certain antibiotics, and the presence of tRNA introns. There is also a similarity in eukaryotic and
archaebacterial transcription ( Keeling and Doolittle, 1995).

The origin of eukaryotes was one of the most complicated events in the evolutionary history of life
( Cavalier-Smith, 1991; Dyer and Obar, 1994; Vellai et al., 1998). The archaebacteria and bacteria
appear to have originated in a hot environment (thermophiles) ( Olsen et al., 1994; Monastersky,
1998). The term progenote has been used for primitive pre-organisms that were in the process of
evolving a translation apparatus, and genote describes modern cells ( Woese and Fox, 1977;
Woese, 1998).
6.2 MOLECULAR VERSUS TRADITIONAL
TAXONOMY
With the increased concern about biodiversity, the disappearance of tropical rainforests, and the
concomitant loss of undescribed species, the importance of taxonomy has increased greatly.
Unfortunately, in this resurgence of popularity, a fierce debate has developed as to whether the
traditional (i.e., wholeorganism based) taxonomy accurately reflects evolutionary relationships.
Some scientists believe that molecular information is sufficient to create a phylogenetic tree using
the pattern of genetic sequences from only one kind of RNA molecule ( Woese et al., 1990).
Discovery of extensive horizontal gene transfer between organisms, however, has undermined
confidence in the accuracy of trees of life based on molecular relationships ( Doolittle , 1999;
Pennisi, 1999). Molecular taxonomists select a gene or its RNA or protein product in an organism
and then study the sequences of the same gene or product in other organisms. At present, the
most popular genes are those that code for ribosomal RNA (16S rRNA). The similarity of these
molecules in all organisms make them useful comparative markers of evolution ( Sogin, 1989;
Schlegel, 1994; Pace, 1997). Molecular taxonomists compare the chemical sequences of genes to
see how they have changed during the course of evolution. The positioning of an organism in a
family tree depends on how much its gene sequences differ from those of other organisms. When
one considers that all life on earth probably arose from a common origin some 3.8 billion years
ago, trying to determine phylogenetic relationships is a challenging task.

Many proteins and certain nucleic acid sequences can be viewed as "living fossils" because they
have been conserved from the dawn of life. They have been found in all cellular life forms and are
believed to have a common ancestor. Margulis ( 1992a, 1996) argues that all living organisms
have at least five different types of nucleic acids and at least 500 different proteins and that one
alone is not an accurate reflector of phylogeny. She believes that whole-organism biology
(genetics, metabolism) should support phylogenetic classification.

6.3 THEORIES ON THE ORIGIN OF EUKARYOTIC

CELLS
Several theories and hypotheses have been proposed to explain the origin of eukaryotic cells:
these include the autogenous (direct filiation) theory, the SET, and the hydrogen hypothesis.

Autogenous Theory
The central assumption of the autogenous theory is that organelles evolved within the cell by
progressive compartmentalization. Cell structures such as the nuclear membrane, endoplasmic
reticulum, Golgi bodies (dictyosomes), vacuoles, and lysosomes are believed to have evolved from
elaborations of the cell and other internal membranes. The theory suggests a primitive ancestor,
the uralga, evolved by gradual selection ( Jensen, 1994). Furthermore, after the eukaryotic cell
developed, endosymbiotic events could have occurred. The cytoskeleton of the cell most likely
originated autogenously ( Cavalier- Smith , 1991, 1992a).

Serial Endosymbiosis Theory

The SET best explains the origin of organelles such as mitochondria, chloroplasts, and microtubular
complexes. The possibility that peroxisomes, hydrogenosomes, and even the nucleus have a
symbiotic origin is under investigation ( Palmer, 1997). These organelles may have arisen by
successive symbioses about a billion years ago. An early step in this process was the invasion of
anaerobic host cells (archaebacteria) by aerobic prokaryotes, which in time were transformed into
mitochondria ( Fenchel and Finlay, 1994). Later, eukaryotic heterotrophs acquired cyanobacteria
and gained photosynthetic capabilities. These symbiotic cyanobacteria eventually developed into
plastids, which include chloroplasts. Some researchers believe that the acquisition of mitochondria
and chloroplasts by phagotropic hosts occurred more or less simultaneously rather than
sequentially ( Cavalier-Smith, 1987b). The endosymbiont hypothesis, first proposed by Margulis
some 30 years ago, has recently been supported by several molecular sequence analyses ( Brown
and Doolittle, 1997; Martin, 1999).

The SET states that eukaryotic flagella, mitochondria, and chloroplasts were acquired by
prokaryotes in that order. These organelles are viewed as transformed bacterial symbionts. This
idea is not new: early scientists such as Ivan Wallin were convinced that mitochondria were
bacteria that lived inside other cells. In 1927 Wallin used the term symbionticism to describe
microsymbiotic complexes such as those of mitochondria with the rest of the cell. Wallin thought
that symbionticism played an important role in the origin of species. As early as 1883, A.F.W.
Schimper proposed that the chloroplasts of plants were cyanobacteria that lived symbiotically
inside plant cells. These insightful ideas were not accepted by the general scientific community
because they were impossible to prove experimentally. As with many scientific hypotheses, the
ideas were eventually forgotten. The revitalization and expansion of these hypotheses is the work
of Lynn Margulis, who has published books and articles promoting the symbiotic origin of
eukaryotic cells. Her persistent and lucid arguments have resulted in the general acceptance of the
symbiotic theory ( Margulis, 1996).

The SET is strongly supported by findings from structural, molecular, and biochemical studies of
eukaryotic cell organelles. The DNA of chloroplasts and mitochondria differs from that of the
nucleus; organelle DNA is circular and histone-free, like that of prokaryotes. Ribosomes of these
organelles are also similar to those of prokaryotes both in size and in the nucleotide sequence of
their RNA. Chloroplasts and mitochondria synthesize proteins, divide by fission, and mutate. The
mitochondria and chloroplasts of algal and plant cells are only semi-independent. For example,
most of the proteins needed for the development and function of chloroplasts are coded for by
genes in the nucleus and synthesized on ribosomes in the cytoplasm. The proteins are then
transported into the chloroplasts. Some proteins are a joint product of genes found in the nucleus
and chloroplast. For example, ribulose bisphosphate carboxylase is an important enzyme in
photosynthesis that catalyzes fixation of atmospheric CO 2 into the organic matter of cells. Such
interaction between the nucleus and chloroplast suggests that the symbiosis between chloroplasts
and plant cells is an ancient one. Similar relationships also exist between mitochondria and the
nucleocytoplasm.

Evidence suggests that during evolution, much of the genome of the bacterial ancestors of
mitochondria and chloroplasts was transferred to the nucleus ( Henze et al., 1995; Lake and
Rivera, 1996; Martin, 1996). These organelles contain less genetic material than free-living
bacteria, and segments of mitochondria and chloroplast DNA are identical to segments of nuclear
DNA. The same type of DNA that occurs in more than one organelle has been called promiscuous
DNA ( Gray, 1989). DNA transfer between organelles appears to be an ongoing process ( Stern and
Palmer, 1984; Knoop and Brennicke, 1994).

The Hydrogen Hypothesis and Origins of

Organelles
The recent hydrogen hypothesis of William Martin and Miklos Muller ( 1998) is based on the
symbiosis between an α proteobacterium and an archaean (methanogen). A similar proposal, the
syntrophy hypothesis, differs mainly in the types of eubacterial symbionts involved ( Lopez-Garcia
& Moreira, 1999). Hydrogen is key to the emergence of eukaryotes, and the classical
endosymbiosis theory has been expanded to include new evolutionary insights ( Martin and Muller,
1998; Doolittle , 1998a) ( figs. 6.1, 6.2 ).

Glycolysis is central to the energy metabolism of eukaryotes, with 1 mol glucose generating a net
gain of 2 mol ATP. In mitochondrial cell respiration, pyruvate is oxidized through the pyruvate
dehydrogenase complex (PDH) to CO 2 and water with an additional 36 mol of ATP generated per 1
mol glucose. In amitochondriate eukaryotes, pyruvate is metabolized through pyruvate-ferredoxin
oxidoreductase (PFO) Fig. 6.2. Type 1 amitochondriate eukaryotes lack organelles, and PFO
decarboxylates pyruvate to produce a) Mitochondrion b) Hydrogenosome Fig. 6.1 Comparison of
(a) mitochondial and (b) hydrogenosomal cell respiration.
ferredoxin and acetyl-CoA. The acetyl-CoA is converted into ethanol and CO 2 or acetate with an
additional gain of 2 mol ATP. Type II amitochondriate eukaryotes have hydrogenosomes, where
PFO converts pyruvate into CO 2, acetate, and reduced ferredoxin. As hydrogenase reoxidizes
ferredoxin, hydrogen is produced and an additional 2 mol of ATP are generated, with CO 2 and
acetate as waste products. Molecular sequence analysis shows that PFO and PDH of
hydrogenosomes and mitochondria share a common bacterial ancestry ( Martin and Muller, 1998).

The proteobacterium excretes hydrogen and CO 2 as metabolic wastes of anaerobic fermentation,

whereas the archaean host sym-

Anaerobic Anaerobic Aerobic Amitochondriate Amitochondriate Mitochondriate eukaryote eukaryote

eukaryote with hydrogenosome Giardia Lamblia Trichomonas vaginalis Saccharomyces
cervisiaeEntamoeba histolytica Neocallimastix (rumen fungus) Fig. 6.2 Three types of eukaryotes
according to their energy metabolism. (A) Amitochondriate eukaryotes that live anaerobically, (B)
anaerobic eukaryotes with hydrogenosomes which produce hydrogen as a metabolic waste product,
(C) aerobic eukaryotes with mitochondria that produce carbon dioxide and water. Adapted from
Martin and Muller ( 1998

biont consumes hydrogen and CO 2, thereby producing a mutually beneficial partnership. Thus, the
first eukaryotes evolved from a methanogen that consumed hydrogen and CO 2 and produced
methane. Its partner, the future mitochondrion, was a bacterium that produced hydrogen and CO
2. Because of the ever-present hydrogen supply, the archaean host became dependent on the α
proteobacterium, and a tight genetic linkage between the two resulted. The degree of dependence
increased following some gene transfer from the proteobacterial symbiont to its host genome. The
host provided the membrane proteins for the import of substrates and enzymes for glycolysis, and
ATP production began under anaerobic conditions. The new host with its completely enclosed
symbiont changed its nutrition from simple molecules such as hydrogen and CO 2 to complex
organic molecules. The symbiont in the course of evolution became (1) lost, as in amitochondriate
archezoons (e.g., Giardia), or (2) a hydrogenosome, as in the protist Plagiopyla, and a
mitochondrion leading to all complex cells ( Martin and Muller, 1998) ( fig. 6.3 ).

Ancestral host cell

What kinds of present-day organisms exist that might be similar to early host cells (uralga) whose
symbiotic associations evolved into the modern eukaryotic cell? The primitive host cell appears to
have been prokaryote-like and probably had the following features: anaerobic, possessed
hydrogendependent metabolism, strictly autotrophic, and could perform endocytosis. Most modern
prokaryotes are unsuitable models for the ancestral host cell because their cell wall is a barrier to
phagocytosis. Early bacteria may have entered host cells in the same way as Bdellovibrio, a
present-day parasitic bacterium that enters its hosts by forcibly penetrating the wall and
membrane of the host cell. (The life history of Bdellovibrio symbionts is described in chapter 3.)
Other organisms that provide insight into possible ancestral host cells include:

1. Giardia lamblia. This one-celled intestinal parasite causes dysentery in humans and is among
the most primitive of the eukaryotes. From an evolutionary perspective, Giardia is "a bacterium in
a eukaryotic cloak" ( Upcroft and Upcroft, 1998, p. 256). On the basis of rDNA phylogeny and
ribosome size, Giardia resembles prokaryotic cells. Giardia is an obligate anaerobe that lacks
mitochondria, peroxisomes, and endoplasmic reticulum. It can undergo endocytosis and so could
have ingested prokaryotic symbionts that were the precursors of mitochondria ( Cavalier-Smith,
1987a; Kabnick and Peattie, 1991; Sogin et al., 1989; Gupta et al., 1994). Unlike eukaryotes,
which possess the enzyme PDH, Giardia has PFO, characteristic of prokaryotes. A 2.4-kb plasmid
has been isolated from Giardia, and like that of some prokaryotes it carries genes for virulence and
antibiotic resistance. The Giardia plasmid is unusual because it behaves like a transposable element
and may be responsible for genomic recombination in an organism that lacks sexual reproduction (
Upcroft and Upcroft, 1998). Some investigators think that Giardia and other primitive organisms
may have had mitochondria at some point and then lost them ( Hashimoto et al., 1998).
2. Thermoplasma acidophilum. This mycoplasmalike, free-living archaean lacks the
characteristic cell wall of the eubacteria. Thermoplasma acidophilum has the smallest genome of
any known free-living organism, and its genome resembles that of eukaryotic cells in having
histonelike proteins associated with its DNA. In addition, actinlike filaments have been reported in
this organism. According to Gupta and Golding ( 1996), the ancestral eukaryotic cell developed
when a Gram-negative eubacterial host engulfed a thermophilic, sulfurmetabolizing archaean.

Mitochondria

The origin of mitochondria differs from that of chloroplasts and appears to be monophyletic ( Gray,
1993; Gray and Spencer, 1996). Determining which living bacterium could represent the ancestor
of mitochondria is difficult. Mitochondria cannot be cultured outside a cell and therefore cannot be
compared directly with other bacteria. Comparisons can be made only through molecular and
biochemical characteristics. Mitochon- dria have double membranes and show greater integration
into a eukaryotic cell than do chloroplasts. The outer membrane is believed to represent the
original ancestor host cell vacuole membrane, and the inner membrane is thought to be that of the
mitochondrial ancestor. New studies, however, have suggested that the outer membrane may be a
remnant of the outer membrane of the Gramnegative endosymbiont which gave rise to the
organelle ( Maynard-Smith and Szathmary, 1995). Mitochondria not only have smaller ribosomes
than those of the eukaryotic cell cytoplasm but also have an antibiotic sensitivity similar to that of
prokaryotic cells. Mitochondrial DNA varies in size; there is two or three times more genome in
plant mitochondria than in animal mitochondria. However, much of the plant mitochondrial DNA
consists of noncoding sequences (introns) ( Stern and Palmer, 1984; Brennicke et al., 1996; Franz
Lang et al., 1999).

Fungal mitochondria have a unique genetic code. The codon UGA, which is a stop codon in
prokaryotes and most eukaryotes, for example, is a second codon for tryptophan in mitochondria.
Again, the sequences of tRNA in mitochondria resemble neither those of prokaryotes nor those of
many eukaryotes. Mitochondrial mRNA genes, like the eukaryotic nuclear genome, have
intervening sequences, or introns, along with post-transcriptional processing (removal of introns
and splicing of exons); these processes are rare in prokaryotes.

The strongest evidence for the endosymbiotic origin of mitochondria comes from the sequencing of
proteins and nucleic acids. The sequences of cytochromes, ferredoxin, and 5S rRNA are similar to
those of photosynthetic prokaryotes such as Rhodopseudomonas. It has been demonstrated from
sequencing rRNA from mitochondria of fungi, mice, and humans, from nuclei of animals, yeast, and
corn chloroplasts, and from Escherichia coli that the greatest similarity in conserved sequences was
between mitochondria and prokaryotes. Molecular evidence indicates that the nonsulfur purple
bacteria (Proteobacteria) are the nearest present-day bacterial relatives of mitochondria ( Gray and
Spencer, 1996). More specifically, the relationship was closest to a subdivision of the a
proteobacteria that includes intracellular parasites such as Rickettsia. Comparison of heat-shock
proteins showed a similar picture ( Viale and Arakaki, 1994; Gupta, 1995a). Mitochondria of the
mud-inhabiting protozoan Reclinomonas americana have characteristics of both free-living bacteria
and host cells ( Sogin, 1997). More than 1000 species of protists and a few fungi lack mitochondria
( Cavalier- Smith , 1987a; 1997).

Chloroplasts

The chloroplast more closely resembles a prokaryotic cell than does the mitochondrion. The
chloroplast probably arose when a phagotrophic host cell engulfed and retained a photosynthetic
prokaryote, which gradually became transformed into an organelle ( Mc Fadden and Gilson, 1995).
This primary endosymbiotic event occurred at least once ( Stiller and Hall, 1997). Other kinds of
plastids arose through secondary endosymbiotic events involving fusions of eukaryotic cells (
Gibbs, 1992; Jensen, 1994; Lewin, 1993; Lockhardt et al., 1992b; Valentin et al., 1992; Palmer,
1993; Douglas, 1994b). There are many different types of plastids, and they probably originated
from multiple symbioses that involved cyanobacteria and algae. Most chloroplasts, like
mitochondria, are surrounded by two membranes. The inner membrane represents the plasma
membrane of the original prokaryotic symbiont, and the outer membrane either represents the
vacuolar membrane formed by the host around the symbiont or is the remnant outer membrane of
the Gram-negative cyanobacterium that was the endosymbiont ( Maynard-Smith and Szathmay,
1995). The chloroplasts of green algae, plants, and red algae have two enveloping membranes.
The chloroplasts of Euglena have three outer membranes, and those of brown algae and diatoms
are surrounded by four membranes. It is clear that these different types of plastids could not have
evolved from a common ancestor ( Cavalier-Smith, 1992b). Cyanobacteria such as Synechococcus
and Gloeobacter ( Pace, 1997) are the most likely ancestors of the plastids of red and green algae,
the latter group giving rise to green plants.

The chloroplasts of Euglena are thought to have arisen when a swimming protist ingested
unicellular green algae that were not digested and subsequently became symbiotic. The first, or
outer, membrane around the Euglena chloroplast is the vacuolar membrane that the host cell
formed around the endosymbiont. The second and third membranes represent the plasma
membrane and chloroplast membrane, respectively, of the original green algae. In brown algae
and diatoms, the outer two of the four membranes around the chloroplast originate from the
endoplasmic reticulum of the host cell. There are more than 27,000 species of algae with four-
membraned chloroplasts, and they are placed in a separate kingdom, the Chromista which includes
brown algae, diatoms, chrysomonads, cryptomonads, and haptophytes. The chloroplasts of
chromistan algae are inside the lumen of the cell's rough endoplasmic reticulum.

Several unusual algae were once thought to be "missing links" in the cyanobacteriachloroplast
evolution. Cyanidium caldarium, Cyanophora paradoxa, and Glaucocystis nostochinearum are
eukaryotic, unicellular algae that contain bluish green plastids, the cyanelles, which are equivalent
to cyanobacteria without cell walls ( Wasmann et al., 1987). How the host cell acquired the
cyanobacteria is not clear. One possibility is that some algae lost their chloroplasts and ingested
cyanobacteria, which were eventually transformed to cyanelles and assumed the role of
chloroplasts. Having once had chloroplasts, such colorless eukaryotic hosts would be better
equipped to maintain externally acquired cyanobacteria. Cyanelles cannot grow separately in
culture, and the DNA content of some, such as C. paradoxa, is reduced, like that of true
chloroplasts. It is not known whether there are genetic interactions between cyanelles and the host
nucleus as there are in the case of true chloroplasts. Thus, although cyanelles have a superficial
resemblance to chloroplasts and have reduced DNA, the present information is too limited to
support the view that they are primitive chloroplasts ( Seckbach, 1994). Similarly, Prochloron, a
phototrophic prokaryote with chlorophylls a and b, was proposed as a missing link between
prokaryotes and chloroplasts. However, studies have revealed species of Prochloron to be more
closely related to cyanobacteria than to higher plant chloroplasts ( Bryant, 1992; Lockhart et al.,
1992a).

Evidence for the endosymbiotic origin of chloroplasts has been collected from molecular data, such
as DNA-RNA hybridization of chloroplasts, nucleocytoplasm, and cyanobacteria and sequencing of
plastocyanin, ferredoxin, and cytochrome c, as well as tRNA and 16S and 5S rRNA. All of these
studies have indicated a close relationship between chloroplasts and blue-green prokaryotes. In
particular, the 5S and 16S sequences of chloroplast rRNA show a greater similarity to those from
cyanobacteria than to those from eukaryotic cytoplasm.

Complete chloroplast genome analyses from Cyanophora, Odontella (a diatom), Euglena, Porphyra
(red alga), Marchantia, Pinus, Nicotiana, Zea, and Oryza indicate that of 210 different protein-
coding genes, 45 are common to each and also to the cyanobacterial genome. Gene loss from
chloroplast genomes occurs in all groups, and 44 different plastid-coded proteins are functional
nuclear genes, thus providing strong evidence for endosymbiotic gene transfer to the nucleus in
plants ( Martin et al., 1998).

Microtubules
Another aspect of the serial endosymbiosis theory involves organelles that are used for motility (
Margulis and McMenanin, 1990; Hinkle, 1991). The term flagella is restricted to motile structures
found in prokaryotes ( Margulis, 1991a). Following the old Russian and French literature, Margulis
calls the organelles of motility found in eukaryotes undulipodia. Bacterial flagella are extracellular
organelles that run by rotation of the motor at their base. The shafts are nonmotile, contain the
protein flagellin, and lack microtubules. In contrast, eukaryotic flagella (undulipodia) are made up
of microtubules, which consist of tubulin and are arranged in characteristic pairs, in a 9+2
arrangement. Margulis ( 1991b) suggests that eukaryotic flagella developed from tubule-containing
spirochetes. Spirochetes are Gram-negative, highly mobile bacteria which are helically shaped and
readily form ectosymbioses with other heterotrophs. They are thought to have formed an
association with Thermoplasma-like host cells and eventually evolved into undulipodia. Symbiotic
associations in the hindgut of termites and wood-eating roaches contain protistan symbionts such
as Mixotricha paradoxa that are covered with a complex arrangement of spirochetes that are
responsible for the swimming of the protist. Margulis ( 1991b) also proposes that symbiotic
spirochetes gave rise, via mutations, to centrioles, chromosomal kinetochores, and the spindle of
the mitotic apparatus. With the development of mitosis came an explosion of many new species of
eukaryotic microorganisms, some of which eventually gave rise to fungi and animals. Nucleus
The origin and evolution of the eukaryotic nucleus is uncertain. The following hypotheses have
been proposed ( Lake and Rivera, 1996):
The nucleus arose autogenously when prokaryotic DNA became enclosed within an
invagination of the plasma membrane, thus creating two compartments, the DNA-containing
nucleus and non-DNA cytoplasm. Each unit then evolved separately ( Maynard- Smith and
Szathmary, 1995).
The nucleus originated endosymbiotically from a prokaryotic cell that contained DNA and RNA.
Gray ( 1992) observed the archaebacterial-like nature of the nuclear genome; Gupta et al. (
1994) and Golding and Gupta ( 1995) proposed that the eukaryotic cell nucleus was formed
by the fusion of an archaebacterium and a Gram-negative eubacterium. Lake and Rivera (
1994) also posit the nucleus as an endosymbiont that took over control of the host cell. Sogin
( 1991) hypothesized an anuclear protoeukaryotic lineage separate from the archaebacteria
and eubacteria that obtained a nucleus through engulfment of archaebacteria. The eukaryotic
lineage, therefore, is a chimera ( Doolittle, 1996; Gray et al., 1999). The presence of many
eukaryotic nuclear genes of bacterial origin may be the result of a gene transfer rachet which
occurs when a bacterial gene replaces a resident homolog of archeal origin ( Doolittle, 1998b;
Katz, 1998).
Peroxisomes

Peroxisomes may be yet another organelle that originated endosymbiotically ( Cavalier- Smith ,
1987a; Maynard-Smith and Szathmary, 1995). Their ancestors most likely were Grampositive
bacteria, which would explain the organelle's single membrane. However, the lack of DNA in
peroxisomes needs explanation.

Hydrogenosomes

Hydrogenosomes are double-membraned organelles. They occur only in eukaryotes that do not
have mitochondria and are facultative anaerobes. Hydrogenosomes produce hydrogen, CO 2, and
acetate, which are substrates for methanogenesis ( Brul and Stumm, 1994; Bui et al., 1996).
Hydrogenosomes appear to have evolved independently from mitochondria in several different
groups, including trichomonads, ciliates, and chytrids ( Embley et al., 1997).

6.4 HOST CELL: AN INTRACELLULAR

ECOSYSTEM
Ecosystems are ecological units of the biosphere, with identifiable boundaries and characteristic
species of organisms. Each organism in an ecosystem generally forms colonies. From such a
perspective, intracellular space represents an extreme in microhabitat. F. J.R. Taylor ( 1983)
defines a cell as a cytocosm and identifies four features of this unique habitat: (1) a stable
environment for the cell following symbiont intrusion (i.e., such a constant environment inhibits
sexuality of the symbiont and encourages it to stay in one phase of its life cycle); (2) slow rate of
succession; (3) exchange of nutrients and materials between the symbiont and host (the exchange
is controlled by selective pemeability of the host membrane); and (4) colonization of cell cytoplasm
by new symbionts, bringing forth processes for evolutionary change. The host cell as an
intracellular ecosystem produces a mutational meltdown and genomic reduction of the resident
symbionts ( Andersson and Kurkland , 1999).

The plant or algal eukaryotic cell, therefore, is one of the oldest, smallest, and most intensive
ecosystems. Individual organisms in the intracellular ecosystem should be genetically independent
in order to reproduce successfully. Interdependent relationships between the individuals of the
ecosystem involved division of labor in cellular functions. Modern-day eukaryotic cells have a
variety of regulatory mechanisms, some in the form of negative feedback systems, that maintain
and perpetuate the ecosystem.
The level of integration achieved by symbionts in the host cell is expressed in terms of its
evolutionary history. Symbioses discussed in this book show a wide range of adaptations between
the symbionts and their ecosystems. Intracellular parasitism can be viewed as a nutritional
relationship in which the parasitic symbiont obtains its food from the host cell. Intracellular
parasites may become lifelong residents in the host cell, but some can also live in the extracellular
environment. An intracellular symbiont must enter the host cell, overcome host defense
mechanisms, reproduce, avoid killing the host cell, and finally exit from the old cell to a new one.
Several examples of intracellular parasitism are discussed in detail in this book.

6.5 SYMBIOSIS AND PARASEXUALITY

Symbiosis is a form of parasexuality, a special type of reproduction found in some fungi that does
not involve the usual stages of the sexual process. Two fungi may fuse, and one may transfer its
nuclei to the other. The nuclei remain together in the hyphae. Each hyphal cell, therefore, may
contain two genetically different nuclei, a condition known as heterokaryosis. The different nuclei
may fuse occasionally and undergo a mitotic reduction that produces nuclei with new genetic traits.
In a symbiotic association the genes of both partners are close together but never actually fuse.
Natural selection acts on both genomes as a unit. Many symbioses are more fit than their individual
partners in given environments, and some have cycles during which the individual and symbiotic
states alternate ( Margulis, 1993b).

6.6 SUMMARY
Basic tenets in biology such as the cell theory (as applied to symbiogenetic cells) and prokaryote /
eukaryote distinction are being questioned as new findings in molecular evolution further confirm
the serial endosymbiosis theory and reveal new relationships between the archaea and the
eukaryotes. The five-kingdom classification of organisms has been criticized as unrealistic, and new
domains (superkingdoms) have been suggested (Archaea, Bacteria, Eukarya) as well as six
kingdoms of Eukarya, i.e., Archezoa, Protozoa, Chromista, Fungi, Animalia, and Plantae. The serial
endosymbiosis theory has been used to explain the origin of six different cell organelles (flagella of
eukaryotic cells, mitochondria, chloroplasts, nucleus, peroxisomes, and hydrogenosomes). The
autogenous theory explains cell membrane systems. A new hydrogen hypothesis is based on the
symbiosis between an α proteobacterium and a methanogen. Ancestral prokaryotic host cells that
may have evolved into modern eukaryotic cells could have been similar to Giardia lamblia and
Thermoplasma acidophilum. Cyanelles are remnants of cyanobacteria that were ingested by
primitive algae that had lost their chloroplasts. Eukaryotic cells have been compared to complex
ecosystems. Parasexuality is a form of symbiosis and is a type of reproduction common to some

7
fungi

FUNGAL ASSOCIATIONS OF PROTOZOA

AND ANIMALS
More than 1 million species of fungi are estimated to exist, but only about 69,000 species have
been described. Most of the undescribed fungi occur in tropical and subtropical regions of the world
( Hawksworth and Rossman , 1997). Some fungi are large, like mushrooms, but most of them are
microscopic. Armillaria bulbosa, a fungal parasite of the roots of conifers and hardwoods, may be
one of the largest and oldest living organisms. One individual strain of this fungus occupies more
than 30 acres of Michigan forest, weighs more than 10 tons, and is estimated to be 1500 years old
( M. L. Smith et al., 1992). Fungi grow practically everywhere and produce large numbers of
asexual spores that float in the air, are buried in soil and water, and cling to plants and animals.
Under favorable conditions, the spores germinate and produce colonies. About two-thirds of all
fungal species live in symbiotic associations with a wide variety of organisms. The major groups of
filamentous fungi are ancient in origin, with a fossil record from the Precambrian and early
Paleozoic ( Sherwood-Pike, 1991).

Fungi have exploited a large number of ecological niches in the biosphere. As saprophytes, they
play a key role in the decomposition of dead plants and animals and thus in the recycling of
carbon. Fungi are an important part of human affairs ( Hurdler, 1998). Some species are beneficial
to humans, but others are extremely harmful. Penicillin, an antibiotic obtained from a fungus, has
saved countless lives; yeasts such as Saccharomyces cerevisiae are fungi that people throughout
the world use to make bread and alcoholic beverages. Some fungi have caused destructive plant
diseases. Phytophthora infestans (Oomycota) was responsible for the potato blight disease that led
to the Irish famine in 1845-1851, during which more than 1 million people died and another million
emigrated to North America. The Bengal famine of 1945-1946 was caused by an infection of rice
crops by the leaf blight fungus Helminthosporium oryzae. The infection reduced rice yields by 75-
90% and resulted in the deaths of an estimated 2 million people. Between 1870 and 1890, the
British switched to drinking tea because Hemileia vastatrix caused a rust infection that destroyed
the coffee trees in Sri Lanka. In the United States large populations of two common trees have
been destroyed by fungal infections. The American chestnut has been virtually eliminated by the
chestnut blight fungus, Cryphonectria parasitica ( Anagnostakis, 1995); the American elm has been
decimated by the fungus Ophiostoma novo-ulmi. Fungi cause many chronic illnesses in humans
and other animals. Ringworm and athlete's foot are familiar fungal infections of skin tissue. Many
types of allergies and food poisoning are caused by common soil and airborne fungi ( Pirozynski
and Hawksworth, 1988; Alexopoulos et al., 1996).

7.1 CHARACTERISTICS OF FUNGI

Fungi, like animals, are heterotrophic, eukaryotic organisms. For their nutrition they break down
and absorb organic molecules produced by other organisms. They store food as glycogen or lipid.
This mode of nutrition differs from that of animals, which ingest food, and from plants, most of
which can manufacture food through photosynthesis. Fungi are biotrophic, necrotrophic, or
saprophytic, depending on whether they obtain nutrients from living cells, dead cells of organisms
they kill, or dead organisms, respectively. Fungi were earlier classified with plants, but the unique
features of these organisms have been recognized and they are now placed in a separate
kingdom.Many fungi form branching filaments, the hyphae, which grow and usually produce a
circular colony, a mycelium. A fungal hypha grows only at its tip and may be partitioned by
crosswalls to form a row of cells. Some fungi lack crosswalls, and a few, such as yeasts, are
unicellular. Fungal cell walls contain chitin.The process of mitosis and meiosis is unique for most
fungi. When the nucleus divides, its membrane does not break down, as in other organisms, but
rather pinches in and forms two daughter nuclei. Further, fungal chromosomes do not align along
the equatorial plate of the cell during metaphase.Many fungi reproduce sexually. Their gametes are
not motile, and the male gametes are carried to the female gametes by wind or water. In some
fungi the male and female sex organs grow toward each other and fuse. Nuclei from these sex
organs form pairs but do not fuse. Each pair of nuclei (N + N) is called a dikazyon. Fungi commonly
have a dikaryotic phase, which may be a major portion of their life cycle. Eventually, the cell nuclei
fuse to produce a diploid (2N) nucleus, which undergoes meiosis. Spores are produced in
distinctive fruiting bodies, which are used in fungal identification.

7.2 CLASSIFICATION
Not all organisms that have been called fungi are closely related and may not share a common
evolutionary history based on morphology, nutrition, and ecology ( fig. 7.1 ). The following is a
brief synopsis of the major groups of fungi as they are recognized today. This list is intended to
familiarize the reader with the groups from which we have selected examples of fungal symbioses.
Kingdom Stramenopila
Phylum Oomycota (zoosporic fungi): blights, downy mildews, and aquatic fungi that
attack rotifers, nematodes, and arthropods
Kingdom Fungi (Mycota)
Phylum Zygomycota (zygosporic fungi)

Fig. 7.1 Diversity of fungal associations, from mutualistic to parasitic, discussed in this chapter and
chapter 8.
Class Zygomycetes: predatory fungi of insects, vesicular--arbuscular mycorrhizal fungi
Class Trichomycetes: obligate symbionts of arthropods
Phylum Ascomycota (sac fungi)
Class Hemiascomycetes: yeasts, fungi that cause leaf curl and witchesbroom disease
Class Plectomycetes: saprophytes and plant pathogens such as powdery mildews
Class Laboulbeniomycetes: obligate symbionts of arthropods
Class Pyrenomycetes: lichen-forming fungi, plant pathogens that cause Dutch elm
disease, ergot of cereals, and plant wilts and rots
Class Discomycetes: lichen-forming fungi, saprophytes and plant pathogens that cause
brown rot of fruits and gray molds of vegetables
 Phylum Basidiomycota (club fungi)
Class Teliomycetes: rust fungi, smut fungi
Class Hymenomycetes: toadstools, jelly fungi, ectomycorrhizal fungi
Class Gasteromycetes: puff balls, stink horns, ectomycorrhizal fungi.
Phylum Deuteromycota (imperfect fungi)
Class Blastomycetes: yeastlike fungi
Class Hyphomycetes: conidial fungi, nematode-trapping fungi
Class Coelomycetes: plant pathogens causing anthracnose of cotton, beans, and flax

7.3 SYMBIONTS OF PROTOZOA AND

ANIMALS
Protozoa
Soil amoebae and testaceous (with shells) rhizopods are common hosts of specialized groups of
zygomycetes. The fungi use adhesive hyphae to capture their hosts. Some fungal species do not
penetrate their host, whereas others are internal symbionts. Each species of fungus produces a
characteristic assimilative organ that digests the host cytoplasm. Stylopage rhabdospora traps soil
amoebae by means of thin, sticky strands of mycelia. At the point of contact with the amoeba, the
fungus produces penetration pegs that enter the protist and branch to form assimilative organs.
Stylopage anomala is commonly found in dung, where it parasitizes amoebae. It reproduces by
spores that are dispersed by mites, which in turn are carried by dung beetles ( Blackwell and
Malloch, 1991). Species of Cochlonema and Endocochlus are obligate endosymbionts of soil
amoebae. Cochlonema explicatum produces adhesive spores that attach to amoebae, germinate,
and then produce germ tubes that enter the host cytoplasm. Once inside the host, the tip of the
germ tube swells and elongates. The amoeba gradually becomes sluggish, and after it dies fungal
spores are released from the host. A few hyphomycetes, such as Dactylella tylopaga, also capture
rhizopods. The morphological adaptations of these fungi, such as adhesive hyphae and assimilative
organs, are similar to those of predaceous zygomycetes.

Fungal attacks on soil amoebae are widespread in nature. These fungi are obligate symbionts
whose axenic culture has not been successful because their spores will germinate only in the
presence of a host. It is not known what specific nutrients the fungi obtain from the amoebae ( fig.
7.2 ).

Nematode-trapping Fungi: Predatory Fungi

An American mycologist, Charles Drechsler, pioneered the study of fungi that trap microscopic
organisms. From 1933 to 1975, Drechsler published papers about fungi that attacked nematodes
and amoebae ( Drechsler, 1934). In England, Charles Duddington book "The Friendly Fungi: A New
Approach to the Eelworm Problem," described the curious behavior of these soil fungi and captured
the imagination of scientists and amateurs. Nematologists have explored the possibility of using
carnivorous fungi to control nematode pests of plants. A soil fungus, Paecilomyces lilacinus, has
been used successfully to control plant disease caused by nematodes. The fungus invades and
destroys the egg masses of the root-knot nematode, Meloidogyne arenaria.

Nematodes are roundworms that are common inhabitants of soil ( Barron, 1992). In addition to
feeding on bacteria and fungi, nematodes parasitize plants and animals. Most

Fig.7.2 Examples of fungal parasitism of soil amoebae. Adapted from Cooke ( 1977).

fungi that trap nematodes are hyphomycetes, but some are zygomycetes. These fungi are
common in decaying organic matter ( Gray, 1987). When a wandering nematode touches a
hypha of the zygomycete Stylopage hadra, the fungal cytoplasm moves rapidly to the point of
contact, where a drop of viscous fluid is released. The nematode sticks to the fluid and, after a
brief struggle, becomes immobilized. At this time, the fungus penetrates the nematode's body
by means of a slender peg, which grows and branches and fills the host body cavity with
hyphae. The hyphae of S. hadra become sticky only after they are touched by a moving
organism.

Hyphomycetes that capture nematodes produce specialized hyphal traps, which may or may
not be adhesive depending on the fungal species. Following are examples of common types of
traps ( fig. 7.3 ).

Fig. 7.3 Three types of mechanisms used by hyphomycetes to capture soil amoebae. Adapted
from Barron ( 1977).

Adhesive nets and branches

Nets and branches are characteristic of many nematode-trapping fungi. Arthrobotrys oligospora
produces a complex of three-dimensional nets and loops. The fungus produces short lateral
branches that grow, curl, and then fuse with the parent hypha. A branch may arise from the
loop hypha and produce another loop. Repeated loop production results in a network of
adhesive hyphae. When a roundworm contacts a trap, it becomes caught, and as it struggles it
entangles itself in other traps until it is no longer able to move. A penetration peg may develop
from any point of the fungal network. In the body cavity of the nematode, the peg produces a
bulbous structure from which the assimilative hyphae arise. Most of the cell contents of the
host are digested and absorbed by the fungus.

Passive rings

Passive rings are typical of Dactylaria candida. Each ring consists of three or four cells and is
supported on a stalk. The diameter of a ring is barely wide enough to accommodate the head
of a nematode. If a nematode tries to pass through the ring, it gets caught and is unable to
retract. The ring often becomes detached from the mycelium during a nematode's struggle to
free itself, but the ring remains viable, and ultimately a peg from one of its cells enters the
host. The killing action of this type of fungus is slow, and frequently a nematode will have
several rings around its body as it moves through additional traps. In addition to passive rings,
D. candida also captures nematodes by means of short, adhesive knobs. The late Harvard
University mycologist William Weston dubbed these fungi the "nefarious nematode noose"
fungus and the "lethal lollipop" fungus.

Constriction rings

Constriction rings are the most sophisticated trapping mechanisms produced by fungi such as
Arthrobotrys anchonia and Dactylaria brochopaga. A typical ring consists of three cells
supported on a one- or two-cell hyphal stalk. As the nematode's head moves through the ring,
it contacts the ring cells and causes them to swell instantly, thereby trapping the nematode by
constriction. Once a nematode is captured, a penetration peg from one of the ring cells grows
into the body cavity and forms assimilative hyphae. Only the inner surface of the ring swells,
and the process is completed in 0.1 sec. The mechanism of ring constriction is poorly
understood. Hot water, dry heat, and glass microneedles can trigger the constriction response.

What stimulates a nematode-trapping fungus to form traps or how it attracts and kills its prey
is not fully understood. Some nematophagous fungi grown in axenic culture form traps only in
the presence of nematodes. In one study, a culture broth in which nematodes had grown
contained substances, collectively called nemin, which induced trap formation in Arthrobotrys
spp. Amino acids such as valine and leucine, carbohydrates such as arabinose and ribose, and
glycerol have stimulated trap formation in some fungi. Some scientists believe that the fungus
produces chemicals that attract wandering nematodes to the traps. It is also possible that
fungal spores may appear as food to nematodes. Toxins produced by the fungus may be the
cause of death of captured nematodes. Fungi use nematodes to supplement nutrients they
obtain from dead organic matter.

Endosymbionts of Nematodes
Endosymbiotic fungi of nematodes have several common features. These include (1) almost
total absence of any hyphal structure outside the nematode, (2) exit tubes for the release of
spores outside of the host, (3) resting spores that allow the fungus to survive in the absence of
a suitable host, and (4) host infection resulting from ingestion of a spore. Following are
examples of fungi that are internal symbionts of nematodes.

Meria coniospora (hyphomycete)

Meria coniospora produces tear-shaped spores that are sticky at their pointed end. The spores
attach to the head of a nematode and produce penetration pegs from which infection hyphae
develop. Young hyphae with a characteristic wavy appearance grow in the host body cavity.
When mature, some hyphae break through the body wall of the host and produce spores ( fig.
7.4 ).

Harposporium anguillulae (hyphomycete)

Spores of H. anguillulae are crescent shaped and have a pointed end. When the spores are
ingested by a nematode, they become embed-

Fig.7.4 Fungal endosymbionts of nematodes. Adapted from Barron ( 1977).

ded in the muscular esophagus and the nematode is unable to feed. The spores germinate and
produce hyphae, which grow throughout the body cavity of the host. At maturity the fungal
colony develops erect hyphae, which produce numerous spores or conidia. The fungus also
produces thick-walled chlamydospores that can withstand unfavorable environmental
conditions ( fig. 7.4 ).

Nematoctonus (basidiomycete)
Some species of Nematoctonus capture nematodes by means of adhesive knobs or aerial
hyphae; other species are endosymbiotic. In the trapping species, each trap consists of a
secretory cell that is swollen at both ends, constricted in the middle, and surrounded by a
viscous, sticky fluid. When a nematode touches the fluid, a bond forms that is so strong that it
rips open the cuticle of a struggling host. The fungus penetrates the captured nematode and
fills its body cavity with hyphae. Endosymbiotic species produce sausage-shaped conidia on
small, stalked hyphae. After dispersal, a conidium germinates and produces an adhesive cell at
the tip of the germ tube. The infection process begins when the cell becomes attached to the
cuticle of a wandering nematode. A toxin produced by the germinating conidium immobilizes
the nematode, which dies soon after it contacts the conidium ( fig. 7.4 ).

Fungal Pathogens in Aquatic Ecosystems

 Water molds play a significant role in decomposition and recycling in aquatic ecosystems.
Species of Achlya, Aphanomyces, and Saprolegnia attack fish and crayfish. Aphanomyces astaci
has devastated freshwater crayfish populations in Europe ( Alderman and Polglase, 1988).
Lagenidium callinectes parasitizes the eggs and larvae of marine crustaceans such as shrimp,
lobsters, crabs, and barnacles. The shrimp embryos become colonized with a bacterium that
produces an antifungal substance called isatin. Shrimp embryos that are surrounded by isatin
are protected from Lagenidium infection ( Gil-Turnes et al., 1989). Lagenidium giganteum
parasitizes mosquito larvae and has been considered for

biological control of mosquitos ( Federici, 1981).

Arthropods
The phylum Arthropoda includes more than 70% of all the known species of animals. Insects, with
an evolutionary history of 300 million years and more than 800,000 species, are the largest class
of animals and represent one of the great success stories in evolution. Insects have occupied
almost every conceivable ecological niche in terrestrial and aquatic habitats and have developed
associations with fungi that range from casual to intimate. Fungal symbionts and insect hosts show
a variety of morphological, physiological, and ecological coadaptations, and they also have some
common characteristics. Both contain chitin as a structural component, which is present in the
walls of fungi and in the exoskeleton of insects. Many fungi and insects are small and produce large
numbers of offspring, and both groups play an important role in the recycling of organic
compounds ( Samson, 1988; Wilding et al., 1989).

Pathogenic fungi of insects

Species of Beauveria, Cordyceps, Entomophthora, Metarhizium, and Verticillium are well-known

examples of fungi that are pathogens of insects. About 750 species of fungi infect insects. About 10
species of these fungi have been used successfully in the biological control of insect pests
(mycoinsecticides). The fungi infect the insect hosts, assimilate their hemolymph, digest soft
tissue, and produce mycotoxins, which kill the host ( Hajek and St. Leger, 1994).

Many characteristics of insect-fungus symbioses can be generalized. Fungal spores usually attach
to the insect cuticle in a specific manner, possibly through physical and chemical compatibilities
between the fungus and its host. High humidity (> 95% in some species) is necessary for spore
germination. The spore produces a germ tube whose tip swells and attaches to the cuticle.
Penetration pegs then develop and enter the host. On agar medium, a germinating spore produces
protease, lipase, and chitinase, enzymes believed to be responsible for the digestion of the host
integument. The fungus may take several days to reach the insect hemocoel, a bloodfilled space in
the tissues. If the insect molts before the fungus reaches the hemocoel, the symbiont is discarded
along with the old cuticle. The fungus multiplies in the hemocoel by small, budding bodies, the
blastospores. The developing fungus produces a toxin that kills the host within 48 h. The host
suffers from tremors and loss of coordination before death. The fungus then enters the mycelial
phase and develops hyphae, which consume all the organs of the host. The insect body becomes
mummified, and hyphae grow out of the body. These hyphae produce conidia that are dispersed by
wind or water.

Molecular mechanisms of fungal pathogenesis in insects

Insect pathogenic fungi produce conidia that germinate and form appressoria as the first essential
step in the infection process. Metarhizium anisopliae can produce appressoria on any hard
hydrophobic surface, and their growth is stimulated by nitrogenous compounds. Intracellular
second messengers such as Ca2+ and cAMP are involved in the formation of appressoria. Enzymes
such as endoproteases, aminopeptidases, and PR1, a serine protease, rapidly degrade the insect
cuticle. Molecular genetic analysis of PR1 and similar proteins has shed new light into how chitin
barriers are breached by fungal pathogens. Within the host tissue, M. anisopliae produces toxins
called destuxins (DTXs) which cause paralysis of muscles by activating their Ca2+ channels.
Destuxinlike secondary metabolites are believed to be widespread among fungi and may also be
involved in their growth and development ( Clarkson and Charnley, 1996). Following are some
well-known examples of entomogenous (insect-inhabiting) fungi.

Entomophthora (zygomycete). Species of Entomophthora have been used to control West African
black flies, which are carriers of a nematode that causes blindness in humans. The fungus is an
obligate parasite of the insect. Aphids, muscoid flies, caterpillars of butterflies and moths, and
grasshoppers are common hosts of other species of Entomophthora. In Russia, pea aphids of
legumes have been suppressed by Entomophthora infections. One major limitation in the
commercial use of this fungus as a biological control agent is the relatively short life of its spores,
about 2 weeks. One curious aspect of Entomophthora is that it produces single-

spored sporangia that are "shot" from the tips of specialized hyphae. An infected insect becomes
surrounded by a halo of white sporangia that are discharged from hyphae that grow out of the
insect's body ( fig. 7.5 ).

Cordyceps (ascomycete). Cordyceps attacks many different types of insects and spiders ( fig. 7.6 ).
The infected hosts shrivel and dry after death but resist decay because of an antibiotic compound,
cordycepin, produced by

Fig. 7.5 Conidial life cycle stages of Entomophthora muscae, parasite of the onion fly. (A) Infected
female fly. (B, C) Lateral view of fly, showing fungal sporulation (arrows). (D) Conidiophores
emerging from insect abdomen. (E) Single conidium attached to insect thorax. (F) Two conidia
attached to an abdominal seta of the host fly. Note the mucilage attached to the conidia. (Courtesy
Raymond I. Carruthers, USDA, Boyce Thompson Institute.)

Fig. 7.6 Cordyceps infection of an insect larva.

the fungus. After the host dies, the fungus produces a cylindrical structure (stroma) that grows
out from the host and produces many fruiting bodies (perithecia), which contain spores. The
ancient Chinese were familiar with the mummified silkworms and cicadas produced by
Cordyceps infections. Caterpillars infected with Cordyceps have been used in Chinese medicine
for curing many illnesses ranging from drug addiction to the effects of overeating. A Chinese
custom was to place precious stone carvings of the infected insects in the mouths of the
deceased in the hope of immortalizing them.

Beauveria bassiana (hyphomycete). Beauveria bassiana is commonly isolated from dead,

overwintering insects. In Russia, large quantities of the fungus are prepared commercially and
used to control the potato beetle. The fungus reduces the longevity of the adult insects and
also causes high larval mortality. Beauveria conidiospores remain viable in soil for up to 2
years. Efforts are being made in several other countries, including the United States, to use
Beauveria to suppress potato beetle infestations.

Metarhizium anisopliae (hyphomycete). Metarhizium anisopliae shows promise for controlling

the rhinoceros beetles that have destroyed many coconut trees in the South Pacific. The beetle
was accidentally introduced into the region in the 1930s. Beetle-infested trees do not produce
fruit, and new plantings are destroyed in the seedling stage. The application of fungus around
the base of an infected tree dramatically improves its health, as the fungus kills the beetles.
 Similar efforts to control rhinoceros beetle larvae with M. anisopliae in southern India have
been successful. The fungus is frequently isolated from soil-inhabiting insects and is also being
used to control mosquito larvae and the sugar cane spittlebug. In Brazil, spittlebug control by
M. anisopliae application has been successfully extended to many acres of sugar cane and
pasture land.

Symbionts of insects: nonpathogenic associations

There are many fungus--insect associations in which the host is not harmed. The associations
are obligatory for one or both partners. The following are examples of nonpathogenic
symbioses.

Laboulbeniomycetes. The Laboulbeniales are a group of microscopic ascomycetes that are

ectosymbionts of insects and other arthropods. The fungi appear as black or darkish bristles

to the naked eye and do not harm the insects. The fungi are obligate symbionts, and although
some species have a wide host range, most live only on certain insect species. The
laboulbeniomycetes became known to the world through the life-long scholarship of Ronald Thaxter
at Harvard University, whose dedication to these insects resulted in a multivolume monograph (
Thaxter, 1896-1931).

Incredibly, a fungus may be restricted to specific locations on the insect such as mouth parts, legs,
or wings. Species of Laboulbenia and Stigmatomyces are common on flies and beetles ( fig. 7.7 ).
Stigmatomyces ceratophorus is the only species that has been grown in axenic culture, on fly
wings kept on nutrient agar. The nutritional requirements of these fungi have yet to be
investigated. A typical fungal life cycle begins with the germination of an ascospore on the host
integument. The

Fig. 7.7 Laboulbenia parasitism of insects. (Top) Schematic drawing of the fungus with its
penetrating organs and perithecia. (Bottom) Scanning electron micrograph of the fungus perithecia
emerging from the insect cuticle. (Courtesy Gerald Van Dyke, North Carolina State University.)

fungus produces a foot cell that anchors the symbiont to the host cuticle and then produces a
thallus that may consist of only a few cells. The thallus produces hairlike appendages, which may
be unicellular or multicellular. Male and female reproductive structures and the fruiting body, or
perithecium, occur interspersed with the appendages. In most Laboulbeniales, the symbiont is
anchored to the host by a foot cell that does not appear to penetrate the cuticle. How these fungi
obtain nutrients from the host, without visible signs of penetration, is not clear. The fungus may
produce minute penetrating structures, or it may be that the cuticle contains sufficient nutrients for
the fungus. In a few species, such as Herpomyces stylopage and Trenomyces histophthorus,
penetrating structures develop from the foot cell and grow through the host integument to the
hemocoel, where fungal assimilative organs are formed. In T. histophthorus, when the fungus
reaches the hemocoel, it produces spherical swellings with nodular branches that penetrate the fat
bodies of the host. In no case do the fungal symbionts cause apparent ill effects in the host (
Tavares, 1985).

Trichomycetes (hair fungi). Trichomycetes are obligate symbionts that usually live attached to the
intestinal wall of marine, freshwater, and terrestrial arthropods ( Lichtwardt, 1986). The fungus
receives nutrients from the contents of the host's digestive tract but does not harm or benefit the
host; thus, the symbiosis is commensalistic ( Moss, 1979). The host becomes infected when it
swallows fungal spores. The spores germinate and the germ tubes attach to the gut lining of the
host by a specialized cell known as the holdfast. The fungal thallus consists of either branched or
unbranched hyphae. Mosquito larvae, when deprived of sterols and vitamin B, survive better if they
are infected with the trichomycete Smittium culisetae ( Horn and Lichtwardt, 1981). Internal
trichomycetes can be identified only by dissecting the insect host. Amoebidium parasiticum is an
exceptional trichomycete in that it attaches only to the outside of aquatic crustaceans and insects (
fig. 7.8 ).

Fungi and ambrosia beetles. Associations between fungi and beetles have been known for more
than 160 years. In 1836, J. Schmidberger observed an association of beetles with a glistening
white material, which he could not identify, in the galleries that beetles excavated in wood. He
thought the material was sap from the wood and called it ambrosia. In 1844, Theodor Hartig
discovered that the "ambrosia" was actually a fungus that was cultivated by the beetles and used
as food.

Ambrosia beetles make up about 36 genera in 2 families of weevils. The beetle genera Platypus
and Xyleborus, each with more than 200 species, are important groups of ambrosia beetles. Other
common genera of ambrosia fungi include Ambrosiella, Cephalosporium, Endomycopsis, and
Fusarium, all ascomycetes. A few basidiomycete ambrosia fungi are also known. The beetles
inhabit the sapwood of weakened trees. Healthy trees and dead trees are usually not infected.
Some beetle species attack only trees in a particular plant family, whereas other species infest

Fig. 7.8 The trichomycete Amoebidium parasiticum attached to Daphnia sp. Adapted from Moss (
1979).
only certain regions of a tree, such as the terminal branches. The beetles excavate galleries in
the wood and feed on the fungus that grows on the gallery walls. The beetles are well adapted
to live in the galleries, being small and dark with elongated bodies and short legs. Their mouth
parts are delicate and covered with flexible setae.

Each species of ambrosia beetle forms its own characteristic tunnel pattern. For example, in
hickory trees, the Xyleborus beetle produces branched galleries from a single entrance into the
bark. The female beetle carries fungal spores into the tunnel and deposits them on a substrate
of chewed wood prepared by the beetle. The spores germinate to produce abundant mycelia
and new spores all over the tunnel walls. The beetles graze on the chains of fungal spores. New
fungal growth appears at regular intervals. The fungal garden, in general, is remarkably free
from bacteria, yeast, and other contaminants. It is not known how the beetle maintains an
almost pure fungus culture. The female beetles tend the fungus gardens, carefully preparing
new beds into which they incorporate larval excrement. The female beetles also tend their
offspring. Larvae with poor survivability are eaten or entombed in a branch tunnel. Larvae
quickly die after the death of the mother beetle. The larvae are unable to maintain the fungus
garden, and bacteria and other contaminants soon overgrow the galleries. Male beetles, which
are wingless and short-lived, have no role in the ecology of a fungus garden, their main
function being to inseminate the females. Some species of ambrosia beetles raise their
offspring in communal galleries; among others, each larva develops singly in a tunnel,
excavating as it grows. The larvae have strong mandibles and can excavate wood. Female
beetles carry the fungus inoculum in an externally attached saclike mycetangium, which is
located on certain parts of the beetle, such as the mandible, elytra, or thorax (fig. 7.9). The
fungus grows within the mycetangium and obtains nutrients from the host's secretions and
excretions. The fungus is protected from drying out throughout the beetle's life and is thus kept
available for seeding new sites during tunnel excavation ( Norris , 1979).

Germinating spores of an ambrosia fungus produce germ tubes that penetrate the wood
parenchyma cells adjoining the tunnel wall. The cells become filled with mycelium, and the
tunnel walls become covered with a velvety mass of hyphae that bear spores. Most ambrosia
fungi are dimorphic, producing a yeastlike ambrosial phase and a mycelial phase. The
ambrosial phase commonly occurs in the mycetangium, in the beetle galleries, and sometimes
in laboratory culture. The mycetangia of beetles often contain axenic colonies of ambrosia
fungi, but many beetles emerging late in the season may carry secondary, foreign fungi that
develop in the tunnels. Oak wilt fungus, Ceratocystis fagacearum, and the Dutch elm fungus,
Ophiostoma novo-ulmi, are two important contaminants carried by ambrosia beetles.

The symbioses between ambrosia fungi and beetles are mutualistic. The fungal symbiont
obtains several advantages from the as-
 Fig. 7.9 Ambrosia symbiosis. (A) Tunnels produced by the ambrosia beetle, Xyleborus, in
hickory tree. (B) Mycetangium of scolytid beetle. From Norris ( 1979).

-1

sociation ( Beaver, 1989): its range is increased by the beetle; its penetration of woody tissue is
facilitated because of the mechanical injury caused by the beetles; it uses insect remains and
excretory products as nitrogen sources; and it multiplies and obtains nutrients from the host within
the mycetangium. The beetle host also benefits from the symbiosis. The fungus produces enzymes
that decompose cellulose and thus break down woody tissues, which makes the excavation process
easier for the beetles. The larvae feed exclusively on fungal conidia, and adults of many species
also feed on the hyphae. The fungus produces steroids which the beetle uses for its development
and for beetle pheromones.

Both the beetle and the fungal symbiont show the consequences of coevolution. The behavior of
the female beetle is the key to the success of the symbiosis. Survival of the larvae depends on her
ability to maintain the fungal garden and to suppress the growth of foreign contaminants in the
galleries.

Wood wasps are similar to ambrosia beetles in their association with fungi. Female wood wasps lay
eggs in the wood of conifer trees that have been weakened by disease or other insects. While the
eggs are moving through the insect's ovipositor, they become coated with spores of a wood-rotting
basidiomycete of the genus Amylostereum or Stereum. The fungal spores are contained in
mycetangia located near the base of the ovipositor. The spores germinate in the wood and produce
a mycelium that breaks down the wood and serves as a food source for the insect larvae. The
larvae bore through the decaying wood, form tunnels and chambers, and develop into pupae and
adults ( fig. 7.10 ). The wasp is an obligate symbiont, but the fungi involved in these symbioses
also occur free-living.

Fungus gardens of termites and ants. Some species of termites and ants cultivate fungi for
food. These termites (Termitidae; higher termites) use the fungi as a supplemental food source, for
vitamins, whereas the ants rely entirely on the fungi for their nutrients. In termites the ingested
fungi also provide enzymes that remain active in the gut and help the insect by digesting cellulose
and other plant polysaccharides ( Martin, 1992). The fungi are grown in specially prepared beds
that contain a mixture of plant material and insect excrement. Termites and ants are similar in
many aspects of their symbioses with fungi. Both insects have a rigid caste system that includes
workers and soldiers, and both build nests that may contain a million or more insects. Termite
nests are called termitaria and they are architectural wonders, with built-in temperature and
humidity controls. Nests are produced in soil and may be up to 20 feet high ( fig. 7.11 ). Fungus-
cultivating termites are common in Africa and Asia and differ from other termites in their lack of
intestinal symbiotic protozoa. The fungi cultivated in termitaria belong to the basidiomycete genus
Termitomyces. White nodules in the fungus gardens are aggregates of conidiophores and conidia (
Wood and Thomas, 1989).

Leaf-cutting ant species of the genera Acromyrmex and Atta maintain fungus gardens in nests
throughout Central and South America. The nests consist of chambers in

Fig. 7.10 Mycetangium of a wood wasp, located next to the ovipositor. Adapted from Cooke (
1977).

TERMITE MOUNDS Fig. 7.11 Termite nests with characteristic tunnels, where fungal gardens are
maintained.
which the fungus is cultivated. The fungal symbionts of leaf-cutting ant species belong to the
basidiomycete Attamyces bromatificus ( Cherrett et al., 1989), an obligate fungus that has never
been found ouside of a nest. Genetic studies have revealed that the ants have propagated the
same fungus for 23 million years ( Chapela et al., 1994; Hinkle et al., 1994). Numerous cultivars of
the fungus have been identified ( Mueller et al., 1998). New nests are started by fertilized females
(queens) that carry some of the fungus from their old nest. Ant fungus gardens are developed by
workers who cut leaf blades into large segments (10 mm in diameter) and carry them to the nest (
fig. 7.12 ). Young leaves are preferred over old ones, and flower parts are also used. Well-worn
trails are evident between ant nests and the bushes and trees that supply leaves. The leaf
segments are cleaned, cut into small pieces (1-2 mm in diameter), and then chewed to a pulpy
consistency. The ant worker mixes the spongy plant material with drops of its own exudates, which
are rich in ammonia and amino acids. A small fragment of vegetation that contains a fungus from
an old garden is then placed on the newly prepared bed. As the new fungus garden grows, ant
workers continue to add drops of anal exudates to the bed. Fungal hyphae, which become swollen
under these culture conditions, and spores are harvested periodically and used as food by the ant
colony. The worker ants continuously remove secondary invaders from the nest, keeping bacteria
and yeast populations at low levels. After the gardens reach an age of several months, they are
discarded and replaced with new ones. Abandoned fungus gardens quickly become overrun with
foreign contaminants. Experimental evidence has shown that the fungus may pass on chemical
signals to the foraging ants regarding the suitability of some substrates as food ( North et al.,
1997).

Septobasidium and scale insects. Septobasidium (teliomycete) is a basidiomycete that

commonly occurs in warm areas of the world. Most species are obligate symbionts of scale insects.
The fungus produces brown-black, flat thalli that adhere firmly to the leaves and bark of living
trees. The thalli have many chambers within which the host insects live. The chambers are
interconnected by tunnels, and each chamber contains one insect. The fungus penetrates the insect
by a few hyphae that form coiled haustoria within the hemocoel. The infected scale insect inside
the fungal chamber inserts its stylets into the plant tissue and feeds on plant sap ( fig. 7.13 ).
Nutrients the insect obtains from the plant are also assimilated by the fungus in the hemocoel.
Infected insects are small and sterile but are seldom killed. A scale insect colony also contains
many uninfected individuals. In the spring, as the fungus produces new mycelial growth, the
uninfected female insects in the fungal chambers produce nymphs. At the same time, the fungus
forms erect fruiting bodies, which produce basidiospores that divide by yeastlike budding. The
spores stick to the bodies of the wandering nymphs. The spores germinate and the germ tubes
penetrate the body hemocoel and form haustoria. Many nymphs settle in the same fungal colony,
but some leave and start new colonies elsewhere. In this mutualistic association, the scale insects
provide nutrients to the fungus and the nymphs disseminate the fungus, while the fungus protects
the insects from temperature extremes and from predation by birds and other organisms ( Couch,
1938). Symbiosis between scale insects and Septobasidium represents an example of kin selection,
where the reproduction of some members of the insect colony is sacrificed when they become
infected with the fungus. The scale insects that reproduce are protected from parasitoid wasps (
Seifert, 1981).

Yeasts as endosymbionts of insects. Many insects whose diet is blood, plant sap, or wood
possess yeast or yeastlike endosymbionts of the genera Candida, Taphrina, and Torulopsis. These
fungi occur in the intestines, gastric cecae, fat bodies, and Malpighian tubules of insects. The
symbionts are housed in specialized cells, mycetocytes, which are grouped together in mycetomes.
An intestinal mycetome has an epithelium that consists mainly of mycetocytes, which are made up
of enlarged fungus-filled cells with irregularly shaped nuclei. The cells lack microvilli on their
absorptive surface, do not divide, and often are polyploid ( fig. 7.14 ). The mycetocytes of some
insects harbor fungi only, but others contain a mixture of fungi and bacteria. Some of the
symbionts can be cultured, but most are obligate. The endosymbionts produce vitamins and
essential amino acids that are used by the host insect and in return receive a stable and favorable
environment in which to live. The endosymbionts are transmitted to new individuals by one of the
following ways: licking and stroking of the offspring by the female parent;

Fig. 7.12 Worker ants of Atta species cutting leaf sections for their fungal gardens.
 Fig. 7.13 Mutualistic symbiosis of the fungus Septobasidium with scale insects. Adapted from
Cooke ( 1977).

the outer surface of the eggs becoming coated with fungi during the egg-laying process by means
of fungus-containing structures attached to the ovipositor; or by the egg cell cytoplasm carrying
the fungus ( Nardon, 1988).

The symbionts in a coccid insect Stictococcus diversisetae enter the ovary and infect some of the
eggs produced by the insect. These eggs develop parthenogenetically and give rise to females;
uninfected eggs develop into males. Loss of a chromosome is involved in the production of males.
Some scientists believe that endosymbionts of infected eggs, in a manner not understood, prevent
the chromosomal loss.

Symbionts of Humans and Other

Vertebrates
Associations of fungi and warm-blooded animals are almost universal. In nature, most animals
have low-grade fungal infections. Some fungi are well known because they cause mycoses in
domesticated animals as well as in humans. Fungal and protozoan infections are now a serious
public health hazard, especially for those who are immunocompromised because of AIDS, cancer
therapy, or drugs taken to prevent organ rejection ( Kasper and Buzoni-Gatel, 1998).

Endogenous fungi make up the normal biota of the skin and mucous membranes and often attack
the host through injured tissue. Exogenous fungi live as saprophytes in the soil and sometimes
produce diseases in animals. Fungal infections of vertebrates are of two main types: systemic
infection, of deep internal tissues and vital organs such as lungs and brains; and superficial
infection, of scalp, skin, or nails from fungi known as dermatophytes. Fungal parasites of
vertebrates have a wide host range and are easily transmitted be- tween humans and animals.
Some fungal infections have become associated with certain occupations and lifestyles of humans.
For example, histoplasmosis is prevalent among poultry farmers and cave explorers because bird
droppings are a common source of fungus. Farmers, gardeners, and agricultural workers often
have mild fungal infections.

Recently, an increasing number of plant pathogenic fungi have become implicated in

inununosuppressed patients. Alternaria alternaria, Aspergillus flavus, and Fusarium oxysporum are
common pathogens of strawberries, tomatoes and other plants that infect the lungs of
immunocompromised patients. Opportunitistc fungal infections in immunosuppressed patients
makes the development of new antifungal drugs critical ( Miller, 1986). Fungi that attack
vertebrates have been reported from all major fungal groups, although only a few infections are
caused by basidiomycetes. Following are some examples of fungal diseases of humans.

Candidiasis

Candidiasis is caused by a yeastlike fungus, Candida albicans. In humans, Candida albicans as well
as true yeasts are residents of the mouth, alimentary canal, and vagina. The fungus normally lives
as a saprophyte receiving nutrients from sloughed-off epithelial cells of the mucous membranes.
Candida albicans causes thrush, in which the fungus invades the superficial tissue and produces
soft, gray-white lesions on the gums, tonsils, and tongue. Candidiasis frequently occurs in
individuals undergoing antibiotic therapy, on prolonged use of birth control pills, during pregnancy,
and with diabetes mellitus. The fungus has a hyphal form during the invasive phase of the
infection. The mechanisms controlling fungus morphology are not known. Candida can also cause
serious systemic infections.

Aspergillosis

Species of Aspergillus cause several types of mycoses. Aspergillosis is increasingly being

recognized as an important complication in individuals suffering from malignant diseases, such as
leukemia and lymphoma, and in those receiving immunosuppressive drugs. Aspergillus fumigatus
is a virulent pathogen that causes pulmonary aspergillosis and can spread to other organs.
Asthmatic symptoms and fever are common in these mycoses. Aspergillus fumigatus may invade
the nasal sinus tissue and can cause endocarditis in patients with heart valve replacements.
Fumigatotoxin, a high molecular weight toxin, has been isolated from the fungus.

Several species of Aspergillus cause avian

Fig. 7.14 Intestinal epthelial cells of insects with mycetome-containing yeast cells. Adapted from
Cooke ( 1977).

aspergillosis. Young birds die from appetite loss, high temperature, and diarrhea. In 1940, many
herring gulls in the Boston Bay area died from infections caused by A. fumigatus that had
developed first on rotting seaweed. Aspergilli have also been involved in equine gutteral pouch
mycosis, mycotic abortion, and mycotic mastitis.

Histoplasmosis

Histoplasmosis is a serious and often fatal infection of the lymphatic system. The disease is
widespread in warm, humid regions of the world and is caused by Histoplasma capsulatum, a
fungus that reproduces like a yeast in host tissue and produces mycelial growth in culture. The
infection begins when spores of the fungus are inhaled into the lungs. After a prolonged exposure
to the fungus, the individual develops chronic, tuberculosislike symptoms of cough, slight fever,
and pulmonary cavitation. Progressively, the disease spreads to the spleen, liver, adrenals,
kidneys, nervous system, and other organs. Histoplasmosis is frequently associated with soils that
become contaminated with fecal material from bats and birds, including chickens. In city parks and
plazas, where birds often roost, the soil and the buildings frequently become contaminated with H.
capsulatum. There is no satisfactory treatment for this disease.

Coccidiomycosis

Coccidioides immitis causes a mild respiratory infection that is called the San Joaquin Valley fever.
The fungus causes lesions in the upper respiratory tract and progressively develops in bone, joints,
and other tissues, including the central nervous system. Symptoms include fever, chills, sweating,
and general weakening. Coccidioides immitis infections are widespread in and parts of the United
States and throughout Central America. The fungus is soilborne and produces spores that remain
viable for long periods. Rodents, domesticated animals, and humans become infected from inhaling
the airborne spores. The fungus is dimorphic; in the invasive phase it produces spherical cells that
function as sporangia at maturity; in the saprobic phase the fungus produces arthroconidia. Most
people and animals living in areas where the fungus is common have mild coccidiomycotic
infections. The chance of becoming infected increases with the length of stay in the area. The
disease is rarely fatal.

Dermatophytic diseases: ringworms

The fungi collectively known as dermatophytes are limited to the keratinized layers of skin, nails,
hair, or feathers of humans and animals. Diseases from dermatophytic fungi include various types
of ringworm. Dermatophytic fungi belong to three genera: Epidermophyton, Microsporum, and
Trichophyton. Species of Epidermophyton are confined mostly to humans and infect only the skin.
These fungi are widely distributed in soil, and most infections are acquired through intimate
contact. Humans contract ringworm disease from infected animals and also from contaminated
combs, hairbrushes, caps, and furniture. Microsporum canis infection is usually acquired from dogs
and cats. Trichophyton species are transmitted to humans from horses, cattle, dogs, and other
animals.

Dermatophytic fungi flourish under warm, humid conditions. Most people during their lifetime
become infected with at least one dermatophytic fungus. Infections are favored under conditions
caused by continuous wet skin (swimmers), moist and oily skin, tight clothing and shoes that
prevent evaporation of perspiration, and obesity, which results in increased folding of the skin
where oil and moisture accumulate.
It is assumed that dermatophytic fungi can digest and assimilate keratinized tissue, although the
mechanism of keratin digestion is not understood. Dermatophytic fungi can be grown in axenic
culture, but in nature most species need a host to grow.

Mycotoxins
It is only since 1960 that we have learned about the significance of mycotoxins in illness and even
death of humans and domesticated animals. In 1960, the deaths of more than 100,000 turkeys
within a 100-mile radius of London led to the discovery of the mycotoxin aflatoxin, which was
isolated from Aspergillus flavus, a fungus that had contaminated the peanut feed of the turkeys.
Mycotoxicoses are now recognized as occurring worldwide, and a number of mycotoxin-producing
fungi have been identified. Scientists in the former Soviet Union were first to appreciate the
significance of mycotoxin-produced diseases. Perhaps the prolonged storage of grains and other
animal feed during the long Russian winters increased the possibility of mold growth. Consumption
of moldy hay was found to be responsible for the deaths of many horses in the Ukraine in 1937. In
1954, A. K. Sarkisov published a detailed monograph in Russian on mycotoxicoses, but the work
remained unknown in Western countries until the mid-1960s.

All mycotoxin-produced diseases are characterized by noncommunicability, unresponsiveness to

antibiotic drugs, and geographically limited outbreaks usually linked to moldy food. The toxic
response in the affected individuals occurs through organs such as the liver, kidney, lung, and
nervous system. The chief mycotoxin-producing fungi belong to the genera Aspergillus, Fusarium,
and Penicillium. Following are some examples of fungi and their mycotoxins.

Aspergillus

Aspergillus flavus and A. parasiticus are two aflatoxin-producing fungi that are prevalent in hot,
humid climates. Aflatoxins have been isolated from edible nuts such as peanuts, walnuts, almonds,
pecans, Brazil nuts, and pistachio nuts and from grains such as wheat, rice, corn, sorghum,
cottonseed, and millets. In a 1960 British study, aflatoxin types B 1, B 2, G 1, and G 2 were
identified. Since then it has been established that dairy products from cows that ingested B 1 toxin
contaminated hay contained M 1 and M 2 types of aflatoxins. The M flatoxins are firmly bound to
casein molecules. The G 1 and M 1 toxins are carcinogenic. Aflatoxin-susceptible hosts such as
rabbits, guinea pigs, rainbow trout, rats, and monkeys develop liver damage with necrotic lesions
and swollen kidneys. The first signs of the disease are loss of appetite and weight, followed by poor
muscular coordination. A high incidence of human liver cancer in parts of Africa, Southeast Asia,
and Japan is correlated with aflatoxin-contaminated food ( Bilbrami and Sinha, 1992).

Claviceps purpurea

Ergotism is caused by the ascomycete Claviceps purpurea and it is one of the oldest recurring
epidemics in European history ( Schumann, 1991). Brownish-black sclerotia are formed in infected
ovaries of grasses such as rye. It has been suggested that women who displayed signs of
"bewitchment" in Salem, Massachusetts, during 1692 may have been suffering from convulsive
ergotism ( Matossian , 1989). Ergot mycotoxins are a complex mixture of alkaloids, and the two
main types are lysergic acid and agroclavine. Disease symptoms of ergotism include gangrenous
necrosis of fingers or toes and convulsions when ergot-contaminated grains are ingested by
humans or cattle. Ergot mycotoxins cause vasoconstriction and have found widespread medical
applications ( Marasas and Nelson, 1987).

Penicillium

Penicillium viridicatum is an important mycotoxin-producing species. It usually infects stored grains

and causes illness and death in humans and domesticated animals. Penicillium citrinum is
widespread in the tropics and subtropics, and its toxin produces a lethal kidney disease. Mycotoxins
of R. islandicum have received attention from Japanese scientists because consumption of rice
contaminated with this fungus produces the disease rice toxicosis. As many as four potent
hepatotoxic carcinogens have been isolated from P. islandicum. One of these toxins, islanditoxin,
causes severe liver damage, hemorrhage, and rapid death. Penicillium cyclopium is commonly
associated with food grains and produces penicillic acid, cyclopiazonic acid, and two tremor-
producing compounds. These fungi usually inhabit soil as saprobes and develop on the seeds only
after harvest.

Fusarium

Fusarium graminearum infects corn and produces trichothecene mycotoxins that induce vomiting in
pigs. Fusarium poa and F. sporotrichioides produce the fescue food syndrome in cattle. The animals
show symptoms of lameness, weight loss, arched back, swelling in the hind legs, separation of the
hoof from the foot, abnormal horn and hoof growth, and dry gangrene in the extremities. If not
checked, the disease can be fatal. Trichothecenes are produced only by Fusarium and related
fungal species. In several Brazilian plant species of Baccharis the female plants produce
trichothecenes which are concentrated in the flowers and fruits. It is speculated that this
represents an example of horizontal gene transfer between the fungal symbiont and Baccharis host
plant ( Kuti, et al., 1990).

Mycotoxins are potent carcinogens that form a DNA adduct, often resulting in mutations as a
result of G to C transversions ( Baertschi, et al., 1989). Recently, a genetic mechanism has
been reported that suggests how these mycotoxins are a causal factor in human
hepatocarcinogenesis ( Aguilar et al., 1993). Aflatoxin has been demonstrated to cause critical
transversions in the p53 tumorsuppressor gene, and such a change has been found in more
than half the liver tumors examined in regions of the world where aflatoxin contamination of
foods is high.

Mycotoxin research is still in its infancy. There are a number of challenging questions that need
additional research, such as the prevalence of mycotoxins in food crops, their relationship with
carcinogenicity, and ways to prevent their occurrence.

7.4 SUMMARY
Fungi have successfully formed associations with all forms of life in most ecosystems and play
a significant role in decomposition. Fungal diseases in plants, animals, protozoa, and even
other fungi have received considerable attention from mycologists. Unique features of fungi,
such as the way they obtain nutrients, their mycelial growth and spore production, and the
chitinous nature of their cell walls have convinced scientists that fungi belong in a separate
kingdom. Fungal taxonomy is changing and organisms traditionally called fungi (chytrids) have
been placed in other kingdoms. In this chapter fungal associations with soil amoebae,
nematodes, insects, and animals were examined in detail.

Some fungi have evolved morphological and physiological adaptations to trap and capture
nematodes. Species of Laboulbenia are microscopic ascomycetes that are obligate
ectosymbionts of insects and arthropods. Some species of these fungi are so highly specialized
that they grow only on certain parts of an insect host. Trichomycetes form commensalistic
relationships with arthropods. Ambrosia beetles and wood wasps form mutualistic associations
with wood-rotting fungi, and some species of termites and ants establish fungal gardens for
food. The basidiomycete Septobasidium is an obligate symbiont of scale insects, with which it
forms a unique mutualistic relationship. Yeasts are endosymbionts of many insects. Fungal
parasites of nematodes and insect pests have provided opportunities for scientists to develop
strategies for biological control. Some fungi cause diseases of humans, such as candidiasis,
histoplasmosis, coccidiomycosis, and ringworms. These opportunistic fungi attack hosts with
weakened immune systems. Mycotoxins such as aflatoxin and islanditoxin produce diseases in
humans and animals. Fungal disease epidemiology is receiving increasing attention from the
medical community.

8
FUNGAL ASSOCIATIONS OF FUNGI, ALGAE, AND
PLANTS
Symbiotic interactions between fungi and phototrophs were instrumental in the colonization of land
by algae and plants. Fungi may have helped the early phototrophs cope with the drying conditions
of the terrestrial environment. Such interactions led to permanent associations now recognized as
lichens, mycorrhizas, mycophycobioses, and endophytes of grasses ( Atsatt, 1988, 1991; Selosse
and LeTacon, 1998).

8.1 MYCOSYMBIONTS OF FUNGI

More than 1000 species of fungi obtain nutrients by infecting other fungi. Most of these
mycosymbionts are conidial fungi (Hyphomycetes), and they have been used as biological control
agents of plant pathogens such as Phytophthora, Pythium, and Sclerotinia. Necrotrophic
mycosymbionts kill their host fungus before the infection process is established, but most fungal
associations are biotrophic and do not appear to injure their hosts. The terms hyperparasitism,
mycoparasitism, and fungicolous are often applied to interfungal associations ( Elad, 1995; Jeffries,
1997).

Necrotrophic Mycosymbionts
Common soil-inhabiting fungi such as Ampelomyces quisqualis, Gliocladium roseum, Rhizoctonia
solani, and Trichoderma viride are necrotrophic mycosymbionts. They can live saprobically on dead
organic matter and also attack a wide range of fungi ( fig. 8.1 ). Many mycosymbionts grow well in
laboratory cultures. When a host fungus is introduced into a culture, the mycosymbiont grows
toward it, and, in some cases, its hyphae coil around the host hyphae. The mycosymbiont produces
diffusible toxic substances that change the host cell's permeability. Nutrients leak out from the
infected cell and it dies. Trichoderma spp. produce hydrolytic enzymes such as β-glucanase,
chitinase, and other volatile metabolites. Rhizoctonia solani is a well-known root pathogen that kills
young seedlings. In culture, R. solani can destroy susceptible fungal hosts such as Rhizopus
nigricans and species of Mucor by penetrating the host mycelium.

Gliocladium roseum attacks a wide range of fungi in nature and in laboratory culture. The fungus
curls around the host hyphae by means of short branches and kills the host on contact. The fungus
then penetrates the cells and absorbs their nutrients. Gliocladium roseum hyphae penetrate cells of
Mucor sp., Rhizoctonia solani, and Rhizopus nigricans while they are still alive.

Ampelomyces quisqualis attacks powdery mildew fungi. At first, the mycosymbiont grows
harmlessly among the host hyphae, but later it kills them. The fungus is believed to overwinter as
a saprobe on mildew-infected leaves. In one study, clover powdery mildew was controlled by
artificial inoculation with A. quisqualis.

Darluca filum attacks only spores and mycelia of rust fungi. Because of this limited host range,
some scientists have speculated that the ancestor of D. filum was a leaf pathogen that lost its
ability to infect leaf tissue and instead parasitized rust fungi.

Tuberculina maxima is often described as the purple mold of rust fungi, and it is a nat-

Fig. 8.1 Three types of interfungal parasitic symbiosis. Adapted from Cooke ( 1977).

ural enemy of pine blister rust. This mycosymbiont infects mostly the pycniospores of the host,
which reduces aeciospore production. Tuberculina maxima also destroys rust mycelium in infected
tissues of white pine. Spores of the fungus remain viable for several years. There has been interest
in using T. maxima as a biological control agent of pine blister rust in the western United States
and Canada ( Schumann, 1991).

Biotrophic Mycosymbionts
Biotrophic fungal symbioses are highly specialized, long-lived relationships, and many fungal
species are obligate symbionts of their host fungi.

Contact mycosymbionts

Contact mycosymbionts were not recognized until 1958 but are now believed to be common. About
six species of hyphomycetes from genera such as Calcarisporium, Gonatobotrys, and Stephanoma
are involved in contact mycosymbioses. Extensive observations have been made on C. parasiticum
and G. simplex.

The mycosymbiont produces short, lateral hyphae that contact the host hyphae and cause them to
release nutrients. Except for arrested growth, the infected fungus shows no adverse effects.
Mycosymbionts will grow in culture only if a growth factor, mycotrophein, extracted from the host
fungus, is part of the culture medium. Contact mycobionts obtain the vitamins biotin and thiamine
from their hosts ( Moore-Landecker, 1996).

Haustorial mycosymbionts

Infection of species of the order Mucorales (zygomycete) by other members of the same order is
common. Spores of the mycosymbiont germinate in reponse to substances that diffuse from a host
mycelium. The germ tube grows toward the host hyphae, and upon contact, the tip of the germ
tube swells to form an appressorium, a flat, hyphal cell. Fine hyphal branches from the
appressorium penetrate the host cell and then enlarge to form inflated haustozia. An internal
mycelium develops from the haustoria, and on maturity hyphae emerge from the host and form
sporangia.

Piptocephalis virginiana attacks a large number of Mucor species. Electron microscopic studies have
shown that the haustorium of P. virginiana is similar in structure to that of fungal symbionts of
plants.

8.2 FUNGAL-ALGAL ASSOCIATIONS

Mycophycobioses
A mycophycobiosis is an obligate symbiosis between a filamentous marine fungus and a large
marine alga in which the alga is the dominant partner ( Kohlmeyer and Kohlmeyer , 1979). The
fungus grows between cells of the algal thallus but never penetrates or damages the cells. The
effect of the fungus on the alga is not clear. The fungus may protect the algae from drying
during low tides when the algae are exposed, and the fungus may be necessary for algal
development. Sporelings of some marine algae, such as Ascophyllum nodosum, will not
develop a thallus unless they are infected by a fungus ( Garbary and London, 1995).

Fungi that form mycophycobioses include species of Blodgettia and Mycosphaerella. Blodgettia
bornetii is a hyphomycete that grows in the walls of tropical species of Cladophora. The fungus
produces spores and is absent only from the growing tips of the alga. The ascomycete
Mycosphaerella ascophylli is always associated with the brown algae Ascophyllum nodosum and
Pelvetia canaliculata. Hyphae grow between cells of the cortex and medulla of the host, and the
fungus undergoes sexual reproduction and produces fruiting bodies while still within the host.

The ascomycete Turgidosculum complicatulum forms associations with the green algae Prasiola
borealis and R. tesselata. Hyphae of the fungus grow throughout the algal thallus and separate
cells of the thallus into groups of four or into rows. These algae can also live without the
fungus. Prasiola thalli infected with fungus are more common than uninfected forms in exposed
or drier parts of the intertidal zone. The fungus protects the alga from drying. Turgidosculum
ulvae is a common symbiont of the green alga Ulva vexata. The fungus grows so extensively
that it separates the layers of the algal thallus.

Lichens
 A lichen is an association between a fungus (mycobiont) and a photosynthetic symbiont
(photobiont) that results in a stable thallus, or body, of specific structure. The photobiont is
either an alga or a cyanobacterium. A remarkable feature of a lichen is the transformation that
the symbionts, in particular the fungus, undergo during the association. A new entity, the
thallus, is formed, and unique chemical compounds are synthesized. The physiological behavior
of the symbionts also changes in symbiosis ( Honegger, 1991; Hill, 1994). There are about
15,000 species of lichens, an indication that this type of symbiosis has been highly successful
and has involved many species of fungi ( Nash, 1996). Surprisingly, only about 30 different
types of algae and cyanobacteria have been reported as photobionts. Taxonomically, only the
fungus and alga of a lichen have Latin names, although commonly the name of the fungus is
also used for that of the lichen. For example, Cladonia cristatella is the name of a mycobiont
but, unofficially, the name of a lichen as well. The photobiont of this lichen is Trebouxia erici.

Types of lichens

A lichen thallus usually consists of layers such as an upper and lower cortex, algal layer, and
medulla. The layers differ in thickness and are better developed in some species than in others.
Fungal hyphae make up most of a thallus; the photobiont cells are only a small percentage
(about 7%) of the total volume ( Ahmadjian, 1993).

There are three main types of thalli: crustose, foliose, and fruticose. A crustose thallus lacks a
lower cortex and is generally considered to be the most primitive type. Thalli of Lepraria
species do not have layers but consist only of powdery granules. There are more species of
crustose lichens than other types, and most of them belong to the genera Lecanora and
Lecidea. Many crustose lichens stick tightly to the substratum and appear to be painted on it.
Some species grow inside rock crevices and bark and still manage to produce separate layers.
Squamules are typical of many species of Cladonia. Squamules are a specialized type of
crustose thallus and are attached at only one end to the substratum.

A foliose thallus has an upper and lower cortex, an algal layer, and medulla and is usually
loosely attached to the substrate by hairlike structures called rhizines. The thallus has many
different sizes and shapes and is often divided into lobes. Common foliose genera include
Anaptychia, Cetraria, Parmelia, Physcia, and Xanthoria. Some foliose lichens, such as
Umbilicaria (rock tripe), have thalli that are attached to the substrate by only one central point.

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Fruticose thalli are upright or hanging, round or flat, and often highly branched. Thalli of Usnea are
hairlike and can reach a length of 5 m, whereas those of Evernia are shorter and strap-shaped. The
layers of a fruticose thallus may surround a central thick cord, as in Usnea, or a hollow space as in
some Cladonia species.

Some lichens, such as Collema, which have Nostoc as a photobiont, do not form a well-organized
thallus. In these cases, fungal hyphae grow inside the thick gelatinous sheaths of the photobiont,
which makes up much of the thallus. Geosiphon pyriforme, once thought to be a primitive lichen
with a Nostoc photobiont, is now considered to be a vesicular arbuscular mycorrhizal fungus (
Gehrig et al., 1996).

Most lichens have an ascomycete as the fungal partner, and only about a dozen are formed by
basidiomycetes. The lichenized ascomycetes are one of the largest groups of the phylum
Ascomycota in the kingdom Fungi. The lichenized habit obviously has had great selective benefits
for fungi.

Distribution

Lichens grow practically everywhere--on and within rocks, on soil and tree bark, on almost any
inanimate object. They grow in deserts and in tropical rainforests, where they occur on living
leaves of plants and ferns. They have been found on the shells of tortoises in the Galapagos Islands
and on large weevils in New Guinea. In the dry valleys of Antarctica, endolithic lichens, such as
Buellia and Lecidea, grow inside sandstone crevices ( Hively, 1997). Dermatocarpon fluviatile and
Hydrothyria venosa grow in freshwater streams, and species of Verrucaria are common in the
intertidal zones of rocky, ocean shores. Verrucaria serpuloides is a permanently submerged marine
lichen that grows on stones and rocks 4-10 meters below mean low tide off the coast of the
Antarctic Peninsula. Verrucaria tavaresiae is another unusual marine lichen with a brown algal
photobiont ( Moe, 1997). Douglas Larson has estimated that about 8% of the earth's terrestrial
surface is dominated by lichens ( Ahmadjian , 1995a). Lichens abound in areas with high annual
humidity, such as the fog belt zones of Chile and Baja California. Extensive lichen populations also
grow in the cool, northern forests of the world, where hundreds of miles of forest floor are covered
with thick carpets of reindeer lichens (Cladonia). Trees along the coasts of the northwestern United
States may be blanketed with beard lichens such as Alectoria and Usnea.

Lichens with organized thalli do not grow well in areas that are continuously wet, such as tropical
rainforests. Only poorly organized species of Lepraria and leaf-inhabiting lichens are found in these
regions. Lecanora conizaeoides and Lecanora dispersa colonize trees and gravestones in industrial
cities and towns, but most lichens cannot tolerate the polluted atmosphere and persistent dryness
of urban areas. The sensitivity of lichens to atmospheric pollutants such as sulfur dioxide, ozone,
and fluorides has made them valuable indicators of pollution in cities and industrial regions. Lichens
have been used to map pollution zones around many of the world's major cities ( Richardson,
1992).

Dispersal and reproduction

Lichens are dispersed by thallus fragments and vegetative diaspores such as isidia and soredia.
Each diaspore consists of a few algal cells and fungal hyphae. Soredia are powdery granules that
originate inside the thallus, as localized overgrowths of algae and hyphae, and break through the
upper cortex. Isidia are cylindrical extensions of the thallus. About 39% of all foliose and fruticose
species of lichens produce isidia. Diaspores are dispersed by water, wind, insects, and birds.

Lichen fungi produce the same type of reproductive structures as other ascomycetes. Only some
aspects of the sexual process have been seen in lichens, such as the fusion of microconidia to the
tips of trichogynes. Dispersal of photobionts occurs by means of motile (zoospores) and nonmotile
(autospores) spores.

Interactions between symbionts

Physical relationships. The basic unit of a lichen thallus is one algal cell with enveloping hyphae.
Fungal hyphae adhere to the surface of an algal cell by means of a mucilage produced by both
symbionts. As the fungus envelops the algal cells, it forms two types of specialized cells,
appressoria and haustoria. These structures are common features of pathogenic fungi. The
appressorium fastens the mycobiont tightly to the photobiont and gives rise to hyphae that grow
into the algal cell and form haustoria. Hyphae penetrate the algal cell by enzymatic and physical
means; that is, they partially dissolve their way through the algal wall and also push their way
through. The plasma membrane of the algal cell always remains intact, no matter how deeply the
hyphae grow inside the cell. In some lichens, hyphae barely penetrate the algal cell wall; in others
they extend more deeply. Lichens with small haustoria are thought to be more highly evolved than
those with large haustoria. Presumably, as lichens evolved, fungal penetrations into the algal cells
were inhibited by stronger algal defenses. Small haustoria are difficult to see because they are
obscured by the chloroplasts that fill many algal cells. An algal cell may have multiple haustoria (
Honegger, 1992).

The frequency of haustoria in lichens shows great variation. Trebouxia photobionts, the most
frequent type of photobiont, commonly contain haustoria. In Cladonia cristatella more than 50% of
the algal cells have haustoria. Crustose lichens generally have the largest haustoria. Large foliose
lichens such as Sticta and Peltigera possess algal cells that are closely associated with fungi but
that are not penetrated by hyphae. This absence of haustoria is unusual among lichens and is
thought to result from the presence of sporopollenin in walls of the Coccomyxa photobionts of
these lichens. Sporopollenin forms a rigid layer in the algal cell wall and appears to stop fungal
penetration. Sporopollenin does not occur in the cell walls of Trebouxia, a fact that may explain
why these algae have many haustoria. The function of haustoria is not clear, but most likely they
absorb nutrients from the algal cells. Details of the interfaces between the symbionts of lichens
have been observed with low-temperature scanning electron microscopy ( Scheidegger, 1994;
Honegger et al., 1996).

Recognition mechanisms. Whether or not organisms recognize each other within a symbiotic
system is a subject of much interest to scientists. In the Rhizobium--legume association, symbiont
specificity is close, and it may be regulated by a recognition mechanism that involves
complementary binding of lectins and polysaccharides. The binding occurs on the external surfaces
of the symbionts. In lichens, the evidence for a similar recognition mechanism is inconclusive.
Although lectins have been isolated from lichens, there are conflicting reports on whether they
function in symbiont interactions ( Molina and Vicente, 1995). If there is a recognition phase in
lichens, it may occur later in the development of the thallus. Initial contacts between fungi and
algae are nondiscriminatory, and mycobiont hyphae will bind to any object, even glass beads.
Similarly, it is not known if there is any type of signal exchange between lichen symbionts ( fig. 8.3
).

Secondary compounds. Lichens contain unique secondary compounds, which are commonly
called lichen acids. These compounds were thought to be products of the symbiosis, but studies of
isolated mycobionts growing in culture with high concentrations of sucrose have revealed that the
fungus alone produces these compounds ( Hamada et al., 1996). In lichens secondary metabolism
appears to be connected to desiccation and aerial growth of the mycobiont ( Armaleo, 1995).

Secondary compounds of lichens may have important ecological roles ( Nash, 1996). Many have
antibiotic activity and may prevent the microbial decay of lichen thalli, which may live for hundreds
and even thousands of years. Lichen substances also inhibit bryophytes, other lichens, as well as
seed germination and seedling development in vascular plants. The compounds protect thalli from
grazing by invertebrate herbivores, such as slugs and insect larvae, and act as light screens to
protect the photobionts from high light intensity. Lichen substances are also chemical weathering
compounds that have a role in soil formation because of their chelating properties.

Synthetic lichens. Some lichens have been produced in axenic laboratory cultures by recombining
their separate fungal and algal symbionts. The most successful artificial syntheses have been with
Cladonia cristatella ("British soldiers"; fig. 8.2 ) and other similar species of Cladonia, and with
Usnea strigosa (beard lichen). The synthetic lichens formed in the laboratory have been identical to
their natural counterparts in morphology and, to some extent, in chemistry.

Synthetic lichens have provided useful information concerning the nature of the lichen symbiosis
and the range of specificity that exists among symbionts. Synthesis studies carried out with
Cladonia cristatella have revealed that the mycobiont will lichenize species of Trebouxia other than

its natural
Fig. 8.2 Early synthesis stages of the lichen Cladonia cristatella. (1) Algal cell enveloped by fungal
hyphae. The hyphae adhere to the surface of the algal cell by means of a gelatinous matrix around
both symbionts. (2) One algal cell enveloped by hyphae and penetrated by a fungal haustorium
(arrow). (3) A group of algal cells completely enveloped by hyphae (a presquamule stage). (4)
Fungal hyphae are forming a network (upper cortex) over the algal groups and are starting to
differentiate into individual squamules.

phycobiont; however, it will not accept "foreign" algae (i.e., those belonging to different genera).
In several experiments, the mycobiont partially lichenized Friedmannia israeliensis, a free-living
alga, i.e. formed soredia. Other algae that were foreign to the C. cristatella mycobiont were
parasitized and killed before the initial stages of lichenization took place. Compatible algae were
also penetrated by the fungus, but they were not killed, possibly because they possessed a
defensive mechanism against the fungus. The relationship between fungus and alga in a lichen is
thought to be a controlled parasitism. The fungus parasitizes the alga, but under natural conditions
the parasitism is slow and infected algal cells may live for years ( Ahmadjian, 1995b).

Many lichens have been cultured in the laboratory by means of the Yamamoto method ( Yoshimura
et al., 1993), which involves grinding thalli and then passing the suspension through different sizes
of sieves. Pieces of the residue are then transferred to culture media and incubated. Experimental
cultivation of lichens has helped clarify our understanding of the developmental stages of lichens (
Stocker-Worgotter, 1995).
Physiology of the symbiosis

Carbohydrate transfer from photobiont to mycobiont. Most of the studies that have dealt with
the physiological interactions between lichen symbionts have focused on the passage of nutrients
from the photobiont to the mycobiont. The reason for this is the availability of radioactive isotopes,
such as 14CO 2, that can be fixed photosynthetically by the photobiont and then traced in other
compounds as they move within the thallus. Studies using radioactive isotopes have revealed much
information about the physiology of the lichen symbiosis ( D. C. Smith and Douglas, 1987).

In a lichen thallus the photobiont excretes more than 90% of the carbon that it fixes
photosynthetically as a polyol or a sugar such as glucose. The polyol excreted by green symbionts
is ribitol, erythritol, or sorbitol; bluegreen photobionts excrete glucose. The fungus may control the
rate of polyol excretion by the photobiont, according to the urease theory. This theory states that
urea in a lichen thallus is hydrolyzed by the enzyme urease to produce CO 2 and NH 3. Carbon
dioxide stimulates photosynthesis of the photobiont, while NH 3 increases its respiration and
carbohydrate release. When the lichen fungus is actively growing, it can increase the flow of
nutrients from the photobiont cells by producing more urease. Lichen acids and certain proteins act
as a feedback control because they inactivate urease ( Perez-Urria et al., 1989). Thus, during
periods of fungal growth there is a greater release of nutrients but also an increase in lichen acids,
which inactivate urease and slow down photosynthesis.

What happens to the large amounts of polyols and glucose released by the photobionts? These
compounds are absorbed by the mycobiont and converted to mannitol, which is a fungal storage
product. Such a conversion creates a sink to which algal nutrients continue to flow. The fungus
uses some of the mannitol for growth and development, but the rest is used to help it withstand
the extreme conditions of its habitat. Some scientists believe that the polyol reserves that
accumulate in the fungus are used up during resaturation respiration. This occurs each time a dry
lichen is rewetted. Lichens cannot control their water content and therefore undergo

Fig. 8.3 Lichen stages that may involve signals between lichen symbionts

frequent cycles of wetting and drying. Each time a dry lichen is wetted, its respiration increases,
remains elevated for several hours, and then returns to normal. In species of Umbilicaria
resaturation respiration lasts for 40 min, but in Cetraria cucullata it may persist for 5 h.
Presumably, during resaturation respiration, the fungus respires polyols instead of proteins and
other vital compounds. A lichen has to compensate for losses resulting from resaturation
respiration as well as for losses that occur when carbon compounds leak out of the photobiont cells
during the first few minutes of wetting.

Photosynthesis and respiration. In a lichen, photosynthesis reflects the activity of the

photobiont, whereas respiration is mostly that of the mycobiont, which makes up the bulk of the
thallus. In Xanthoria parietina, the fungus constitutes about 43% of the thallus volume, while the
alga makes up only 6.7%; extracellular substances and air spaces make up the rest of the thallus.
This wide difference in the proportions of symbionts in a thallus makes measurements of metabolic
processes difficult. Moreover, the close physical relationship between the symbionts makes it
impossible to obtain pure fractions of the algal symbiont, thereby limiting the types of studies that
can be performed.

The metabolic rate of a lichen is influenced by light, temperature, day length, and water content.
Some lichens can adjust their metabolic responses to different environments and seasons. For
example, Caloplaca trachyphylla and Peltigera rufescens can acclimate their photosynthetic rates to
winter and summer temperatures, thereby achieving near maximum rates throughout the year.
Similar adaptive responses may occur with light intensity, water content, day length, and season (
Ahmadjian, 1993).

The net photosynthetic rate of many lichens depends on the amount of thallus water. If a thallus is
saturated with water, diffusion of CO 2 to the phycobiont is much slower than when a thallus has air
spaces. In some lichens, crystals of chemical substances coat the outer surface of the fungal
hyphae and prevent a thallus from becoming waterlogged. Many chlorolichens (those with green
algal photobionts) can achieve maximum rates of photosynthesis by absorbing only water vapor
from the atmosphere. Lichens kept at 9598% relative humidity will reach water contents of up to
70% of their dry weight, and their rate of CO 2 uptake will be about 90% of the rate achieved when
their thalli are wetted with liquid water. In contrast, cyanolichens are at an advantage when wetted
with liquid water ( Green et al., 1993). Populations of desert coastal lichens, such as those in Baja
California and in Chile, are never wetted by liquid water and depend on daily fog to carry out
photosynthesis and respiration. Photosynthesis also depends on the amount of light that reaches
the algal cells. Most lichen thalli have an upper cortex, which covers the algal layer. The cortex
frequently contains lichen acids and pigments that shade the photobiont.

A CO 2 -concentrating mechanism has been found in cyanolichens with Nostoc photobionts and in
lichens with Trebouxia photobionts, but not in lichens whose photobionts (e.g. Coccomyxa) lack
pyrenoids ( Palmqvist, 1993).

Nitrogen fixation. About 8% of the known lichen species have cyanobacteria as their photobiont,
usually species of Calothrix, Fischerella (Stigonema), Nostoc, or Scytonema ( Rai, 1990).
Cyanobacteria may be primary symbionts, as in Collema and Peltigera, or secondary symbionts as
in Lobaria, Stereocaulon, and Sticta. As secondary symbionts the photobionts are housed in
cephalodia, which are gall-like structures, or in separate regions of a thallus that occur on or inside
a primary thallus that has a green photobiont. Most lichen-forming cyanobacteria fix atmospheric
nitrogen inside specialized cells, the heterocysts. The percentage of heterocysts to total vegetative
cells is much greater in cephalodial cyanobacteria (i.e., 20-30%) than in those that are primary
photobionts (i.e., 4%). The reason for this wide difference is not known.

Rates of nitrogen fixation in lichens are affected by light intensity, darkness, thallus moisture,
temperature, pH, desiccation of the thallus, and season. Seasonal variation in nitrogenase activity
has been reported for Peltigera canina and Stereocaulon paschale.

Cyanobionts of lichens release more than 95% of the nitrogen they fix as ammonia, which is
converted by the mycobiont into amino acids and proteins. Hyphae near the photobiont layer of
Peltigera canina have been found to contain high concentrations of glutamic dehydrogenase, an
enzyme that assimilates ammonia. The specific activity of nitrogen-assimilating enzymes of the
cyanobionts, such as glutamine synthetase, is in-

hibited by compounds produced by the mycobiont ( Ahmadjian, 1993). Thus, the cyanobionts are
unable to use most of the nitrogen they fix, and their growth is slowed because of nitrogen
deficiency. This deficiency, in turn, causes an increase in the number of heterocysts.

Lichens with nitrogen-fixing cyanobionts are important contributors to the nitrogen economy of
different ecosystems. Lobaria oregano is a large, foliose lichen that contains a blue-green
photobiont in cephalodia. The lichen is abundant in the Douglas fir forests of the Pacific
Northwest of the United States, and when its thalli fall off the trees onto the ground, they decay
and release significant amounts of nitrogen (2 lb N per acre per year). Organic nitrogen
compounds may also leach out from lichen thalli during heavy rains.

Molecular biology of lichens

Molecular studies have provided new and important information toward understanding lichens.
The focus of these studies has been on evolution and systematics. Some of the findings from
molecular studies include the following: (1) the variable occurrence of group I introns in the
Cladonia chlorophaea complex suggests that lichen fungi have evolved rapidly, a view that is in
sharp contrast to the traditional belief that lichen evolution has been static ( DePriest, 1993,
1995); (2) at least five independent origins of the lichen habit occurred among different groups
of fungi ( Gargas et al., 1995); and (3) fungi that adopted a mutualistic lifestyle, (e.g., those that
formed lichens) evolved faster than nonmutualists ( Lutzoni and Pagel, 1997). Species
relationships in Trebouxia, the most common lichen photobiont, are being determined ( Friedl
and Rokitta, 1997).

8.3 FUNGI AND PLANTS

 Mycorrhizas
Mycorrhizas are symbiotic associations between fungi and the roots of terrestrial plants ( Ruehle
and Marx, 1979). These associations were first described in 1885 by the German botanist A.B.
Frank, who named the infected roots mykorrhizen. Seven types of mycorrhizas are recognized:
vesicular-arbuscular mycorrhiza, ectomycorrhiza, orchid mycorrhiza, ericoid mycorrhiza,
ectendomycorrhiza, arbutoid mycorrhiza, and monotropoid mycorrhiza. The first four types are
the most common. In each type of mycorrhiza the fungal hyphae grow extensively through the
soil around the infected roots and provide a greater area of absorption of nutrients than
nonmycorrhizal roots.

Mycorrhizas occur in practically all terrestrial plants and are an important reason for the well
being of plants, especially those growing in nutrient-poor soils ( Allen, 1991; Varma and Hock,
1995; S. E. Smith and Read, 1997). Mycorrhizal plants are better able to withstand drought,
disease, pests, high soil temperatures, toxic metals, and transplantation than plants without
mycorrhiza. Fungi benefit from the association by receiving organic compounds from the plant.

Vesicular-arbuscular mycorrhizas are the most common type of mycorrhiza. They occur in most
families of angiosperms, especially in herbaceous plants, in all gymnosperms except the
Pineaceae (pine family), and in ferns and liverworts ( Varma, 1995). The next most common
type, the ectomycorrhizas, occur only in trees and shrubs, in about 3% of the known
gymnosperms and angiosperms ( fig. 8.4 ).

Vesicular-Arbuscular mycorrhiza

Fungi that form vesicular-arbuscular mycorrhizas are zygomycetes that belong to the order
Glomales and to the genera Acaulospora, Gigaspora, Glomus, and Sclerocystis. About 80 species
of these fungi are found throughout the world. The species have wide host ranges, although
some sporulate better with some hosts than others. The fungi are present in the soil or on nearby
roots, and they infect the developing roots. This type of mycorrhiza is difficult to recognize
because there are no structural changes in the root. The fungus grows between the cortical cells
of the root and also penetrates the cells. The fungus does not form an outer sheath around the
root, but the hyphae do grow out from the root into the soil.

Fungal hyphae that penetrate the cells of the cortex form shrublike growths called arbuscules
that fill most of the host cell. Arbuscules are elaborate, branched haustoria that remain
surrounded by a plasma membrane of the host ( fig. 8.5A ). These structures develop in
response to fungitoxic compounds produced by the plants. An arbuscule may live for up to 15
days before it degenerates.

The host cell may then become infected by new arbuscules. The fungus also produces thick-walled
swellings, the vesicles, both within and between the plant cells ( fig. 8.5C ). Arbuscules and
vesicles are so characteristic of these fungi that the associations they form are called vesicular-
arbuscular mycorrhizas. Species of Gigaspora and Scutellospora, however, do not form vesicles,
leading some to suggest that the name "arbuscular-mycorrhiza" would be more appropriate for this
group ( Walker, 1995). Vesicles store lipids, whereas arbuscules are structures through which
nutrients pass between the fungus and plant cell. An infected host cell has an enlarged nucleus and
lacks starch granules. Host cells that digest the arbuscules return to their preinfected condition;
that is, their nuclei return to a normal size and starch granules appear.

Vesicular-arbuscular mycorrhizas differ widely with respect to the extent of their infection in the
root and the types of structures they form within the host cells. Many of these fungi form spores
and fruiting bodies of different kinds. The fungi infect only young, living root cells, generally in an
area directly behind the growing root tip. As the root matures, its cells are no longer susceptible to
outside fungal infection. Once infection occurs, new root tissue may be infected by hyphae that
grow out from the infected plant cells or by new fungi from the soil.
Fungi that form vesicular-arbuscular mycorrhizas are obligate symbionts. They cannot live
independently, and they receive their organic compounds from the host plant. The fungi lack
enzymes, such as cellulase and pectinase, that are necessary to degrade vegetable litter and
humus. The obligate nature of these fungi may explain in part why they do not grow in axenic
laboratory cultures in the absence of plant roots. Modern techniques of molecular biology are
providing new insight into the cellular dynamics of the mycorrhizal symbiosis ( Harrison, 1998;
1999).

Curious structures that resemble bacteria have been found in the hyphae of some vesicular-
arbuscular mycorrhiza. The origin and function of these structures is not known ( Perotto and
Bonfante, 1997).

The earliest known vascular plants, such as Rhynia and Asteroxylon, contained vesicular-arbuscular
mycorrhizas, according to a study of the fossil remains of these plants. The presence of these
associations in such early vascular plants and their widespread occurrence among present land
plants indicate that mycorrhizas played an important role in helping plants colonize the terrestrial
environment. The fungi may have protected the early plants from drying and helped them obtain
nutrients and water from the soil. An increase in the concentration of atmospheric CO 2 stimulates
not only plant growth but also that of its mycorrhizal fungi ( Staddon and Fitter, 1998).

Vesicular-arbuscular mycorrhizas are extremely common in tropical forests and in agricultural

crops. These associations have immense potential as biological fertilizers and can be used to
reclaim vast areas of barren soils in the world, particularly in the tropics, which have low levels of
available phos-
phate. These mycorrhizas interlink different plants and are important in the transfer of
nutrients between plants.

Some plant families, such as the Cyperaceae (sedges), Caryophyllaceae (carnation), Cruciferae
(mustard), and Juncaceae (rush), lack mycorrhizal infections. It is not clear whether this
absence of mycorrhiza results from the environmental habitats of these plants (many grow in
wet habitats), which suppress fungal growth, or from a type of root epidermis and cortex that
prevents fungal penetrations.

Ectomycorrhiza

In ectomycorrhizas the fungus forms a sheath or mantle around the root. The mantle gener-

Fig. 8.5 Vesicular-arbuscular mycorrhizal symbiosis. (A) Scanning electron micrograph showing
arbuscules of Glomus mosseae in root cell of Liriodendron tulipifera. (B) Electron micrograph
showing part of a vacuolate arbuscule of Glomus mosseae inside the root cell of yellow poplar.
(C) Vesicles of Glomus fasiculatus in stained soybean roots. (Parts A and C, courtesy Merton F.
Brown, University of Missouri; part B, courtesy Gerald Van Dyke, North Carolina State
University.)

ally is 20-40 µm thick, completely surrounds the root, and consists of either a tissuelike mat or
a loose web of hyphae. From the mantle, individual hyphae or strands of hyphae grow
outwardly into the soil and inwardly into the root cortex. Hyphae grow through the middle
lamella that separates the cortical cells and form a network between the cells called the Hartig
net. Hyphae do not penetrate the cortical cells or the inner core of the root where the vascular
system is located. Fungal infection generally inhibits the development of root hairs and
produces structural changes in the root. The changes are a result of growth hormones, auxin
and cytokinins, produced by the fungus. Infected roots often assume a stumpy, coral-like
appearance, which is typical of many ectomycorrhizas.
In contrast to vesicular-arbuscular mycorrhizas, which involve relatively few species of
zygomycetes, more than 5,000 species of basidiomycetes, mostly hymenomycetes and
gasteromycetes, and even a few ascomycetes, form ectomycorrhizas. Ectomycorrhizas are
common in temperate and tropical forests. A fungus may be specific to one type of tree, or,
more usually, it may form associations with different trees. For example, a fungus may
associate with a beech tree as well as a spruce tree. Conversely, one tree can associate with
different fungi. A Douglas fir tree can form mycorrhizas with as many as 100 different fungi.
Different fungi may form mycorrhizas simultaneously on the roots of one tree without
competing with each other. Fungi present in the roots of a seedling are often replaced by other
fungi as the tree matures.

Ectomycorrhizas vary considerably in shape. They may be club-shaped or nodular, simple or

branched. They start to develop 1 to 3 months after a seed germinates. Differences between
ectomycorrhizas include the color of the outer fungal sheath or mantle, shape of the infected
roots, and the abundance and texture of the hyphae that are in the soil around the root ( fig.
8.6 ).

Ectomycorrhizas protect the root from other parasitic organisms. The fungal sheath around the
roots is a barrier through which pathogenic organisms cannot penetrate. Mycorrhizal fungi also
secrete antibiotics that inhibit the growth of potential parasites. Fungitoxic compounds
produced by cortical cells as a result of mycorrhizal infection also inhibit growth of pathogens.

Ectomycorrhizas develop best when plants are growing under suboptimal nutritional conditions.
The fungi absorb and make available the nutrients the plants need. Because the absorbing
surface of the root is surrounded by a fungal sheath, all nutrients must pass through the
fungus before they enter the root. In this way the fungus controls the amount of nutrients that
pass into the root. The fungus provides phosphorous, nitrates, potassium, and other minerals
to the plant. The most important mineral is phosphorus, which moves more rapidly through the
fungal hyphae than through the soil. The sheath that a fungus forms around the root stores
carbon compounds and minerals that can be used by both symbionts during adverse periods. If
abundant nutrients, such as nitrogen, are present in the soil, the development of
ectomycorrhizas is inhibited, and established associations revert to their nonsymbiotic
condition. High nitrogen concentrations inhibit the fungus from producing auxin, which is
necessary for mycorrhizal formation.

The plant provides sucrose to the fungus, which converts it into carbohydrates such as
trehalose, mannitol, and glycogen. Because the plant cells cannot use these carbohydrates, the
sheath around the roots becomes a sink to which there is a steady flow of sucrose from the
plant.

Other mycorrhizas

There are several variant forms of ectomycorrhiza, one of which is the ectendomycorrhiza. In
this type of association, which is common in pine seedlings, the outer fungal sheath is greatly
reduced or absent. The Hartig net is present and the hyphae penetrate the host cells. Another
variant form is the arbutoid mycorrhiza. This type occurs in members of the heather family
(Ericaceace), such as trees and shrubs of the genus Arbutus (Madrone), in Pyrola
(wintergreen), and in the small, creeping plant Arctostaphylos uva-ursi (bearberry). This
mycorrhiza has a sheath, a wellformed Hartig net, and extensive penetrations of the host cells.
Ectendomycorrhizas and arbutoid mycorrhizas are produced by fungi that also form
ectomycorrhizas. The kind of association produced depends on environmental conditions and
the type of host and fungus ( Brundrett, 1991).

The ericoid mycorrhiza and monotropoid mycorrhiza occur in the heather family and related
families. Most species of these families produce ericoid mycorrhizas, in which neither a sheath
nor a Hartig net is formed.

-120-
The fungus penetrates the root cells and forms hyphal coils. When the hyphae in a host cell
degenerate, the cell dies, a situation that is unlike that of other mycorrhizas in which the host cells
remain alive even after their internal hyphae degenerate or are digested. The fungus that forms
ericoid mycorrhizas is usually an ascomycete, such as Pezizella, although the basidiomycete
Clavaria may also be involved.

Monotropoid mycorrhizas occur in the genus Monotropa (Ericaccae), in plants known commonly as
Indian pipe and pinesap. These plants lack chlorophyll and thus cannot manufacture their own
food. They live as heterotrophs and also receive nutrients from nearby trees by means of
mycorrhizal connections. Boletus, a common forest basidiomycete, simultaneously forms
ectomycorrhizas with the roots of forest trees such as beech, oak, pine, and spruce, and
monotropoid mycorrhizas with species of Monotropa. Organic compounds pass from the tree roots
through connecting fungal hyphae to the chlorophyll-less plants, which some scientists regard as
epiparasites on the host trees. The monotropoid mycorrhiza has a thick sheath and Hartig net, and
the host cells are penetrated by haustoria.

Orchid mycorrhizas differ from those of other plants. The fungi that associate with orchids are
basidiomycetes, and they are unique because they can break down complex carbohydrates, such
as cellulose and lignin, into simpler carbon compounds, which they transport to the orchid
seedlings. Other mycorrhizal fungi can use only simple carbohydrates and depend on the plant to
provide them with these compounds. Orchid seeds have very little stored food, and when they
germinate the embryos rely on mycorrhizal fungi to supply them with carbohydrates and other
nutrients during their early development. The fungi that infect the cortical cells of adult orchids are
often digested by the plant cells. In this way, the plant obtains nutrients and also controls the
extent of the fungal infection. Some tropical orchids that are epiphytes and some that lack
chlorophyll are epiparasites on neighboring plants by means of mycorrhizal fungi, which connect
the orchids to the trees.

The relationship between the fungus and the cells of a root is similar to that between a fungus and
the photobiont of a lichen. From a casual inspection, mycorrhizas appear to be mutualistic
associations, one in which both partners benefit ( F. A. Smith and Smith, 1996). The fungus
absorbs water for the plant, provides it with minerals, and also protects it from pathogenic
organisms. In return, the fungus receives simple organic compounds such as sucrose and also
growth factors from the plant. What appears to be an idyllic association, however, may conceal a
different situation. Elias Melin, a pioneer of mycorrhizal research, has proposed that the
relationship in ectomycorrhiza is one of controlled parasitism. Melin ( 1962) considers the
mycorrhizal fungus to be a pathogen whose intrusion into the plant root is checked by fungitoxic
compounds produced by the plant cells. Thus, a mutual standoff between fungus and plant results
in a long-lasting symbiosis. If conditions change (e.g., if the plant is given an optimal nutritional
supply from external sources), then the plant cells kill the fungus and end the mycorrhizal
association. Controlled parasitism also describes the symbiosis of lichens, where the parasitism of
the photobiont cell is slow enough to allow for the development of a symbiotic relationship (
Ahmadjian, 1993).

Identifying the fungi that form mycorrhizas is difficult. Ectomycorrhizal fungi that are isolated and
grown in axenic cultures develop slowly, produce compact colonies similar to those of lichen fungi,
and do not have any of the characteristics of natural mycorrhizas. The cultured fungi have not
revealed any unique nutritional needs or differences from other fungi to explain their symbiotic
habit. The best way to identify ectomycorrhizal fungi is to see if fruiting bodies of basidiomycetes
growing near the trunks of trees are connected by means of hyphal strands with the mycorrhizas of
the trees.

Plant Pathology: The Study of Plant

Diseases
Fungi cause millions of dollars worth of damage each year to agricultural crops and ornamental
plants. Principal fungal diseases of plants include wilts, rots, rusts, smuts, and cankers. Since the
days of Anton de Bary, plant pathologists have studied life cycles, taxonomy, biochemistry, and
now the molecular biology of host-fungal relationships. In collaboration with plant geneticists, they
have successfully developed disease-resistant varieties of plants. Other control measures include
disease-free seeds and seedlings, fungicides, plant protection quar- antine, and manipulation of
agricultural practices. How to control fungal pathogens of plants has been a persistent problem for
humankind, one that will never be completely solved because of the changing nature of fungi. As
resistant strains of plants evolve or are developed, new strains of the pathogen arise naturally to
infect them. Understanding the life cycle of a fungus and the physiological and genetic basis of
pathogenesis is the first step in any effort to control plant diseases ( Kombrink and Somssich,
1995; Valent, 1999). Pathogen-triggered host cell death in a variety of plant--microbe interactions
has become a focus of current research in plant pathology ( Dangl et al., 1996; Gilchrist, 1998).

Fungal phytotoxins

Fungi produce toxins in infected plants and in culture medium. Toxins are cellular poisons that are
effective at low concentrations. Host-selective toxins and non--host-selective toxins are two classes
of toxic substances that pathogens produce in plants. Host-selective toxins affect only the host of
the toxin-producing pathogen and are required to determine pathogenicity. Mutants for host-
selective toxins lose their ability to infect and cause disease. Nonselective toxins cause disease
symptoms in host plants and in other plant species that are not normally attacked in nature. They
are not essential for a pathogen to cause disease.

Host-selective toxins. Victorin, or HV toxin, is produced by the fungus Cochliobolus

(Helminthosporium) victorae in oat plants. The fungus infects the basal parts of oat seedlings, and
its toxin is carried to the leaves, causing leaf blight and plant death. HV toxin is a cyclic
pentapeptide that binds to several host cell membrane proteins. Its production is controlled by a
single gene.

T-toxin causes southern corn leaf blight and is produced by Cochlibolus heterostrophus. The
disease first appeared in 1968 and spread rapidly throughout the corn belt in the United States.
The pathogen attacks only corn varieties with Texas male sterile (Tms) cytoplasm. Resistance and
susceptibility to C. heterostrophus and its toxin are maternally inherited. T toxin destroys
mitochondria of susceptible cells and inhibits host ATP synthesis. Corn varieties with Tms
cytoplasm contain a mutation in their mitochondrial DNA for URF13 protein, which is absent in corn
with normal cytoplasm. In the presence of T toxin, protein URF13 causes pores to be formed in the
mitochondrial membrane of corn with cytoplasmic male sterility.

HC toxin is produced by Cochilobolus (Helminthosporium) carbonum, a pathogen of corn, and is

responsible for a leaf spot disease. HC toxin affects some corn varieties, but the biochemical and
genetic mechanisms of resistance are not understood.

AM toxin is produced by the fungal pathogen Alternaria alternarial, which is responsible for
alternaria leaf blotch of apples. The toxin is highly selective for susceptible apple varieties and
causes structural changes in the host cell membrane and chlorophylls and a loss of electrolytes.

Other host-specific toxins are known, and new discoveries are giving plant pathologists more
insight into the molecular mechanisms that govern pathogenicity.

Non--host-selective toxins. Tenotoxin is produced by Alternaria alternaria and causes chlorosis

in plant seedlings. Tenotoxin binds to the chloroplast protein and inactivates ATP synthesis.
Examples of other non--host-specific toxins include fumaric acid produced by Rhizopus sp. and
oxalic acid produced by Cryphonectria parasitica.

Hydrophobins and fungal infection

Hydrophobins are structural proteins that can aggregate into rod-shaped structures. Hydrophobins
facilitate mycelial growth and spore dispersal. Mutants lacking hydrophobins are easily wettable
and do not disperse easily. Large amounts of hydrophobins are produced by Cryphonectria
parasitica (chestnut blight). Virulent strains of Ophiostoma ulmi (dutch elm disease) accumulate
the hydrophobin cerato-ulmin as the disease progresses ( Wessels, 1997).

Magnaporthe grisea, the fungus that causes rice blast disease, develops appressoria, which give
rise to hyphae that penetrate the host cells. Appressorial formation is accompanied by increased
levels of cAMP and MPG1 hydrophobin. Hydrophobins may also play an important role in root
colonization by ectomycorrhizal fungi. For example, Pislothus tinctorius, a fungus associated with
Eucalyptus, undergoes a complex developmental process that includes hydrophobin gene
expression ( Ebbole, 1997). Floral mimicry by plant pathogens

Pseudoflowers are fungus-infected leaves that occur among regular flowers and mimic them in
form, color, smell, and sugar content. The bright yellow surface of the infected leaves with sugary
fluid forms patterns under visible and ultraviolet light that closely resemble those of natural
flowers. Insects are attracted to pseudoflowers and contribute to the reproduction and
dissemination of their fungal pathogens. Floral mimicry deceives insects as well as naturalists, who
unknowingly collect pseudoflowers ( Roy, 1993). Rust pseudoflowers attract bees, butterflies, and
especially flies, which bring about sexual reproduction in the fungal pathogen by transporting the
rust spermatia to compatible hyphae ( Roy, 1993).

On blueberry plants, Monilinia sp., known as the mummy berry fungus, causes blight on leaves,
flowers, and twigs and mummifies berries into white, hard structures. Suzanne and Lekh Batra
described the deceit used by the fungus to attract bees to infected leaves. Mummified berries
overwinter in moist debris and in the following spring form cups on their surface which release
ascospores that infect young leaves. The infected leaves become discolored in a uniform fashion,
with leaf edges remaining green while the middle of the leaf turns bluish brown with a central gray
patch of fungal conidia. The diseased leaf looks and smells like a flower to an insect, which is
rewarded with sugar-coated conidia. The mummy berry fungus succesfully modifies its host plant
to trick its pollinators to spread the pathogen ( Batra and Batra 1985).

Fungal castration of plants

Rusts, smuts, downy mildews, and endophytes are fungi that castrate (suppress sexual
reproduction) plants. Fungi that sterilize the plants they infect prevent the host's coevolutionary
response (i.e., to produce resistant progeny). In addition to affecting sexual reproduction, these
fungi stimulate clonal reproduction and dispersal of their hosts, thereby producing more individuals
which their spores can infect ( Clay, 1991).

Rust diseases in plants

Rusts are basidiomycetes that cause many plant diseases ( Yamamoto, 1995). The fungi get their
name from the brownish red or black spores that cover the infected areas of the plant. There are
about 5,000 species of rust fungi, which infect many flowering plants, conifers, and ferns. Human
concern over rusts is due to their ability to infect important crops such as wheat, corn, barley, oats,
and other cereals. Up to 10% or more of a crop may be lost because of these destructive pests.

Rusts are unusual basidiomycetes because they lack fruiting bodies and have a complex life cycle
during which they produce five different types of spores. Each type of spore has a particular
function in the life cycle and is produced at a different time of the year. Some rusts complete their
life cycle on only one plant, but many need two host plants, usually unrelated to each other. For
rusts needing two hosts, eradication of the least important plant is the best way to control the
spread of the disease. For example, Puccinia graminis (black stem rust of wheat) infects cultivated
wheat and has the common barberry as its alternate host. The sexual process of the fungus takes
place on the leaves of the barberry, and the spores produced can infect wheat. If barberry plants
are eradicated in wheat-growing areas, the life cycle of the fungus is broken, and the wheat will
not become infected. Unfortunately, fungal spores are light and can travel on air currents for
hundreds of miles. Thus, even a single infected barberry plant miles away from cultivated wheat
crops may serve as a source of infection. On barberry plants, the rust pathogen can evolve virulent
races that can break down resistance in existing wheat varieties.

The life cycle of P. graminis begins in the spring, when basidiospores of the fungus infect the leaves
of a barberry plant ( fig. 8.7 ). The basidiospore produces a hyphal filament that penetrates the
epidermis and forms branches in the leaf tissues. Hyphae grow inside the cells as well as between
the cells. Within 1-2 weeks, the fungus produces pycnia on the upper leaf surface. Pycnia produce
pycniospores that serve as gametes. The spores are contained in a thick, sugary fluid that covers
the top of the pycnia. Small insects, attracted to the nectarlike fluid, pick up the sticky spores and
transfer them to receptive hyphae that grow out from the pycnia. Rust fungi have plus and minus
 mating strains. Sexual compatibility occurs when pycniospores of one strain fuse with receptive
hyphae of another strain. After fusion, the nucleus of a pycniospore migrates through the receptive

hypha to the lower surface of the leaf, where the fungus has started to form ae-
Fig. 8.7 Life cycle of wheat stem rust fungus, Puccinia graminis tritici.

cia, structures that produce another type of spore. The pycniospore nucleus pairs with a nucleus in
the aecial primordium, and the resulting hyphae contain both types of nuclei. These dikaryotic
(two-nuclei) hyphae give rise to aeciospores, each of which has two nuclei. Numerous aeciospores
are released from the lower surface of the barberry leaf and are carried away by the wind. If an
aeciospore lands on a wheat plant, it germinates and gives rise to a dikaryotic mycelium that
infects the wheat tissue. Several weeks later, the fungus produces another type of spore, the
urediniospore. These spores are formed in such large numbers that they rupture the plant
epidermis and produce rusty streaks on the stems and leaves of the infected wheat. Urediniospores
infect other wheat plants and, if unchecked, can infect an entire field of wheat. Continued
urediniospore production in the wheat-growing regions can provide a source of inoculum for new
wheat seedlings. In the autumn, the fungus produces a fifth type of spore, the teliospore. These
black spores have thick walls and allow the fungus to survive the winter. In the spring, teliospores
germinate and form basidiospores, which then infect the barberry plant and complete the life cycle
of this rust.

Rust infections occur in many plants other than wheat. The infections usually result in abnormal
growth of the host plants and the formation of galls. "Cedar apples" are galls on juniper plants and
are caused by the rust fungus Gymnosporangium juniperi-virginianae. The galls are made up of
plant tissue that contains fungal hyphae and teliospores. Apple trees serve as the alternate host,
where the fungus reproduces sexually.

The fungus that causes white pine blister rust, Cronartium ribicola, produces urediniospores and
teliospores on the leaves of currants and gooseberries. The teliospores form basidiospores, which
infect white pine trees. In the bark of the pine tree the fungus produces pycnia and aeciospores in
such large numbers that they rupture the bark surface and form blisters. The quality of pine wood
is affected adversely by the rust fungus.

Ustilago maydis

Smuts are basidiomycetes that lack fruiting bodies. Unlike some rusts, they do not need an
alternate host to complete their life cycle. Smuts produce large numbers of brown chlamydospores,
which are often packed together into large spore balls. Smuts also produce basidiospores that
behave like yeast cells; that is, they divide by budding and often fuse to form a dikaryotic stage.
Each spore eventually gives rise to a dikaryotic mycelium, which infects the host plant. There are
more than 1,000 species of smuts. They infect many plants including agricultural crops such as
corn, wheat, and oats ( Hargreaves et al., 1995).

Corn smut disease is characterized by yellowing of leaves, stunting, and the formation of tumors in
the leaves, stems, tassels, and ears. Infected ears of corn are called huitacoche in Spanish and are
considered to be a culinary delicacy. Some corn growers cultivate corn smut for gourmet food
markets ( Schumann, 1991). The fungus Ustilago maydis is dimorphic; its life cycle includes a
yeastlike form that multiples by budding and is haploid and saprobic, and a filamentous form which
is dikaryotic and requires a corn plant for its growth. The filamentous form results from fusion of
two compatible haploid cells. In the U. maydis life cycle, dimorphism and pathogencity are
controlled by two incompatibile loci, a and b. The gene a locus controls a pheromone response
pathway; gene b locus codes for a regulatory protein. Three other genes called fuz1, fuz2, and rtf1
are involved in the development of the filamentous form ( Banuett, 1992, 1995). Ustilago maydis
can synthesize auxin and cytokinin and cause tumor induction in the infected corn plant. In this
respect it is similar to Pseudomonas olive knot disease and Agrobacterium crown gall disease. One
gene (fuz7) from the b locus is required for tumor formation. Some interesting questions on the
molecular nature of the U. maydis--corn symbiosis include how karyogamy (nuclear fusion) is
regulated in the life cycle, how pheromones encoded by one mating type stimulate filamentous
growth, how the fungus transforms the host growth, and how the host plant regulates fungal
meiosis.
Mildews

Downy mildews are obligate pathogenic symbionts of flowering plants. They attack young leaves,
twigs, and fruits, and their development depends on the film of moisture on the plant surfaces. The
epidemic downy mildew of grapes (Plasmopara viticola) during the late nineteenth century in
France led to the discovery of the first successful fungicide, Bordeaux mixture, in 1885. In 1979,
Peronospora tabacina caused the downy mildew of tobacco throughout the United States and
Canada, with losses to growers in millions of dollars.

Powdery mildews are ascomycetes that are obligate parasites of many plants, including lilacs,
apples, grapes, and roses. These fungi form a thin mycelial layer on the surface of leaves and
penetrate the epidermal cells by means of haustoria ( fig. 8.8 ). The powdery mildews produce
large numbers of asexual spores called conidia and these spores, together with the mycelium, form
a powderlike coating over the infected leaves ( Kunoh, 1995).

The fungal haustorium

Haustoria are specialized hyphal cells that are produced inside living cells of other organisms by
parasitic fungi. In the case of plants, fungi penetrate the cell walls by enzymatically digesting the
outer wall and then mechanically pushing through the inner wall. After the fungus has entered a
cell, it forms a haustorium. Haustoria have many different shapes. Those of lichen fungi are
clubshaped, and those produced by mycorrhizal fungi are branched. Haustoria of powdery mildews
have fingerlike lobes and are associated with specialized structures ( fig. 8.8 ). The fungal
haustorium is a branch of an extracellular hypha and terminates in the host cell. In some fungi the
haustorium develops from an appressorium, another specialized cell of the fungus that attaches
closely to the outer surface of the host cell by means of a mucilaginous substance. Haustoria of
rust fungi have a narrow neck region at the site of penetration and a swollen region inside the host
cell; most contain a nucleus, mitochondria, and other organelles and are surrounded by a dense,
granular matrix. The host cell forms a new plasma membrane, different in structure from that of
the uninfected cell, around the penetrating fungus.

Although there is little direct experimental evidence to reveal the function of haustoria, it is
presumed that they absorb nutrients from the penetrated cells. Whether haustoria kill the infected
cells depends on the type of relationship the fungus has with the host. In rust and powdery mildew

diseases, infected cells

Fig. 8.8 Schematic diagram of haustorium of powdery mildew fungus. Adapted from Spencer (
1978).

may be more active and live longer than uninfected cells. In lichens, haustoria kill the cells of the
photobiont but only after a period of time, because the parasitism is gradual and under control.

An infected plant cell may have one or many haustoria inside its protoplast, depending on its
resistance to the invading fungus. Some plant cells produce wall material around haustoria and
limit their penetration into the cell.

Fungal Endophytes of Grasses

Many species of grasses, both wild and cultivated, are infected with ascomycetes of the genera
Atkinsonella, Balansia, Epichloe (Acremonium), and Myriogenospora ( Ball et al., 1993; Latch,
1995; Redlin and Carris, 1996; Schardl, 1996). Acremonium coenophialum, the fungal endophyte
in tall fescue, has coevolved with its host to their mutual benefit. The fungus produces only
mycelia, spreads by infected seeds, and does not sporulate ( fig. 8.9 ). The infected grass is
resistant to insects and nematodes and is also toxic to grazing animals ( Ball et al., 1993;
Saikkonen et al., 1998).

Symptoms of toxicity in livestock that eat the grass are caused by ergot alkaloids produced by the
fungus in the leaf sheaths, where the fungus concentrates, and translocated throughout the plant.
Nitrogen-fixing microorganisms (diazotrophs) have been reported as endophytes of sugarcane and
rice and are believed to significantly contribute to the nitrogen requirement of the host plant.

Hypovirulence
Some strains of pathogenic fungi are less virulent to their hosts than are normal pathogenic
strains. Hypovirulent strains of Czyphonectria parasitica have reduced the blight of the European
chestnut (Castanea sativa) in many parts of Europe, and similar strains offer the first glimmer of
hope of controlling the devastating fungal blight of the American chestnut (Castanea dentata).

Hypovirulent strains can spread by means of hyphal fusions among a normal pathogen population
and transmit to the normal strain an agent that may be a dsRNA virus. This virus somehow
diminishes the virulence of the pathogen. In effect, the hypovirulent strains cause a disease of the
normal strains. Because the virus is present in the asexual spores (conidia) of the hypovirulent
strains, the disease can spread rapidly and over great distances. The effectiveness of hypovirulent
strains depends on the ability of their hyphae to fuse with those of normal strains. If there are
genetic incompatibilities among the strains that prevent hyphal fusions, then the virus cannot be
transmitted and the pathogen is unaffected. Thus, a disease may be controlled in some
geographical areas, but not in others.

8.4 SUMMARY
Mycosymbionts are fungi that parasitize other fungi, either killing their hosts or obtaining food from
living hosts. These fungi are of interest to researchers who want to develop biological controls for
fungal pathogens. Mycophycobioses, associations between marine fungi and marine algae in which
the alga is the larger partner, are being studied by scientists interested in the early stages of
symbiotic evolution.

Lichens are associations of fungi that are mostly ascomyetes and photobionts (photosynthetic
partners), which are generally unicellular green algae and cyanobacteria. The lichen thallus is the
result of a morphological transformation of the fungal symbiont and to a lesser extent the
photobiont. The main types of thalli are crustose, foliose, and fruticose. Lichens produce secondary
compounds that have a variety of roles in the symbiosis. Lichens are dominant species in some
parts of the world, except in cities, where air pollution inhibits their growth. Studies on synthetic
lichens have provided information about symbiont specificity, thallus development, and the
physiological nature of the symbiosis. Many studies have examined carbohydrate transfer between
lichen symbionts, photosynthesis and respiration of lichen thalli and nitrogen fixation and
metabolism. The photobiont excretes most of its photosynthetic products and, in the case of
cyanobacteria, much of the nitrogen they fix. The fungus exerts some control over the photobiont
and can regulate the amount of excreted compounds. Molecular studies are revealing important
information about lichen evolution and systematics.

Mycorrhizas are mutualistic associations of fungi and the roots of terrestrial plants. There are seven
different types of mycorrhizas. The most common type is the vesicular--arbuscular mycorrhiza,
formed by zygomycetes, which produces arbuscules and vesicles inside root cells. Basidiomycetes
form ectomycorrhizas, the second most common type, which are characterized by a sheath or
mantle of fungal tissue around the roots. Other types of mycorrhizas are ectendomycorrhiza,
arbutoid mycorrhiza, ericoid mycorrhiza, monotropoid mycorrhiza, and orchid mycorrhiza. Mass-
scale inoculations of pine seedlings with the ectomycorrhizal fungus Pisolithus tinctorius have been
successfully carried out at several locations around the world. The prospect of developing a means
for the host plant to obtain minerals without having to depend on chemical fertilizers has
encouraged reforestation efforts on marginally suitable lands.

The role of fungi in causing diseases of agricultural crops has been a major factor in the growth of
the science of plant pathology. Rusts and smuts are obligate parasitic symbionts of plants. These
fungi have complex life cycles and inflict heavy crop losses in many parts of the world. Efforts to
control mildew and blight infestations of plants have played a historic role in the development of
fungicides. Fungal endophytes are common in wild and cultivated grasses. Fungus-infected leaves
appear as flowers to some pollinating insects (floral mimicry), which helps to disperse the fungal
spores. Some fungi can suppress sexual reproduction of their plant hosts, thereby limiting their
coevolutionary response and the production of resistant progeny. Fungi produce different types of
toxins in infected plants.

Hypovirulence, a reduced pathogenicity in strains of some fungi, may be caused by a virus.

Hypovirulent strains of pathogenic fungi are being used to control blights of chestnut trees.

Axenic culturing of obligate parasitic fungi has had limited success. Some species of rusts and
smuts have been cultured, but powdery mildews have not yet been grown in axenic culture, nor
have fungi that form vesicular-arbuscular mycorrhizas and many lichen fungi. Some lichen fungi
and ectomycorrhizal fungi can be cultured, but their growth rates are extremely slow. The slow
growth as well as the unpredictable nature of symbiotic fungi in culture has limited the types of
studies that can be conducted with them. Why symbiotic fungi are difficult to grow axenically is not
clear. Some species may need nutrients that are not contained in the culture medium. More likely,
the slow growth of these fungi is a result of their natural symbiotic adaptations. Long-lasting
associations such as lichens and mycorrhizas require a balanced relationship between the
symbionts because unregulated growth of the fungus would quickly kill the host. In both lichens
and ectomycorrhizas, an abundance of external nutrients inhibits the symbiotic transformation of
the fungus and causes a breakdown of established associations.

9
PARASITIC AND MUTUALISTIC
PROTOZOANS
Protozoans live in oceans, lakes, streams, soils, and around the roots of plants. They also live in
the alimentary canal, bloodstream, liver, lungs, brain, and other organs of animals. Some
protozoans cause serious human illnesses, such as malaria, African sleeping sickness, and
Chagas's disease ( Roizman, 1996; Wills, 1996); others are pathogens of domesticated animals,
such as horses, cattle, and poultry. Flagellate protozoa cause diseases in plants such as coffee,
coconut palm, and oil palm ( Dollet, 1984). The red tides that kill fish along coastal areas of the
eastern United States are caused by large populations of toxin-producing dinoffagellates (
Anderson, 1994). A newly discovered killer of fish is the dinoflagellate Pfiesteria piscicida. Its
toxins have killed millions of fish along the North Carolina coast ( Russell, 1998; Burkholder,
1999).There are an estimated 200,000 protist species, which is more than twice the number of
chordates. The populations of some protists may be extremely high. For example, there may be
thousands of these organisms in only 1 cm3 of soil and billions in the rumen of herbivores.
Millions of flagellate symbionts inhabit a termite's intestine and may make up as much as one-
third of the insect's weight. The White Cliffs of Dover in England consist entirely of the fossil
remains of foraminiferan amoebae.Photosynthetic protists are of immense importance in the
ecology of marine organisms because they are the first link in the food chain. In this chapter, we
examine symbioses that involve the nonphotosynthetic protists, the protozoans; in the chapter
10 we consider the role of photosynthetic protists in animal symbioses.Studies in protistan
biology have been concerned with how symbionts infect a host, overcome its defense
mechanisms, and reproduce successfully. How are the host animal's antibody mechanisms and
phagocytic activities of cells such as macrophages neutralized during infection ( Goodenough,
1991)? In this chapter, we focus on the advances in our understanding of protozoan
pathogenesis and the conditions under which a symbiont--host relationship becomes
nonpathogenic.
9.1 KINGDOM PROTOZOA
Characteristics
Protozoa are aerobic or anaerobic organisms that lack organized tissues and embryos. They are
unicellular or multicellular, heterotrophic; and reproduce both sexually and asexually. Protists
may be immobile or may move by means of pseudopodia, eukaryotic flagella, or cilia.
Classification
The following is a taxonomic grouping of organisms that are discussed in chapters 9 and 10.
Kingdom Protozoa
Phylum Sarcomastigophora
Subphylum Mastigophora
 Class Zoomastigophora
Order Kinetoplastida; Phytomonas, Leishmania, Typanosoma
Order Diplomonadida; Giardia

Order Trichomonadida; Histomonas, Trichomonas

Order Hypennastigida; Trichonympha
Class
Phytomastigophora
Order Dinoflagellida
(dinoflagellates);
Gymnodinium
Class Opalinatea;
Opalina
Subphylum Sarcodina
Class Rhizopoda;
Acanthamoeba,
Naegleria, Entamoeba
Phylum Apicomplexa
Class Sporozoasida
(gregarines and
coccidians);
Toxoplasma,
Plasmodium, Theileria
Phylum Ciliophora
Class
Kinetofragminophorea;
Dasytricha,
Diplodinium,
Entodinium Epidinium,
Isotricha
Class
Oligohymenophorea;
Paramecium
Phylum Foraminifera
(forams)

9.2 PROTOZOANS AS SYMBIONTS

Amoebae
Amoebae occur in all environments that support life. They are frequently ingested by potential
hosts from drinking water or food. Many species of amoebae have adapted to a symbiotic existence
in the alimentary canal of animals, where they are often referred to as "harmless commensals."
Some amoebae, however, are human pathogens. Entamoeba histolytica causes dysentery, and
species of Acanthamoeba and Naegleria can invade the cerebrospinal fluid of mammals. Amoebae
are characterized by pseudopodia and trophozoites that feed by phagocytosis and multiply by
binary fission. Some amoebae produce resistant cysts at certain stages of their life cycles.

Entamoeba
Entamoeba histolytica infects about 10% of the world's population, with about 100 million people
suffering from invasive colitis or liver abscess. There may be 50,000-100,000 deaths per year in
endemic areas, making E. histolytica the third major cause of death, behind malaria and
schistosomiasis ( Ravdin, 1990). Pathogenesis of invasive amoebiasis includes (1) attachment to
the intestinal mucus barrier, (2) mucus breakdown, (3) cytolysis of epithelial cells due to the
parasite's enzymes and toxins, (4) penetration of trophozoites through the intestinal mucosa, and
(5) the host inflammatory response ( Ravdin, 1990; Montfort and Perez-Tamayo, 1994). An
amoebic adherence lectin, N-acetyl-D-galactosamine (Gal/GalNAc) plays a significant role in the E.
histolytica attachment mechanism. Several proteolytic enzymes, which are released on contact
with the host tissue, have been isolated from E. histolytica. An enterotoxin has also been isolated
that may interfere with the function of calcium channel proteins ( Gitler and Mirelman, 1986).

Entamoeba has also been reported from monkeys, apes, and other mammals. Infection usually
begins when a host ingests fecal-contaminated food or drink, which contains cysts of the amoeba.
The cysts hatch and produce amoebae, which feed on bacteria and other food particles in the large
intestine. After several cycles of growth and multiplication, some amoebae encyst; that is, they
become spherical, secrete a thick wall, and store glycogen. Cysts are excreted in large numbers in
the feces. An infected individual may excrete an estimated 45 million cysts in one day. If the cysts
are ingested by a new host, they hatch, and each cyst liberates four trophozoites. Amoebic
infection may occur without symptoms, or the amoebae may attack the epithelial lining of the large
intestine and cause ulcers and dysentery. In more serious cases, amoebae may penetrate the
intestinal wall and infect organs such as the liver and brain.

Understanding the pathogenicity of Entamoeba histolytica has been controversial. What was
previously identified as E. histolytica is now believed to be a composite of at least two species, E.
dispar and E. histolytica. Entamoeba dispar is the smaller of the two species and is nonpathogenic.
At one time it was believed that all cases of E. histolytica infection required medication, and,
consequently, individuals were treated with dangerous drugs. Eye disorders, allergies, migraine,
rheumatoid arthritis, and liver disease were often falsely attributed to the presence of amoebae in
infected persons.

Pathogenic strains of E. histolytica lose their virulence when they are cultured in the laboratory.
Electron microscopic observations have revealed that amoebae from an infected person have a
"fuzzy coat," the nature of which is not understood, that is absent in laboratory strains. Surface-
active lysosomes that might be responsible for the breakdown of host cell membrane have been
discovered in the amoebae. Cytoskeletal rearrangements in response to signals from external
stimuli appear to characterize virulence of E. histolytica ( Guillen, 1996). Golgi bodies and
mitochondria are absent. Some scientists have observed that E. histolytica alone cannot produce
disease and that the presence of bacterial or viral symbionts is essential for its virulence (
Mirelman, 1987). Viruslike particles have been detected in the cytoplasm and nucleus of E.
histolytica.

Entamoeba invadens is a common pathogen of turtles and snakes. Morphologically, the amoebae
resemble E. histolytica, and scientists have increasingly used it as a model to investigate the
pathogenesis of invasive amoebiasis ( Eichinger, 1997).

Acanthamoeba and Naegieria

Species of Acanthamoeba and the amoeboflagellate Naegleria are common in stagnant lakes and
ponds and may cause primary amoebic meningoencephalitis (PAM) in humans and other mammals
( Ferrante, 1986). Young children swimming in such areas are most susceptible to Naegleria
infections. Amoebae of N. fowleri enter the nasal passages, where they penetrate the olfactory
epithelium and move along the olfactory nerve. Electron microscopy of trophozoites of N. fowleri
has revealed unusual "feed cups" or amoebostomes, which are used in the phagocytosis of host
cells ( John et al., 1985; Marciano-Cabral, 1988).

There are several species of Acanthamoeba that cause meningoencephalitis, inflammation of

internal organs, corneal ulcers, gastritis, and diarrhea. Bacterial contamination of contact lens
cleaning solution enhances multiplication of Acanthamoeba that are associated with eye infection (
Bottone et al., 1992). In the 1920s, when acanthamoebae were isolated from diseased tissues,
they were dismissed as contaminants by scientists. Later it was shown that intranasal inoculation
of mice with Acanthamoeba killed the mice, and the amoebae could be observed in the brain
tissues. Worldwide, more than 100 cases of PAM have been identified, most of which were fatal.
Pathogenicity of these amoebae has been attributed to their ability to release a phospholipolytic
enzyme that demyelinates nerves.

Ciliates
Ciliates are well adapted to live in aquatic habitats. Most ciliates are free-living heterotrophs, but
some live in the alimentary canals of vertebrates, insects, and annelids. Ruminants maintain a
large number of ciliate species. Ciliate symbionts also inhabit the liver, blood vessels, and gonads
of animals. Two different types of nuclei, micro- and macronuclei, are always present. Asexual
reproduction is by binary fission, and conjugation is involved in sexual reproduction. The ciliate
trophozoites feed on host fluids by pinocytosis or through cytostomes. Balantidium spp. are
commensalistic symbionts of humans, pigs, and monkeys.

Rumen ciliates: the mutualistic protozoans

Mutualistic associations have evolved between ciliates and herbivorous mammals. The rumina of
cattle and sheep contain, in each 1 ml of gut contents, an estimated half million ciliates consisting
of 30-50 species, whose role is controversial. Ciliates form a large proportion of the rumen biomass
and have a slow turnover. They survive by attaching firmly to the gut and thus washout is reduced.
Ciliates ingest rumen bacteria, starch, and chloroplasts and have been blamed for the low
efficiency of rumen fermentation ( Van Soest, 1994).

Rumen ciliates are obligate anaerobes. New hosts acquire them through a mother licking her calf or
from eating grass that is dropped after being regurgitated from a rumen. The environmental
conditions of the rumen are complex and have not been duplicated in the laboratory. The rumen
has a temperature of 39°C, is anaerobic, contains particulate matter that is resistant to the host's
digestive enzymes, and is deficient in glucose and amino acids. Although the alkaline pH of the
rumen favors growth of the ciliates, the posterior portion of the stomach, the omasum, has an acid
environment, which is lethal to the ciliate symbionts. Ciliates may supply their host with about one-
fifth of its protein and with acetic, propionic, and butyric acids, and also with a better balance of
amino acids than do the bacterial symbionts of a rumen.

Two kinds of ciliates live in the rumen: entodiniomorphs and holotrichs ( fig. 9.1 ). Ento-

-133-

D Epidinium caudatumIsotricha intestinalisDasytricha ruminatium Fig.9.1 Types of mutualistic

rumen ciliates. (A) and (B) External and internal morphology of a typical entodiniomorph. (C) and
(D) Two examples of holotrich symbionts. Adapted from Furness and Butler. ( 1983).

diniomorphs have a semirigid pellicle that covers the cell and gives each species a distinctive
morphology. They also have unique internal plates, which form a type of cytoskeleton. Around the
mouth of each symbiont is a band of cilia, which function in a coordinated fashion. The number and
location of ciliary bands are used to identify species of entodiniomorphs. In most species, the ciliary
zone can be retracted and the pellicle drawn over it. Diplodinium, Entodinium, and Epidinium are
important genera of symbiotic ciliates and have been the subject of many physiological studies.
Entodinium bursa is the largest of the entodiniomorphs, and it consumes large quantities of plant
material. Rumen ciliates produce hydrogen and volatile fatty acids. The hydrogen they produce
serves as a substrate for methanogens, which live as ectosymbionts of ciliates. Entodiniomorphs
digest cellulose and starch, as well as rumen bacteria. Many scientists have attempted,
unsuccessfully, to grow rumen ciliates in the laboratory.

Rumen holotrichs resemble free-living ciliates. Their number in sheep and goats has been
estimated to be 160,000-200,000/ml of rumen content. Some common holotrichs are Dasytricha
ruminatium, Isotricha intestinalis, and L prostoma. Holotrich ciliates, in contrast to
entodiniomorphs, can incorporate large quantities of sugar and convert it to starch for storage.
Holotrich starch is indistinguishable from plant starch.

Opalinata
Opalinids possess many cilialike organelles in oblique rows that cover the body surface.

Their phylogeny is a matter of dispute ( Patterson , 1985).

Opalina ranarum: an amphibian symbiont

About 150 species of the genera Cepedea, Opalina, Protoopalina, and Zeleriella inhabit the large
intestines of frogs and toads. The symbiosis of Opalina ranarum and frogs has been studied
extensively in Europe ( Smyth and Smyth, 1980). Opalina obtriganoidea is found in frogs and toads
of North America. The symbionts have flat or cylindrical cells, up to 1mm long, and their
reproductive physiology is controlled by gonadal hormones of the host. Opalinids do not harm the
host, which may harbor thousands of these symbionts in its large intestine.

The symbionts have 2-200 nuclei. Electron microscopy of opalinids shows a complex of cortical
folds, ribbons of microtubules, and pinocytotic vesicles forming at the bases of the cortical folds (
Patterson and Delvinquier, 1990). A cytostome is lacking. Generally, adult individuals of O.
ranarum multiply by binary fission in the rectum of frogs and toads. The symbiont life cycle is
synchronized with that of the host. In the spring, at the onset of the frog's breeding period, the
symbionts divide rapidly and produce small, precystic forms. These become transformed into cysts
and are eliminated with feces from the host. Cysts ingested by tadpoles hatch in the duodenal
region, and the symbionts then migrate to the large intestines. Microgametes and macrogametes
are formed, and fusion of the sex cells results in zygotes, which give rise to a new generation of
opalinids (fig. 9.2). Researchers have induced encystation of O. ranarum by injecting
hypophysectomized and gonadectomized frogs with pregnancy urine, nonadotrophin, testosterone,
and adrenaline ( Smyth, 1994).

Flagellates
Flagellated protozoa differ widely in their morphology and physiology. Some have evolved a high
degree of intracellular differentiation and possess unique structures whose functions are not fully
understood. All flagellates have flagella and can swim, which is a distinct advantage over an
amoeboid lifestyle. Some flagellates colonize the digestive, circulatory, and lymphatic systems of
animals. Flagellate symbionts can be divided into three broad groups: intestinal flagellates, which
inhabit the alimentary canal and genital tract of the host; hemoflagellates, which inhabit the blood,
lymph, and other tissues of vertebrate hosts and the intestines of insects; and phytoflagellates,
which cause plant diseases.

Intestinal flagellates

Intestinal flagellates are obligate anaerobes and do not reproduce sexually. In some species, the
trophozoite stage alternates with a resting stage, the cyst. Intestinal flagellates ingest food by
phagocytosis and pinocytosis, and some species have cytostomes. Little is known of the nutrition
and metabolism of these flagellates, and only a few species have been cultured. Most species are
nonpathogenic, but some cause disease.

Most animals acquire flagellate symbionts from contaminated food or drink. Both the cysts and
trophozoites can spread the infection. The symbionts usually occupy the lower portion of the host's
alimentary canal, where digestive activities are lacking (e.g., the cecum in mammals and birds and
the cloacal cavity in amphibians and reptiles). Below are descriptions of some intestinal flagellates.

Trichomonads. All trichomonads have an anterior tuft of three to five flagella, a rigid median rod,
an axostyle and an undulating membrane. They lack mitochondria but have hydrogenosomes.
Asexual reproduction is by longitudinal binary fission, and it produces large numbers of
trophozoites which are present in the digestive and reproductive systems of most animals.
Following are some examples of trichomonad symbioses with vertebrates and humans.

Trichomonas gallinarum causes avian trichomoniasis in pigeons, chickens, turkeys,

Fig. 9.2 Life cycle of Opalina ranarum. Adapted from Smyth

and other domesticated birds. The symbiont inhabits the upper intestinal regions of an infected bird
and may cause diarrhea, weight loss, and ruffled feathers. The trichomonad does not produce
cysts. Young birds become infected by their parents. Different strains of the flagellate have proved
almost 100% infective in pigeons. Trichomonas gallinarum has been used extensively to study
mechanisms of pathogenicity. Trichomonas foetus causes a serious genital infection in cattle and
often results in abortions.

Trichomonas vaginalis occurs commonly in the genital tract of humans and is sexually transmitted.
Under certain conditions, which are still not fully understood, it becomes pathogenic on the
epithelial cells of the urethra and vagina and erodes the mucosal lining. The flagellate also infects
rats, mice, hamsters, and guinea pigs and has been cultured in vitro.

Pathogenicity of trichomonad symbionts is correlated with their migration from the large intestine,
where they are nonpathogenic, to other sites in the body. The pathogenicity of trichomonads is
greatly reduced under axenic conditions. For example, T. gallinae, normally a highly virulent
pathogen of pigeons, loses its pathogenicity after 4-5 months of axenic culture. Some unknown
cytoplasmic factors may become diluted when the symbiont is grown in culture. In addition,
cultured flagellates stimulate less antibody production in rabbits. Pathogenic strains have either
small amounts of antigens or incomplete antigens and thus trigger only a limited antibody response
from the host. Although great advances have been made in understanding trichomonad
pathogenesis, many intriguing problems still remain ( Honigberg, 1989).

Giardia. Giardia lamblia is a common inhabitant of the small intestine of humans, monkeys, and
pigs, especially in warmer climates. Unique traits of Giardia include (1) it can grow in axenic
culture, (2) it is one of the earliest branches of eukaryotes, and (3) it has symmetrically arranged
two sets of organelles ( Sogin et al., 1989; Kabnick and Peattie, 1990). The symbiont attaches to
the intestinal wall of the host by means of an adhesive disc and feeds on secretions of the
intestinal mucosa. Trophozoites of this symbiont multiply by binary fission. A heavy infestation of
an individual can result in the production of more than 14 billion cysts per day. Some forms of
Giardia produce the disease giardiasis, symptoms of which include diarrhea, vomiting, and poor
vitamin B12 absorption. At least 10% of the human population in the United States carries a milder
form of Giardia, which usually is harmless and asymptomatic, although the feces from an infected
person may contain large amounts of mucus and fats.

Giardia has a convex dorsal and a concave ventral side that contains an adhesive disc. The cell has
two nuclei, each with a prominent nucleolus. Mitochondria, smooth endoplasmic reticulum, and
Golgi bodies have not been observed in the cells ( Gillin et al., 1996). The symbiont has four pairs
of flagella, which are positioned to help the symbiont swim as well as to make the initial contacts
with the host. The middle portion of Giardia contains microtubules that form a unique and
distinctive cytoskeleton, which is involved in both attachment and contractibility. Giardia attaches
to the host cell surface by means of its adhesive ventral disc ( fig. 9.3 ). The indented margin of
the disc penetrates the cell and forms an interlocking system with microvilli of the host cell. Giardia
microtubule cytoskeletons can be isolated by means of sonic treatments. Giardia and giardiasis
have been studied and reviewed extensively ( Adam, 1991; Thompson et al., 1993).

Unlike other organisms, Giardia lamblia and Trichomonas vaginalis cannot synthesize purines and
pyrimidines. They obtain the nucleic acid components from their host cells by phagocytosis and
lysosomal activities. Enzymes for the purine and pyrimidine pathways are similar in both of these
species ( Jarroll et al., 1989; Wang, 1989). Giardia shows surface antigenic variations and the
nature of its variant-specific surface proteins is being explored ( Nash, 1995).
Histomonas meleagridis. Blackhead disease of turkeys was first identified in Rhode Island in
1895 and by 1920 it was killing up to 45% of the commercial turkey flocks in the United States. In
the 1920s, Ernest Tyzzer, a Harvard University parasitologist, identified Histomonas meleagridis as
the cause of the disease and described its biology and pathology. The pathogen first infects the
intestine and then spreads to the liver and causes death of the host. Histomonas meleagridis alone
is unable to cause blackhead disease and requires the participation of intestinal bacterial species
such as Escherichia coli and Clostridium perfringens ( Roberts and Janovy, 1996). Tyzzer
discovered that chickens and nematodes were

Fig. 9.3 Giardia ultrastructure. (Left) Scanning micrograph of Giardia trophozoites attached to
surface of intestinal villi of rat. Note dome-shaped lesions, which were previous sites of parasite
attachment. (Right) Electron micrograph of sectioned Giardia attached to microvillous border. Note
the microtubular cytoskeleton of adhesive ventral disc (Courtesy Stanley L. Erlandsen, University of
Minnesota).

carriers of the flagellate. The disease declined by 1940 primarily because of improved sanitation
and management practices such as not housing chickens and turkeys together.

Histomonas meleagridis forms two types of cells. One type of cell is round, lacks flagella, and
infects the liver and mucosal lining of the cecum. The other type of cell has one or two flagella and
occurs in the intestine.

Birds that are susceptible to the cecal nematode Heterakis gallinarum have a high incidence of
infection by H. meleagridis. Nematodes of both sexes as well as eggs of the female contain the
flagellate, and when the nematodes infect poultry, the eggs hatch and produce infected larvae.
Disintegration of some of the larvae by the host's digestive enzymes frees the parasite, which then
invades the cecal mucosa and migrates to the liver. The symbionts cause prominent lesions on the
cecal walls, a thickening of the cecal mucosa, and coagulation necrosis of liver parenchyma.
Earthworms also have a role in blackhead disease of poultry. Infected nematode eggs are often
eaten by earthworms, which in turn are eaten by poultry.

Histomonas meleagridis can be cultured in the presence of bacteria from the cecum of a natural
host. The cultured symbiont loses some or all of its pathogenicity, but this can be restored, either
fully or partially, by serial passages through poultry. Because of the unusual nature of the
flagellate's life cycle and the involvement of another parasitic symbiont, a nematode, as an agent
of transmission, H. meleagridis continues to be studied by scientists.

Flagellates of termites and roaches. Flagellate symbionts of the wood-eating roach

Cryptocercus punctulatus and termites of the genera Reticulitermes and Zootermopsis have been
the focus of much research. Lemuel R. Cleveland, a pioneer in this field, who devoted more than 40
years of his life to research on this subject. The flagellates inhabit the posterior portion of the lower
termite's alimentary canal and belong to the orders Hypermastigida, Oxymonadida, and
Trichomonadida.

An estimated 30-50% of the total weight of a wood-eating termite consists of protozoa. Some,
such as Trichonympha, can digest cellulose and thus allow the host insect to subsist on a diet of
wood. The symbionts break down cellulose anaerobically by means of the enzyme cellulase to
glucose, which is then converted to acetic acid, carbon dioxide, and hydrogen in hydrogenosomes.
The nature of this symbiosis is complicated because the host insects also have bacterial and fungal
symbionts, which provide vitamin B complex to the hosts. These organisms occur in the alimentary
canal, in the Malpighian tubules, or within mycetocytes. Nitrogen-fixing bacteria also occur in the
termite and provide nitrogen as well as growth factors and vitamins for the host and its flagellates.
Other termites also have methane-producing bacteria. Termite gut shares many similarities with
rumen of cattle and are often described as the world's smallest bioreactors ( Brune, 1998).

There are more than 2000 species of termites in the world. They occur mostly in warm climates,
but 41 species are known from North America. Wood-digesting, mutualistic protozoa are
characteristic of the lower termite families (Hodotermitidae, Kalotermitidae, Mastotermitidae, and
Rhinotermitidae). Only about 10% of termites are colonial insects with a high degree of social order
and a distinctive "caste system" based on morphological and physiological features. Termites build
complex nests consisting of galleries and chambers, and each species produces a nest of specific
shape and size that may be built underground or above ground as mounds ( fig. 7.11 ).

Termites undergo simple metamorphosis from egg to four nymphal instar stages and finally to the
adult stage. The first nymphal instar acquires its flagellate symbionts by feeding on anal droppings
of infested termites. During each molt, the entire epithelial lining of the alimentary canal is shed,
resulting in the loss of intestinal flagellates. Each newly molted instar also acquires its symbionts
from anal droppings. The termite digestive tract consists of three parts: foregut, midgut, and
hindgut. Most cellulose digestion takes place in the paunch region of the hindgut, which is dilated
and contains the bacteria and flagellates. The flagellates digest cellulose to simpler compounds that
can be used by the termites, and the host in turn provides an anaerobic chamber and food for the
symbionts. Termite symbionts can be eliminated by starving the host or by maintaining it at high
temperatures or high oxygen pressure. Termites that lose their symbionts do not survive on their
normal wood diet and die within weeks. Survival of these symbiont-free termites can be prolonged
by feeding them glucose or fungus-decomposed wood. Termites that reacquire their flagellates will
recover and live normally. Not all flagellates in the termite hindgut have the cellulose enzyme
system. Only the cellulolytic strains are mutualistic symbionts of termites, although the remaining
flagellates have diverse lifestyles.

Mixotricha paradoxa is a large wood-eating flagellate that lives in the gut of the termite
Mastotermes darwiniensis. At first the symbiont was thought to be a ciliate because its outer
surface is covered with structures that look like cilia. However, studies have shown that M.
paradoxa is covered with spirochetes and rod-shaped bacteria, not with cilia. The spirochetes
undulate in a coordinated manner and propel the symbiont through the intestinal fluid. The four
flagella of the symbiont are believed to be used only for steering ( fig. 9.4 ). The association is
considered to be mutualistic because the spirochetes receive nutrients from the flagellate. Lynn
Margulis has suggested that the flagella of eukaryotic organisms evolved from similar
spirochetelike prokaryotes that were ectosymbionts ( Margulis, 1991b). The flagellate symbionts of
termites possess a variety of bacteria as intracellular symbionts. The bacteria are responsible for
the flagellate's ability to digest cellulose.

There are many similarities between anaerobic fermentation in ruminants and cellulose degradation
in termites. Both ruminants and termites ingest plant cellulose and degrade it to usable compounds
by means of mutualistic symbionts.

Hemoflagellates
Intestinal flagellates of bloodsucking insects have successfully invaded the reticulo endothelial
tissue of vertebrates. In a vertebrate host, the flagellates swim in the bloodstream or become
intracellular inhabitants of macrophages. Hemoflagellates assume different morphological forms
during their life cycle, with their mitochondria also changing shape. Some of the flagellates can

resist the host's antibodies. Diseases produced by hemoflagel-

FLAGELLATES OF TERMITES Fig. 9.4 Common genera of symbiotic flagellates of termites. Adapted
from Cleveland and Grimstone ( 1964).

lates in humans and domesticated animals have cost millions of lives and include African sleeping
sickness, Chagas's disease in South America, and leishmaniasis in Asia and Africa. Hemoflagellates
have been studied extensively by parasitologists, cell biologists, immunologists, and evolutionary
biologists.

Trypanosoma brucei rhodesiense: antigenic variation. African sleeping sickness affects

thousands of people throughout equatorial Africa. Both sexes of the tsetse fly, Glossina sp., are
vectors of the trypanosome that causes the disease. The symbiont develops into the infective form
in the insect's salivary glands. When the insect feeds, it injects trypanosomes into the lymph and
bloodstream of the host. The trypanosome population increases rapidly and reaches a peak in
about 5-7 days. The flagellates are of the slender-form type ( fig. 9.5 ). A week after the initial
infection, two classes of host antibodies appear in the blood serum: IgG antibodies, which are
directed against cell antigens, and IgM antibodies, which are active against cell surface antigens.
The surface antigens of trypanosomes are believed to be glycoproteins. The antibodies cause
agglutination and lysis of the trypanosomes, and the disease then enters a remission phase as the
number of flagellates declines sharply. During this phase, large numbers of flagellates of the short,
 stumpy form appear in the blood. These forms cannot develop further in the mammalian host. A
few slender-form trypanosomes develop new antigenic properties and are unaffected by the host's

antibodies. These flagellates then multiply until the host's im-

Fig. 9.5 Schematic diagram of developmental stages of Trypanosoma brucei in mammals and
tsetse flies. (Top) Changes in cell surface and mitochondria. Note presence of surface antigens
(arrows) around forms of the parasite occurring in bloodstream. From Vickerman ( 1971), with
permission. (Bottom) Scanning electron micrograph of trypanosome among mammalian red blood
cells. (Courtesy Steven Brentano, University of Iowa).

mune system manufactures new antibodies that are specific for them. The antibodies destroy most
of the new trypanosomes, but again a few symbionts alter their antigens, and the process is
repeated. Each new antigenic variant of the symbiont is matched by the host's production of
antibodies against it, but eventually the host is overwhelmed. After the insect has a blood meal
from an infected individual, the stumpy-form symbionts undergo several transformations before
they migrate to the salivary glands, where they develop into the infective form. The insect may
inject as many as 40,000 trypanosomes into a host during a blood meal. The developmental stages
of a symbiont last from 17 to 50 days, depending on biotic and abiotic factors, and the insects
remain infective for their lifetime, which is about 2-3 months.

Symbionts in the mammalian blood have a distinguishable coat on the outside of their cell
membranes. This surface coat is present only in the infective form of the trypanosomes that
emerge from the insects. Antigenic variation of the trypanosome is associated with changes in its
surface coat. Surface coat antigens are phenotypic expressions of trypanosome genes ( Deitsch et
al., 1997). Up to 10% of the trypanosome genome may consist of genes that code for antigens. A
single trypanosome can produce at least 100 antigenic variants, and up to 1000 genes may be
involved. The surface coat of the trypanosome is composed of about 10 million glycoprotein
molecules known as variant surface glycoproteins (VSG). Each VSG molecule is made up of about
500 amino acids, to which are linked two types of oligosaccharide side chains. One of the
carbohydrate chains links the VSG molecule to the trypanosome cell membrane. Variations in the
amino acid sequence of the VSG molecule results in new antigenic properties. Several VSG genes
have been sequenced ( Mansfield, 1995). Three mechanisms of genetic rearrangement involved in
a VSG gene expression are (1) duplicating and transposing a gene to another position near the
telomeric region of a chromosome, replacing the resident gene, (2) duplicating and transposing an
unexpressed telomeric VSG gene to another telomeric site where it is expressed, and (3)
expressing an inactive telomeric gene. There is some evidence that there may be two or more
expression sites for VSG genes ( Borst, 1991).

The system of antigenic variation in trypanosomes is an innovative evolutionary adaptation that

allows the symbionts to survive in the host and makes the task of developing effective vaccines
against the diseases they cause a difficult one.

Trypanosoma cruzi: intracellular parasite. To live and multiply in host cells such as
macrophages, whose function is to recognize and kill foreign invaders, requires a great deal of
evolutionary audacity. Yet, some flagellates have successfully conquered the macrophages, one of
the most hostile intracellular environments. Trypanosoma cruzi causes Chagas's disease in nearly
8% of people living in Central and South America ( Schofield, 1985). The flagellate enters the host
through cuts in the skin or mucus membranes. The vector of T. cruzi is a brightly colored bug of
the insect family Reduviidae. The insect voids the trypanosomes along with its feces during a blood
meal. The flagellates appear in the blood and periodically invade the reticuloendothelial tissues,
especially those of the heart. Inside the tissues, the symbiont assumes a spherical form, multiplies
as an intracellular parasite for about 5 days, and forms a cystlike cavity, the pseudocyst. Some of
the flagellates break through the pseudocyst membrane and reenter the bloodstream to infect
other tissues. The symbionts that remain in the pseudocyst disintegrate and cause a lesion.
Reduviid bugs ingest T. cruzi during the blood meal from an infected individual. The trypanosomes
multiply and undergo several transformations, first in the midgut and later in the hindgut.
Trypanosoma cruzi is a versatile parasitic symbiont that can invade and replicate inside a wide
variety of host cells, entering them by forming membranebounded vacuoles. Host cell lysosomes
gather at the attachment site of the parasite and fuse with the plasma membrane of the vacuole.
Destruction of the vacuolar membrane is brought about by release of a pore-forming toxin by T.
cruz (Tc-tox). The symbionts escape from the vacuoles into the host cytoplasm, where they
multiply ( Burleigh and Andrews, 1995; Mauel, 1996).

Unlike T. brucei, which defeats the host's immune system by sequentially changing its surface
antigens, T. cruzi expresses several glycoproteins simultaneously. Some of the principal surface
antigens are neuraminidase, GP-72, and GP-85. GP-72 is a major surface glycoprotein that seems
to regulate the host's complement activation. GP-85 is a lectin that binds to fibronectin receptors
and helps the parasite invade the host cell. Neuraminidase of T. cruzi controls the infection process
by a negative mechanism that may check pathogen replication and allow it to remain latent ( De
Souza , 1984; Pereira, 1995).

Molecular biology of Trypanosoma. Trypanosomes have an elongated, flat body with a single
nucleus that has a prominent, central nucleolus. The flagellum arises posteriorly, extends along the
free margin of the body to form an undulating membrane, and continues anteriorly as a free
flagellum. The flagellum originates from a basal body and kinetoplast, which are enclosed in a
mitochondrion. The mitochondrial DNA is in the form of a kinetoplast, which is a mass of circular
DNA that consists of thousands of maxicircles and minicircles. Of the total cellular DNA of
Trypanosoma, 10-25% is kinetoplast DNA (kDNA). Studies have suggested that maxicircles have
genes that are similar to the mitochondrial DNA of other eukaryotic cells. The minicircles, which
make up approximately 95% of the KDNA, encode for small-guide RNAS, which control editing
specificity ( Stuart and Feagin, 1992). RNA editing is a type of RNA processing that regulates only
the mitochondrial genes of kinetoplastid flagellates ( Stuart, 1995; Cavalier- Smith, 1997).
Mitochondrial morphology varies a great deal in hemoflagellates. The mitochondria are usually in
the form of a single or branched tube that runs the length of the cell, and they possess
characteristic cristae. In Trypanosoma brucei, the mitochondrial shape changes along with the
transformation the symbiont undergoes when it passes from the vertebrate bloodstream to the
insect's alimentary canal. In the bloodstream, the symbiont has a slender form and a long, tubular
mitochondrion with a double membrane but only a few cristae. In the alimentary canal of the
insect, the symbiont assumes the stumpy form and has a well-developed spherical mitochondrion
with a tubular network of prominent cristae. The mitochondrion of the slender form is inactive,
lacking enzymes of the Kreb's cycle and electron transport chain, while that of the stumpy form is
active.

Trypanosomes without functional mitochondria depend on glycolysis and substratelevel

phosphorylation for their ATP production. The parasite has acquired a novel way of increasing its
level of glycolysis by compartmentalizing various glycolytic enzymes into a membrane-bound
organelle, the glycosome. The high concentration of enzymes in glycosomes allow glycolysis to
proceed at a rate 50 times that of a mammalian cell. Unlike mitochondria, a glycosome does not
contain nucleic acids and is bounded by a single phospholipid bilayer membrane. There may be
200-300 glycosomes per cell. Glycosomal proteins in T. brucei are encoded by nuclear genes (
Boothroyd, 1995; Cavalier- Smith, 1997).

Leishmania: intracellular survival in macrophages. Several species of Leishmania cause

diseases in humans. Leishmania tropica is the causal agent of oriental sore, which is endemic to
Africa, the Middle East, and India; L. mexicana causes New World cutaneous leishmaniasis, or
chiclero ulcers, a disease common in Mexico, Guyana, and other countries of Central and South
America; L. braziliensis causes an infection called espundia, a disease endemic to the jungles of
South America, which involves mucous membranes of the mouth, nose, and pharynx; and L.
donovani is the causal agent of visceral leishmaniasis in most of the tropical and subtropical
countries of the world.

Leishmania has two different morphological forms depending on the type of host: an oval-shaped
body without an external flagellum when it occurs in a vertebrate host, and an elongated body with
a flagellum when it inhabits the intestines of sand flies. In the mammalian host, Leishmania lives
exclusively in macrophages. The establishment of the symbiosis involves recognition, intracellular
entry, surviving the host's lyric enzymes, and growth and multiplication of the symbiont ( Chang,
1995).

As soon as flagellates from the insect host enter the mammalian bloodstream, a complex process
of cell recognition takes place. Some type of chemotaxis may take place because the flagellates are
strongly attracted to macrophages and, to a lesser degree, to lymphocytes, but show no affinity
with red blood cells. The symbionts possess cell surface components that fit receptors in the
macrophage cell. Macrophages permit internalization of the symbiont by phagocytosis. The flagellar
end of the symbiont may play a significant role in its interaction with the host cell. Entry of the
symbiont into the macrophage is accompanied by chemiluminescence and the release of hydrogen
peroxide. Macrophages treated with cytochalasin B fail to phagocytize the symbionts, which
suggests that the cytoskeletal proteins actin and myosin participate in the process of symbiont
entry. Leishmania may develop a resistance to the lysosomal enzymes of the macrophage, or it
may release substances that inactivate the enzymes. Once inside the macrophage, the symbiont
assumes its nonflagellated form, which is better suited for intracellular existence. The symbiont
grows within the membrane-bound vacuole of the macrophage and multiplies slowly. Some
Leishmania strains have developed stable associations with the host.

Leishmania interaction with macrophages

provides an excellent model for several important aspects of cell biology such as receptor-mediated
endocytosis, membrane transport, and molecular signals and gene expression and also intracellular
targeting and movement of internalized molecules. There is a common surface glycoprotein (gp63)
found on surfaces of several pathogens, and its role in leishmanial virulence has been studied
extensively ( Chang, 1995).

Phytomonas: plant pathogenic flagellates

Flagellate protozoans were first observed in 1909 in latex-bearing cells of Euphorbia, the rubber
plant. Since then the pathogen has been found to cause disease in plants that belong to the
families Asclepiadaceae (milkweed), Moraceae (figs), Rubiaceae (coffee), and Euphorbiaceae
(cassava). Recently, flagellate protozoa have been isolated from tomatoes ( Agrios, 1997). All
species of Phytomonas belong to the family Trypanosomatidae and require a plant and an insect to
complete their life cycle. In plants, some Phytomonas flagellates live in the phloem sieve tubes,
while others live in latex-containing cells. Phloem necrosis of coffee commonly occurs in South
American coffee-growing regions and is caused by Phytomonas leptovasorum. The infected plant
shows symptoms of wilt, which is accompanied by yellowing and dropping of leaves. The plant dies
within 36 weeks. Vectors of this disease are several species of pentatomid insects of the genus
Lincus. In the early 1980s more than 15,000 coconut trees were killed by Phytomonas staheli in
Trinidad ( Dollet, 1984).

Apicomplexans
Members of the phylum Apicomplexa have an apical complex that consists of a polar ring, conoid,
rhoptries, micronemes, and subpellicular microtubules ( fig. 9.6 ). The polar ring is located at the
anterior end of the symbiont, just beneath the cell membrane. The conoid is a spirally coiled
structure within the polar ring. There may be two to seven rhoptries that lead to the apex of the
cell. Micronemes are small, convoluted structures that extend over most of the anterior end, and
their ducts join those of the rhoptries. Subpellicular microtubules radiate from the apex and extend
most of the length of the symbiont. A mouth is located laterally, and along its edge are two
concentric rings of some yet-unknown material that may help the symbiont acquire nutrients. The
apical complex participates in the penetration of the host cell by secreting proteolytic enzymes.

Apicomplexan life cycles may require two host organisms and involve several kinds of asexual and
sexual stages. The following are examples of symbionts included in the phylum.

Gregarines

Gregarines are extracellular symbionts that inhabit the alimentary canal and body cavity

Fig. 9.6 Ultrastructural details of a typical apicomplexican trophozoite

of invertebrates, especially arthropods, molluscs, and annelids. The life cycles of several gregarine
species are synchronized with those of their hosts. Sporocysts are produced only in the sexually
mature host and are liberated along with the host sex cells, so that the next generation of larvae
will become infected with the symbionts. The host's hormones may play a significant role in the
development of the symbiont.

Toxoplasma gondii: a cosmopolitan

coccidian
The coccidian symbiont T. gondii occurs in many animal species throughout the world. In humans,
toxoplasmosis is generally a mild or asymptomatic infection, but in some cases it may cause
blindness and damage to the central nervous system. The symbiont invades the macrophage and
circumvents its lysosomal system. Sexual stages in the life cycle begin after cats ingest cysts of the
symbiont from tissues of an intermediate host such as a rodent or bird ( fig. 9.7 ). The cysts
contain hundreds of sporozoites, which infect the host epithelial cells and then transform into
merozoites. Merozoites multiply rapidly and are released into the intestinal lumen when the host
cells rupture. Each merozoite can infect another epithelial cell. After several such cycles, some
merozoites become gametocytes. Two gametocytes fuse together to form a cyst, which is then
expelled with the feces.

Asexual reproduction of T. gondii begins when a host other than a cat ingests cysts from the meat
of an infected animal. Spores liberated from the cysts penetrate the intestinal wall and enter the
circulatory system and infect the macrophages. In the macrophage, spores are called tachyzoites.
Living tachyzoites use macrophages as an intracellular habitat, divide repeatedly by binary fission,
and eventually kill the host macrophage. When the host defense mechanism is mobilized,
macrophages surround large numbers of symbionts and form a wall around them and form
pseudocysts that contain dormant symbionts. Pseudocysts occur in all tissues of the host but are
most common in the brain, retina, and liver. Raw hamburger is believed to be an excellent source
of pseudocysts. Most infections of T. gondii occur when undercooked or raw meat containing
pseudocysts is consumed. Tachyzoites will cross placental barriers and infect the developing
embryo.

Tachyzoite invasion of macrophages. Early workers had reported that when tachyzoites were
liberated in a host environment, they exhibited various types of movement such as gliding or
rotating in a somersault fashion. Penetration of the macrophage was thought to be achieved
through a collision with the host membrane. Later evidence, however, supports the view that a
symbiont's penetration is mainly the result of the host cell's phagocytic activities.

The sequence of events that results in the destruction of a pathogen in a macrophage is called the
respiratory burst and involves a number of metabolic activities. When a macrophage is unable to
degrade the pathogen contained in a vacuole phagosome, there is no respiratory burst.
Toxoplasma gondii strains differ in their pathogenic abilities. A proteinaceous substance has been
isolated from one strain of lysed Toxoplasma that enhances the virulence of another strain.
Toxoplasma penetration of macrophages was greatly facilitated when lysozyme or hyaluronidase
was added to the culture medium. The process of entry is initiated when the symbiont attaches to
the macrophage by its anterior end and forms a small depression in the host cell membrane. Once
inside the cell, the symbiont becomes enclosed in a phagosome, which is a membrane-bound
vacuole ( Lingelbach and Joiner, 1998). The host cell lysosomes fail to fuse with the phagosome,
suggesting that T. gondii contains a substance that interferes with the lysosome--phagosome
fusion ( Dubremetz, 1998). Toxoplasma gondii also changes the phagosome to allow the gathering
of endoplasmic reticulum and mitochondria along the vacuolar membrane. Macrophages can
degrade previously killed tachyzoites, but living symbionts gain immunity to the host's destructive
enzymes by altering their properties. Newly developed tachyzoites escape from the host cell by
twisting through the host cell membrane.

Malarial parasites

Malaria is caused by the multiplication of the parasite Plasmodium in the blood and tissues of a
vertebrate host. Plasmodium species are obligate intracellular parasites that have several
morphologically different developmental stages. Although there are more than 100 species of
Plasmodium, which affect a wide range of vertebrates, each species has a narrow host range
Four species of the malarial parasite infect humans: R. falciparum, R. malariae, R. ovale, and R.
vivax. Plasmodium vivax accounts for 43% of all cases of malaria and occurs primarily in Asia and
Africa. Only people with Duffy blood groups are resistant to the disease. Plasmodium vivax can
reoccur many years after the first infection due to the presence of dormant hypanozoites in the
liver of infected individuals. Plasmodium faiciparum is the most virulent form of malaria and
accounts for more than 50% of all cases. It occurs primarily in tropical and subtropical parts of the
world. The malarial parasite of chimpanzees and monkeys, R. knowlesi, occasionally infects
humans and produces mild symptoms. Plasmodiumberghei in rodents and R. gallinaceum and R.
lophurae in birds have been used extensively as experimental models to explore the ultrastructural
details of the symbiont--host interaction.

History of the disease. Malaria is one of humankind's oldest diseases and has played a significant
role in history as well as in the development of modern science. Almost half the world's population
lives under the threat of malaria. Each year an estimated 150 million people contract the disease
(80% of them in Africa), and about 1 million die of it. Malaria remains as one of the greatest
threats to childhood survival in Africa. Global warming raises the specter of bringing even more
areas under the threat of malarial infections.

Discovery of the malarial parasites and understanding of the intricate details of their life cycle
represent the golden age in parasitology. Ronald Ross, and Battista Giovanni Grassi, working with
human malaria, showed in 1898 that the parasites developed in the mosquito and that humans
became infected when bitten by mosquitoes. In 1880, Alphonse Laveran described the parasites in
blood cells, but it was not until the early 1950s that the role of the liver in the disease was
resolved. Both Laveran and Ross were awarded Nobel Prizes for their discoveries.

The first drug to be used against malaria was quinine, which was extracted from the bark of the
chinchona tree. Peruvian Indians knew of the therapeutic value of quinine, and when the European
colonists learned of it, the use of quinine became global.

The malarial symbiont is transmitted by Anopheles mosquitoes, and the development of the
pesticide DDT in the 1940s was an important breakthrough in controlling the spread of the disease.
Unfortunately, as mosquito control programs spread throughout the world, DDT-resistant strains of
mosquitoes began to appear ( Crampton, 1994).

During the Second World War, hundreds of thousands of American soldiers contracted malaria. The
U.S. Army launched a project to combat the disease, eventually developing antimalarial drugs such
as chloroquine, artemisinin, and mefloquine. A malaria eradication program was developed, based
on the use of DDT and chloroquine. During the 1960s the incidence of malaria declined sharply in
all parts of the world, with expectations that it would be totally eradicated. Then came the
realization that Plasmodium falciparum and other species were producing strains that were
resistant to chloroquine, while the mosquitoes were becoming resistant to DDT. The reemergence
of malaria after more than 2 decades of relative success in controlling it has alarmed scientists,
who are now trying to understand the molecular basis of the disease. Scientists are using
techniques of immunology and molecular biology in attempts to develop an effective vaccine
against the malarial symbiont ( Facer and Tanner, 1997), a difficult task considering the complexity
of the disease and the variability of the parasite ( Good et al., 1998). Much has been learned from
the malarial parasite about the molecular mechanisms, genetics, immunology, and biochemistry of
symbiont-host interactions. Efforts by several collaborating laboratories are underway to sequence
the complete genome of Plasmodium falciparum. Recent research has once again focused on the
mosquito vector in an attempt to create, with DNA technology, strains of the mosquito that would
be incompatible hosts for the malarial parasite ( Collins and Paskewitz, 1995).

Life cycle of Plasmodium. In humans, infection begins when a female Anopheles mosquito
penetrates the host skin and injects into the blood saliva that contains sporozoites of Plasmodium (
fig. 9.8A ). Sporozoites are covered by a single protein, the circum-sporozoite protein (CSP). Both
the genes involved and the protein have been cloned and sequenced. Sporozoite antigens have
been considered as one of the principal antigens for the antimalarial vaccine. The parasites migrate
to the liver, where they multiply in the parenchyma cells. The sporozoites are transformed into
merozoites, and when all the liver host cells rupture, they liberate 100,000-300,000 merozoites. In
most mammals, merozoites infect red blood cells and grow and multiply intracellularly. The mature
stage of the symbiont in a red blood cell is called a schizont. The schizont divides to form
merozoites, which are released when the red blood cell ruptures and then infect other red blood
cells. This asexual part of the life cycle is the schizogony. The clinical signs of malaria are
associated with the cyclical blood stage of schizogony. In each cycle of schizogony, some of the
invading merozoites differentiate into male (micro-) and female (macro-) gametocytes. The
gametocytes mature in about 10 days and do not develop further in the vertebrate host.

When a female mosquito bites an infected individual, micro- and macrogametocytes are ingested
with the blood. In the mosquito's stomach, each microgametocyte forms eight microgametes,
which fertilize the macrogametes formed by the macrogametocyte. The resulting zygotes become
transformed into motile, wormlike structures, the ookinetes, which penetrate the stomach wall and
form sporocysts. Each sporocyst breaks open and releases sporozoites, which migrate to the
salivary glands. The sexual part of the parasite life cycle is the sporogony ( Beier, 1998).

Merzoites: intracellular symbionts of erythrocytes. Within the vertebrate host, sporozoites

and merozoites have to invade specific cell types. Proteins associated with their outer surfaces
have been implicated as ligands for receptors on the host cell surfaces. Liver cells possess
receptors for CSP. Therefore, the CSP protein is involved in the initial interaction and attachment to
the hepatocyte surfaces.

On the merozoites, a surface protein 1(MSP1), along with rhoptry-associated protein, plays a role
in the invasion of the erythrocytes ( Wakelin, 1996). These proteins undergo a complex series of
modifications between their synthesis and successful erythrocyte invasion.

Glycophorins are major surface proteins of the human erythrocytes, and Plasmodium merozoites
depend on these molecules for invasion of erythrocytes. The thick cell surface coat is removed
during the invasion process. The coat consists of fine filaments that stand erect, like hairs, on the
plasma membrane. The filament tips are either Y or T shaped and can stretch when attached to the
cell. The filaments may attach to the underlying cytoskeleton of the merozoite. Two subplasma
membranes make up a system of interconnected cisternae that lie under the apical complex. The
polar ring of the apical complex is responsible for a particular merozoite shape, and it forms a
convergent point for rhoptries and micronemes.

It is not known how merozoites are attracted to erythrocytes, but once contact is made the
parasite adheres to any part of the erythrocyte surface. The invasive process begins, however, only
when the symbiont's apical complex contacts the host cell surface, which has specific receptor
molecules (figs. 9.8B, 9.9A,B ). Merozoites can attach to a glass surface, to other merozoites, and
to white blood cells. If the merozoite--erythrocyte attachment is incorrect, the merozoite detaches
itself and makes another attempt. The host membrane shows a characteristic bending when the
apical complex of the merozoite contacts the erythrocyte. Secretions released from rhoptries and
micronemes cause the erythrocyte membrane to invaginate and form a cup, into which the
merozoite advances. The surface coat of the merozoite becomes detached as the symbiont moves
into the cup ( fig. 9.8C ). Finally, the entire merozoite becomes surrounded by the host membrane
and enclosed within a vacuole. It has now been established that the apical complex of the parasite
is responsible for the invasion of the erythrocyte. The rhoptries and micronemes are thought to be
derived from the Golgi bodies and to possess lysosomic enzymes. After the initial contact, the
movement of a merozoite into the host cavity is rapid. This is interesting because merozoites lack
obvious motility mechanisms. It has been suggested that suction produced by the invagination of
the erythrocyte pulls the symbiont into the vacuole ( Lingelbach and Joiner, 1998). In the vacuole,
microspheres near the cell surface of the merozoite protrude into the host cytoplasm. The contents
of the microspheres are released in the vacuole and cause it to enlarge. The cisternae that lie
underneath the plasma membrane of the merozoite, along with the microtubules in the cortex,
disappear. The merozoite loses its spherical shape and becomes transformed into a trophozoite (
Aikawa, 1988).

Malarial pathogenesis. Plasmodium infections cause severe anemia and cerebral malaria.
Plasmodium sp. produce toxins that stimulate the release of cytokines such as tumor necrosis
factor a. (TNF-α) from host T-cells. It is now believed that excess release of nitric oxide by the
vascular endothelium in response to TNF-α diffuses through the brain tissue and disrupts neuronal
activities. Rising levels of TNF-α are associated with body temperature during a malarial paroxysm.
When children are given anti-TNF-α therapy, they soon recover in intermediate steps. The
development of malarial pathogenesis has been a subject of intense research ( Miller et al., 1994).
 A Fig. 9.8. Malarial parasitic symbiosis. (A) Developmental stages in the life cycle of Plasmodium.
(B) Stages in the invasion of an erythrocyte by a Plasmodium merozoite. (Courtesy Lawrence
Bannister, Guy's Hospital, Medical and Dental Schools, London.)

Theileria parva
The piroplasm Theileria parva causes theileriasis in cattle and water buffalo. The symbiont is an
intracellular inhabitant of erythrocytes and leukocytes and is spread by ticks ( Mehlhorn and
Schein, 1984). The host--symbiont relationship is unique. The parasite causes the host cell to
proliferate and undergo synchronous S-phase and nuclear division as they share a common
cytoplasm ( Dyer and Tait, 1987). Theileria becomes a permanent resident of the macrophage
cytoplasm by escaping from the vacuole. The symbiont synchronizes its growth and duplication
with that of the host cell. The schizont of T. parva segregates along with the host chromosomes
into the two daughter cells. Electron microscopic evidence suggests that the schizont of T. parva is
closely involved with the host's mitotic apparatus. The symbiont becomes encased in microtubules
during centriole replication and aster formation and becomes compressed between the
microtubules during elongation of the spindle. By the time the nuclear membrane disintegrates, the
schizont is fully integrated into the mitotic spindle along with the host chromosomes. During
anaphase, the schizont elongates with the polar microtubules, and when the host cell constricts in
the middle to produce two new cells, the schizont is also partitioned into two. Schizont-microtubule
attachments have not been observed ( Carrington et al., 1993).

-148-

B Fig. 9.8. (continued)

35-Kilobase Apicomplexan DNA: A Ghost

of Photosynthesis Past
Malarial parasites have 14 chromosomes in addition to two extrachromosomal DNAS, 6-kb
linear and 35-kb circular molecules. The 35-kb circular DNA was originally thought to be the
mitochondrial DNA of Plasmodium. The 6-kb fragment carries the genes for cytochromes and
cytochrome oxidase that are typical of mitochondrial DNA. Recent sequence evidence, however,
suggests that 35-kb Plasmodium DNA is more like that of plastid DNA ( R. J.M. Wilson et al.,
1991; Palmer, 1992). Similar 35kb circular DNAs have been identified in Babesia, Eimeria, and
Toxoplasma and are believed to be common in other species of this group. The 35-kb DNA in
apicomplexans is contained within a vestigial plastid ( Feagin, 1994). In Toxoplasma, the
plastid is surrounded by four plasma membranes, and during cell division of the parasite the
plastid divides into two in a way similar to that of plastids in plants and algae. Apicoplast is the
name given to the vestigial plastids of apicomplexan protists. The 35-kb apicomplexan DNA
appears to be a plastid genome that has "forgotten" how to photosynthesize ( Vogel, 1997).
These findings suggest that apicomplexans are closely related to dinoflagellates. As discussed
in the next chapter, dinoflagellates are common symbionts of invertebrates.

Apicomplexan plastids offer an excellent opportunity to develop drug therapy against diseases
that affect millions of humans. We can now understand how antibiotics such as doxcycline
(antimalarial), rifampicin, thiostrepton, and spramycin control toxoplasmosis and
Cryptosporidium ( McFadden and Waller, 1997).

Fig. 9.9 Electron micrographs of Plasmodium merozoites invading red blood cells. (Top left) A
merozoite showing nonspecific type of attachment to an erythrocyte. Note the presence of surface
coat (arrow). (Top right) Long-range attachment between parasite and host cell involving
merozoite coat filaments (arrow). (Bottom) A partially depleted rhoptry is visible at the anterior
end of the parasite (arrow). Note the formation of a vacuole that surrounds the symbiont (Courtesy
Lawrence Bannister, Guy's Hospital, Medical and Dental Schools, London).

9.3 SUMMARY
Protozoans cause diseases in millions of humans and domesticated animals. Many of these
parasites have complex life cycles that involve two hosts and several types of spores. Species of
amoebae range from harmless commensals in a host's alimentary tract to virulent pathogens.
Entamoeba histolytica causes dysentery, and species of Acanthamoeba cause meningoencephalitis
in humans and mammals. Opalinids are commensals in the large intestine of frogs and toads.

Two kinds of ciliates, entodiniomorphs and holotrichs, live mutualistically with herbivorous
mammals. The ciliates live in the rumen of the host along with bacteria.

There are two groups of flagellated protistan symbionts: intestinal flagellates and hemoflagellates.
Representative intestinal flagellates include trichomonads, Giardia lamblia, Histomonas meleagridis,
and various genera that inhabit the alimentary canal of wood-eating termites and roaches.
Mixotricha paradoxa, a wood-eating flagellate found in the gut of termites, is covered with
spirochetes that propel the flagellate. Hemoflagellates include trypanosomes and Leishmania,
which cause devastating diseases in humans and animals such as African sleeping sickness,
Chagas's disease, and leishmaniasis. Apicomplexans include gregarines, which are extracellular
symbionts of invertebrates, Toxoplasma gondii a symbiont of many animal species, Plasmodium, a
parasite that causes malaria, and the piroplasm Theileria parva, which causes theileriasis in cattle
and water buffalo. Apicoplasts are vestigial plastids that have been found in apicomplexans.

Symbiotic associations may be more complex than first imagined and may involve several layers of
organization. For example, scientists now believe that Entamoeba histolytica cannot produce the
disease amoebiasis alone; rather, the presence of bacterial and viral symbionts in the cytoplasm
and nucleus of the amoeba may be necessary for its virulence. Similarly, the ability of the termite
symbiont Mixotricha paradoxa to digest cellulose may be the result of intracellular bacterial
symbionts. Symbioses in the rumen of herbivorous animals and in wood-eating termites consist of
complex interrelationships between bacteria, ciliates, and fungi. Similar patterns may exist in other
associations.

The life cycles of the symbionts of many protistan associations are synchronized with each other,
thereby ensuring continuity of the symbiosis from one generation to another.

10 PHOTOSYNTHETIC ASSOCIATIONS
OF PROTOZOANS AND INVERTEBRATES
Many invertebrates, such as sea anemones, corals, flatworms, and protozoans, have formed
mutualistic associations with photosynthetic microbes ( Saffo, 1992b; Trench, 1993). The
photobionts supply nutrients to their hosts, which allows them to colonize habitats they
normally could not because of limited supplies of prey. Two groups of algae (protists),
collectively called zoochlorellae and zooxanthellae, are the most common photobionts. These
names do not have taxonomic status but are useful for identifying the general type of
endosymbiont in a host. Zoochlorellae are mainly species of Chlorella, a freshwater, unicellular
alga which is a wellknown symbiont of at least 30 genera of ciliates, amoebae, hydras,
sponges, and clams ( D. C. Smith, 1991). About 10 species of Chlorella are recognized. DNA-
DNA hybridization studies have revealed that many of the species are not closely related and
that the Chlorella-type morphology evolved in several lineages of the Chlorococcales.
Zooxanthellae are mostly dinoflagellates, which appear yellow-brown in the many marine
organisms they colonize. The dinoflagellate chloroplasts have efficient light-harvesting
complexes that include chlorophyll a, chlorophyll c, and large amounts of xanthophylls.
Reproduction and genetics of dinoflagellates are unusual and not well understood. They have
more nuclear DNA than other eukaryotes. Their chromosomes remain condensed during
interphase, lack centromeres, and unwind only for DNA replication. The nuclear membrane
does not break down during mitosis, as in animals and plants. Microtubules within the spindle
 coordinate the segregation of chromosomes. Dinoflagellates are diverse in form. Their cell walls
are composed of two interlocking plates; two flagella are inserted in the cell wall at about the
same location. A transverse flagellum wraps around a groove in the cell, and a longitudinal
flagellum is perpendicular to the cell. Dinoflagellates are surrounded by a twomembrane
structure, the amphiesma.

A common dinoflagellate of marine invertebrates is Symbiodinium microadriaticum ( fig. 10.1 ).

Comparison of small ribosomal subunit RNA sequences show that Symbiodinium-like
zooxanthellae represent a collection of distinct species of the genus Symbiodinium ( Rowan and
Powers, 1992; Rowan et al., 1997). Symbiodinium microadriaticum is greatly modified when it
lives inside animal cells. The algal cell wall becomes thinner, which allows for close contact with
the animal's cytoplasm. The cell loses its grooves and flagella and divides only by binary
fission. The algal cells also change physiologically. In the animal, zooxanthellae excrete large
amounts of glycerol, which the host uses to synthesize lipids and proteins. In some
invertebrates, almost 50% of the carbon fixed by the algae is translocated as glycerol within 24
h to animal tissue. Zooxanthellae also excrete glucose, alanine, and organic acids, and perhaps
other compounds in amounts too small to detect. When the algae are isolated from animals
and grown in culture, they stop excreting substances. But when cultured algae are exposed to
homogenates of their host, they once again excrete glycerol and other compounds. Although
the nature of the stimulatory host factor is not known, it seems to be a general one because
homogenates from one type of animal, such as a clam, affect zooxan-
Fig. 10.1 Symbiodinium microadriaticum, a common photosynthetic symbiont of marine
invertebrates.

thellae from another animal, such as coral, and vice versa. The homogenates are effective only if
they are taken from animals that contain symbiotic algae ( D. C. Smith and Douglas, 1987).

In some marine animals there is a reciprocal exchange of substances between the symbionts and
their hosts. The host animals supply the algae with acetate, which the algae use to synthesize fatty
acids. The fatty acids are translocated back to the animal, which uses them to synthesize waxes
and other compounds.

The number of algal cells in each animal cell varies. The hydroid Myrionema amboinense has 1-56
algal cells in each endodermal cell, whereas tentacle cells of the golden brown sea anemone,
Aiptasia pallida, contain about 10 algal cells. Each algal cell is housed inside a vacuole
(symbiosome) formed by the animal cell. The algae are located in translucent parts of the animal,
where they receive light for photosynthesis. The number of photosynthetic symbionts within host
cells is controlled by nutrient supply, factors for cell division, and pH ( Douglas and Smith, 1984;
McAuley, 1985).

Although aposymbiotic invertebrates can be reinfected with strains of S. microadriaticum other

than their natural ones, the foreign algae grow poorly in the host. How an animal recognizes a
specific algal strain is not known, but it appears that a recognition process takes place after the
algae are phagocytized. Algal strains that are compatible with the host are not digested because
they either avoid or resist the host's digestive enzymes.

Algal symbionts pass from one generation of invertebrates to another in different ways. Animals
that reproduce asexually by budding transmit some of their algae directly to their offspring. In
sexual reproduction, algae are found in or on the eggs produced by the parent. In some
invertebrates, offspring ingest free-living algae from their surroundings. Larvae of the jellyfish
Cassiopeia xamachana will not develop normally unless they become infected with algae. Certain
marine nudibranchs obtain their algae after they eat anemones and corals, which contain symbiotic
algae.

10.1 ALGAE AND MARINE PROTOZOANS

AND INVERTEBRATES
Living Sands: Foraminiferans and Radiolarians
Foraminiferans and radiolarians are shelled amoebae that commonly contain dinoflagellates as
symbionts. The amoebae use carbon compounds excreted by the algae and also digest algal cells.
Foraminiferan amoebae float or attach to objects on the ocean floor and make thin, translucent
shells that consist of calcium carbonate. The shells have pores through which strands of cytoplasm
project and inside of which the algae are contained. Algae that associate with foraminiferans
include unicellular green algae, diatoms, and several unicellular red algae. One foraminiferan may
associate with different algae, sometimes simultaneously.

Foraminiferan algae are enclosed in vacuoles and lack cell walls. This makes it difficult to identify
the algae to species, as the cell wall is an important taxonomic trait. If grown free from the host,
the algae will produce cell walls.

The functional relationship between foraminiferans and their symbiotic algae is not fully
understood. In addition to providing the host with carbon compounds, algal photosynthesis
increases the foraminiferan's rate of calcification by removing excess CO 2. In return, the algae
receive nitrogen, phosphorus, and vitamins from the host ( Lee, 1995).

Radiolarians have siliceous shells with holes through which strands of cytoplasm project. A network
of the cytoplasm surrounds the outer part of the radiolarian and contains numerous algal cells,
each within a vacuole. Algal symbionts of radiolarians are yellow-brown and include dinoflagellates,
prasinomonads, and a pyrmnesimonad. Each radiolarian has only one type of alga, but the number
of algal cells in each host differs. Small radiolarians that are part of a colony may contain 30-50
algal cells, whereas large, solitary radiolarians may contain up to 5000 algal cells. Some hosts
maintain their algal population at a constant level by digesting some of their algal cells.

Colonial radiolarians are good experimental subjects for the study of algal-protozoan relationships (
Anderson, 1992). The colonies can be exposed to radioactive compounds and then separated into
algal and host fractions. The central part of the radiolarian is algae-free and detaches easily from
the outer network of cytoplasm, which contains the algae. The separate fractions can then be
analyzed for radioactivity to determine the movement of compounds from alga to host. Studies
have shown that most of the carbon compounds produced by the algae during photosynthesis pass
to the host within the central capsule. The host may also obtain nutrients from prey. Compounds
released from the digestion of prey may be used by the algae. The symbiotic algae assimilate
waste products of the host, such as ammonia and carbon dioxide, thus enabling the radiolarian to
conserve energy that it would have used to dispose of wastes. Because of their symbiotic
relationship with algae, radiolarians flourish in nutrient-poor waters such as the Sargasso Sea.

In some planktonic foraminiferans and radiolarians, the dinoflagellates are moved out of their
shells during the day and back into them at night. The algae are carried in and out of the shells by
the cytoplasmic streaming of the host cell. This daily rhythm exposes the algae to optimum light
during the day. At night the algae are brought close to the main body of the host, which facilitates
nutrient transfer.

Sponges
Marine sponges contain a variety of endosymbionts, including bacteria, dinoflagellates, diatoms,
and cryptomonads. The symbioses are especially common among tropical sponges. Many sponges
contain endosymbiotic cyanobacteria that are unicellular or filamentous. The cyanobacteria are
either intercellular, in sponge tissue directly below the outer surface, or within vacuoles inside the
host cells. The sponge obtains nutrients from the digestion of bacteria or from the excretion of
compounds such as glycerol and nitrogen from the bacteria ( Wilkinson, 1987).

Cyanobacterial symbionts are thought to shade sponge tissue and thus protect it from the
damaging effects of intense light. Sponges have not changed much since the Precambrian and may
be refuges for primitive kinds of algae and cyanobacteria ( Wilkinson, 1992).

Sea Anemones and Jellyfish

The sea anemone Anthopleura xanthogrammica contains two types of symbiotic algae:
zoochlorellae and zooxanthellae. The relative proportion of each type of algal symbiont in the
animal depends on the water temperature of the anemone's habitat. At high temperatures (26°C)
zooxanthellae are more common, and at low temperatures (12°C) zoochlorellae are predominant.
At intermediate temperatures the anemone has somewhat equal proportions of the symbionts.
Curiously, the zoochlorellae excrete only small amounts of fixed carbon. Since in some anemones
the zoochlorellae predominate, the algae must supply something other than carbon compounds,
possibly nitrogen and phosphorus, which stimulate the anemone to grow.

Cnidarians position themselves in ways to increase the exposure of their symbionts to light. For
example, when tentacles of Anthopleura pallida are relaxed and fully stretched, the algae in their
gastrodermal cells lie in a single layer and are exposed to maximum daylight. When the tentacles
contract, the gastrodermal cells shrink, and the algal cells lie on top of each other, the uppermost
cells shading the lower ones. Cultured algal cells isolated from Aiptasia developed thicker cell walls
that related to increased division rates. Algal cells in the anemone are surrounded by multiple host
membranes ( Palincsar et al., 1988).

Cassiopeia xamachana is a jellyfish that has been used to study how an invertebrate selects its
symbiotic algae. The life cycle of Cassiopeia includes a sexual medusoid stage, containing algae,
and an asexual polyp stage, which lacks algae. The polyps mature only after they establish an
association with S. microadriaticum and/or bacteria ( Hofmann and Brand, 1987). By exposing
polyps to different strains of algae, it is possible to determine which algae are able to colonize the
animal. Robert K. Trench and his co-workers (see Trench, 1993) found that although a Cassiopeia
polyp could phagocytize different strains of algae, only one strain would survive and form the
symbiosis. Thus, the host recognized its symbiont only after it was phagocytized. Cassiopeia
xamachana does not swim freely, but rather lies upside down on the sea floor, a behavioral
adaptation that allows the algae in its tentacles to receive maximum daylight for photosynthesis,
and gives the animal its common name, the upside-down jellyfish.

Reef-building Corals
The symbiotic association between algae and reef-building coral animals (Scleractinia) is of great
importance in tropical ecosystems and has been the subject of much study ( Roberts, 1993). Coral
reefs support large communities of organisms. Coral animals are similar to sea anemones, except
that their polyps are small, only 10mm in diameter, and they excrete a calcium carbonate shell
around their body. As the polyps die, their shells do not decay, and new polyps grow over them.
After many years of this process, coral reefs are formed. Symbiotic algae live inside nutrient-rich
cells of the digestive cavity of the coral polyp. In some corals more than 90% of photosynthate
may be released by the symbiont to its host cell ( Muscatine, 1990). Photosynthate release is one
of the most studied aspects of symbiosis but still is not fully understood. It may be triggered by the
action of coral digestive enzymes that increase the permeability of the algal cells.

Corals with symbiotic algae deposit calcium carbonate around their bodies much faster than
animals without algae. The algae stimulate calcification through their photosynthetic fixation of CO
2. Removal of CO 2 by the algae increases the overall reaction rates of the calcification process.
High calcium ATPase activity is also linked to coral growth ( Marshall, 1996). It is believed that
calcium ATPase helps convert carbonate into CO 2, which is used in photosynthesis.

The algae supply the coral with oxygen as well as with carbon and nitrogen compounds. The animal
obtains vitamins, trace elements, and other essential compounds from the digestion of plankton.
Animal waste products, such as ammonia, are converted by the algae to amino acids, which are
translocated to the animal. Such a recycling of nitrogen is an important feature in the nitrogen-
poor habitats of coral.

The vast reefs that are formed in tropical waters result largely from the stimulatory effect of the
zooxanthellae on the corals, which appear to be highly flexible in terms of the kinds of
zooxanthellae they can take in. The high productivity of coral reefs is noteworthy, considering that
they occur in shallow (less than 100 m deep) tropical waters that are low in nutrients. Pigments
produced by symbiotic corals protect both the host and the algae from harmful effects of ultraviolet
radiation. How symbiotic corals evolved is not known. Although all of the reef-building corals
contain symbiotic algae, many other types of corals do not form associations with algae.

Coral bleaching

Coral bleaching is caused by the loss of symbiotic algae from the host or the loss of algal pigments.
Bleaching may be caused by environmental stresses such as global warming, pollution, and
increased ultraviolet radiation. In some cases, small reservoirs of algae survive the bleaching and
can recolonize the coral ( Brown and Ogden, 1993; Buddemeier and Fautin, 1993).

Understanding coral immunology is important to appreciating the nature of coral diseases. One
model used to explain coral bleaching is that of the coral disease rapid tissue necrosis. A wide
variety of marine bacteria live within the coral mucus ( Ducklow and Mitchell, 1979), including
Vibrio alginolyticus and V. vulnificus, which can release endotoxins and proteases and degrade the
coral mucus ( Bigger, 1988). These bacteria appear to cause rapid tissue necrosis of corals (
Richardson, 1998). These and other bacterial species multiply rapidly under elevated temperatures.
Phagocytosis is the primary host defense in all invertebrates. Molecules analogous to vertebrate
antibodies have been found in cnidarians. In an evolutionary sense, coral bleaching may be viewed
as an opportunity to create a symbiotic reshuffle that produces new associations which may be
more tolerant of environmental stresses.

Convoluta roscoffensis: Green Flatworms

Convoluta roscoffensis (Turbellaria, Platyhelminthes) is a small, marine flatworm that lives in the
intertidal zones of beaches in the Channel Islands of the United Kingdom and in western France.
The worms are 2-4 mm long and deep green from the algae they contain; a large worm may
contain up to 40,000 algal cells. During high tide, the worms are buried in the sand, but at low
tide, during the daylight, they move up to the surface. During this time the algae photosynthesize
until the next high tide, when the worms burrow back into the sand. When the worms are on the
surface of the sand, they secrete a gelatinous substance that holds them together in colonies and
protects them from drying.

The algal symbiont of Convoluta roscoffensis is Tetraselmis convolutae, a motile, unicellular green
alga. The algae are ingested by digestive cells of the worm and become greatly modified. They lose
their cell walls and flagella and develop fingerlike lobes that fit between the animal's muscle cells
and greatly increase the surface area between the symbionts. The algal lobes are also near the
host's cilia, which, like muscle cells, have high energy demands. Because of these close contacts,
photosynthetic products of the alga pass directly to the most active regions of the animal. Despite
their irregular shapes, all the algal cells face the same direction. The anterior end, which normally
has four flagella, faces the inner part of the animal, while the lobed posterior end, which contains
the chloroplast, faces the outside. The algal cells are separated from the host cytoplasm by
membranes produced by the animal ( Douglas, 1988).

When the worms mature, they produce eggs that hatch into larvae, which lack algae and thus are
aposymbiotic. Free-living cells of T. convolutae are attracted to the egg cases of the worms
through an unidentified chemical stimulus. The algae swim to the egg cases and attach to them.
When female worms lay eggs, they also extrude algal cells and mucus that coats the eggs. Larvae
that hatch from the eggs ingest the algae, which then divide until their normal population in the
animal is reached.

After the worm matures and has its full complement of algae, it stops feeding and depends on the
algae for nutrients for the rest of its life. Aposymbiotic larvae that do not become infected with
algae will starve to death, regardless of how much food they ingest.

The physiological relationships between C. roscoffensis and its symbiotic algae have been
determined from various studies. The algae provide alanine, glutamate, fatty acids, and sterols to
the animal. In return, the algae use uric acid, a waste product of the animal, as a source of
nitrogen.

Convoluta roscoffensis can be infected with algae that are related to its normal symbiont. These
foreign algae undergo the same modifications as the natural symbiont, but the animal usually does
not grow as vigorously with these secondary symbionts. If an animal that is infected with a
secondary symbiont is exposed to cells of its natural, primary symbiont, it will discard or kill the
foreign alga and replace it with its natural one.

The Convoluta--alga symbiosis is an early example of detailed physiological studies conducted by

Keeble and Gamble during 1903-1907 that attracted public attention to the broader significance of
symbiosis in nature ( Keeble, 1910).

Tridacnid Clams: Climax Forests of Coral

Reefs
Among the molluscs, only tridacnid clams form symbioses with algae. These large clams grow
among coral reefs in shallow, tropical waters of the Indo-Pacific. Taxonomically, giant clams belong
to two Hippopus species and six Tridacna species. Tridacna gigas can weigh up to 1000 pounds and
produce shells more than 3 feet long ( Lucas, 1994). Like Cassiopeia, the clams have undergone a
behavioral adaptation, and they attach to the coral reef in such a way that their valves face upward
and are open. This position allows their symbiotic algae to receive optimum illumination.

Symbiodinium lives within a tube system which develops from a diverticulum duct of the stomach
that branches several times and finally terminates in the mantle. The tubules are thin and narrow
and allow close contact with the hemolymph that circulates through the mantle. Presence of the
tubules near the surface of the mantle allows Symbiodinium to capture sunlight ( Norton et al.,
1992). Young clams ingest free-living algae, which move from the alimentary tract to the mantle.
As with other associations, the symbiotic algae supply the clams with substantial amounts of
glycerol. Clams kept in the dark in nutrientrich waters die quickly, whereas those kept in sunlight
flourish.

Giant clam farming

Tridacna dersa and Tridacna gigas are two large and heavily harvested species in coral reef
environments of Indonesia, the Philippines, Micronesia, and southern Japan. The commercial
exploitation of these giant clams has caused a decline in their populations. By using farming
techniques of land-based nurseries in coral reef environments, scientists are exploring the
possibility of raising giant clams as a sustainable food source ( Heslinga and Fitt, 1987).

The clams reach sexual maturity in about 5 years and produce large quantities of sperm and eggs.
Spawning can be synchronized by using pheromones and environmental cues ( Knop, 1996). The
planktonic phase of a tridacnid life cycle lasts about a week, during which there are significant
losses from predation. The surviving larvae attach to corals and develop into juvenile clams, which
orient their mantles toward the sun. Only young clams can form symbioses with zooxanthellae.
Young clams up to about 3 years old may be eaten by reef carnivores that include crabs, fish,
snails, and octopuses. Adult tridacnids appear to be immune to predation ( Heslinga and Watson,
1985). Giant clams have been compared to climax forests because they are sedentary, grow
slowly, resist predation, and depend on photosynthesis.

The hatchery at the Micronesian Mariculture Demonstration Center in Palau produced more than
250,000 juvenile clams in 1985 for introduction on reefs of Guam, American Samoa, the
Philippines, and Hawaii ( Heslinga and Fitt, 1987). The successful culture of giant clams may
provide another experimental system to study the molecular biology of marine symbioses (
Yellowlees et al., 1993)

Sea Slugs: Animals that Express

Chloroplast Genes
An unusual type of marine relationship, which strictly speaking is not a symbiosis because it
involves only part of an organism, is that between sea slugs, such as Elysia sp., and the
chloroplasts of algae. Sea slugs ingest the large cells of siphonaceous, or tubular, algae, especially
Caulerpa and Codium, and the digestive cells of the animals phagocytize the algal cells. The
chloroplasts resist digestion and continue to function for several weeks or even months within the
host cells. The slugs contain so many chloroplasts that they appear green. A chloroplast may be
enclosed within a vacuole or lie free in the cytoplasm of the animal.

Chloroplasts photosynthesize inside the animal cell and release glucose, which the slug uses for
some of its nutrient needs. Functional chloroplasts are not digested by the animal, but when a
chloroplast stops photosynthesizing, it is quickly digested. How the animal cell distinguishes
between active and inactive chloroplasts is not clear. Chloroplasts cannot divide inside the animal
because they are unable to synthesize chlorophyll without the enzymes that are present in the
algal cytoplasm. A slug constantly replenishes its chloroplast supply by feeding on new algae. In E.
chlorotica some chloroplast proteins were synthesized in the host animal cell cytoplasm and
transferred to its chloroplasts, thereby extending their survival for up to 9 months ( Mujer et al.,
1996; Pierce et al., 1996).

The factors that cause chloroplasts to release glucose inside the slug's digestive cells are not
known. Chloroplasts that are removed from the animal stop excreting glucose or only excrete small
amounts. But if homogenates from symbiotic slugs are added to the isolated chloroplasts, they will
once again excrete glucose. This stimulation of excretion by unknown host factors is like that in
other marine symbioses.

In a similar relationship, some planktonic foraminiferans and other protozoans do not readily digest
the chloroplasts of diatoms and other algae on which they prey. These chloroplasts also continue to
photosynthesize while they are inside the protozoans ( Laval-Peuto, 1992).

Tunicates
Some tropical marine tunicates contain an unusual photosynthetic symbiont, Prochloron, that has
characteristics of both cyanobacteria and green algae ( Lewin and Cheng, 1989) ( fig. 10.2 ).
Prochloron cells are green, but their cell structure is prokaryotic; that is, the cell lacks a nucleus,
has a wall like that of cyanobacteria, and has organelles such as polyhedral bodies. The cells,
however, lack phycobiliproteins and phycobilisomes, which are universal in cyanobacteria. Like
green algae, Prochloron cells have chlorophylls a and b, whereas cyanobacteria have only
chlorophyll a. Prochloron has such a unique mix of characteristics that it has been placed into a
separate phylum, Prochlorophyta ( Matthijs et al., 1994).

All strains of Prochloron are unicellular and have spherical cells. In some tunicates the symbiont
cells lie within a cellulose matrix that surrounds the outer surface of the animal, whereas in other
tunicates the symbionts are loosely attached to the cloacal wall. Prochloron cells always have an
extracellular position in the animal.

The larvae of some tunicates have specialized pouches that carry Prochloron cells obtained from
the parent. As the tunicate develops, it is infected at an early stage by the symbiont. Thus, the
continuity between Prochloron and the tunicate is uninterrupted from one generation to the next.

Experimental studies on these associations are limited because Prochloron does not grow in
laboratory culture. All studies, therefore, must be carried out on symbionts newly squeezed out of
the tunicates or from dried or preserved specimens. Preliminary findings have revealed that the
symbiont photosynthesizes in the tunicate and releases carbon compounds. Presumably, these
compounds, as well as the oxygen produced during photosynthesis, are used by the animal.

Prochlorophytes have been found in symbiosis with marine invertebrates and they are also
dominant members of the phytoplankton of open oceans ( Chisholm et al., 1988; Mullineux, 1999).
They may have been common in earlier periods, such as the Upper Cambrian, when the low
concentrations of oxygen in the sea and atmosphere were more conducive for their free-living
growth. Prochlorophytes in marine invertebrates may represent relict populations, isolated
remnants of a once widespread group.
10.2 ALGAE AND FRESHWATER
INVERTEBRATES
Many freshwater invertebrates contain symbiotic Chlorella (Chlorophyceae) ( table 10.1 ). The
algae are intracellular inhabitants of host cells that are capable of endocytosis. The algae are
enclosed within perialgal vacuoles, which protect them from the host's digestive enzymes.
Symbioses range from temporary, as in Coleps, to complex hereditary associations, as in
Paramecium bursaria. In R. bursaria, growth rates of both the host and symbiont are coordinated.
The Chlorella symbionts of these different invertebrates appear similar, although some algal strains
excrete glucose instead of maltose.

The presence of similar strains of Chlorella in unrelated invertebrates suggests that these
associations developed independently on different occasions. The associations probably began
when cells of Chlorella were ingested by invertebrates and resisted not only the host's attempts to
eject them but also the digestive enzymes of the host. Once the algae became permanent
residents in the animal cell, relationships between the symbionts evolved. Why some species of
invertebrates were more susceptible to algal infection than others is not known. For example, there
are species of Hydra and Paramecium that resist colonization by algae.

Paramecium bursaria: green paramecia

Paramecium bursaria is one of the best studied freshwater symbiosis because both the paramecia
and algal cells are easily maintained under laboratory conditions ( Reisser, 1992b, 1993). Each
algal cell is contained inside a perialgal vacuole (or symbiosome) that is formed by the host ( Meier
and Wiessner, 1989) ( fig. 10.3 ). The alga somehow changes the vacuole so that it does not fuse
with lysosomes, and the alga therefore avoids being digested. When an algal cell divides, each of
its four daughter cells becomes enclosed in its own vacuole. After the algal cells are ingested, they
move from the center of the animal cell to the periphery. Paramecia normally feed on bacteria, and
when the food supply is low, those animals with algae live longer than those with none. When food
is plentiful, however, both types of paramecia multiply at the same rate. Only certain strains of
Chlorella sp. form symbioses with R. bursaria. All other Chlorella-like algal species do not trigger
the formation of perialgal vacuoles and are digested in food vacuoles. Chlorella symbionts may use
a lectinlike mechanism to become enclosed within the perialgal vacuole of the host ( Reisser,
1992a).

Algae of R. bursaria excrete large quantities

Fig. 10.2 Prochloron, a photosynthetic symbiont of tropical marine tunicate, Didemnum midori. (a)
Scanning electron micrograph of Prochloron in the host tissue. (b) An electron micrograph of
Prochloron showing cellular details of organized chloroplast and the absence of nucleus. (c) Details
of the stalked thylakoids with concentric regions of stroma that contain carboxysomes, large
crystalloids and dense granules. (a Courtesy of Ralph A. Lewin, Scripps Institution of
Oceanography, University of California, San Diego; b and c Courtesy of Hewson Swift, University of
Chicago).

of maltose and glucose and receive ammonia and glutamine as nitrogen from the host ( Reisser,
1993). About 45% of the total photosynthetically fixed carbon is released by the algal symbiont.
The excretion is not triggered by any host factors. The amount and type of substances that are
excreted is pH dependent, and with increasing acidity, more sugars are released. At alkaline pH,
amino acids such as alanine are excreted. The excretion of metabolites from symbiotic Chlorella is
an altruistic behavior which is not seen in free-living Chlorella.

When fed bacteria in the dark, paramecia divide more rapidly than their symbionts, and
aposymbiotic paramecia develop. Aposymbiotic strains can be reinfected by different algae,
including free-living strains, but only those that have been removed from a previous symbiosis
grow well in the animal. During the establishment of the symbiosis, algae in the perialgal vacuoles
divide slowly until there is a population of about 600-1000 algal cells in each host cell.
Hydra-Chlorella Symbiosis

Hydra is a common inhabitant of freshwater ponds and lakes. It remains attached to living or
decaying vegetation and feeds on protists and on small animals such as shrimp. Some species of
Hydra contain the green alga Chlorella ( fig. 10.4 ). The algae divide asexually, by mitosis, and
each mother cell produces four daughter cells. The animal appears green because its cells are
packed with algae. The animals obtain nutrients from the algae and can live in areas where food is
scarce and survive periods when the external food supply is low. The animals ingest algal cells, as
they do other particles of food. Algae that become symbiotic escape digestion and are retained
inside vacuoles. When the animal divides, some of the algal cells are transmitted to the offspring,
so that every individual in the animal population contains algal cells ( Muscatine and McNeil, 1989).

Green hydras live in the same habitats as brown hydras. When the natural food supply is
abundant, both types of Hydra multiply at about the same rate. When the external food supply is
scarce, however, green hydras, because of their symbiotic algae, have an advantage over brown
hydras.

Hydra viridis is a common species of green hydra. Several strains of this species have been
established in laboratories throughout

Fig. 10.2 (continued)

the world. These strains have been studied in terms of the relationships between the algae
and their animal hosts. Hydra grows well in laboratory cultures. They are fed brine shrimp,
kept in the light to allow the algae to photosynthesize, and maintained in clean, bacteria-free
water. Under these conditions a hydra population doubles by asexual budding about every 2
days.

When H. viridis is exposed to high light intensity in the presence of 3-(3,4-dichlorophenyl)-

1,1-dimethylurea, a chemical that in-
Carbohydrate
Host
Released
Protozoa
Amoeba
Acanthocystis turfacea Maltose
Amphitrema flavum
Chaos zoochlorellae
Diffugia oblonga
Heleopara sphagni
Hyalosphenia papilio
Mayorelia viridis

Ciliates
Acazyophyra sp.
Glucose,
Climacostomum virens
fructose, Xylose
Coleps hirtus Xylose
Disematostoma butschlii
Fructose,
Euplotes daidaleos
xylose
 Frontonia leucas
Frontonia vernalis
Halteria bifurcata
Holosticha viridis
Malacophrys sphagni
Ophzydium versatile
Glucose,
Paramecium bursaria maltose,
trehalose
Prorodon viridis
Prorodon ovum
Spirostomum viridis
Stentor polymorphus Maltose
Stentor niger
Stentor roeseli
Teutophrys trisulca
Vorticella sp.

Porifera
Ephydatia fluviatilis Glucose
Heteromeyenia sp.
Spongilla lacustris

Cnidaria
Hydra virdis Maltose
Hydra magnipapillataa

Platyhelminthe
s
Turbellarians
Castrada viridis
Dalyellia vizidis
Phaenocora typhlops
Tyrphloplana viridata

Mollusca
Bivalves
Anodonta sp.
Linnaea sp.
Unio pictorum
Adapted from Reisser ( 1992b).
a
Algal symbiont: Symbiococcum hydrae

hibits photosynthesis, the animal loses its green algae and becomes aposymbiotic or bleached. A
bleached hydra will survive and grow if it has sufficient food. Chlorella symbionts from several
strains of H. viridis have been grown separately from the animal. The cultured symbionts differ
from the symbiotic forms in that they excrete smaller amounts of photosynthetic products. When
the algae are placed back into the animal, they once again excrete compounds at the rate of
symbiotic forms. If provided with suitable algae, bleached Hydra will ingest them and become
green again ( Rahat, 1992). A foreign species of Chlorella was shown to infect bleached H. viridis,
but the animal did not multiply as rapidly as normal strains.
Reinfection of aposymbiotic H. viridis occurs through five stages. During the contact stage, the
algae attach to the membrane of a hydra cell. During engulfment, the animal cell ingests the algae
by means of membrane projections, or microvilli. The animal cell takes in anything that contacts it,
including latex spheres and foreign algae. In the recognition stage, by means of an unknown
process, the digestive cells recognize potentially symbiotic algae. Several factors may be important
in the recognition process, including antigens and surface charges on the algal cells and the type of
microvilli produced by the animal cell during the engulfment stage. Algae that the cell rejects are
expelled from the cell; acceptable algae are retained and surrounded by vacuoles. In the migration
stage, algae that are accepted by the animal are moved to the base of the digestive cell. Because
the algal cells are nonmotile, their movement is controlled by the animal cell, possibly by means of
microtubules. Finally, during the repopulation stage, algal cells divide asexually until the normal
algal population of an animal cell is reached. Further division of the algal cells is closely linked to
that of the host cell. When the host cell divides, so do the algal cells.

A single hydra contains about 150,000 al- gal cells as well as numerous bacteria that occur in
vacuoles with or without algae. The bacteria may help the algae take up and store phosphate. The
number of algal cells in each hydra cell varies depending on the location of the digestive cell within
the animal. In the central region of the animal, each digestive cell has about 20 algal cells, and in
the head and tentacles each cell has about 10 algae.

Algal and bacterial symbionts are transmitted to hydra progeny during asexual and sexual
reproduction of the animal. Cells of the symbionts are attached to the outer surface of the eggs
and are ingested by the young hydra when they emerge from the eggs. In some hydra strains
extensions of endodermal cells carry algae into the developing eggs.

Under normal conditions symbiotic algae are not digested by hydra. There are two main reasons for
this. First, cell walls of the algae contain sporopollenin, a compound that resists digestive enzymes.
Second, vacuoles containing algae do not fuse with lysosomes, organelles that contain digestive
enzymes and normally fuse with food particles the animal ingests. But if a digestive cell takes in
more algal cells than its normal algal population, the extra cells are either digested or ejected.

A bilateral movement of nutrients takes place between the symbionts of H. viridis. The algae
supply the animal with photosynthetic products such as maltose ( McAuley et al., 1996). At pH 4.5,
almost 60% of the carbon fixed by the algae is excreted as maltose, but at neutral pH very little
maltose is excreted. It is not known whether the host can change the pH of its cytoplasm to control
maltose release by the algae. The animal rapidly hydrolyzes maltose to glucose and forms the
storage compound glycogen. Algae also provide the animal with oxygen, which they produce during
photosynthesis. The animal provides the algae with nutrients, including precursors of proteins and
nucleic acids. These compounds and others that are breakdown products from the hydra's food
enable the algae to survive if the animals are kept in darkness.

The Hydra-Chlorella symbiosis appears to be a finely tuned, nonpathogenic equilibrium between

the way the symbionts avoid digestion and the ability of host cells to regulate algal reproduction (
Muscatine and McNeil, 1989).

10.3. PARASITIC ALGAE

Pathogenic Green Algae
Cephaleuros is a common pathogen of vascular plants in tropical and subtropical regions. It is a
member of the family Trentepohliaceae, which also includes the familiar epiphytic alga
Trentepohlia. There are about a dozen species of Cephaleuros, the most common one being C.
virescens ( Chapman and Waters, 1992).

Cephaleuros causes red rust disease on commercial plants such as citrus fruits, coffee, and tea.
The alga forms a flat, disclike orange thallus, 5-10mm in diameter, that is usually subcuticular in
leaves and has hairlike filaments that break through the cuticle and have a velvety appearance;
the alga also grows on fruits and stems. The damage caused by Cephaleuros depends on the type
of host and its condition, the site of infection, the season, and the number of algal thalli on the
plant. Algae that penetrate leaves often kill the host cells that lie beneath the thallus, either by
shading them or by toxic substances they release. The infection does not extend beyond the algal
thallus, however, and the overall damage to the plant is slight. In leaves of the coffee plant, the
infected host produces thick-walled cells that form a barrier between the parasite and healthy leaf
tissue. Algae may girdle young stems and kill the plants. Infection of fruits such as guava and
mango reduces their attractiveness and marketability.

Cephaleuros does not penetrate the host cells, regardless of the extent of the infection. The alga
obtains water and minerals from the host and a substrate on which to grow. It also changes the
carbon and nitrogen metabolism of the infected leaves, but whether it receives organic compounds
from the host is not known.

Cephaleuros produces motile zoospores that spread the infection. The zoospores swim along the
leaf surface or are washed by rain onto other leaves, and when they come to rest, they divide and
form a thallus, which penetrates the host epidermis.

An interesting aspect of Cephaleuros is that it may be parasitized by a fungus to form a lichen. The
fungus forms haustoria inside the living algal cells and eventually kills them. This type of host-
parasite relationship is an example of hyperparasitism. The most common lichen formed by a
fungus-Cephaleuros association is the genus Strigula, which occurs on leaf surfaces throughout the
tropics. Lichenized and nonlichenized Cephaleuros may occupy the same habitats, but the
lichenized form does not harm the leaves and grows more slowly than the unlichenized form.

Several parasitic green algae, not as common as Cephaleuros, cause limited damage to their hosts.
Chlorochytrium is an endophyte of aquatic plants, such as duckweed, and also of several mosses.
Rhodochytrium occurs in various angiosperms, such as milkweed and ragweed. Rhodochytrium
lacks chlorophyll, and when first discovered it was mistaken for a primitive fungus.

Parasitic Red Algae

The phylum Rhodophyta consists of about 6000 species of mostly marine organisms with complex
life cycles. More than 100 species, within 50 genera, of red algae parasitize other red algae. About
80% of the parasites occur on closely related species (i.e., ones in the same family or tribe)
whereas others parasitize unrelated hosts. Parasitic red algae occur only in the subclass
Florideophyceae, especially in the orders Ceramiales and Cryptonemiales, and not in the more
primitive subclass Bangiophyceae.

Parasitic red algae are small (0.2mm to 4cm long), colorless or with reduced pigments, and host
specific. They attack only one or two genera, to which they are closely related, and they penetrate
the host tissue by means of rhizoids. Some parasites consist only of a rhizoidal system and do not
form a vegetative thallus external to the host. Some parasites complete their life cycle 3-5 weeks
after their spores germinate on the host; other parasites take about 18 weeks to complete their life
cycle.

Red algal parasites generally do not cause disease and thus are not considered pathogens. Their
parasitic nature, however, has been confirmed from radioisotope studies, which have demonstrated
movement of organic compounds from the host to the parasite. For example, in one study, a
segment of the red alga Gracilaria, with its attached parasite Holmsella, was placed in sea water
that contained 14 C-labeled bicarbonate and allowed to photosynthesize for several hours. The
segment was removed from the labeled solution, washed, and either separated immediately into
host and parasite plants or allowed to photosynthesize for several more hours in an unlabeled
solution, before host and parasite were separated and analyzed for radioactive compounds. The
results of this study clearly showed that the amount of radioactivity in the parasite increased with
time, indicating a movement of compounds. The compound that moved from host to parasite was
the alcohol glycoside floridoside. When this compound reached the parasite, it was converted to
mannitol and floridean starch. Similar results were obtained with the alga Polysiphonia lanosa and
its parasite Choreocolax polysiphoniae ( Goff, 1983). In this case, sodium mannoglycerate, another
alcohol glycoside, was translocated to the parasite and converted to an unidentified sugar. Thalli of
Choreocolax that were detached from their host fixed CO 2 more rapidly than thalli that remained
on the host. This finding suggested that the host inhibited photosynthesis by the parasite.
Morphological relationships between parasitic red algae and their hosts vary considerably. Some
parasites attach to the host by only one or two short rhizoids, whereas others develop extensive
rhizoids in the host tissue. Parasites that penetrate deeply usually damage the host cells (e.g., they
become plasmolyzed), but such damage remains localized. Many parasites are linked with host
cells by means of pit connections. When pit connections develop, nuclei of the parasite are
transferred to the host cell. The red alga Leachiella pacifica inserted some of its nuclei into the cells
of its host, Polysiphonia confusa. The foreign nuclei caused the host cell to enlarge to 20 times its
original size and to develop a thick cell wall. The number of host nuclei, mitochondria, and
chloroplasts increased, as did the DNA content of the host nuclei; products of photosynthesis also
accumulated in the cell ( Goff et al., 1996).

How parasitic red algae evolved is a matter of speculation ( Goff et al., 1996). One hypothesis is
that they arose when mutated spores of a red alga produced small gametophytes that were unable
to live independently but had the ability to penetrate cells of the parent plant. There are present-
day red algae whose tetraspores germinate and give rise to dwarf gametophytes that attach to the
parent plant. The gametophytes form rhizoids and develop pit connections with the parent cells. A
second hypothesis is that the algae were originally epiphytes that became parasites. The first
hypothesis seems more plausible because of the close taxonomic relationships that exist between
parasitic red algae and their hosts.

Some scientists believe that ascomycetous fungi may have evolved from parasitic red algae that
lost their plastids. Ascomycetes and red algae have many common morphological and cytological
features.

10.4 SUMMARY
Symbiotic algae live in the cells of many marine invertebrates. The algae excrete glycerol and other
compounds that the host uses to produce lipids and proteins; waste products of the host are used
by the algae. Anthopleura xanthogrammica, a sea anemone, has two types of algae, zooxanthellae
and zoochlorellae; the former is more prevalent when the anemone grows in warm waters and the
latter is more common in anemones found in cold waters. The life cycle of the upside-down
jellyfish, Cassiopeia xamachana, cannot be completed until its asexual polyp stage becomes
infected with algae. Symbiotic algae greatly enhance the activities of reef-building corals, tridacnid
clams, foraminiferans, and radiolarians. A single marine flatworm, Convoluta roscoffensis, may
contain up to 40,000 unicellular green algal cells. The algae live inside the digestive cells of the
worm and are greatly modified. A mature worm is completely dependent on the algae for food.
Larvae of the worm are aposymbiotic and must be infected with algae in order to develop further.
Giant clams are being cultivated as a source of food. Marine sponges contain symbiotic
cyanobacteria. Sea slugs obtain glucose from chloroplasts of algae they ingest. Prochloron occurs in
tropical marine invertebrates and has characteristics of both green algae and cyanobacteria. Hydra
viridis and Paramecium bursaria contain symbiotic algae that belong to the genus Chlorella.
Infection of H. viridis involves different stages of contact, engulfment, recognition, migration, and
re- population. Several green algae are parasitic on vascular plants, and many small red algae
parasitize other red algae.

Symbioses between algae and invertebrates involve common features such as integration of the
symbionts, regulation of the algal population, specificity, bilateral nutrient exchange, and
recognition mechanisms. Integration of a symbiont into a host generally results in reduction of
parts, thin walls, loss of sexuality, and slow growth. Algal populations are regulated by the host by
several mechanisms, including digestion or ejection of surplus algae and inhibition of symbiont cell
division. Anemones and corals eject surplus algae, and in other symbioses algal division is
restricted. Studies of recognition systems between symbiont and host in different associations have
not revealed a common pattern, a finding that is consistent with the different lines of evolutionary
development among symbioses.

11 ANIMAL PARASITISM
Flukes, Tapeworms, Nematodes, and Parasitoids
 Scientists estimate that up to 50% of all animal species are parasitic symbionts. Some phyla such
as the Platyhelminthes, Nematoda, and Arthropoda contain large numbers of parasitic species.
Hosts and parasites have evolved together and under natural conditions many have become
mutually tolerant. Host organisms can live independently, but, in many cases, the parasite's
association with its host is obligatory ( Price, 1980; Jenzen, 1988; Thompson, 1994).Animal
parasites affect the health of humans and domesticated animals throughout the world. In most
warm climates parasitic infections from flukes, nematodes, and arthropods greatly diminish the
quality of life. Scientists estimate that three out of four people in the world are infected with
parasites, and many people carry multiple infections. The prevalence of parasitic diseases is
extremely high among people who are poor and illiterate ( Evans and Jamison, 1994). Because of
public health concerns, the science of parasitology continues to attract public support to study
host--parasite relationships and to develop effective strategies to control and cure parasitic
infections. In this chapter, we examine symbioses that involve flukes, tapeworms, nematodes,
and arthropods. We describe the morphological adaptations of parasites, how the host defends
itself against parasitic invasion, how parasites evade a host's defenses, and coevolution of
parasites and hosts.Helminths are widely distributed parasites of plants and vertebrates and are
surpassed only by insects in the variety and diversity of hosts that they parasitize. There are
more than 100,000 species of helminthic parasites, but only a relatively few species are of
economic and public health concern. Infections caused by helminths such as Schistosoma,
hookworms, and filarial nematodes are the major causes of sickness in humans inhabiting
tropical regions ( Warren, 1988). Several species of nematodes are parasites of vegetables,
fruits, and other crop plants. Some free-living nematodes, such as Caenorhabditis elegans, are
being used to study metazoan genetics and developmental biology because of the ease with
which they can be cultured and analyzed biochemically.Helminths have complex life cycles. They
live for a long time within the host animals, and they often possess a remarkable ability to evade
the host's defense mechanisms. The prevalence of helminthic infections in some areas is high;
however, only a few either very young or old hosts develop disease. Helminths do not multiply in
humans, and therefore the severity of the disease depends on the extent of the original infection.
Some helminths may accumulate after repeated infections of a host.
11.1. CLASSIFICATION
We have included in this chapter a broad classification of helminthic symbionts:
Phylum Platyhelminthes (flatworms)
Class Trematoda (flukes): Clonorchis, Diplozoon, Fasciola, Paragonimus, Polystoma,
and Schistosoma.
Class Cestoda (tapeworms):
Diphyllobothrium, Echinococcus, Hymenolepis, Lingula,

-166

Schistocephalus, Spirometra, Taenia, and Taeniarhynchus.

Phylum Nematoda (roundworms) Human and vertebrate parasites: Ancylostoma, Ascaris,
Brugia, Dirofilaria, Dracunculus, Haemonchus, Litomosoides, Necator, Onchocerca, Trichinella,
and Wuchereria.
Insect parasites: Deladenus, Mermis, Neoaplectana, Romanomermis, and Sphaerularia.
Plant parasites: Anguina, Bursaphalenchus, Globodera, Heterodera, and Meloidogyne.

11.2. TREMATODES: THE FLUKES

Some Fluke Symbioses
Adult flukes are obligate endoparasites of vertebrates and include the liver fluke, the lung fluke,
and the blood fluke of humans. After sexual reproduction, the female fluke produces eggs that exit
through the genital pore into the host environment and then are passed out of the host with the
feces or urine. There are several larval stages, which multiply asexually in snails serving as the
intermediate host. A larval stage (cercaria) with a characteristic tail emerges from a snail and
either penetrates a vertebrate host immediately or encysts and attaches to grass blades, which
may be ingested by a herbivorous vertebrate ( fig. 11. 1 ).
Polystoma integerrimum
Polystoma integerrimum inhabits the urinary bladder of frogs throughout Europe and North
America. Up to 50% of the frogs in the United States carry the fluke. The life cycle of the fluke is
unusual in that its maturation is synchronized with the sexual maturation

of its host. When the frog spawns, the flukes become sexually mature and release their eggs. The
synchronized cycles enable the parasite to produce larvae at a time when tadpoles are abundant.
The fluke larvae attach to the tadpole gills and feed on mucus and other materials. The mature
larvae migrate from the gills to the bladder. Experimental evidence shows that fluke maturation is
regulated by host hormones. Pituitary extracts injected into an immature frog induce sexual
maturation of the parasite. The exact nature of the hormonal control of fluke development is not
known. Sexual maturity of the protozoan Opalina is also synchronized with reproduction of its frog
host ( Smyth, 1994).

Diplozoon paradoxum
Diplozoon paradoxum is a parasite of freshwater fish and has a unique life cycle that involves
permanent fusions of pairs of flukes. Such fusions raise interesting questions about the
biochemical, genetic, and immunological compatibility of the fused individuals. The attachments
begin when the larvae pair and fuse with their posterior suckers. The reproductive openings of each
worm exit on the opposite end of their partner in order to permit cross-fertilization. Larvae that do
not form pairs die before becoming sexually mature. In D. nipponicum, the reproductive cycle is
synchronous with that of the fish host ( Smyth, 1994).

Fasciola hepatica
Fasciola hepatica, the sheep liver fluke, commonly inhabits the bile duct, liver, or gall bladder of
cattle, horses, pigs, and other farm animals. The parasite's life cycle, ultrastructure, nutrition, and
biochemistry have been studied extensively. Upon hatching from metacercariae, young flukes
penetrate the intestinal wall, move through the body cavity, and enter the liver. During their
migration, the young flukes feed on muscle, intestinal, and liver parenchyma cells. When the
parasites reach the bile duct, they feed on epithelial cells, blood, and liver tissue. The fluke also
absorbs amino acids, glucose, and fatty acids through its tegument. Epithelial cells of the bile duct
become hyperplastic during the fluke's infection. The fluke somehow stimulates production of the
host tissue on which it feeds ( Roberts and Janovy, 1996).

Clonorchis sinensis
The Chinese liver fluke, Clonorchis sinensis, is an important parasite of humans and other fish-
eating mammals in Far Eastern countries. The fluke makes its way to the liver and bile duct directly
from the alimentary canal and feeds on epithelial and blood cells. In endemic areas, the prevalence
of fluke infection may range from 14-80%, and the number of flukes in an infected individual varies
from 20 to 200. Adult flukes can live for more than 8 years in humans. Fish become infected with
larvae that are liberated from a snail host. People acquire these flukes by eating uncooked or
smoked fish whose muscle contains metacercariae of the parasite. Fish farming in East Asia is a
major source of fluke infections in people. In other areas, dogs and cats serve as reservoir hosts of
C. sinensis ( Komiya, 1966).

Paragonimus westermani
Species of Paragonimus inhabit different organs of vertebrate hosts and are excellent examples of
zoonosis. Paragonimus westermani, the lung fluke, infects humans as well as cats, dogs, and rats.
In humans the flukes become encapsulated in the lung tissue and produce eggs, which pass
upward into the trachea to the mouth and then down the intestinal tract to exit with the feces. This
fluke is extremely prevalent in the people of China, the Philippines, Thailand, and other Asian
countries. Humans become infected with the fluke by eating raw crabs and crayfish. Crab juice is
frequently used to prepare food in Korea and the Philippines.

Schistosoma
Next to malaria, schistosomiasis is the most important parasitic disease in the world, affecting
more than 200 million people in more than 75 countries ( Webbe, 1981). Schistosomes are blood
flukes, and they reside in the mesenteric blood vessels of humans. Adult schistosomes have a
unique morphology and physiology. Male and female flukes are elongated and wormlike, and the
female fluke is held permanently in the ventral groove of the male fluke ( fig. 11.2 ). The presence
of eggs of blood flukes in various host tissues triggers an immunological response, causing the
affected person to show disease symptoms that include enlargement of the liver and spleen,

bladder calcification, deformity of the ureter, and kidney disorders. Schistosomiasis is common
among poor people, especially farmers who live and work under unsanitary conditions, and is
increasingly being recognized as an anthropogenic disease. The development of irrigation projects
increases the range of the snail species that are the intermediate hosts of Schistosoma.

Three important blood flukes that infect humans are Schistosoma mansoni, S. japonicum, and S.
haematobium. Schistosoma mansoni causes intestinal bilharziasis in South America, Central
America, and the Middle East and has been studied extensively by scientists because of the ease
with which it grows in laboratory mice, hamsters, and rats. Schistosoma japonicum affects millions
of people in Asia and is also prevalent among rats, pigs, dogs, cattle, and horses. Urinary
schistosomiasis is caused by S. haematobium and occurs in about 40 million people in Africa and
the Middle East. It is a disease of young adults between the ages of 10 and 30 years, and rarely
occurs in older persons. Scientists in the Sudan have noted a correlation between bladder cancer
and urinary schistosomiasis. Research in the development of an effective vaccine in the control of
schistosomiasis is making significant progress ( Cherfas, 1991; Bergquist and Colley, 1998).

Fluke--Host Relationships
Studies of the biology of host--parasite relationships of flukes have focused on structure and
function of the body wall (tegument), nature of the host's immune response to the fluke, and
evasion of the host's immune mechanism by the parasite. On penetration, the cercaria's tegument
undergoes profound changes in structure and physiology.

Until the early 1960s, it was believed that all flatworms were covered with a cuticle, a tough, inert
structure. Ultrastructure of the fluke body covering, however, has revealed it to be a unique and
biologically active structure. The term tegument is now applied to the outer covering of flatworms
instead of cuticle, which is characteristic of roundworms.

The tegument is composed of (1) an outer region, the distal cytoplasm, which consists of
cytoplasmic extensions of tegumental cells forming a syncytium, and (2) an inner proximal
cytoplasm, which consists of tegumentary cells ( fig. 11.3A ). The tegument is bound externally
and internally by a plasma membrane. The tegumentary matrix contains many mitochondria,
ribosomes, vacuoles, endoplasmic reticulum, and Golgi bodies and is capable of nutrient uptake,
osmoregulation, and excretion and can support various sensory structures. The tegument contains
secretary products believed to have an immunological role in protecting the parasite from host
digestive enzymes. The tegument is also covered with a carbohydrate--protein complex, the
glycocalyx, which is continuously produced by the tegument ( McLaren and Hockley, 1976).

The free surface of the tegument is folded into ridges or fingerlike projections. In blood flukes the
tegument has an elaborate network of channels that open to the outside. Although flukes have a
well-developed digestive system, the tegumentary uptake of nutrients is significant. In vitro studies
have shown that small molecules are absorbed through the fluke tegument, and large nutrient
molecules are taken in by pinocytosis ( Wakelin, 1996).
The tegument of the adult Schistosoma is unusual because it does not have a glycocalyx. Scanning
electron microscopic observations have shown that the tegument of the male schistosome has an
irregular, spiny surface, whereas that of the female is mostly smooth with spines concentrated at
the tail end. When larvae of Schistosoma mansoni emerge from the intermediate snail host, they
are surrounded by a glycocalyx ( fig. 11.4 ). Immediately after penetration of a vertebrate host,
the larvae become transformed into the schistosomulum. The larval surface membrane and
associated glycocalyx are replaced by mem-

A FLUKE BTAPEWORM Fig. 11.3 Ultrastructure of the helminthic tegument. (A) Fluke tegument with
the outer surface covered with a glycocalyx. (B) Tapeworm tegument with characteristic
microtriches and glycocalyx.

-1

Fig. 11.4 Tegumental changes in Schistosoma mansoni. (A) Schematic representation of outer
surface changes in cercaria following penetration of host skin. Note relative amounts of glycocalyx
on cercaria, schistosomulum, and adult. Adult worms have a characteristic multilayered membrane
system. (B) Electron micrograph of the fibrillar nature of the glycocalyx of a schistosome. (C)
Electron micrograph of an adult schistosome and the three membrane outer covering (B and C:
Dianne J. McLarren, National Institute for Medical Research, London). A

branes derived from the host and membranous inclusions that are formed near the surface of the
larval tegument within minutes of skin penetration ( McLaren, 1989) ( fig. 11.4 ).

The fluke migrates from the skin to the lungs and then to the liver, and in the process loses its
tegumental spines. The lung stage of the fluke is particularly resistant to the host's hormonal and
cellular immune mechanisms. The schistosomulum changes or masks its original surface antigens
and thus does not trigger the host's immune responses. In 1964 Raymond Damian, a pioneer in
parasitic immunology, proposed the concept of molecular mimicry to describe how the parasitic
symbiont has surface antigens similar to those of its host ( Damian, 1964). These antigens disguise
the parasite against the host's immune response. The question of whether the hostlike antigens are
synthesized by the fluke or obtained from host cells has been a subject of intense research.
Evidence has confirmed that schistosomula can acquire A, B, and H bloodgroup antigens from the
host's red blood cells, along with glycoproteins of the major histocompatibility complex. These
antigens and others required by the symbiont seem to play a central role in the phenomenon of
molecular mimicry ( Damian, 1987; 1989).

Although the first parasites successfully evade the host's immune responses, they induce the host
to reject all further parasites of the same kind. This phenomenon is called concomitant immunity (
Hagan and Wilkins, 1993).

Adults of Fasciola, like Schistosoma, can live in the host's bile duct for long periods of time, and
mechanisms of molecular mimicry fail to explain how the liver fluke evades the host's antibodies.
Scientists have suggested that Fasciola periodically sloughs off its glycocalyx along with associated
host antibodies and replaces it with a new coat ( Coles, 1984).

11.3 CESTODES: THE TAPEWORMS

Tapeworms represent the ultimate in biological adaptation to live in another organism. All
tapeworms are obligate symbionts of vertebrates and arthropods. The adult tapeworm body

consists of a head, the scolex, which

Fig. 11.4 (continued)

may possess anchoring devices such as suckers and hooks, a neck or zone of proliferation, and a
body made up of segments called proglottids that may number from 3 to 3000. Sexually mature
tapeworms live in the small intestines of vertebrates; their larval stages develop in the visceral
organs of an alternate host, which may be a vertebrate or an arthropod. The development of larval
stages in the muscles and nervous tissue of the vertebrate host produces serious disease. Some
scientists view the adult tapeworms in the alimentary canal as endocommensals living in a
nutrient-rich environment. Adult tapeworms are well known for their rapid growth and production
of large numbers of eggs.

The extent of the disease in the host depends on the number of larvae involved, their survival, and
the host's immune response. Infected individuals rapidly become resistant to further infection, and
this immunity is passive and can be transferred to healthy hosts. Scientists have noted the
similarity between the antigens of various tapeworm species. Scientists hope to collect
immunogens for developing vaccines from a nonpathogenic tapeworm such as Taenia hydatigena,
which is easily maintained and propagated in the laboratory.

There is increasing evidence that the establishment and survival of adult tapeworms in the host
intestine is immunologically con- trolled. The crowding effect, often noted in tapeworm infections,
means that the size of individual parasites is inversely related to the number of parasites in the
host. Solitary tapeworms are commonly found in humans, for reasons that are not clear. It is
possible that an established tapeworm secretes substances that inhibit the development of other
tapeworms. Alternatively, the host may tolerate only a certain amount of parasitic load before its
immunological mechanism becomes operative. Scientists believe that the competition for
carbohydrates and nucleotides plays an important role in producing the crowding effect ( Zavars
and Roberts, 1985).

The Tapeworm Tegument

Tapeworms lack a digestive system and absorb all their nutrients through their tegument, which is
remarkably similar to that of the flukes. A unique feature of the tapeworm tegument are minute
projections, called microtriches, that cover the outer surface ( fig. 11.3B ). Microtriches are points
of attachment for the tapeworm in the host intestines, and they significantly increase the
absorptive surface of the tegument. The glycocalyx of the tapeworm tegument enhances the host's
amylase activities while inhibiting the host's production of trypsin, chymotrypsin, and pancreatic
lipase. The glycocalyx protects the tapeworm from the host's digestive enzymes. Both the larvae
and adult tapeworms live in an environment rich in amino acids, fatty acids, glycerols, acetates,
and nucleotides. These molecules are absorbed by diffusion and active transport, which take place
at specific sites on the tegument ( Threadgold, 1984).

Some Tapeworm Symbioses

Diphyllobothrium latum: the fish tapeworm

The adult form of the fish tapeworm is a common inhabitant of the alimentary canal of fish-eating
mammals, birds, and fish. In temperate climates, people who eat fish often carry D. latum in their
small intestine. The fish tapeworm is well known for its ability to absorb vitamin B 12, thereby
causing the host to be deficient in a vitamin that is essential for the development of red blood cells.
As a rule, frogs, fish, or snakes are the intermediate hosts of this tapeworm. Inhabitants of
southeast Pacific Islands often use crushed frogs to prepare food and in this way acquire the
tapeworm larvae. Humans infected with adult tapeworms pass eggs in the feces. Ciliated larvae
(coracidia) emerge from the eggs and infect animals such as water fleas, in which the larvae
become transformed into procercoid larvae. Fish acquire the tapeworm by eating water fleas. The
tapeworm larva penetrates the alimentary canal of the fish and develops into the final larval stage,
the plerocercoid, in the fish musculature.

Diphyllobothrium (Spirometra) mansonoides: the

beneficial parasite
The adult Spirometra mansonoides occurs in the alimentary canals of dogs and cats. From eggs
passed in host feces, larvae hatch and develop in water fleas. Frogs become infected with the larval
stage by eating water fleas. Mice that eat frogs become infected with the final larval stage of the
tapeworm. Scientists have observed that infected mice showed increased growth and efficiency of
nutrient uptake. J. F. Mueller ( 1974) considers D. mansonoides to be a beneficial parasite because
the larger size of the infected mice makes them sluggish and therefore an easy catch for the final
host, a cat. The plerocercoid larvae of D. mansonoides produce plerocercoid growth factor (PGF),
which is similar to human growth factor (HGF). The gene for PGF in the tapeworm came from a
human gene for HGF that became sequestered in the symbiont during the course of its evolution (
Phares, 1987).

Taenia solium: the pork tapeworm

Humans become infected with pork tapeworm when they eat undercooked pork which has a
speckled appearance because of the presence of larvae. Taenia solium is a dangerous tapeworm
because of autoinfection with cysticercus larvae. The larvae may occur in every organ and tissue of
the body, but they are most commonly found in subcutaneous tissues, eyes, brain, and muscles.
The incidence of infection is high in parts of Europe, the Middle East, East Africa, and Mexico.

Taeniarhynchus saginatus: the beef tapeworm

Humans acquire the beef tapeworm by eating raw hamburger or other uncooked beef. Adult
tapeworms occur in the human small intestine, where they may grow to lengths of > 20 m.
Humans pass the eggs of T. saginatus with feces, which are ingested by cattle. The larvae hatch in
the digestive tract of the cattle, penetrate the intestinal wall, enter the circulatory system, and
finally become lodged in the muscles and other tissues as cysticeri. The flesh riddled with
tapeworm larvae is called "measly" beef.

Hymenolepis diminuta: the rat tapeworm

Hymenolepis diminuta has been a favorite experimental subject to investigate the nutrition,
biochemistry, immunology, and developmental biology of tapeworms. The adult tapeworm
commonly occurs in the alimentary canals of rats, mice, and hamsters. Insects such as beetles,
fleas, cockroaches, and flies are the intermediate hosts. Rat tapeworms can live for an indefinite
period and show no signs of aging. Adults of H. diminuta have been kept alive for more than 14
years by using serial surgical transplantations. The host shows no symptoms. The tapeworm has a
diurnal migration in the intestine, moving anteriorly in the morning and returning to the posterior
portion of the small intestine in the evening. The migration is correlated with the feeding behavior
of the host ( Arai, 1980).

Schistocephalus solidus: a case of parasitized host

behavior
Schistocephalus solidus inhabits the alimentary canal of fish-eating birds and mammals. The birds
pass tapeworm eggs, which hatch and produce swimming larvae that are ingested by water fleas.
When a fish such as the three-spilled stickleback eats infected water fleas, the final larval
(plerocercoid) stage of the tapeworm develops in the fish's body cavity. The stickleback-
Schistocephalus association has been widely used as a model to study the effects of parasitic
symbiosis on host behavior. The plerocercoid larvae metabolize energy more efficiently than the
host ( Milinski, 1990). A tapeworm that closely resembles S. solidus is Ligula intestinalis, whose
plerocercoid larvae are > 1 m long. Ligula intestinalis-infected fish show parasitic castration, which
is characterized by suppressed growth of the gonads ( Smyth and McManus, 1989).

11.4 NEMATODES: THE ROUNDWORMS

Roundworms are second only to insects as the most abundant animals on earth. Most nematodes
are free-living. They occur in freshwater, marine, and soil habitats and feed on microorganisms and
decaying organic matter. Many nematodes are adapted for a parasitic lifestyle in plants, fungi, and
animals. Nematode parasites of plants have syringelike mouthparts, which they insert into plant
tissue to absorb nutrients. Some nematodes are ectoparasites on plant roots; others are
endoparasites and cause abnormalities of the host tissue. Scientists estimate that every kind of
animal is inhabited by at least one parasitic nematode. Nematodes parasitize organs such as eyes,
tongue, liver, and lungs, often causing destruction of the host tissues.

Roundworms are elongated, cylindrical helminths ranging in length from microscopic to several
inches. Sexes are separate, and in most species mature females lay resistant eggs. The nematode
life cycle consists of four larval stages and the mature adult. Each larval stage is transformed into
the next by a molting process, during which the old cuticle is shed and a new one is formed.
Sexually mature roundworms do not molt. The study of nematodes has provided some significant
insights into modern biology. For example, the process of meiosis was first elucidated in Ascaris
megalocephala by Edouard van Benden in 1883, and later in 1899, Theodor Boveri , using the
same roundworm, described the role of egg and sperm nuclei in fertilization ( Roberts and Janovy,
1996). Modern developmental genetics is based on studies of Caenorhabditis elegans. Nematodes
also show the phenomenon of eutely or nuclear constancy, which is characterized by the presence
of all cells of the adult in the fully developed embryo. This gives biologists the best documented
case of germinal lineage in the animal kingdom ( Crofton, 1966). A major landmark in modern DNA
biology has been the complete genomic sequencing of Caenorhabditis elegans.

Parasites of Humans and Vertebrates

Intestinal roundworms

Ascaris: large roundworms. Ascaris lumbricoides is one of the largest intestinal nematodes of
humans and is prevalent throughout the warmer climates. More than 1 billion peo- ple are infected
with this nematode but most are asymptomatic carriers ( Crompton, 1988). The nematode may
cause intestinal obstructions and interfere with host nutrition. Adult worms live in the small
intestine, and their eggs are passed with host feces to the outside. When eggs are ingested with
contaminated food or drink, the larvae hatch in the small intestine, penetrate the gut lining, and
are carried by the bloodstream to the liver, heart, and lungs. From the lungs the nematodes travel
to the mouth and then back into the alimentary canal. It is not known why the nematode adopts
such a circular path in its development in a host. During the larval migration, the parasite molts,
feeds on host tissue, and grows. The young adult roundworms in the small intestine increase in
size and become sexually mature in about 2 months. A mature female roundworm produces about
200,000 eggs each day. Most infected humans carry only one to a few nematodes, but some may
have hundreds of worms. Individuals are susceptible to ascarid infections because of genetic
predisposition and social, behavioral, and environmental factors either alone or in combination (
Holland et al., 1992). The adults of A. lumbricoides can remain in a host for up to 1 year. Because
of its size and simple onehost life cycle, A. lumbricoides has been studied for its morphology,
physiology, biochemistry, and host-parasite relationship ( Nadler, 1987).

Haemonchus contortus: sheep stomach worm. Haemonchus contortus is a bloodsucking nematode

that resides in the abomasum of sheep, goats, cattle, and other ruminants and causes severe
anemia in heavy infections. Female nematodes pass their eggs with host feces onto soil, where
under favorable conditions the eggs hatch and the larvae undergo three developmental stages. The
last larval stage does not feed and has to be ingested by a grazing host animal before it can
develop further. In the host's stomach, the parasite resumes its growth, undergoes a final molt,
and develops into an adult. A single sheep may carry thousands of adult worms, which attach
firmly to the stomach villi and feed on blood.

A phenomenon called self-cure takes place in the host sheep. When an infected host receives a
challenge infection, a large number of previously acquired adult worms are expelled, and the host
becomes resistant to reinfection. The worm expulsion has many characteristics of an immediate
hypersensitive response to antigen release by the incoming larvae ( Lloyd and Soulsby, 1987). In
other words, parasites acquired early in life make the host resistant to heavy infections by the
same parasite. From an evolutionary perspective, according to the welcome mat hypothesis, both
the host and the parasite avoid mutual extinction.
Research on the mechanism of the selfcure phenomenon has shown that newly ingested larvae
produce substances that trigger a localized allergic reaction in the host. Host animals that have
expelled adults of H. contoitus have high levels of histamines in their blood. The host animal
becomes sensitized against the parasitic antigens after repeated exposure to H. contortus.

The self-cure phenomenon has been studied extensively in rats infected with Nippostrongylus
brasiliensis because both organisms can be maintained under laboratory conditions ( Kassai, 1982).
Infected rats release eggs of the parasite in the feces by day 6 after infection and show peak egg
production on day 10. Within the following week, egg production drops to almost zero, and there is
a massive expulsion of adult worms from the host intestine. A small number of nematodes survive
the expulsion phase and remain in the host intestine for a long time. The host animal becomes
resistant to further reinfections. It is believed that the host's high levels of IgE, along with the mast
cell activities, trigger an immediate hypersensitive response and are responsible for the worm
expulsion from the intestines ( Rothwell, 1989). An increase in oxygen radicals has also been linked
with worm expulsion, suggesting that the expulsion phenomenon may involve many components of
the host's immune system. The nematodes release larger amounts of acetylcholinesterase, which
interferes with the host neuromuscular transmission preventing peristalysis and thus helping the
worms survive. The hosts produce specific antibodies against acetylcholinesterase activity of the
nematodes. The worms that survive expulsion respond by producing larger amounts of
acetylcholinesterases. During the entire process, the host's intestinal cells become highly inflamed
and damaged, and it is unable to feed ( Sanderson and Ogilvie, 1971).

The phenomenon of lactational rise (or spring rise) has been observed in Haemonchus and other
gastrointestinal nematodes of herbivores. For example, the number of eggs in a lamb's feces
greatly increases 4-8 weeks after the young are born. Several weeks later, egg production falls to a
low level. Studies have confirmed that the host's cellular immunity becomes depressed during
pregnancy and lactation. From an evolutionary perspective, the lactational rise may be viewed as
an adaptation for the survival of the nematode because it facilitates transmission of the parasite to
the offspring ( Smyth, 1994).

Ancylostoma and Necator: hookworms. An estimated 1 billion people who live in the warm climates
of the world are infected by hookworms ( Crompton, 1988). The two most important hookworms
are Ancylostoma duodenale, the oriental hookworm of China, Japan, Asia, North Africa, the
Caribbean Islands, and South America, and Necator americanus, the American hookworm which is
prevalent primarily in South and Central America but also occurs in Africa and Asia.

The mature female hookworm resides in the small intestine of its host and lays about 10,000 eggs
each day, which pass out of the host with the feces. The eggs hatch in warm, moist soil and
develop into infective larvae. The larvae penetrate the human skin and enter the blood circulation,
traveling to the lungs, trachea, and down the esophagus to the small intestine, where they become
sexually mature. The nematodes damage the host intestine as they feed on mucus, blood, and
associated tissues. Infected persons with poor nutrition often show symptoms of anemia and iron
deficiency. Epidemiologists have found that during the first decade of their lives humans steadily
acquire hookworms. The level of infection remains stable from the second to the fifth decade but
increases again from the sixth decade. During much of the adult host's life the static parasitic
population is believed to result from acquired immunity ( Shad and Warren, 1990; Gilles and Ball,
1991).

Filarial nematodes

Filarial nematodes are obligate parasites with complex life cycles involving humans and other
vertebrate and arthropod vectors. The adult female worms give birth to larvae, the microfilariae,
that invade new tissues and develop to the third larval stage. Larvae in this stage migrate to the
skin or peripheral blood circulation. A bloodsucking insect becomes infected with these larvae
during a blood meal. Wuchereria bancrofti and Brugia malayi occur in the human lymphatic system
and cause the disease elephantiasis. These nematodes affect about 200 million people throughout
the tropics ( Sasa, 1976). In Africa, the same mosquito that transmits malaria is also the vector of
W. bancrofti. The filarial worm Onchocerca volvulus causes skin tumors and blindness in some 20%
of the people of Africa and the Middle East. The nematode is spread to humans by bloodsucking
black Ries ( Evered and Clark, 1987).
The heartworm, Dirofilaria immitis, is parasitic in the heart and pulmonary artery of dogs and other
mammals. The worm is transmitted by several species of mosquitoes. The disease is produced
from lesions caused by dead or dying worms.

The Guinea worm, Dracunculus medinensis (which, strictly speaking, is not a filarid) has been
known since the early recorded history of the Middle East, India, and Africa. The adult female
nematode may be > 12 m long and takes more than 1 year to reach the skin of the ankles, where
it causes a blister and emerges to lay eggs. The eggs are ingested by water fleas, such as
copepods, where they develop into the infective larval stage. Ancient physicians learned the
importance of extracting the entire worm by slowly winding it on a stick. In the Bible, there is a
reference to God sending "fiery serpents" among the people of Israel if they disobeyed his
commandments. Many scholars believe that the "fiery serpents" were Guinea worms. Humans
become infected with Guinea worms by drinking water that contains copepods. A number of
countries where dracunculiasis is endemic are making significant efforts to eliminate this disease (
Hopkins, 1990).

A typical filarial life cycle begins when humans acquire the parasite from the bite of an infected
bloodsucking insect. The parasites show a high degree of host specificity as well as tissue
preference. Once in the bloodstream or lymphatic vessel, the nematode larvae become sexually
mature. Adult worms of Wuchereria and Onchocerca live in the lymphatic vessels for many years;
Guinea worms live for little more than a year. The mature female gives birth to microfilariae, which
are the larval stages still ensheathed in the egg membrane. Microfilariae of various nematodes
have a 24 h periodicity. The number of microfilariae of Wuchereria bancrofti reaches a peak in the
peripheral blood circulation late at night. The microfilariae are acquired by night-feeding
mosquitoes along with their blood meal. In the insect host, the larvae de- velop in the thoracic
muscles, undergo two molts, and then migrate to the mouth parts. Laboratory studies on
Litomosoides carinii, a filarial parasite of the cotton rat, have provided fundamental insights into
the hostparasite relationship of filarial worms. This nematode infection of the cotton rat is prevalent
throughout the open grasslands of North and South America.

The development of disease from a filarial parasite varies a great deal among individuals, for
reasons that are not clear. Some humans develop acute filarial disease with recurrent episodes of
fever, yet the microfilariae are not always detected. Other individuals are asymptomatic but
produce microfiliae, thus contributing to the spread of the infection. Some individuals are immune
to filarial infections.

Scientists generally agree that symptoms of filarial disease are the result of host immune response.
Obstructive lesions are produced by a delayed hypersensitivity reaction to dead or dying
nematodes. Elephantiasis results from the host's immune reaction to adult worms, and the skin
and eye disease of onchocerciasis is a reaction to the microfilariae. Acute disease symptoms
develop after treatment with antifilarial drugs, which suggests that the sudden release of dead
parasites disturbs the equilibrium between the host and parasite. A nonpathogenic relationship
results when the host's immune response to the parasitic antigens is suppressed ( Kazura et al.,
1993).

It is ironic that treatment to eliminate nematode parasites from a host precipitates a disease
condition that does not occur in stabilized infections. The number of filarial parasites in the
lymphatic system remains constant for many years, indicating that the host somehow manages to
control the intensity of infection ( Maizels and Lawrence, 1991).

Trichinella spiralis
Trichinella spiralis, the largest known intracellular symbiont of vertebrates, has been described as
"the worm that would be a virus" ( Despommier, 1990, p. 163). The adults of this parasite reside in
the intestinal epithelium, while the larvae occur in parasite-induced nurse cells associated with
skeletal muscles ( Despommier, 1993). The nematode has been studied extensively by physicians,
public health officials, experimental biologists, and ecologists. The severity of the disease
trichinosis depends on the intensity of infection in a host. Symptoms include intestinal discomfort,
diarrhea, and nausea from the presence of adult nematodes in the small intestine, and edema,
fever, fatigue, and muscle pain caused by the migration of larvae to muscle tissue. Trichinella
spiralis has a wide host range among carnivorous vertebrates. Human trichinosis is associated with
the consumption of undercooked pork containing parasitic larvae. The nematode has a short life
cycle, does not need an intermediate host, and is easily maintained in laboratory animals.

Trichinella infection begins with the ingestion of meat containing the infective firststage larvae ( fig.
11.5 ). After the meat is digested, the larvae become liberated and enter their intracellular habitat,
a row of columnar epithelial cells in the small intestine. A larva may occupy more than 100
epithelial cells without damaging the host cells. The columnar cell membranes fuse with each other
and form a syncytium. It is not known how the parasite enters the columnar cells; the larvae do
not possess any special organs for cell penetration. After about 30 h the larvae undergo four molts
and become adult worms. The speed of the molting is unusual and might be related to the host
immune response. The adult nematode increases in size and occupies an average of 400 columnar
cells. During the molting process, the symbiont undergoes extensive morphological changes in its
cuticle, hypodermal gland cells, and alimentary canal. Hypodermal glands produce secretions that
are believed to possess unique antigenic properties. Little is known about how the adult worms
obtain their nutrients. Stichosomes are unique discoid cells that occur in the anterior half of the
infective larval stage. These cells produce secretory granules that are strongly antigenic. Some
scientists speculate that the host immune response is directed against the secretory granules.

Sexually mature worms copulate and the females give birth to 200-1600 larvae, depending on the
host species. The newborn larvae migrate to the lymph, and some move into the general
circulation, infecting organs such as the liver and lungs. Recent studies have shown that striated
muscle cells are the preferred intracellular habitat for newborn larvae, which are attracted to the
muscles by chemical and electrical stimuli. A larva penetrates the muscle cell by a mechanical
process and then redirects the host cell metabolism to its own survival. The infected muscle cells
lose their myofilaments, undergo

nal size. The muscle cell containing the coiled larva is transformed into a nurse cell. Muscle
cells function within a narrow range of physical and chemical conditions; few parasites have
successfully invaded the intracellular muscle environment. Trichinella spiralis survives in
skeletal muscle cells by transforming the structure and biochemistry of the host cell. The nurse
cells lose their ability to contract like normal muscle cells. Calcification of the nurse cell is a
complex host response, often developing several years after the initial infection, and results in
the worm becoming encysted. For reasons not yet understood, the muscles of the eye, tongue,
jaws, diaphragm, and the ribs are most susceptible to Trichinella infection.

Trichinella spiralis evokes a powerful host immune response. As a new generation of larvae
becomes established in the muscles, adult worms residing in the small intestines are expelled
from the host ( Appleton and McGregor, 1984). Animals previously exposed to T. spiralis resist
further infection, and a host mother transfers the immunity to her offspring. In an immune
host, T. spiralis grows slowly and does not reproduce. Different larval and adult stages of T.
spiralis in the same host produce different antigens. The host immune system responds by
developing antibodies against the three stages of the parasite's life cycle: adult, infective
larvae, and newborn larvae. Multiple antigen-antibody interactions facilitate the migration of
newborn larvae from the intestine to the muscle. Trichinella antigens have been used to
develop a vaccine to control trichinosis in pigs.

Nematode-Insect Symbioses

Insects are the dominant form of life on earth, and nematodes have successfully evolved
symbioses with many of them. There are an estimated 3000 nematode--insect associations.
Nematodes of insects have intricate life cycles that are synchronized with those of their hosts.
The symbionts cause changes in the insect's morphology, physiology, and behavior and reduce
the reproductive potential of the host. Heterorhabditis and Steinernema are nematodes that
parasitize insects and transmit bacteria that kill their hosts. There has been renewed interest in
nematode-insect symbioses because of the potential for controlling insect pests by means of
pathogenic symbionts ( Poinar, 1979; Kaya and Gaugler, 1993).

Deladenus
 The complex life cycle of Deladenus involves two hosts, a wood wasp and the fungus
Amylostereum. The fungal part of the life cycle occurs in dying or dead pine trees, where the
fungus grows inside galleries dug by the wasp larvae. Nematode larvae feed on the fungal
hyphae and grow and molt until they become sexually mature adults. Old fungus colonies
somehow trigger the parasitic behavior of the nematodes. A fertilized female nematode
penetrates a wasp larva and enters its body cavity, where it increases in size and molts. The
old nematode cuticle ruptures, and the hypodermal cells become transformed into absorptive
cells with microvilli. When the wasp larvae pupate, the female nematode gives birth to live
larvae, which migrate to the reproductive organs of the newly developed female wood wasp.
The wasp disperses the nematodes by depositing them on a suitable tree along with its own
eggs.

Mermis nigrescens
Mermithid nematodes are well-known obligate parasites of insects, spiders, and other
invertebrates. Mermis nigrescens is a common parasite of grasshoppers and katydids. The
parasite usually kills its host. The nematode larvae are found in all stages of the insect,
obtaining nutrients from the host body cavity. The female nematode is > 10 cm long and lives
in the soil. After a rainfall, the nematode climbs onto vegetation and lays eggs on the foliage.
The eggs remain viable for up to 1 year. Grasshoppers and other susceptible hosts ingest the
eggs along with the foliage. The larvae hatch in the grasshopper's alimentary canal and reach
the body cavity by penetrating the gut wall. The parasite increases in size from 3mm to >
10cm. In the last larval stage, the parasite leaves the host body and burrows in the soil. During
the escape process, the insect host is killed. The larvae remain dormant until the following
spring, when they molt and become adults. A mature female produces more than 14,000 eggs
and can overwinter and resume egg laying the next spring ( Nickle, 1984).

Romanomermis
The possibility of developing a biological control for mosquitoes has heightened inter

est in the mermithid nematode Romanomermis, which kills mosquito larvae. Sexually mature
adults of the nematode live for up to 6 months in the shallow waters of lakes, ponds, and streams (
fig. 11.6 ). The female lays many eggs that hatch in about 7 days. The larvae are strong swimmers
and seek out mosquito larvae at the water surface. The nematode penetrates the cuticle of the
larvae and develops in the host for 5-10 days. The parasite is not carried through the pupal or
adult stages of the mosquito because either the infected larvae usually die or the immature
nematodes leave the host. The nematode larvae return to the bottom mud, where they develop
into adults and the life cycle is repeated. Once introduced into an area, the nematode becomes a
permanent resident of the ecosystem. Scientists have developed methods to mass produce the
eggs of Romanomermis for largescale applications ( Petersen and Cupello, 1981).

Attempts are being made to control black flies in Africa by using parasitic nematodes that kill the
insect larvae. Black flies are vectors of the filarial nematode Onchocerca volvulus, which causes
blindness in humans.

Neoaplectana
The nematode Neoaplectana carries a bacterial symbiont in a specialized intestinal pouch.
Nematodes either are ingested by an insect or enter the host's body through natural openings.
Once inside the host alimentary canal, the nematode releases its bacterial symbionts, which move
into the body cavity and multiply, causing death of the host ( fig. 11.7 ). Nematodes develop
rapidly in the dead host and become sexually mature. The females produce larvae that emerge
from the host carcass carrying the bacteria and burrow into the soil, where they can survive for 3
years or more. Scientists have attempted to use this parasitic nematode as a way to control insect
pests ( Maggenti, 1981).
Nematode-Plant Symbioses: Plant Nematology
Most nematodes that attack plants are obligate parasites and include root-knot nematodes and cyst
nematodes, which cause destructive infections in crop plants. The nematodes have a hollow,
needlelike structure, the stylet, which they insert into plant tissue, like a syringe, to draw out
nutrients. Most plant nematodes feed on roots but some invade parts of a plant above ground.
There are more than 2000 described species of plant parasitic nematodes, but only a few species
are pests. Scientists are beginning to realize that serious nematode infestations are the result of
unsound agricultural practices such as monocropping and reliance on genetically uniform plants.

Life cycles of plant nematodes are simple, consisting of adult and juvenile stages associated with
the same host. Cell proliferation, giant cell formation, suppression of cell division, and cell wall
breakdown are some host responses to nematode parasitism. Arabidopsis thaliana, a small crucifer,
is a host to several plant parasitic nematodes, including Heterodera and Meloidogyne species.
Molecular biology approaches have opened new perspectives to understanding host gene
expression and accompanying molecular changes in the plant--nematode interaction dynamics (
Niebel et al., 1994). The infected plants are stunted and have yellow leaves, and in the case of
crop plants, yields are significantly reduced. Most plant nematodes are ectoparasites, but a few
species are endoparasites ( Atkinson, 1995; Zacheo and Bleve- Zacheo, 1995).

Since the end of the Second World War, soil fumigation has been the preferred method for control
of nematode pests. Unfortunately, when fumigation is discontinued or suspended, the nematode
populations increase to destructive levels. Because many of the common soil fumigants are now
considered to be harmful to humans and other organisms, scientists are searching for alternative
methods of nematode control, such as the use of biological control methods, resistant varieties of
plants, crop rotation, and agricultural practices that maintain the stability of the soil-microbe
equilibrium.

Meloidogyne: the root-knot nematode

The root-knot nematode infects more than 2000 cultivated plants throughout the world,
contributing to an estimated annual loss of $77 billion. Plant nematologists have conducted
extensive studies on the biology, physiology, and host--parasite interactions of the Meloidogyne--
tomato symbiosis. Juvenile nematodes penetrate the root tips of a host plant and move between

the cells to the

Fig.11.6 Romanomermis parasitism of mosquito larvae. (A) Stages in the life cycle of
Romanomermis culcivorax. Adapted from Nickle ( 1984). (B) Nematodes collect at the bottom of
tank after emerging from mosquito larvae. (C) Nematodes in the thorax of mosquito larvae (Parts
B and C courtesy James Peterson, USDA, Lincoln, Nebraska.)

vicinity of the vascular tissues. Plant cells surrounding the nematode undergo hypertrophy and
hyperplasia and form a gall. Most galls contain giant cells that are formed in response to the
nematode's feeding. The juveniles in the gall molt rapidly and become adults. The female
nematode becomes enlarged and spherical and lays eggs, which are extruded in a protective
gelatinous matrix ( fig. 11.8A ). Under favorable conditions, the juveniles hatch from the eggs and
infect other roots. Plants parasitized by root-knot nematodes are less resistant to infections from
soilborne fungi and bacteria and more susceptible to environmental stresses such as temperature
change and drought. Yields of heavily parasitized plants are greatly reduced.

Heterodera and Globodera: the cyst

nematodes
The cyst nematode Heterodera is a major parasite of agricultural plants in temperate regions. A
unique feature of these parasites is that the female body containing the eggs is transformed into a
resistant cyst. The infection begins when juveniles penetrate root tips of the host and migrate
through the cortex. The juvenile begins the feeding process by orienting its head toward the
vascular tissue. Giant cells are formed, but galls do not develop. After egg production the female
dies and becomes a resistant cyst, which remains in the soil for several years. The eggs in the cyst

hatch when a specific stimulus is received from a host plant. Cyst nematodes

Fig. 11.6 (continued)

cause extensive losses in crops such as sugar beets, soybeans, potatoes, and cereals. Scientists
have studied the hatching factors in the hope of developing an effective cyst nematode control (
Niebel et al., 1994).

Anguina tritici: the wheat gall nematode

In 1743, Turbevill Needham, an English naturalist, claimed support for the spontaneous generation
of life when he crushed wheat galls in water and observed that the contents of the galls became
alive. The galls crushed by Needham contained large numbers of juveniles of Anguina tritici in a
state of anhydrosis. Wheat galls are unknowingly sown along with wheat seed. Under warm, moist
conditions, the galls break open and liberate juveniles, which infect nearby wheat seedlings. The
juveniles first feed as ectoparasites and later penetrate the plant tissues as the wheat flowers
begin to develop. After the nematodes mate, the female migrates to the ovary of the flower and
lays thousands of eggs, which hatch to produce juveniles. The ovary is transformed into a resistant
gall. Galls are also produced on the leaves and stems. Nematodeinfected plants are usually stunted
or wrinkled and have twisted leaves. Anguina tritici is prevalent throughout Asia, the Middle East,

Fig.11.7 Wax moth larva parasitized by Neoaplectana carpocapsae (Courtesy W.R. Nickle, USDA,
Beltsville, Maryland).

and Brazil. Because the seed galls float in water, they are easily separated from wheat seeds. With
improved agricultural practices, the incidence of A. tritici is declining in many areas ( Maggenti,
1981).

Bursaphalenchus: beetle-pine mutualistic

symbiosis
Bursaphalenchus xylophilus is a nematode that lives in weakened or dead trees. The nematode is
spread by a beetle that may carry up to 15,000 juvenile nematodes to a new location. The
nematodes feed on wood tissue and are suspected of killing pine trees previously weakened by
environmental stresses. Their life cycle is intertwined with that of the bark beetle. The beetle
normally lays eggs under the bark. Upon hatching, the larvae dig tunnels in the wood tissue, where
they pupate and overwinter. As the adult beetles emerge from the pupae, the nematode juveniles
develop and gather near pupal cases. The nematodes hang from the roof of the tunnel gallery by
their tails. As the adult beetle emerges from the pupal case, its vigorous movements cause the
nematode juveniles to attach to it. The juveniles migrate swiftly to undersurfaces of the wings or to
the air passages of the host. The infected beetles emerge and find other pine trees to feed on.
During the feeding process, nematode juveniles leave the beetle to invade the new substrate. The
beetle lays eggs in the areas softened by nematode activity. The ecological relationship between
the plant-parasitic nematode and the bark beetle is thought to be mutualistic.

11.5 PARASITOIDS: NEGLECTED SYMBIOSES

Parasitoids are restricted to the orders Hymenoptera (ants, bees, and wasps) and Diptera (flies)
and are defined as insects that live freely as adults but as larvae they feed in or on the body of an
another arthropod. There may be 1.6-2 million species of parasitoid insects, many remain
undescribed ( Godfrey, 1994). Parasitoids are common in all terrestrial ecosystems and are
categorized by the host they parasitize and where their offspring develop. Environmental and
scientific interest in these insect symbioses has been heightened by their exploitation as biological
control agents. An adult parasitoid must find a suitable host and lay eggs on or in it by using its
ovipositor. The parasitoid Cotesia marginventris is attracted to plants damaged by its host and uses
chemicals in the host's saliva and feces as orientation cues. Wasp parasitoids, whose females
control the sex ratio, must decide whether to lay male or female eggs. Parasitoid development is
greatly influenced by the quality of its host in addition to coping with defense mechanisms with
counteradaptations. Adult parasitoids also must find a mate and avoid predation. Evolutionary
aspects of the physiological interactions between parasitoid and host provide new opportunities to
study the biological basis of biodiversity ( Godfrey, 1994). The coevolution of host synchronization
and diapause strategies of parasitoids is receiving attention from evolutionary biologists because of
the emergence patterns for males and females that maximize their chances of finding mates and
hosts. Evidence from comparative morphology and DNA segment analysis shows that parasitoids
evolved from fungus-feeding ancestors ( Whitfield, 1998).

Parasitoid-polydnavirus mutualism is also drawing the attention of scientists because in some

parasitoids virus particles are injected in the host along with the wasp's eggs, and either venom or
virus or both suppress the host immune response (see fig. 2.4). The polydnaviral genome consists
of several pieces of circular DNA, and the nucleocapsid is surrounded by a double-membrane
envelope. The virus multiplies in the wasp ovary cells and has no adverse effect on it. But in a
lepidopteran caterpillar, the virus suppresses its cellular immune response and disrupts the normal
endocrine functions that control insect development, and when the caterpillar dies, the new
generation of wasp that emerges carries the virus (see chapter 2). Some studies have shown that
viral DNA is required for a successful parasitism of caterpillars by parasitoid wasps. The
polydnavirus and wasp genomes have become so well integrated that the virus depends on the
wasp for its spread and reproduction ( Whitfield, 1990).

Figs, Wasps, and Nematodes

Figs and wasps are one of the best studied species-specific examples of mutualism and
coevolution. There are more than 1000 species of figs that grow throughout the tropics. Allen Herre
( 1993) explored the complex relationship of fig trees with wasps that polli-

Fig. 11.8 Plant root parasitism by the root-knot nematode Meloidogyne incognita. (Left) A root gall
has formed around the female nematode. Note protruding egg mass. (Right) Cross-section of an
infected root, showing giant cells near the head of a feeding nematode (Courtesy J.D. Eisenback,
Virginia Polytechnic Institute).
nate them and nematodes that infect the wasps. All fig species are pollinated by chalcidoid
wasps, which develop as mutualistic symbionts in galls within the figs. (We discuss the
complexity of fig pollination in chapter 12.) In fig wasps, the nematodes are species specific and
often act as a virulent pathogen in some wasp species, while in others they have no effects.
Herre was one of the first to provide experimental evidence on the evolution of virulence. He
predicted that if a disease spreads easily among hosts as a function of higher contact rates, then
the infection will be virulent and destructive ( Herre, 1993). This idea is supported by the earlier
observation of Paul Ewald ( 1993) that emergence of virulence can be a more successful
evolutionary strategy than the traditional view that evolution favors the emergence of
nonvirulence in various host--pathogen interactions. For an estimated 40 million years, figs,
wasps and nematodes have coevolved into relationships that allow all three species to coexist.

Nonpollinating Fig Wasps

There are many nonpollinating wasp species that visit fig trees and interfere with the established
mutualism between the host plant and its pollinator. Many wasp species stimulate gall formation
by altering the ovules on which their larvae feed. Some wasps use ovules already initiated by
other wasp larvae. It is believed that fig-pollinating wasps evolved from parasitoid ancestors. The
parasitoid wasps have identical life histories to the pollinating wasps, but they carry no pollen
 and have long ovipositors. The larvae of pollinating wasps are parasitized by other species of fig
wasps that are true parasitoids. These species are potential competitors of pollinating wasps.
Some species have evolved large mandibles and fight among themselves for females of their
species within the fig synconium. Males of other wasp species resemble females. In general,
wasp communities associated with the fig trees are specialists and show a high degree of host
specificity. Fig-wasp mutualism is sustained by a number of characteristics of each partner, such
as direct adaptations to mutualism, byproducts of selection on another trait, and preadaptations (
Anstett et al., 1997; Godfrey, 1994).

11.6 SUMMARY
Helminths are metazoans with complex life cycles. Humans in many parts of the world are
infected with helminthic parasites, which include flukes, tapeworms, and roundworms such as
hookworms and filarial nematodes. Flukes are common in vertebrates, where they infect different
organs such as the liver and urinary bladder as well as the blood. In some cases, the life cycle of
a fluke is synchronized with that of its host. Schistosomes are blood flukes that cause the
diseases schistosomiasis and bilharziasis. The outer body wall of a fluke or tapeworm, the
tegument, is a complex structure that protects the parasite from the digestive enzymes of the
host and also absorbs nutrients. Some parasites disguise themselves by producing surface
antigens similar to those of their hosts, a phenomenon called molecular mimicry.

Tapeworms are highly specialized parasites of vertebrates and arthropods. In the tapeworm life
cycle, water fleas and various types of insects are common intermediate hosts. Nematodes, or
roundworms, are parasites of plants, animals, and fungi. Some animals become resistant to
reinfection after their initial infection by a parasite. This phenomenon is called self-cure.
Hookworms infect almost a billion people in regions with warm climates. Filarial nematodes have
complex life cycles that involve humans and other animal vectors. Filarial diseases such as
elephantiasis are the result of the host's immune responses to the parasites. The nematode
Trichinella spiralis causes the disease trichinosis in humans and other vertebrates and results in
multiple antigen-antibody interactions between host and parasite. Insects have many nematode
parasites, some of which are being studied as possible biological controls of pests. Plants also are
infected by different roundworms.

Parasitoids are common in all ecosytems. Figs and wasps have coevolved complex relationships.
Nonpollinating wasps also visit fig trees. The use of parasites to control other parasites has had
much appeal among scientists. Such biological control methods, when successful, are much
better alternatives than the indiscriminate spraying of pesticides. In this respect, it is important
that the life cycles of parasites be thoroughly understood in order to realize the full potential of a
biological control agent.

12
FLOWERING PLANT SYMBIOSES
Angiosperms, or flowering plants, make up more than 80% of all living plants. Although their origin
remains shrouded in what Darwin called the "abominable mystery," the explosive diversity of
flowering plants during the past 110 million years is an evolutionary success story. The fossil
record confirms that many flowering plant families existed 100 million years ago. Flowering plants
have shown great flexibility in the morphology of their roots, stems, leaves, flowers, fruits, and
seeds. As a defense against herbivorous animals, many plant species have evolved prickles and
spines, hairy leaves, and a wide range of secondary metabolites ( Jones and Firn, 1991). The
growth--differentiation balance (GDB) hypothesis deals with plant resource allocation patterns.
Primary metabolism supports the growth of new tissues, while secondary metabolism supports the
production of specialized tissues, organs, and chemical compounds. According to the GDB
hypothesis, growth and differentiation require energy that comes from photosynthesis ( Herms and
Mattson, 1992; Lerdau et al., 1994). Thus, in a coevolutionary arms race, a cost-benefit equation is
central to the allocation of a host's limited energy resources. One reason for the success of
flowering plants may be their ability to form mutualistic symbioses, such as those with bacteria
that fix nitrogen, fungi (mycorrhizae), and insects, birds, and mammals, which pollinate plants and
disperse seeds.

12.1 PLANT-ANIMAL INTERACTIONS

Plants and Grazing Herbivores
Herbivores depend on plants for food and have been a major influence in the evolution of leaf size
and shape ( Brown and Lawton, 1991). In response, some plants have evolved structural and
chemical defense mechanisms that discourage herbivory. Other plants encourage herbivory and
regenerate quickly. Plants that are under grazing pressure often store food in roots and rhizomes
and can regenerate when growing conditions are favorable.

Grasslands constitute about 25% of the earth's surface and vary widely in geography and climate.
All grasslands are dominated by a low, perennial ground cover formed by species of the family
Poaceae and some dicotyledonous plants. Grasses and dicots have evolved different strategies
against herbivores. Grasses store food in roots or stems, and their leaves have heavily lignified
midribs. Herbivores grazing on grass must contend with siliceous leaf tissues, to which they have
responded by increased consumption and low retention time. Elephants, horses, and pandas follow
this strategy. In general, mammalian herbivores of forests and woodlands have short-crowned
teeth and feed on leaves, fruits, and seeds of trees and shrubs. Grazing mammals, however, have
continually growing, elongated teeth to com-pensate for the abrasive action of grass leaves (
MacFadden, 1997).

Grazing ruminants extract energy molecules by slowly degrading plant cell walls through anaerobic
microbial fermentation. Their rumen detoxifies secondary metabolites and retains plant food longer
in the digestive system. One way herbivores lessen a plant's secondary metabolite defense is by
consuming different plant species. Tropical and temperate forest herbivores favor this strategy.
Some animals, such as the koala bear, which feeds exclusively on leaves of eucalyptus trees, have
specialized biochemical pathways in their liver cells and can detoxify plant poisons ( Van Soest,
1994).

Cultivated crop plants and domesticated animals are the result of associations with humans during
the past 10,000 years. Most crop plants have been selected for their increased nutritional contents
and absence of antiherbivory features.

Carnivorous Plants
Three principal publications, Darwin Insectivorous Plants ( 1875), Lloyd The Carnivorous Plants (
1942), and Juniper et al.'s The Carnivorous Plants ( 1989), provide historical aspects and insights
into the functioning and evolution of these curious plants. About 500 species of carnivorous plants
grow in wet or waterlogged, acidic soils ( Zamora et al., 1998). In response to nutrient-poor
habitats, carnivory is believed to have evolved in at least six independent evolutionary lineages (
Thompson, 1981) ( table 12.1 ). Carnivorous syndrome is a conceptual description of plants that
(1) attract, (2) retain, (3) trap, (4) kill, (5) digest, and (6) absorb useful substances ( Lloyd, 1942;
Juniper et al., 1989). These plants eat mainly insects, but spiders, woodlice, slugs, earthworms,
tadpoles, and fish are also prey. Carnivorous plants obtain nitrogen, phosphorus, sulfur, potassium,
calcium, and magnesium from insect carcasses ( Adamec, 1997).

In recent studies, hydrolytic enzymes have been discovered in the digestive juices that are
produced by carnivorous plants ( Juniper et al., 1989). Some of these digestive enzymes may be
produced by bacteria living in the modified leaf traps ( fig. 12.1A ). Carnivorous

Table 12.1 Major carnivorous plants

Order/genus Common name Distribution
Sarraceniales
Sarracenia Pitcher plant or trumpet leaf Eastern North America
 Order/genus Common name Distribution
Darlingtonia Cobra plant Northern California
Heliamphora Marsh pitcher Venezuela, Brazil
Nepenthales
Nepenthes Dutchman's pipe Sri Lanka, Madagascar
North and South Carolina,
Dionaea Venus fly trap
USA
Drosophyllum Portuguese sundew Portugal, Spain, Morocco
Drosera Sundews Worldwide
Violales
Triphyophyllum West Africa
Saxifragales
Byblis Australia
Cephalotus Australian pitcher plant Southwest Australia
Scrophulariales
Pinguicula Worldwide
Utricularia Bladderwort Worldwide
Brazil, tropical Australia,
Genlisea
Guyana
Bromeliales
Brocchinia Venezuela, Guyana
Catopsis Southern Florida to Brazil

plants can now be grown axenically, so future studies may determine the nature of these digestive
enzymes and the role of other microbial symbionts in the release of animal nutrients.

Most carnivorous plants have a weak root system and form clonal colonies by stolons or rhizomes.
They capture prey by using one of four trapping strategies: adhesive traps (e.g., Byblis,
Drosophyllum, Drosera, Pinguicula, Triphyophyllum), snap traps (e.g., Dionaea), pitcher traps
(e.g., Catopsis, Cephalotus, Darlingtonia, Heliamphora, Nepenthes, Sarracenia), or suction traps
(e.g., Utricularia) ( fig. 12.1b ).

The carnivorous traps, because of their highly developed visual and olfactory signals, are
considered examples of "aggressive mimicry" to attract insects ( Pasteur, 1982). Pitcher plants,
with their flowerlike appendages, attract insects that are inexperienced pollinators ( Williamson,
1982; Joel, 1988). Nectaries are common to all pitcher plants and, unlike floral nectaries, are
designed to deceive insects. By providing nectar in certain habitats, pitcher plants support insect
communities. In such a mutualism, the pitcher plants can grow in nutrient-poor habitats by
obtaining nutrients from prey; in return they provide food to insects that escape capture ( Joel,
1988).

Ant-Plant Mutualism
Ant-plant symbioses are significant in that they are formed between a highly developed metazoan,
the zoobiont, and a flowering plant, the photobiont. The importance of ant-plant mutualism was
recognized from the pioneering research of D.H. Janzen ( Beattie, 1985; Janzen, 1985).

Ant-plant mutualism is based on four major themes in which ants (1) defend the host plant against
herbivory, (2) disperse seeds, (3) provide plants with nitrogen, phosphorus, and potassium, and
(4) pollinate plants. In return, plants provide ants with nutrients and nest sites. Plant structures
used as nesting sites are called domatia and consist mostly of hollow stems and twigs. Domatia are
formed by tissue-boring insects and their larvae. Symbioses between Acacia plants and ants of the
genus Pseudomyrmex have been well studied ( Janzen, 1967). Ants chew out a hole at the tip of
one thorn in each pair of thorns and live in the hollow cavity. In addition to providing nest sites for
the ants, the host plant secretes nectar from large nutritive organs called Beltian bodies. Acacia
nectar is a rich mixture of proteins, carbohydrates, and lipids, and ant workers feed it to the
larvae. Acacia ants continuously patrol the host plants and their immediate surroundings, repel
insect or mammalian herbivores and destroy any invading vines by cutting through their petioles (
Beattie, 1989).

In southeast Asia, the fast-growing Macaranga tree has evolved associations with ants of the genus
Crematogaster. The host plant provides nesting space and food for the ants, which in turn protect
the host against herbivores by removing eggs of other insects and removing any competing plants
that are near the host ( Fiala et al., 1991).

Phloem-feeding aphids are tended by ants, which harvest their energy-rich excretions (honeydew)
in a symbiosis described as conditional mutualism. Ants protect aphids from predators. About 25%
of aphid species have developed associations with ants, and host plants play a central role in that
they allow aphids to attract ants. How mutualistic interactions are sustained remains to be
determined ( Cushman and Addicott, 1991a; Gaume et al., 1998).

In tropical forests, epiphytic plants offer conspicuous nesting sites for arboreal ants. The ants live
in swollen petioles, leafy pouches, stipules, and hollowed-out stems. Each ant garden may be
inhabited by two or more ant species. Seeds of the host plant are collected by the ants and
deposited in their nest walls. Domatia of epiphytic ant gardens not only house ant colonies but also
absorb nutrients from ant waste. Ant garden mutualistic symbioses are often exploited by ant
species that parasitize resident ant colonies ( Letourneau, 1991).

Many ant species, such as harvester ants, store seeds in their underground nests. Ant seed
dispersal is commonly found in plant species inhabiting nutrient-poor soils. Ants are attracted to a
food body (elaiosome) which is attached to the seeds. When the food body is eaten, the seed
remains intact and subsequently germinates ( Beattie, 1985). Harvest ant symbioses are diverse in
Australia, where the host plants include herbs, shrubs, and trees ( Andersen, 1991).

Though ant pollination has been reported several times, there are only two well-documented cases
( Peakall et al., 1991). Ants that visit flowers are more likely to take nectar than to transfer pollen.
Well-designed experiments are exploring ant pollination in differ- Fig.12.1 Carnivorous plant
symbiosis. (A) Schematic representation of the microenvironment of a generalized pitcher plant
showing exchange of nutrients. (B) Four types of capturing strategies used by carnivorous plants:
(1) pitcher trap (Nepenthes), (2) snap trap (Dionaea), (3) suction traps (Utricularia), and (4)
adhesive traps (Drosera). Note the structure of the digestive glands. Adapted from Juniper et al. (
1989). A Microenvironment of Sarracenia (Pitcher Plant)

ent ecological habitats, and it may be more common than thought.

12.2 PLANTS AND POLLINATORS

Coevolution of Insects and Plants
Coevolution of insects and plants over the past 200 million years has resulted in relationships that
have influenced the development of both groups of organisms. An example of such a relationship is
pollination in flowering plants. A primary reason that flowering plants are the dominant group of
plants is their exploitation of insects as pollinators. Insects transport pollen from one plant to
another, while plants provide insects with a food source such as nectar or pollen. Nearly 70% of
flowering plants rely on insects for pollination ( Kearns and Inouye, 1997), and 30% of our food
comes from bee-pollinated crops. Steep declines in populations of pollinators, particularly bees, is a
cause for concern ( Norstog, 1987; Barth, 1991; Pellmyr, 1992; Proctor et al., 1996; Kearns et al.,
1998).

Pollination is the transfer of pollen from an anther to a stigma or similar receptive surface.
Evolutionary adaptations of flowers promote cross-pollination, which results in greater hybridization
and, subsequently, greater variation on which natural selection can operate. The mechanisms that
plants have evolved to prevent self-pollination and to facilitate cross-pollination are many and
varied. They include different maturation times for anther and stigma of the same flower,
morphological adaptations of the flower, such as different style lengths in flowers of individual
plants, and a self-incompatibility that is genetically determined.

Flowers of the orchid family, one of the largest families of flowering plants, are highly

Fig. 12.1 (continued)

adapted to promote cross-pollination. Such adaptations have resulted in thousands of orchid

hybrids.

Some insect-plant relationships are so highly evolved that neither symbiont can reproduce
independently of the other. Examples are the yucca and the pronuba moth and the fig and its
attendant wasp. Many plants have flowers whose morphological features are adaptations to specific
types of animal pollinators. Animals have also adapted to specific types of flowers.

Many plants and animals have coevolved biochemically. Protective chemical compounds produced
by plants to ward off predators have resulted in some insects being resistant to these compounds
or even using them for their own defense against other animals. For example, larvae of the
monarch butterfly feed on milkweed plants and ingest a latex from the plant that is poisonous to
other insects but tolerated by the larvae. Adult monarch butterflies retain this poison and, as a
consequence, are not eaten by birds ( Buchmann and Nabhan, 1996).

Floral Features that Attract Pollinators

The primary reason insects visit flowers is that flowers provide a source of food (pollen and nectar).
Pollen is high in proteins, fats, and sugars, and probably was the original food source for many
insects. Most flowers produce sufficient pollen to compensate for that eaten by insects.

When insects visit a flower, they generally become covered with pollen grains. Because they cannot
clean all the grains from their bodies, they transport the pollen to the next flower they visit and
pollinate it. The anthers of most plants release pollen through longitudinal slits, but in some plants,
such as blueberries and tomatoes, pollen is released through terminal pores. In these flowers, bees
grasp the stamens, and by moving their bodies they cause the anthers to vibrate and release
pollen. This phenomenon is called buzz pollination. Pollen released from anthers may be attracted
to an insect because of electrostatic conditions around the insect and flower. Bees develop positive
charges when flying to a flower, which has a negative charge, and when a bee lands on a flower
the charges are dispelled.

Nectar is a sugar solution consisting of sucrose and its breakdown products, glucose and fructose.
Nectar is secreted from small strands of phloem that terminate in specialized nectar glands, the
nectaries, located at the base of the pistil, or in protected areas such as tubes or spurs. The sugar
concentration of the nectar of different plants as well as the amount of nectar produced in a flower
varies and is usually related to the energy needs of the pollinators. For example, flowers pollinated
by birds produce more nectar than flowers pollinated by insects. Rare plants and those with
specialized pollinators have flowers that produce nectar with a high sugar concentration. For
example, some gentians have closed flowers and are pollinated by large bumblebees. The insect
forces open the petals in order to enter the flower. Because of the work involved in opening the
flower and the rarity of the plant, the number of gentian flowers that a bee can visit is limited.
Thus, in this insect-plant relationship, flowers with a concentrated nectar are favored over those
with a less concentrated nectar.

Flowers that bloom at night produce nectar only at night, and day-blooming flowers produce nectar
only in the morning. In addition to nectar and pollen, flowers may have oil-secreting glands. The oil
is gathered by pollinating insects and mixed with the pollen they collect.

Many insects use flowers as a place to lay eggs or to hide from predators. The classic relationship
between the Yucca plant and the yucca moth ( Tegeticula yuccasella) and the fig plant and its wasp
illustrates the intimate nature of some insect-plant symbioses. In both relationships, the symbionts
have developed traits that are a result of their coevolution. The flowers are pollinated by the
insects, which lay their eggs in the flowers. Larvae hatch from the eggs and use some of the
developing seeds for food.

The life cycles of the Yucca plant (some 30 species of Yucca grow in North America) and moth are
synchronized in several respects. For example, when Yucca flowers blossom, the female moths are
ready to lay their eggs, and when seeds of the flower mature the insect larvae hatch from the
eggs. The female moth visits the white flowers of Yucca at night, and by means of specialized
mouth parts gathers the sticky pollen from the anther, rolls the pollen into a ball, and carries it to
another flower. In the second flower the moth lays its eggs in the ovary, among the ovules or
immature seeds. Then, in what seems to be almost purposeful behavior, the moth carries the ball
of pollen it took from the first flower and deposits it on the stigma of the second flower, thus
causing pollination and ensuring that the ovules develop into seeds. The larvae eat only about one-
third of the seeds in the ovary. When the larvae are mature, they bore out of the ovary and drop to
the ground, where they pupate until the next flowering season, when the adult moths emerge
again ( Powell, 1992).

Pollination of the fig plant is one of the most sophisticated examples of plant-insect symbioses
known and is an excellent example of coevolution ( Anstett et al., 1997) ( fig. 12.2 ). The
symbiosis is an obligate mutualism between a gall wasp, Ceratosolen arabicus, and the sycamore
fig, Ficus sycamorus. Fig trees have been propagated from stem cuttings for centuries throughout
the Mediterranean region, but in East Africa they grow wild, reproducing sexually and producing
fruits throughout the year.

The fig fruit is actually an inflorescence called a syconium, with its margins folded to a pear-shaped
structure that has a small opening, the ostiolum, which is lined with hairlike scales. The inner
fleshy surface of the inflorescence contains numerous small flowers; those close to the ostiolum are
male, whereas those deep within the interior are female. When fertilized, each female flower
produces a hard, grainy seed.

The female gall wasp deposits an egg into the ovary of a female fig flower with its ovipositor and
induces the ovary to form a tumorlike gall, which encloses the insect egg. As female wasps enter
the fig fruit through the ostiolum, they frequently lose their wings, antennae, and other body parts
because of the dense scales around the opening. Wasps that pass through the ostiolum are
attracted to the female flowers, of which there are two types, those with long styles (1.5 mm) and
those with short styles (0.8 mm). Long-styled flowers are sessile, and short-styled flowers are
borne on stalks. The wasp ovipositor is about 0.8 mm long and can reach the ovary of a short-
styled flower, into which it deposits an egg. On a long-styled flower, however, the female wasp
inserts its ovipositor through the style, but because it cannot reach the ovary, it quickly withdraws

the ovipositor. Nonethe-

Fig.12.2 Diagrammatic representation of the mutualistic symbiosis between the sycamore fig and
the gall wasp. Adapted from Meeuse and Morris ( 1984).

less, the wasp still places pollen collected from other flowers onto the stigma. The female wasps
die shortly after they have deposited their eggs. Larvae emerge from the eggs a few days after
they have been laid and feed on the gall tissue. Larvae then transform into pupae from which adult
wasps with strong mandibles develop and eat their way through the gall. Male wasps develop first
and search for galls containing female wasps. The males puncture the galls and fertilize the
females by inserting the tips of their abdomens into the gall. After they are fertilized, the female
wasps emerge from the galls. The wasps then gather near the ostiolum and begin to tunnel
through the syconium. Because of their sensitivity to light, the male wasps withdraw into the
interior of the fig fruit, where they eventually die.

As the tunneling activities near completion, the male flowers mature and produce pollen. Before
the female wasps leave the syconium they fill specialized pouches on their thoraxes with pollen
from anthers of male flowers. On emerging from a fig fruit the females search out new syconia,
and the entire cycle is repeated. The symbiosis between the fig plant and wasp is obligatory and
mutualistic. Neither symbiont can reproduce sexually in the absence of the other ( Addicott et al.,
1990).
Insects sometimes are attracted to flowers for reasons other than food and shelter. Orchids of the
genus Ophyrs produce flowers that so closely resemble female bees or wasps in morphology and
fragrance that the male insects attempt to mate with the flowers and in doing so transfer pollen
from one flower to another. Similarly, flowers of the orchid Oncidium resemble enemies of some
insects, and when the male insects attempt to fight the flowers they bring about pollination. These
forms of floral deception also occur in plants other than orchids.

Pollinators are also attracted to flowers be cause of their odor, color, shape, and nectar guides.
Color is an important attractant over long distances (distances > 1 m), whereas odor is more
effective at shorter distances. The various features of a flower are generally closely related to the
characteristics and behavior of its pollinators. Flowers can be grouped according to the types of
insects that pollinate them.

Types of Flowers
Insect-pollinated flowers

Bees are the most common insect pollinators, and they are also important pollinators of crop plants
such as fruit trees. Bee-pollinated flowers are generally brightly colored and often have a sweet
fragrance. The classic studies of the Austrian scientist Karl von Frisch in the 1920s showed that
bees can discriminate between colors and scents ( Frisch, 1950). Von Frisch found that bees can
distinguish the colors blue and yellow, a finding that correlates well with the fact that most bee-
pollinated flowers are blue or yellow. Bees also detect ultraviolet light, and many flowers have
pigments near their nectar glands that absorb ultraviolet light and create patterns directing
pollinators to the stored nectar. Bees cannot discern red flowers, except from their ultraviolet
markings. The pleasant odors of bee-pollinated flowers are usually the result of essential oils, such
as peppermint and lavender oils.

Nectar guides are color markings or a pattern of ridges on a flower that signal to an insect where
nectar is stored. In violets and orchids, the nectar guides are streaks of color on the lower petals.
Bee-pollinated flowers usually have modified petals that act as platforms to allow insects to alight
on the flower. Bees tend to concentrate their visits on one type of flower at a time.

Beetle-pollinated flowers are usually white or dull in color and smell like fruit or carrion. Beetles
have poor vision but a keen sense of smell. They eat different parts of a flower, and the flowers
they normally pollinate have their ovules embedded in protective tissues away from the regions
where the beetles forage.

Moth- and butterfly-pollinated flowers usually produce their nectar at the base of long petal tubes
or spurs. The nectar is out of the reach of many insects but available to the long tongues of
butterflies and moths ( Nilsson, 1998). Some butterflies pollinate red flowers in addition to blue
and yellow flowers. Because moths feed only at night, the flowers they pollinate are white or
yellow, and their fragrance develops strongly at night. Bird- and bat-pollinated flowers Bird-
pollinated flowers are large, usually red or yellow, and odorless. Large flowers are necessary to
hold the amounts of nectar birds require. Birds do not have a good sense of smell, but their vision
is well developed and they can see brightly colored flowers. Hummingbirds are the most common
bird pollinators in North and South America, and nectar is their primary energy source. The nectar
is produced in floral tubes away from the reach of smaller insects. Hummingbirds do not land on a
flower but hover before it when they are drinking nectar. Mainly tropical plants such as hibiscus
and bird of paradise have flowers that are pollinated by birds.

Bat-pollinated flowers such as those of some tropical cacti open only at night. They have dull colors
and fruity odors and are large enough to withstand the bat's foraging. As the bats drink the nectar
and eat some of the floral parts, pollen collects on their fur and is thereby transferred to other
flowers. Similarly, the colors of nectar guides become duller, and fragrant flowers lose their odor
and stop producing nectar. Moth-pollinated flowers change their position after pollination and thus
make it impossible for the moths to fit their proboscises into the usual channels in the flower.

12.3 PARASITIC PLANTS

In 1969, publication of Job Kuijt The Biology of Parasitic Flowering Plants was a major landmark in
the study of parasitism among flowering plants and inspired a generation of scientists to explore
this phenomenon. A recent publication edited by Malcolm Press and Jonathan Graves, Parasitic
Plants ( 1995), describes some of the advances made since Kuijt's work.

Plants that parasitize other plants have been recognized for many years. The parasitic habit has
developed independently in at least 11 unrelated families of dicotyledons. In some families, all the
plants are parasitic; in others only one genus is parasitic. How and why this habit arose in plants,
particularly in families whose other members are autotrophic, is not clear ( Musselman and Press,
1995). Parasitic plants are not found in the ferns, gymnosperms, or monocotyledons. A
classification of some common parasitic plants is given in table 12.2.

There are about 3000 species of parasitic plants, ranging from trees to small, herbaceous plants
and some are important weeds in Mediterranean and subtropical Asia and Africa. Holoparasites
obtain all of their nutrients from other plants, whereas hemiparasites still retain photosynthetic
leaves and can manufacture food. About 40% of plant parasites attack the shoots of host plants,
while 60% are root parasites ( Norton and Carpenter, 1998).

Plant Parasite Haustorium

A haustorium is a complex system that not only transports but also digests nutrients that it
extracts from the host. The parasite and host contain different sugars and amino acids, and
haustorial cells are responsible for converting nutrients from the host into a form suitable for the
parasite. The origin of haustoria in parasitic plants is obscure, and they may have evolved from
roots. Haustoria have highly modified xylem cells and lack phloem. Haustoria are initiated when the
roots of a parasitic plant intertwine with a host. The cortical portions of the host and parasite fuse,

Table 12.2 Some common parasitic flowering plants

Family Genus Common name
Cuscutaceae (dodders) Cuscuta Devil's thread
Grammica
Hydnoraceae Hydnora
Loranthaceae (mistletoes) Arceuthobium Dwarf mistletoe
Nuytsia
Phrygilanthus
Orobanchaceae (broomrapes) Conopholis
Epifagus
Orobanche
Rafflesiaceae Rafflesia arnoldii
Santalaceae (sandalwoods) Phacellaria
Santalum album
Thesium
Scrophulariaceae (figworts) Alectra
Lathraea
Pedicularis
Striga Witchweed
Tozzia
Viscaceae (mistletoes) Dendrophthora
Viscum album European Christmas mistletoe
Phoradendron American Christmas mistletoe

but their vascular systems remain separate. The next step in the process involves the penetration
of parasitic root tissue into the host and the formation of a single vascular system. Penetration of
host tissue is facilitated by mechanical force and enzymatic action of parasitic roots.
Mistletoes
The largest and best known group of parasitic plants are the mistletoes. There are about 800
species, most of which occur in the tropics and subtropics. All mistletoes in North America belong
to the family Viscaceae and belong to two important genera, Phoradendron (American mistletoe)
and Arceuthobium (dwarf mistletoe) ( fig. 12.3 ). Viscum album, the traditional European
mistletoe, has a wide range of hosts and is an important pest in orchards and forests. A few
mistletoes are specific to only one host; for example, the dwarf mistletoe Arceuthobium douglasi
attacks only Douglas fir trees. Mistletoes of the genus Arceuthobium show parallel phylogenetic
patterns of cospeciation with that of the host, and speciation occurs by host switching. Mistletoes
can even parasitize themselves ( Press and Graves, 1995).

Most mistletoes parasitize tree branches, but some, such as the giant mistletoe, Nuylsia floribunda,
have a terrestrial habit. This mistletoe forms a tree that grows as high as 10 m and parasitizes
roots of nearby grasses and other plants. It is common in West Australia and is called the
Christmas tree because of the bright flowers it produces in December. The Brazilian mistletoe,
Phrygilanthus acutifolius, is a vine that parasitizes several different trees simultaneously by means
of a large network of underground roots.

Mistletoes that grow on trees and shrubs have roots that grow along the host's branches and
develop haustoria. The roots are not influenced by gravity and grow up and down the branches. In
some mistletoes, new shoots develop from these roots and allow the parasite to compete for light
with the developing canopy of the host plant.

Many mistletoes are dispersed by birds,

Fig. 12.3 Dwarf mistletoe, a common parasite of white spruce throughout the northeastern United
States and Canada. Note small staminate and pistillate flowers Adapted from Hawksworth and
Wiens ( 1972).

which eat the fruits and void the seeds onto tree branches. The fruit of a mistletoe is a oneseeded
berry. Some mistletoes have fruits that explode and disperse their seeds, such as the dwarf
mistletoe, whose seeds travel up to 15 m and stick to any surface they contact. The dwarf
mistletoe is a serious pest of forest trees in the western United States because of the ease with
which it spreads from tree to tree.

When a mistletoe seed germinates, the emerging root forms a disclike structure that presses
against the surface of the host. A column of cells arises from inside the disc, penetrates the host
tissue by means of mechanical and enzymatic action, and forms a haustorium. The tissues of the
haustorium fuse with the host's vascular tissue and form a passageway through which water and
minerals move. A few mistletoes fuse with the host phloem tissue and in this way obtain
photosynthetic products from the host. Actual penetration of a host branch by a haustorium
appears to be a rapid process that follows a series of slow, preparatory stages. If a haustorium fails
to penetrate the branch, the parasite forms another disclike structure from which a new
haustorium develops. The parasite may make numerous attempts to penetrate the host.

In many mistletoes the haustorium stimulates the host tissue to divide, and a structure called a
placenta forms along the haustorial surface. The shape of the placenta varies according to the
species of mistletoe. Some Mexican species produce an elaborately furrowed placenta that is
commonly called woodrose and is prized for its beauty. Mistletoe evolution shows the importance of
host specialization in speciation either through host switching or cospeciation ( Norton and
Carpenter, 1998).

Sandalwoods
Sandalwoods are similar to mistletoes in that they are hemiparasitic, mostly tropical plants, and
may be trees, shrubs, or herbs. Most sandalwoods are root parasites, but some attack tree
branches. The sandalwood tree, Santalum album, has a fragrant wood that is used in cabinetry and
wood carving. Its oil is used to manufacture perfumes and soaps. Phacellaria is a sandalwood that
parasitizes mistletoes and other sandalwoods. The parasite grows inside the host tissues and
produces small external, leafy branches that emerge from swollen nodes of the host.

Some sandalwood species are destructive parasites of cultivated crop plants. Thesium humile on
wheat and barley, T. australe and T. resedoides on sugarcane, and Osyris alba on almonds, pears,
and grapes are well-known destructive plants.

Dodders
Dodders are holoparasites that consist only of twining stems and scalelike leaves. They belong to
the genera Cuscuta and Grammica and are members of the Cuscutaceae family. Dodders occur
throughout the world and attack a variety of hosts, both wild and cultivated. A dodder seed is
devoid of cotyledons, and the embryo is coiled. The seeds are passed through the digestive tracts
of grazing animals and can remain dormant for 5 years. When a dodder seed germinates, it gives
rise to a seedling that must find a suitable host within a few days or die. The seedling first grows
upright and then in a spiral fashion until it contacts a suitable host. After contact, the dodder forms
haustoria that grow toward the vascular cells of the host. When xylem cells of the host are
reached, the haustorial filaments differentiate into tracheids or vessels. After the haustorial
connections to the host have been established, the small roots of the dodder die and the parasite
loses all connection to the earth. In Cuscuta, haustoria are unusual in that the possess a group of
transfer cells that form fingerlike branches to clasp the host sieve cells. This results in a 20-fold
increase of surface area to obtain host nutrients. The haustorium contains a high concentration of
abscisic acid, which may have a role in sucrose uptake ( Riopel and Timko, 1995). The tips of the
dodder are phototrophic so the parasite always grows upward along the host plant. The Cuscuta
stem shows nastic movements that allows the parasite to "forage" the host and searches out
shoots with highest nutritional contents. Host secondary metabolites may be responsible for the
location and development of haustoria ( Kelly, 1992). Cuscuta's survival strategy is based on
continual attachments to new hosts so that overexploitation of the same individual is minimal.
Cuscuta is a metabolic sink which can attract more than 80% of the host's photosynthate and most
of its nitrogen, causing devastating losses from the host tissue. Late in the growing season the
dodder produces flowers and sets seeds to repeat the cycle.

The Cuscuta symbiosis provides an opportunity to study the molecular evolutionary steps
associated with the loss of photosynthesis. Cuscuta europea and C. reflexa have provided insights
into the biochemistry of photosynthesis.

Broomrapes and Figworts

Broomrapes and parasitic figworts are mostly herbaceous root parasites of temperate regions.
Broomrapes are holoparasites and belong to the family Orobanchaceae; figworts are hemiparasites
and are in the family Scrophulariaceae. In broomrapes and in some advanced parasitic figworts,
such as Alectra, Latraea, Striga, and Tozzia, the seeds germinate only in the presence of exudates
from the roots of specific host plants. The exudates cause the parasite to grow toward the host and
also to develop thicker roots with more root hairs. Striga (witchweed) species are obligate
parasites of cereal crops, such as sorghum and millet, in Africa and India. Seeds of these parasites
remain viable in the soil for many years and germinate only when near a suitable host. Compounds
called sorgoleons that stimulate germination of Striga asiatica seeds have been isolated from
sorghum roots. Sorgoleons may be part of a host's chemical defense system to inhibit the growth
of competing plants. Once contact with the host is made, initiation and development of haustoria
are triggered by secondary metabolites like phenolics, kinetins, and quinones which are present in
host cell walls ( Press & Graves, 1995).

Tozzia (big wort) lives underground much of the time, takes several years to mature, and then
produces a green flowering shoot above ground. After flowering and releasing seeds, the plant
dies. Common broomrapes include beechdrops ( Epifagus virginiana), found only on roots of the
American beech, squawroot ( Conopholis americana), a parasite of the roots of red oaks, and
cancer root ( Orobanche uniflora), a root parasite of a variety of plants.

Hydnora

Hydnora is a genus of subterranean, parasitic plants and a member of a small, unique family, the
Hydnoraceae, all of whose members are root parasites. The plants generally grow in remote areas
of South Africa and in East African countries. Hydnora produces thick, coarse roots that are bright
red inside and grow through the soil and small, thin roots that parasitize the host roots. The coarse
roots produce white, fleshy flowers that have a foul odor and are pollinated by beetles. Hydnora
fruits are large and fleshy and mature underground. They have a fruity odor and are eaten by
humans and other animals. The dried and powdered roots of Hydnora abyssinica, called tartous,
are used by traditional African healers to treat diarrhea and other sicknesses. Seeds of the parasite
germinate only in the presence of exudates from the host roots.

Rafflesia

The most exotic and spectacular parasitic plant is Rafflesia arnoldii, a native of the jungles of
Sumatra. The plant is a holoparasite on the roots of plants and produces the largest known flower
(about 1 m in diameter) in the plant kingdom. The parasite lacks stems and leaves and consists
only of thin, filamentous haustoria that are so fine they have been confused with fungal hyphae.
The flower of Rafflesia is fleshy, purplish brown with white patches, and has highly fused sexual
parts ( fig. 12.4 ). The inner parts of the petals are raised to form a circular rim or diaphragm. The
flower smells like carrion and is pollinated by flies. Rafflesia belongs to a small family,
Rafflesiaceae, which consists of about 8 genera and 30 species of tropical plants. The fruits are
large and fleshy with small seeds approximately 1 mm in diameter. How the seeds are dispersed
and the conditions under which they germinate are not known. The family parasitizes only tropical
members of the Vitaceae, or grape, family, which form woody vines.

12.4 SUMMARY
One reason angiosperms have been so successful is their ability to form mutualistic symbioses with
bacteria, fungi, and animals. Plants have evolved various defenses that discourage herbivory.
Carnivorous plants capture prey by using different trapping strategies. Ants help plants to disperse
seeds, pollinate flowers, supply nutrients, and defend against herbivores. Insects and flowers have
coevolved for millions of years, and pollination is the result of such a coevolution. Adaptations of
flowers have been toward changes that promote cross-pollination, as this leads to greater

hybridization and variation among the offspring. A few plant-insect

Fig. 12.4 Rafflesia arnoldii, a parasitic plant, produces the largest known flower in the plant
kingdom. Adapted from Kuijt ( 1969).

relationships such as that between the Yucca plant and a moth and the fig plant and a wasp are so
highly evolved that neither symbiont can complete its life cycle in the absence of the other one.
Some plants and animals have undergone biochemical coevolution.

Flowers attract insects with pollen and nectar as sources of food. Buzz pollination is a means by
which bees cause anthers to release their pollen. The sugar concentration of different plant nectars
varies and is related to the energetic needs of the pollinators. Birds require more nectar than
insects. Insects are also attracted to flowers because of their color, shape, odor, and nectar guides.
Some orchids produce flowers that resemble female bees or enemies of the pollinator, attracting
male bees, which try to mate or fight with the flowers and in the process bring about pollination.

Flowers have features that relate to the behavior of their pollinators. Bee-pollinated flowers have
bright colors, a sweet smell, and nectar guides, whereas flowers pollinated by beetles are white
and have a fruity or carrion smell. Bird-pollinated flowers are large and odorless, those pollinated
by bats open at night and have dull colors and a fruity fragrance. After pollination a flower
undergoes changes that serve as signals to pollinators that food is no longer available at the
flower.
Parasitic plants occur in many different families of angiosperms, especially in tropical and
subtropical regions, and range in size from trees to small herbs. Mistletoes are the largest group of
parasites, with about 800 species. Most mistletoes have a wide host range, parasitizing tree
branches by means of haustoria that fuse with the vascular tissue of the host. A plant parasite
haustorium is a complex system that extracts and digests nutrients from the host. Sandalwoods
are mostly root parasites, and dodders consist mainly of thin stems that wind around the host.
Broomrapes and figworts are root parasites that grow in temperate regions. Their seeds germinate
only in the presence of exudates from the host plant. Hydnora is a root parasite that grows and
flowers underground. Rafflesia arnoldii parasitizes the roots of other plants and produces the
largest flower among the angiosperms.

The parasitic habit is found in all kingdoms including plants. It may seem surprising that organisms
that can manufacture their own food through photosynthesis should revert to parasitism and take
nutrients and water from other organisms. Why this habit has evolved among separate lines of
plants is unknown. In the first chapter we indicated that over a long period of time some parasitic
as- sociations have evolved into mutualistic ones. Whether this will occur among plant parasites in
the future is not clear. At present, there is no evidence to suggest that plant parasites might confer
a benefit on their hosts. Because the parasitism is between members of the same taxonomic
group, angiosperms, the defenses of the host plant may not be sufficiently strong to slow or stop
the growth of the parasite.

The relationships between flowers and their pollinators are of great interest to ecologists and
evolutionary biologists, who are using pollination systems to test various hypotheses related to
general ecological and evolutionary principles. The process of coevolution and the adaptive
response of plants and animals to each other are seen clearly among a myriad of examples of
flowering plants and their pollinators. There appears to be a continuum of relationships from plants
whose flowers are accessible to many different insect pollinators to the extreme examples of the
yucca and fig plants that depend on only one type of insect for pollination. Pollination biology has
changed dramatically during the past 15 years, from a purely descriptive subject to one in which
computer and mathematical models are being used to simulate gametic competition in flowers and
various aspects of plant-pollinator relationships.

13 BEHAVIORAL AND SOCIAL SYMBIOSES

Darwin book The Expression of the Emotions in Man and Animals ( 1872) was a turning point in the
study of behavior. For the first time, behavior was considered from an experimental view, and it
was recognized that adaptive behavior of an organism fosters its survival and reproduction. Today,
behavioral scientists study four aspects of behavior: causation, development, evolutionary history,
and function. Observation and description are central to behavioral studies, but the problems
involved in carrying out such studies are great. For example, observations cannot be made
continuously during the lifetime of an organism under study. Further, behavior is complex, and a
scientist can study only one aspect of it at a time. Therefore, personal biases of the scientist
become an important consideration in behavioral analysis. Causation is studied by examining the
factors that influence behavior over relatively short periods of time in the life of an organism.
These factors include environmental as well as sensory and physiological stimuli. Regulation of
behavior in an interorganismic association involves internal as well as external factors. Changes in
hormone levels or brain abnormalities may result in behavioral modifications of the host. Similarly,
a symbiont's behavior may be influenced by developmental signals from the host.

The development of behavior results from the interplay between the genetic and environmental
components of an organism's life cycle. Many behavioral patterns deteriorate or are modified
because of symbiotic associations. Scientists also study the role of natural selection in the evolution
of behavior within a species. Advances in sociobiology have brought about new insights into the
evolution of social behavior. Genetic fitness and natural selection are two central tenets of
sociobiology. Genetic fitness is measured as a contribution of a particular genotype to the next
generation in a population. Parasitic symbioses, infectious diseases, pathogenicity, and host
resistance are important factors that increase the genetic fitness of the associating species. Natural
selection always maximizes fitness by increasing the chance for survival and reproduction of a
species ( Moore and Gotelli, 1996).
The functional aspect of behavior relates to an organism's adaptiveness to its environment. The
processes of natural selection mold behavior to a particular environment. Examples of specific
behavioral patterns include migration patterns among birds, fishes, and mammals; food selection
among specialized feeders; and symbiotic associations between different species. Based on
symbiotic interactions, behavioral ecology today is gathering a new perspective that includes
population regulations ( Anderson and May, 1978; Price, 1980), maintenance of genetic
polymorphism ( Clarke, 1979), structure of the ecological communities ( Dobson and Hudson,
1986), phenotypic determination as in bird coloration ( Hamilton and Zuk, 1982), and social
organization ( Hausfater and Watson, 1976).

Although behavioral patterns can be recognized in most symbioses, including those involving
bacterial and fungal symbionts, the best known patterns are those of animals. In this chapter, we
consider some examples of cleaning symbioses of marine organisms, symbioses in which parasites
modify host behavior, and symbioses of social birds and insects.

13.1 BEHAVIORAL SYMBIOSES

Cleaning Symbioses
Many marine fishes are cleaned regularly of ectoparasites and diseased or damaged tissues by
specialized fish or shrimp called cleaners. The cleaners provide a valuable service by keeping fish
free of parasites and disease, and, in turn, they acquire food and protection from predators. The
behavioral patterns associated with these symbioses are remarkable and include posing by host
fishes to expose to the cleaners that part of their body that needs attention. Posing is a means of
communication between the host and the cleaner. Host poses include opening mouths and gills to
allow the cleaner to enter, or assuming an unnatural vertical position ( fig. 13.1 ).

Cleaning symbioses have been observed in many parts of the world but are most common in
tropical waters. The fish involved in this symbiosis first establish cleaning stations near prominent
parts of the ocean floor, along the margins of kelp beds, or even in ship wreckage. Some stations
remain constant for several years, whereas others are used only for a short time. Tropical cleaners
have bright colors in patterns that contrast sharply with their background. The colors, along with
ritualistic displays ("dances") put on by the fish, attract host fish to the cleaning stations. Tropical
cleaners work alone or in pairs. In contrast, cold-water cleaners have dull colors and live in
schools. Many species of fish are known to be cleaners, especially in their juvenile stages. These
fish generally live along the coast or in coral reefs. Deep ocean fishes, such as tuna, may also have
cleaners, but this has not been firmly established. Even marine turtles, manta rays, and sharks
visit cleaning stations.

Parasites that are removed from the host include small crustaceans such as copepods and isopods,
as well as mats of bacteria and fungi that grow from infected host tissue. Parasitic crustaceans are
usually transparent or the same color as the host, which makes it difficult for the cleaner to remove
them all. The food cleaners obtain from their hosts is only part of their diet; they also eat small
animals from the surrounding water or from the ocean floor.

Although many different types of fish have evolved the cleaning habit, some of their adaptations
are similar. Many cleaners, for example, have long, pointed snouts that enable them to probe into
small crevices of the host's skin or gills and tweezerlike teeth that are used to remove parasites
embedded in the skin.

The small wrasse or senorita, Oxyjulis californica, is a cleaner common off the southern California
coast. It cleans a wide variety of fishes including the ocean sunfish (Mola mola), black sea bass
(Stereolepis gigas), and blacksmith (Chromis punctipinnis). In the Caribbean a genus of goby
(Gobiosoma) includes species that are cleaners even as adults and live off what they receive from
their hosts. Many fish may crowd around a single cleaner and assume various poses as they wait
their turn to be cleaned. Several other wrasses such as the bluehead wrasse (Thalassoma
bifasciatum) and the Mexican rainbow wrasse (Thalassoma lucasanum) are also cleaners.

The more highly coevolved the symbiosis between a cleaner and host, the less likely that the
cleaner will be eaten by the host. Senoritas are rarely eaten even though they enter the mouths
and gills of some large hosts. Other cleaner fish, and especially shrimp, are not as fortunate and
may be eaten by their hosts. The tiny neon goby (Elaca-tinus oceanops) lives among coral reefs
and cleans a variety of fishes including large groupers, parrot fishes, grunts, angels, and morays.
The goby swims in and out of the mouths and gills of its hosts with impunity. Several gobies may
group together to clean a large fish. The large barracuda, Sphyraena barracuda, which grows to a
length of 6 feet, is cleaned by a blue and yellow wrasse (Bodianus rufus) that is less than 1 inch
long. Remoras are sucker fish that spend most of their lives attached to sharks and turtles and not
only share in their meals but also clean them of parasites.

Several species of cleaner shrimp are common in tropical waters. The Pederson cleaning shrimp
(Periclimenes pedersoni) is 4 cm long and has antennae that are even longer. The shrimp generally
lives with an anemone, which protects the shrimp and serves as its cleaning station. When a fish
approaches the anemone, the shrimp waves its antennae and body back and forth until the fish
gets close enough for the shrimp to climb on it. The shrimp then crawls over the fish's body and
removes parasites. In some cases, cleaner shrimps and cleaner fishes occupy the same station.

The boxer shrimp (Stenopus hispidus) lives in tropical waters throughout the world. It grows up to
8 cm long, has a white body with red stripes, and long, white antennae. The shrimp lives among
gray corals and is easily visible to fish seeking its services.

Experiments designed to test the importance of cleaner fish and shrimp to marine fishes have
shown that cleaners control the spread of host parasites and infections. In a field experiment
conducted in the Bahamas, Conrad Limbaugh ( 1961) removed all the cleaning organisms from two
small, wellpopulated reefs. Limbaugh noticed that within a few weeks the number of fishes in the
reefs had declined sharply and the fishes that remained were infected by fungi and other parasites.
A marked difference was seen between the health of fishes of reefs without cleaners and the health
of those with cleaners. Other experiments have not been as conclusive, and some investigators
believe that the cooperative behavior of the hosts could be a response to the tactile stimulation
given by the cleaners ( Losey, 1987; Poulin and Grutter, 1996).

Some fishes change color while they are being cleaned. For example, the black surgeonfish
(Acanthurus achilles) turns bright blue, and other fishes assume a bronze color. The color changes
may help to increase the contrast to ectoparasites, thereby making parasites more visible to the
cleaners.

Often when fishes are being cleaned, they become alarmed and signal their alarm to their cleaners,
who then quickly retreat. Cleaners working in a host's mouth during an alarm are ejected by the
host or given a signal that causes them to leave.

Some fishes in the Indo-Pacific region where cleaners may have first evolved mimic the color
patterns of cleaners. Such a disguise protects a fish from predators or allows it to approach prey
without alarming them. Because the saber tooth blenny (Aspidontus taeniatus) closely resembles
the common cleaner Labroides dimidiatus, it can approach other fish, particularly juvenile ones,
which are less wary, and bite off pieces of their fins. The blenny also removes parasites from larger
fishes. The blenny may be in an intermediate stage of evolution, between a predator and a cleaner.

Much of the information on cleaning symbioses comes from observations made by divers. For this
reason, studies have concentrated along coastal areas and around tropical islands and reefs.
Observations of deep-sea fish populations and those of colder waters such as in the Arctic and
Antarctic are limited.

Cleaning symbioses also occur with land animals. The red rock crab, Grapsus grapsus, is a cleaner
of the marine iguana Amblyrhynchus cristatus, which lives on the Galapagos Islands. Various types
of birds, such as oxpeckers, remove parasites and infected tissue from large animals such as water

buffalo, crocodiles, cattle, and zebras ( Breitwisch, 1992). Anemone-

Clownfish Symbiosis
Fishes of the genera Amphiprion, Dascyllus, and Premnas, commonly called clownfish, form
mutualistic associations with giant sea anemones that live in coral reefs throughout the Pacific
Ocean. The associations are obligatory for the fish, but facultative for the anemones. The
anemones eat prey they have paralyzed by means of poisonous nematocysts discharged from
specialized cells in their tentacles. The clownfishes are immune to the stinging nematocysts and
can nestle within the tentacles and contact them frequently without harm. The question of how
clownfish develop their immunity to the anemone poison has intrigued scientists ( Fautin, 1991;
Fautin and Allen, 1994).

Some clownfish go through a period of acclimation before they become protected from the
anemone. Studies designed to determine how acclimation is achieved appear to show that the
mucous coating around the fish is changed by association with the anemone and the fish is no
longer recognized as prey by the anemone.

The change in the mucous coat was first thought to result from internal secretions from the fish,
but scientists now believe it is a result of the addition of mucus from the anemone ( Mariscal,
1971). When the fish acquires this mucus, the anemone no longer recognizes it as foreign and thus
does not discharge its nematocysts when its tentacles are contacted by the fish. To obtain mucus
from the anemone, the fish undergoes an acclimation behavior. The first stage of this behavior is
recognition of the specific type of symbiotic anemone by the fish. The clownfish approaches the
anemone cautiously, swims around its column and oral disc, and comes as close to the tentacles as
possible without touching them. After frequent passes over the oral disc, the fish makes the initial
contacts with the anemone by means of its tail and fins. At first, the tentacles adhere to these
structures and the fish pulls away and frees itself. More extensive body contacts then follow until
the fish becomes fully acclimated and can bury itself in the anemone's tentacles. The acclimation
time varies but may take up to 1 h. If a clownfish becomes separated from its host for longer than
one hour, it must again perform the acclimation behavior. The immunity a fish acquires from one
anemone does not protect it against another anemone should the fish decide to move on.

Studies by Richard Mariscal have demonstrated the importance of the protective cover around the
clownfish ( Mariscal, 1971). When Mariscal presented pieces of a grouper and an acclimated
clownfish, skin-side down, to a giant anemone, the tentacles of the anemone adhered strongly to
the grouper skin but not to that of the clownfish. When both pieces were presented flesh-side
down, the tentacles of the anemone adhered to both pieces, which were then eaten.

Once a clownfish acclimates to its anemone, it stays there as long as the food supply is adequate.
Clownfish are brightly colored and marked, and they attract larger fish to the anemone. These fish,
if they venture too close, are stung by the tentacles and eaten by the anemone. The clownfish
share in the meal and also remove fragments of the prey and wastes from the anemone. The fish
may also bite off and eat pieces of the anemone's tentacles that contain symbiotic algae and eat
the crabs and shrimp that also live among the anemone's tentacles. Thus, in addition to protection
from predators, the clownfish obtain food.

A similar relationship exists between the Portuguese man-of-war (Physalia physalia) and the horse
mackerel (Trachurus trachurus). The fish live between the tentacles of this cnidarian and somehow
avoid being stung by its nematocysts. The bright blue and silver colors of the fish, as well as its
small size, attract prey for the man-of-war.

Host-Parasite Interactions and Behavior

Modifications
Many parasites, such as protozoans, helminths, and nematodes, have complex life cycles. They
spend their early life in one animal species, the intermediate host, and reach sexual maturity in
another animal species, the definitive or final host. A cost-benefit equation is key to understanding
the evolution of behavior. From a parasite's perspective, its behavioral evolution depends largely
on enhancement of its transmission. The evolution of host behavior is influenced by efforts of the
host to avoid contact and to live with the infection. Therefore, ecological and behavioral models for
habitat choice and patch settlement of host organism may be determined by the parasitic symbiont
( Curio, 1988; Barnard and Behnke, 1990). For example, male fleas are more likely to be found on
juvenile male ground squirrels, which are the part of the
host population that leaves its home range to colonize new areas. By selecting juvenile male
squirrels, male fleas can move to new resource patches and also avoid inbreeding ( Hanley et al.,
1996).Host behavior determines parasitic infections by factors such as host exposure, host
susceptibility, and parasitic virulence. A parasite may support features of another species that
differs from its host. The larvae of the swallowtail butterfly, Danaus chrysippus, taste bad to
insectivorous birds because the larvae feed on toxic plants. A parasitoid lays its eggs only on
butterfly larvae that are not eaten by birds, and thus an advantage gained from the plant toxin is
neutralized by the parasitoid ( Gibson and Mani, 1984).A cleaning fish's grooming behavior on its
host fish decreases the number of ectoparasites it carries. Fever in an infected host is also an
adaptive feature, as shown by desert iguanas, which raise and lower their body temperature to
slow the growth of parasites ( Kluger, 1979).Parasitic symbionts have a variety of physiological
and ecological mechanisms to ensure their survival and reproduction. Some parasites modify the
behavior of their intermediate host in such a way that the host becomes more vulnerable to
predation by the definitive host species ( Giles, 1983; Moore, 1984a). The life cycles of symbionts
and their hosts may show parallel evolutionary development ( Beckage, 1997).It is argued that
parasites evolve toward reduced virulence against the host in order to prolong survival of both
host and parasite ( Holmes, 1983). A parasitic symbiont with its larval stages in an intermediate
host must allow its survival until it passes to the next host in the parasite's life cycle. For
example, larvae of the tapeworm Schistocephalus solidus inhabit three-spined sticklebacks,
whereas the adult tapeworms occur in the intestines of fish-eating birds. The tapeworm larvae
interfere with stickleback reproduction, slow its growth, increase its oxygen consumption, and
hamper its movement by distending the host belly. Parasitized fish swim close to the water
surface, making it easier for birds to eat them. This form of behavior is called adaptive
manipulation. Another example is host castration and prolonged life of fluke parasitized snails,
which allow longer periods for the parasite to reproduce ( Minchella et al., 1985). Following are
further examples of parasites that alter their host behavior:
1. Larvae of the nematode Tetrameres americana invade the muscles of grasshoppers, causing
the host to become sluggish and therefore easily caught by chickens in which the nematodes
live as adult worms.
2. Larvae of the tapeworm Taenia multiceps parasitize the central nervous system of sheep
and cattle. The infected animal staggers in circles and becomes separated from the herd.
Carnivores such as wolves and wild dogs constantly search for such weakened prey. The
tapeworm becomes sexually mature in the digestive tract of the second host species.
3. The fluke Leucochloridium macrostomum occurs in the rectum of European birds such as
crows, sparrows, jays, and nightingales. The birds acquire the flukes in a highly unusual
manner. Fluke eggs are ingested by snails, where they develop into the larval, sporocyst
stage. The parasite then produces a second sporocyst generation, which migrates to the
head and tentacles of the snail. These structures then enlarge, and the tentacles become
green, brown, or orange with prominent banding patterns. The sporocysts pulsate, and this
movement attracts birds, which peck on the sporocysts and ingest them. Inside the host
birds the larval cysts rupture and liberate the adult parasites. This is an example of
aggressive mimicry. Sporocysts acquired by a parent bird are transferred to nestlings. In a
similar manner, pulsating sporocysts of the fluke Ptychogonimus megastoma are ingested
by shore crabs, and the fluke larvae become encysted in the crab tissue. Fish acquire the
parasite by ingesting the infected crabs.
4. The lancelet fluke Dicrocelium dendriticum inhabits the gallbladder of sheep, deer, rabbits,
and other grazing mammals. Eggs of the fluke are eaten by land snails, in which the
cercarial larval stages develop. The mature larvae exit the snail in a slime ball, which is
eaten by ants. The "brainworm" larvae of the fluke forms cysts in the brain of an infected
ant

and radically transforms its behavior. The affected ant climbs to the tip of a blade of grass and
hangs on tightly. This abnormal behavior increases the chances of the ant being transferred to
a grazing host ( Holmes and Bethel, 1972). One cercaria of many ingested by an ant becomes
the brainworm, and it sacrifices future growth and reproduction, thereby leaving other
cercariae in the ant to assume the evolutionary burden of the species. This is an example of a
parasitic altruism, where a cercaria sacrifices its own life to alter an ant's behavior but gains
many times over in the next generation ( Smith Trail, 1980).
5. Acanthocephalan worms are endoparasites that inhabit the alimentary tracts of vertebrates,
where they attain sexual maturity. The intermediate host of these parasites is an arthropod,
whose behavior is transformed after infection. The altered behavior significantly increases the
chances of the parasite being acquired by the final host through preferential predation of the
infected intermediate host. There are several well-known examples of this phenomenon.
a. Polymorphus paradoxus is an endoparasite that inhabits the alimentary canals of
beavers, mallards, and muskrats. Aquatic crustaceans such as amphipods are the
intermediate hosts of the parasite. Healthy amphipods normally move away from light
and rarely appear at the surface of a lake or pond. When disturbed, they dive and burrow
into the mud at the bottom. Parasitized amphipods, however, are attracted to light and
are commonly observed clinging to vegetation on the surface of the water. This altered
behavior increases the likelihood of the amphipods being eaten by a final host species (
Moore, 1984a; 1995). Similar anomalous behavior of amphipods infected with
Polymorphus marilis allows ducks to catch the crustaceans.
b. Songbirds preferentially feed on pill bugs or sow bugs (isopods) that have become
infected with the acanthocephalan Plagiorhynchus cylindraceus. Songbirds do not feed on
uninfected pill bugs, but infected bugs may make up more than 40% of a bird's diet. The
behavior of the parasitized pill bugs is altered so that they seek out exposed areas, where
they are quickly eaten by birds.
c. Cockroaches parasitized with Moniliformis moniliformis are attracted to light and become
hyperactive. The roaches are less likely to remain hidden and therefore more likely to be
caught by rats, the final host for the parasite.

13.2 SOCIAL SYMBIOSES

Among insects and birds, symbioses occur not only between individuals of different species but also
between individuals and societies and between different societies ( Abrahamson , 1989; Price et
al., 1991; Choe and Crespi, 1997).

In insect societies, each individual has only a limited role in relation to the society as a whole.
Because of this limitation, other species of insects can easily intrude into the society, and this leads
to symbiotic interactions. A similar situation exists in birds whose young mature slowly and do not
form close bonds with their parents. These parents are easily deceived when eggs are laid in their
nests by other birds and will incubate the foreign eggs and care for the young along with their own.
Among other birds, such as ducks, and among mammals, social symbiosis is rare because the
parents form close recognition bonds with their young at an early stage and repel all apparent
intruders.

Edward Wilson of Harvard University is the leading authority on social symbioses. His studies of
insect societies and their complex internal and external interactions have greatly increased our
understanding of the subject. Wilson groups social symbioses among animals into three major
categories: social parasitism, social commensalism, and social mutualism ( Holldobler and Wilson,

1990; Crozier and Pamilo, 1996). We consider only these three major groups Social
Parasitism
Parasitic ants, wasps, and other insects

More than 200 species of ants have evolved different types of social parasitism with other ants,
ranging from the loosely organized to the highly integrated. The simplest form of social parasitism
is one in which a colony of ants builds a nest next to a colony of a different species and steals food
from and preys on the neighboring workers. Some ants may even occupy the nest of another
species and maintain a separate colony within the nest ( Bourke and Franks, 1995).

A highly evolved form of social parasitism among ants is shown in colonies containing different ant
species living together. For example, in several subfamilies a fertile queen may enter a colony of
ants of a different species and by some unknown mechanism be accepted by the workers. The host
queen is killed, either by its own workers or by the intruder queen. As the host workers die of old
age or injury, there are no offspring to replace them, and the nest becomes fully colonized by
offspring of the new queen. Wilson calls this example temporary social parasitism ( Holldobler and
Wilson, 1990).
Another type of social parasitism among ants involves slavery. Some ant species raid the nests of
different ant species, steal pupae, and return with them to their own nest, where the young ants
are reared as slaves. The slave ants do all of the work in the nest such as foraging, building, and
rearing the brood ( Topoff, 1990, 1994).

One of the most highly integrated examples of social parasitism involves the parasitic ant
Teleutomyrmex schneideri, which is found in the Swiss and French Alps. Teleutomyrmex schneideri
parasitizes a close relative, Tetramorium caespitum. The ant parasite lives only in the nests of its
host and lacks a worker caste. The parasite queen is much smaller than the host queen and spends
most of its time clinging to the back of the host queen or workers. The parasite ants are fed by the
host workers. Both queens lay eggs; those of the host produce only workers and not sexual forms,
while those of the parasite produce only sexual forms. When the parasite ants mate, the new fertile
queens either remain in the same nest or fly away and invade other nests of the host ant. It is
thought that Teleutomyrmex represents an end point in the various evolutionary pathways of
parasitic ants. The morphological adaptations of the ant and loss of the worker caste are traits of
other highly evolved parasitic ant species.

Parasitic wasps behave in a manner similar to parasitic ants. The queen wasp penetrates the nests
of other wasps, kills the host queen, and assumes her role. Some wasps are permanent residents
in the nests of other wasps and, like ants, have evolved to a point where they do not have a
worker caste and depend on the host workers to care for them. A similar type of behavior has been
reported for parasitic bumblebees.

The origin of parasitic ants is of great interest to scientists. In 1909, Carlo Emery developed a
hypothesis, known as Emery's rule, that states that parasitic ants and slave-making ants evolved
from closely related species that now have become their hosts. Wilson suggests that parasitic ant
species and their host species developed from a common parent species through geographical
isolation and genetic differences. When the range of the two new species later overlapped, one
became a parasite on the other. Wilson believes that Emery's rule applies also to parasitic wasps
and bumblebees.

Termites do not exhibit the same type of social parasitism as other social insects because termite
queens form new nests after fertilization rather than returning to their old nests or attempting to
invade established nests.

Some beetles are accepted into an ant or termite colony because they produce substances that
attract the host insects. The substances are similar to pheromones, which the host normally
produces, and have a calming effect on the hosts after they lick these substances. Wilson calls
these compounds appeasement substances because they help the beetle become adopted into an
ant or termite population. After the beetle obtains entry into a nest, it feeds on the host larvae,
and by stroking worker ants with its antennae can stimulate them to regurgitate food. Many of the
beetles that live with ants and termites have evolved morphological features, such as a swollen
abdomen, that mimic features of their hosts. Loss of eyes and wings are also common changes the
beetles have undergone.

Brood parasitism in birds

Social parasitism is also present among birds, especially cuckoos and cowbirds. About 100 species
of cuckoos are obligate brood para-sites, which means that they depend on other birds to hatch
their eggs and rear their young ( Payne, 1998). The cuckoos lay their eggs among those of the
host birds, and when the eggs hatch, the young cuckoos destroy the host's eggs and kill any young
of the host ( fig. 13.2 ). This association is so highly evolved that the eggs and young of the cuckoo
resemble those of the host bird in color and size. One of the best studied social parasites among
birds is the European cuckoo (Cuculus canorus). This species consists of different populations, each
having a different host bird, whose eggs are mimicked. How the cuckoo populations maintain their
specific relationships with their host birds is not clear, particularly since the different cuckoo
populations interbreed. Cuckoos have evolved other adaptations in addition to egg mimicry for
their parasitic habit. Their eggs have thick shells that resist damage when the eggs are dropped
into host nests, and the birds have extendible cloacae, which enables them to deposit their eggs
into small nests ( Lyon, 1993). Another remarkable adaptation is the ability of some cuckoos to fly
like hawks and in so doing to distract the host bird from its nest so that the cuckoo may deposit its
eggs undisturbed ( Lotem and Rothstein, 1995). Relationships between cuckoos and their hosts
afford excellent examples of coevolution ( Davies and Brooke, 1991; Rothstein and Robinson,
1998).

Cowbirds parasitize more than 200 bird species and have caused population decline in many of
these species ( Rothstein and Robinson, 1994). A coevolutionary arms race produced some
interesting behavioral outcomes: avian brood parasites have evolved special mechanisms to obtain
parental care from their host species, while the hosts have evolved mechanisms or traits to avoid
and reject parasites. For example, the host may remove its own eggs along with those of the
parasite. The presence of brood parasites can be advantageous if they remove ectoparasites from
the host nestlings.

In response to wide-ranging parasitic symbionts, many hosts have adopted behavioral

countermeasures. Hamadryas baboons and Hadza tribesmen in Tanzania avoid schistosome-
infested waters and collect water by digging holes in dry river beds ( Holmes, 1983). Other
examples of behavioral countermeasures include grooming, cleaning symbioses of marine animals,
sunning of frogs and salamanders to dislodge leeches, and dust-bathing of birds ( Murray, 1990).

Social Commensalism
The nests of social insects are home to many different scavengers such as mites, beetles, and
millipedes. The host insects either ignore these commensals or accept them as part of

Fig. 13.2 Brood parasitism of cuckoo and cowbird. Adapted from Wilson ( 1975

the colony. Some silverfish and millipedes live with army ants, participate in their forays, and even
share their prey.

Among insect-eating birds, several species may flock together to better avoid predators and to
forage more efficiently. Similar groups of mixed species have been reported among marine fishes,
such as dolphins and whales, and African mammals. Mixed groups of animals often congregate to
increase the efficiency of early warning of predators.

Social Mutualism
Mutualistic symbioses are common between ants and various insects such as aphids, scales,
mealybugs, and treehoppers. The ants tend these insects like cattle, milking them for food and
protecting them from predators ( Davidson and McKey, 1993) ( fig. 13.3 ).

Aphids penetrate the food-transporting system (phloem) of plants with their stylets and feed on the
plant sap, which is rich in sugar and minerals. The aphids digest only a small amount of the plant
sap they take in and excrete the remainder in a form called honeydew. Aphids that are tended by
ants excrete the honeydew slowly so that it accumulates as drops around the anus. Mutualism
between ant and aphid has evolved to such a high degree that their life cycles have become
coordinated, and the aphid has lost various structures that it normally has to defend itself, such as
a hard exoskeleton and jumping legs. When an ant is ready to feed, it strokes an aphid with its
antennae and stimulates it to exude honeydew. Some ants carry the aphids they tend back to the
nests each evening to protect them from predators and return them to the plants each morning.

To ensure a steady source of food, worker ants carry aphids and other honeydew insects to
different plants if the original ones are disturbed or uprooted. Some ant species maintain aphid
eggs in their nests during the winter and when the aphids hatch in the spring carry them to nearby
plants. The queens of some ant species even carry scale insects during their nuptial flights. Bees
and wasps also keep honeydew insects.

Some butterfly species have caterpillars that are tended by ants. The large blue butterfly Maculinea
arion cannot complete its life cycle without help from red ants. Until the caterpillars pupate and
hatch, the ants protect the caterpillars in their nests and feed on their sugary excretions. When the
chrysalis opens, the butterfly leaves the ant's nest. Other symbioses between caterpillars and ants
have been described ( DeVries, 1992).

Some South American ant species appear to have a mutualistic relationship with each other. The
ants form adjacent nests, share common trails, and collect honeydew from the same population of
treehoppers. The different ants are friendly to each other and seem to thrive because of the
association.

The giant cowbird (Scaphidura oryzivora) of Central and South America is a brood parasite that
lays its eggs in the nests of oropendolas, which are members of the grackle family. Oropendolas
have a natural enemy, the parasitic botfly (Philornis), which infests their nests and kills their
young. Studies have shown that oropendola nests containing young cowbirds have fewer botflies
than nests without cowbirds. Young cowbirds instinctively attack and kill maggots of the botflies
and remove them not only from the nests but also from the young host birds. Benefits to

Ants tending aphids Fig. 13.3 Ants licking honeydew from aphids. In this mutualistic symbiosis, the
ants obtain food from aphids, which they protect from predators

cowbird nestlings occur only when the host environment lacks stinging wasps and biting ants,
which provide some protection against botflies ( N. Smith, 1968). Cowbirds, unlike cuckoos,
tolerate the eggs and young of the host birds. Thus, an association that began as one of strict
parasitism has evolved into mutualism ( Rothstein and Robinson, 1994).

13.3 SUMMARY
Behavior in a symbiotic association is influenced by external and internal factors. Symbiosis
commonly modifies the behavior of the associating partners. Cleaning symbioses are common in
tropical, marine waters, where specialized fish and shrimp clean other fish of external parasites and
damaged tissues. In return for this service, the cleaners obtain food and protection from predators.
Cleaners and hosts communicate with each other by means of various behavioral patterns such as
posing or by means of bright colors and unusual color patterns. Some common cleaners include
wrasses such as the senorita, the neon goby, remoras, Pederson shrimp, and the boxer shrimp.
Experiments have shown that cleaners have an important function in tropical ecosystems by
controlling the spread of parasites. Some fish have evolved color patterns similar to those of
cleaner fish. This disguise protects the fish from predators and allows them to get close to prey
fish. Cleaning symbioses are also present among land animals.

Clownfish live in a mutualistic association with giant anemones that grow in tropical coral reef
communities. The fish acquire immunity to the anemone by means of an acclimation process
during which the fish coats itself with mucus from the anemone. The mucus disguises the fish in
such a way that it is not recognized as foreign by the anemone. Clownfish attract other fish to the
anemone and share the prey caught by its host.

Some animal parasites such as nematodes, tapeworms, flukes, and acanthocephalan worms modify
the behavior of their intermediate hosts in a manner that makes them more likely to be preyed on
by the final hosts.

Social symbioses occur among insect societies and birds and include social commensalism, social
mutualism, and social parasitism. In social symbioses each individual has only a limited role. Social
parasitism is highly evolved among ants and may involve simple predation, slavery, or the
integration of a foreign queen into an existing ant colony. According to Emery's rule, parasitic and
slave-making ants evolved from closely related species that now serve as their hosts. Interestingly,
a similar situation may exist among parasitic red algae, many of which occur only on other closely
related red algae.
Certain beetles become accepted into ant or termite colonies because of appeasement substances
produced by the beetles that appear to calm the hosts. The beetles feed on host larvae and food
that is regurgitated by worker ants.

Cuckoos are highly developed social parasites that lay their eggs in the nests of other birds. When
the eggs hatch, the young cuckoos kill the host's young and are raised by the host. Adaptations of
the cuckoo include mimicking the eggs of the host, extendable cloacae, and eggs with thick shells
that resist breaking when they are dropped into the host nest.

Silverfish and millipedes are commensals in the nest of social insects. Ants form mutualistic
associations with aphids, scales, and mealybugs, milking them for the food (honeydew) they obtain
from plants. Ants tend aphids like cattle, protecting them and carrying them from plant to plant.
Ants have a similar relationship with the caterpillars of some butterflies.

Cowbirds and oropendolas have a mutualistic relationship in which the young cowbirds kill botfly
maggots that are serious parasites of young oropendolas and thereby protect their foster nest and
host.

14 SYMBIOSIS AND COEVOLUTION

Although Darwin's theory of organic evolution has provided an intellectual framework for
understanding the processes of speciation and the unity of life forms, the theory has never fully
satisfied evolutionary biologists. In Darwin's view, evolution is the descent, with modification, of
different lineages from common ancestors. Population genetics, natural selection in gene pools,
adaptation, and isolating mechanisms have traditionally been considered by many biologists as
significant factors in the evolutionary process. Evolutionary ideas are now being refashioned in the
language of punctuated equilibria, genetic polymorphism, molecular evolution, and sexual
selection. To this list of new approaches, one can add the concepts of coevolution and symbiosis.
Evolutionary ecologists are convinced that evolutionary processes cannot be understood fully until
the close interactions between organisms are examined. Indeed, the term symbiogenesis was
introduced by Mereschkovsky in 1920 to recognize a process whereby new species arise as a result
of symbiotic interactions between different organisms ( Margulis 1991a, 1993a, b; Khakhina,
1992).

Coevolution is a kind of evolutionary "arms race" between organisms living together in intimate
associations. Adaptations that give one organism an advantage are countered by neutralizing
adaptations of the other organism. For example, evolution of virulence in a pathogenic organism is
often matched by a concomitant evolution of resistance of the host species. Reciprocal genetic
exchange, often occurring simultaneously in interacting populations, is the basis of coevolution.
Traits of one species evolve in response to those of another species, whose traits, in turn, were
influenced by the first species. Evolution of the eukaryotic cell has involved a series of genetic
interactions between ancient prokaryotes. Scientists have recently discovered that DNA exchange
has taken place among the nuclei, mitochondria, and chloroplasts of eukaryotic cells, attesting to a
complex evolutionary process. Coevolution that takes place between two different species is
pairwise coevolution. On a larger scale, one or more species may evolve a trait in response to traits
of several other interacting species, a phenomenon called diffuse coevolution. For example, a plant
species may develop toxins or other defenses in response to herbivory, and various animal
populations evolve means to overcome these defenses. The plant species then develop new
defenses, and the evolutionary process is repeated. In some interacting species, a coevolutionary
equilibrium appears to take place. Thompson has introduced a new "geographic mosaic theory of
coevolution," which differs from approaches that focus only on local populations ( Thompson,
1994).

The traditional theory of coevolution includes cospeciation as well as coadaptation among

interacting organisms. Long-term, close contacts between organisms and the reciprocal influences
that take place between them often result in the formation of new species. The phylogeny or
evolutionary histories of organisms involved in symbioses are closely related to each other. The
many relationships between insects and plants, in particular those involved in pollination, are clear
examples of the adaptation and speciation that can occur in symbiotic associations. The lichen
symbiosis is an example of how freeliving fungi (ascomycetes) become transformed into new
species during the course of symbiotic associations with algae. Parasitologists have often observed
a close relationship between the phylogeny of parasites and their host species. For example,
Enterobius, a genus of pinworms that infect primates, has a phylogeny and geographical
distribution that closely parallel those of its primate hosts. Evolutionary insights have been made
on the taxonomic relationships of species of bats, squirrels, and pocket gophers from the studies of
their ectoparasites, such as lice and fleas.Ecologists have applied the following rules to better
understand the evolution of host parasite interactions:
• Farenholz's rule states that the phylogeny of many parasites mirrors that of their hosts.
Because most parasites are obligate symbionts, it is assumed that parasites of present-day
hosts had ancestors that were parasites of earlier hosts.
• Szidat's rule states that the more primitive the host, the more primitive its parasites.
• Eichler's rule states that because the host and parasite share a parallel evolution, parasites
can be used to understand the phylogeny and origin of host species. Large host groups will
have more genera of parasites than small host groups. Parasitic symbionts may evolve in two
ways. Speciation may occur if some parasitic species become established in different hosts, or
if the parasites invade new tissues or organs in the same host species.

Additionally, Dieter Ebert ( 1998) used serial passage experiments to study the evolution of
parasites and their adaptation to their hosts. He found that within a host, competition among
pathogens resulted in increased virulence, whereas pathogens tended to become avirulent to their
former hosts.

14.1 GENETIC CONFLICTS AND

SYMBIOSES
From an evolutionary perspective, symbiotic relationships are an uneasy balance between
conflicting interests of host and symbiont genomes ( Werren and Beukeboom, 1998). Genetic
conflicts are believed to be the underlying cause for major evolutionary phenomena that include
(1) evolution of sex, its maintenance and its absence, (2) evolution of bacterial genomes, (3)
emergence of linkage groups (chromosomes), (4) sexual selection and mate choice, (5) meiosis
and crossingover, (6) multicellularity, (7) diploidy, (8) senscence, (9) sexual and somatic
incompatibility, (10) eusociality, (11) speciation and biodiversity, (12) genome size, and (13)
organelle behavior ( Bell, 1993; Hurst et al., 1996; Partridge and Hurst, 1998). The persistence of
one-cell stages in multicellular life histories may have a role in reducing conflicts of interest among
genetically different replicators within an organism ( Grosberg and Strathmann, 1998). Almost
10% of presentday angiosperms show male sterility due to one or several mitochondrial genes. In
Thymus vulgaris, all individuals contain male sterility mitochondrial genes and depend on nuclear
genes to counteract their effects by restoring pollen production ( Belhassen et al., 1991).

Genes, like organisms, may cooperate because they benefit from such gene-to-gene cooperation.
Pathogenic virulence may be viewed as the opposite of genetic cooperation. As the transmission of
pathogenic symbionts increases, they become less virulent to their hosts. In general, a pathogen
that has co-transmitted with its host is selected to minimize harm to its host. Enhanced host fitness
favors the symbiont's well being ( Frank, 1996).

Self-promoting genetic elements, also called ultra-selfish genes, were first recognized in 1945 as
parasitic chromosomes ( Ostergren, 1945). Various self-promoting genetic elements are vertically
transmitted, and they manipulate their host so as to increase their spread at the cost of other
genes within the genome. In Chlamydomonas reinhardtii, mitochondria are inherited from the
minus parental strain, while chloroplasts are inherited from the plus types. Likewise, in some
gymnosperms, mitochondria are inherited from one parent and chloroplasts from the other. Such
uniparental inheritance of mitochondria or chloroplasts may be viewed as an adaptive response to
counter self-promoting cytoplasmic genes.

Mitochondria and Programmed Cell Death

Recent studies suggest that the endosymbiotic origin of mitochondria and aerobic me- tabolism in
eukaryotes formed the basis of apoptosis, programmed cell death, which is common throughout
metazoans ( Green and Reed, 1998). Apoptosis plays a crucial role in animal development as well
as in the normal functioning of the immune system. Malfunctions of apoptosis result in cancers,
autoimmune diseases, and the loss of brain cells in Alzheimer's disease. In the origin of eukaryotic
life, the ancestors of present-day purple bacteria that were to become mitochondria formed
symbioses that benefited both the symbionts and host cell and allowed them to exploit energy
resources in the emerging oxygen atmosphere. The symbiosis also carried the seeds of potential
genetic conflict between the two genomes. The new mitochondrial symbionts controlled the life and
death of the host cell by providing critical antioxidants. The conditions that favored early
mitochondria could also have threatened the host cell's life by causing the release of mitochondria
to a free-living state. The potential genetic conflict and instability continued until some of the
mitochondrial genes were laterally transferred to the host nucleus. In this way the symbionts
became totally dependent on the host cell ( Kobayashi, 1998). Mitochondrial mechanisms involved
in apoptosis include (1) disruption of electron transport oxidative phosphorylation and ATP
production, (2) release of proteins such as cytochrome c that trigger activation of intracellular
cystein protease known as caspase, and (3) changes in cellular reduction-oxidation potential. Once
cytochrome c is released, the cell becomes committed to apoptosis. A group of proteins known as
Bcl-2 are channel-forming proteins that regulate mitochondrial activities ( Green and Reed, 1998) (
fig. 14.1 ).

Genetic strategies for reciprocal coadaptation have been examined for a number of host-symbiont
relationships. The strategies include gene-for-gene relationships, epistatic interactions, and
polymorphic variation.

Fig. 14.1 Role of mitochondria in cell death caspase activation. Oxidants and Ca2+ overload trigger
mitochondria to release caspase-activating proteins such as cytochrome c, which ultimately leads
to apoptosis. Redrawn from Green and Reed ( 1998).

Gene-for-Gene Interactions
The gene-for-gene hypothesis was first proposed by Harold Flor and was based on his studies with
flax (Linum usitatissimum) and the rust fungus Melampsora lini. Flor found that two independent
genes for resistance in the host plant were matched with two genes for virulence in the fungal
pathogen ( Flor, 1956). The resistance gene is inherited as a dominant trait, whereas virulence is
inherited as a recessive trait. The host plant and its fungal pathogen have thus evolved a
complementary genetic system in which each gene determining a host response is matched by a
gene or genes for a certain behavior in the pathogen ( Innes, 1995).

Since 1942, gene-for-gene coevolution has been documented or strongly suggested in more than
three dozen cultivated plants and the fungi that infect them. These include wheat-Puccinia graminis
tritici; wheatPuccinia recondita; barley-Ustilago hordei, barley-Erysiphe graminis hordei;
wheatErysiphe graminis tritici; apple-Venturia inaequalis ( Frank, 1992). Gene-for-gene
interactions have also been reported between the snail Biomphalaria glabrata and the blood fluke
Schistosoma mansoni; resistance in the snail results from a single, dominant gene, which is
matched with an avirulent gene of the fluke. Resistance or susceptibility of the individual snails is
controlled by the relative frequencies of genes. Gene-for-gene interactions are present in animal-
plant symbioses such as the cyst nematode parasitism of potato, barley, and soybean and aphid
infestation of rushberry and alfalfa. Resistance to the Hessian fly, Mayetiola destructor, in wheat
plants is governed by genes that are matched by corresponding genes for virulence in the fly (
Callow et al., 1988).

Scientists suspect that there may be a clustering of genes in host chromosomes that confer
resistance to more than one pathogen. For example, barley chromosome number 5 has genes that
control resistance to the powdery mildew fungus Erysiphe graminis hordei and also to the rust
fungi Puccinia hordei and Puccinia striiformis.

Gene-for-gene coevolution does not explain all types of host-parasite interactions ( Innes, 1995).
Gurmel Sidhu ( 1984) has described the complex interplay of two or more symbionts associated
with a host plant. For example, he has shown that the plant nematode Meloidogyne incognita alters
the resistance of tomato plants to the wilt fungus Fusarium, which alone cannot parasitize the
plant. Similar interactions between the fungal pathogens Fusarium and Verticillium on tomato
plants have been described. Sidhu has explained these observations in terms of the concept of
epistatic parasitism. Disease expression by the hypostatic parasite depends on the establishment of
the epistatic parasite ( Sidhu, 1984).

Molecular Approaches to Plant Pathogenesis

Since 1992, disease resistance genes have been cloned and characterized from several plant
species. Avirulence (avr) genes have been isolated from several plant pathogenic bacteria and
fungi since 1984. In the presence of an avr gene a plant pathogen is unable to induce disease on a
host plant and thus determine the host range of the pathogen. Some avr genes, when transferred
to virulent pathogens, cause the hypersensitive response in host plants ( DeWit, 1995). For
example, an avr gene (avrBsT) in the bacterium Xanthomonas compestris causes the
hypersensitive response in peppers, and loss of the avrBsT gene in the pathogen causes the
disease of bacterial leaf spot on resistant pepper varieties. Avr genes encode for hydrophilic
proteins that lack long stretches of amino acids known as signal sequences, which allow avr
proteins to be secreted to the outside medium. Avr proteins are responsible for plant disease
symptoms such as angular leaf spots on cotton and citrus canker. Hypersensitivity response and
pathogenicity (Hrp) genes that encode for the bacterial membrane pore-forming proteins allow for
the passage of the avr proteins to the outside. Resistance gene products are able to recognize the
avr proteins, and plant defense blocks the pathogen's further advance. Molecular biology of the
host resistance gene Hm1 was first described from corn in 1992. Lack of Hm1 in corn makes it
susceptible to the leaf spot fungus Cochliobolus carbonum. Although the fungal pathogen produces
a virulence factor, the HC toxin, which is responsible for the disease symptoms, the host Hm1 gene
encodes for an enzyme, HC toxin reductase, which destroys the toxin. Since 1992, more than a
dozen resistance genes have been characterized from crop plants. The PTO gene of tomato confers
resistance against the bacterial speck disease caused by Pseudomonas syringae, which car- ries the
avirulence pto gene (see fig. 5.4). The host PTO gene is a protein kinase, which is a signal
transducer in the hypersensitive response. Other resistance genes isolated include Cf2, Cf4, and
Cf9 from tomato against the fungus Cladosporium fulum, tobacco N' gene for resistance aginst
tobacco mosaic virus; and the flax L6 gene against the rust fungus Melampsora lini that carries the
avr6 gene ( fig. 14.2 ).

Genetic Polymorphism
Genetic polymorphism is common among species that participate in symbiotic associations,
particularly those involving host-parasite interactions. J. B.S. Haldane ( 1949) was the first to
suggest that parasitic symbionts might be the reason for polymorphism in their hosts. For example,
the variety of antibodies produced by a host organism has evolved in response to the many
different antigens produced by trypanosome parasites. Multiple genes for resistance and virulence
in hostparasite symbioses have resulted in a high degree of genetic polymorphism in the
interacting species. Some scientists have suggested that parasitic associations may be the principal
cause of protein polymorphism within a population. Stephen I. Gould and Richard Lewontin ( 1979)
have argued that evolutionary changes increase polymorphism within a population. Such a view is
a radical departure from Darwin's theory, which states that genetic changes of an organism are
subject to selective pressures of the environment and that natural selection conserves one genetic
type best suited to a particular set of environmental conditions and eliminates the others.

In humans, malaria has been responsible for the selection of three or more types of genetic
variation with respect to resistance to the disease. Resistance caused by the sicklecell hemoglobin
gene (HbS) has been studied extensively. Red blood cells containing sickled hemoglobin collapse
and assume a sickle shape under low oxygen tension. The HbS gene is inherited as a recessive
gene, and individuals who are homozygous for it die prematurely. Individuals heterozygous for that
trait survive because they contain a normal hemoglobin gene. In Africa, where malaria is endemic,
heterozygous individuals have a survival advantage because Plasmodium is unable to develop in
the sickle cells. Such an evolutionary strategy on the part of a host is
 Fig. 14.2 Gene-for-gene interactions are central to plant disease resistance. Resistance is
expressed only when the host plant contains a specific resistance gene product that recognizes the
corresponding avirulence gene product. All other combinations fail and disease results. Adapted
from Staskawicz et al. ( 1995).

called heterozygote advantage ( Friedman and Trager, 1981).

Duffy blood groups, MN antigens, and glucose-6-phosphate dehydrogenase (G6PDH) deficiency are
other biochemical variations in humans that have evolved in response to Plasmodium infection.
Individuals deficient in G6PDH, whether heterozygous and homozygous, are resistant to the
parasite because Plasmodium, an obligate intracellular symbiont of red blood cells, depends on the
host to supply G6PDH. The gene for G6PDH is highly polymorphic, with more than 50 alleles coding
for hundreds of variant forms of the enzyme. Individuals with low levels of this enzyme are
susceptible to anemia and are also sensitive to drugs such as sulfanilamides and the antimalarial
drug primaquine. The G6PDH deficiency gene occurs with high frequency among people from Africa
and the Mediterranean region. The absence of Duffy antigens from African people may also be
related to resistance to Plasmodium ( Mathews and Armstrong, 1981). Duffy antigens occur on the
surface of red blood cells at the sites to which Plasmodium vivax binds in order to invade the cell.
The presence of MN antigens is also thought to be a significant example of genetic polymorphism in
response to Plasmodium infection.

14.2 IMMUNOPARASITOLOGY: CONFLICT

OR COMPROMISE?
From an evolutionary perspective, host-parasite relationships may begin in conflict but eventually
they move toward compromise, and the immune system plays a central role in the complex system
of checks and balances ( Dawkins, 1990; Mitchell, 1991; Read, 1994). Distinctions between self
and nonself exist in all animals and show phylogenetic complexity and adaptive immune responses.
Mounting an immune response is metabolically expensive; a host may compromise between the
available resources for its growth and development and for defense ( Behnke et al., 1992). A
balance between the beneficial and potential harmful effects of immune responses to infection has
to be considered in terms of a series of evolutionary trade-offs. For the host it involves resistance,
pathology, and loss of resources, and for the parasite it involves reproduction, immungenicity, and
pathogenicity. Compromise strategies have led to stable equilibria between many parasites and
their hosts ( Wakelin, 1996). Genetic structure, evolution of microparasites, mechanisms of
pathogenesis, and the evolution of immune response have consequences for public health,
treatment, and prevention ( Levin et al., 1999).

Resistance to an infection may be nonspecific, or innate, immunity--for example, where parasites

are prevented from entering the body tissues by barriers such as the skin. Mucus secreted by
epithelial cells also is a protective barrier because it prevents infectious agents from attaching to
the epithelial cells. Again, secretions such as tears, saliva, nasal fluids, and milk contain
antibacterial substances. Specific acquired immunity is another type of host resistance that
involves the production of antibodies and a complex interplay of T- and B-cells. The acquired
immune response results in the recovery of the host from a disease and is followed by the host
acquiring a specific memory, with which it responds vigorously to an infection by the same
parasite.

Immunological responses of vertebrates involve organs (thymus, spleen, and bone marrow),
lymphocyte cells (T- and B-cells), and antibody molecules. T-cell receptors recognize antigens and
release cytokines that coordinate the immune response. Major cytokines released by T-cells include
interleukins, tumor necrosis factor, and interferons. B-cells recognize antigens and produce large
amounts of immunoglobulins (Ig) that circulate throughout the body. Interactions between the
host's antibody molecules and the parasite's antigens constitutes one important phase of a host's
defenses.
Each Ig molecule is composed of four peptide chains: two identical heavy chains are linked by a
disulfide bond to two identical light chains. There are an estimated 1 billion variations of Ig
molecules in humans. Each type of Ig molecule is a product of a single clone of B-cells.
Immunologists recognize five classes of Ig molecules: IgG, IgA, IgM, IgD, and IgE.

Immunoglobulin G (IgG) is the most abundant antibody in the interstitial tissues and plays a major
role in destroying bacterial toxins and enhancing the phagocytosis of bacteria by lymphocytes.
Because IgG can cross the placental barrier, it provides the first level of immunological defense
against invading organisms during the first few weeks of an infant's life.

Immunoglobulin A (IgA) is generally found in saliva, tears, nasal fluids, sweat, and secretions
produced in the lining of the trachea, lungs, and urogenital and digestive tracts. IgA plays a vital
role in defense against skin-inhabiting microorganisms and strongly inhibits the attachment of
microbes to the surface of mucosal epithelial cells.

Immunoglobulin M (IgM) antibodies are large molecules that are efficient agglutinating and
cytotoxic agents. These antibodies are confined mainly to the bloodstream and are an effective
defense against blood parasites.

Immunoglobulin D (IgD) antibodies are unique because of their susceptibility to proteolytic

breakdown and their short life span. Most IgD molecules and IgM antibodies are confined to the
surface of lymphocytes. Some scientists suspect that IgD and IgM molecules interact to form
receptor sites for antigens.

Immunoglobulin E (IgE) molecules remain bound mostly to the surfaces of large tissue cells (mast
cells) and occasionally are found in the serum. IgE molecules protect the external mucosal surfaces
of the body by causing an inflammatory reaction in response to pathogenic agents. Parasitologists
have often noted high levels of IgE following infection by helminthic infections or skin response to
feeding of ectoparasites. When IgE molecules beome bound to the receptors on the mast cells,
biologically active mediators such as histamine and serotonin are released, which trigger
hypersensitivity responses in local tissues. Increased permeability of blood vessels, changes in
muscle contractions, and secretion of fluids often accompany the inflammatory hypersensitivity
response. This can harm the parasites, but the host tissue may also be damaged in the process.

The adaptive immune response in a vertebrate host consists of the following stages: lymphocytes
are first activated and undergo a phase of clonal proliferation, then some become effector cells,
while the remainder constitute memory cells. The secondary immune response in a host originates
with the activation of the memory cells. B-lymphocytes mature in the bone marrow of mammals
and become the antibody-producing cells. This constitutes the humoral immunity. Lymphocytes
mature under the influence of the thymus and form the basis of cellular immunity. Cellular
immunity, the principal host response against intracellular symbionts, is a complicated process. In
brief, cellular immunity involves the following steps. Different surface antigens of a parasite are
recognized by the T- and Bcells. Macrophages bind to the antigen molecules and carry them to
helper T-cells, which in turn transfer the antigens to B-lymphocytes. The activated B-cells then
proliferate and mature to manufacture antibodies. Some activated T-cells kill parasites via
phagocytosis from the action of destructive mediators such as amines and cytokines. The
antibodyantigen combination activates complement, a complex series of proteins that operate as a
cascade system and can bring about lysis on the parasite surfaces.

Interferons are antiviral molecules secreted by virus-infected host cells. Interferons attach to
neighboring healthy cells and make them resistant to infection by other viruses. The phenomenon
of viral interference or cross-protection has been noted in bacteria, plants, and animals. By
continuously changing the structure of their surface antigens, viruses such as the influenza virus
render previously manufactured antibodies ineffective. In addition to humoral antibodies, the host
uses sensitized cytotoxic T cells to destroy virus-infected host cells. Examples of such responses to
viral infection include mumps, measles, herpes, pox, and rabies. Children with T-cell immune
deficiency are unable to recover from viral infections.

Parasitologists have often described the phenomenon of premunition, which is the immunity of a
host to reinfection following recovery from disease. The exact mechanism of premunition is not
understood. Humoral antibodies are effective against the forms of parasites that occur in the host's
bloodstream ( Mitchison, 1990). Some parasites, however, have evolved novel strategies to
counter a host's immune system and are able to establish long-term chronic infections. A few
wellknown examples of such strategies are described below.

Immunity against malarial parasites is primarily a premunition, and it is effective only if a small,
residual population of the parasite is present in the host individual. In endemic areas, infants are
protected against malaria by maternal antibodies ( Roberts and Janovy, 1996)

Trypanosomes circumvent the host's humoral antibodies by changing the structure of their surface
antigens with each cycle of reproduction. When the host produces new antibodies, the parasite also
changes its antigens.

Some parasitic symbionts such as Trypanosoma cruzi, Leishmania, and Toxo-plasma escape the
host's antibodies by taking up residence in macrophages. Adults of Schistosoma mansoni living in
the host blood vessels disguise themselves by covering their surfaces with antigens obtained from
red blood cells. This mimicry successfully masks the parasite, and the host's humoral antibodies fail
to recognize it as foreign.

Sequestration is beneficial to both host and parasite. The invading organism becomes isolated and
stands apart from the host. Parasites also avoid the host immune response by living in tissue or
cells of immunologically isolated zones. For example, larvae of Trichinella spiralis reside in muscle
cells, where they are protected from the host's antibodies.

14.3 ROLE OF PHYTOALEXINS AND

CROSSPROTECTION IN PLANT DEFENSES
The biochemical basis of plant resistance to fungal parasitism is not entirely understood ( Kombrink
and Somssich, 1995). When a fungus infects a plant, it stimulates the plant to produce
phytoalexins that inhibit growth of the fungus ( Hammerschmidt, 1999). Phytoalexins are produced
in response either to complex carbohydrates that are present in the walls of the invading fungus or
to compounds excreted by the pathogen. The fungal compounds are called elicitors because they
stimulate the plant cell to synthesize enzymes that bring about the production of phytoalexins. If a
fungus lacks specific elicitors, then the infected plant cannot form phytoalexins and therefore has
no defense against the pathogen. The relationship between phytoalexins and elicitors is extremely
complex ( fig. 14.3 ). Many different compounds and conditions stimulate phytoalexin production,
and there is no clear pattern to the findings of many studies on this subject. There is also a
continuing controversy over the general effectiveness of phytoalexins in determining plant
resistance to pathogens. More than 300 chemicals with phytoalexinlike properties have been
isolated from plants belonging to more than 30 families. Phytoalexins produced by plants in each
family have similar chemical structures. For example, in most legumes the phytoalexins are
isoflavonoids, whereas the members of the potato family produce terpenoids. Some well-studied
phytoalexins include phaseolin in beans, pisatin in peas, glyceolin in soybeans, rishitin in potatoes,
and gossypol in cotton ( Kuc, 1995; Ebel, 1998).

Instead of causing severe disease, some strains of plant viruses confer immunity against more
virulent strains of the same virus on a host plant. This phenomenon is called cross-protection. The
viruses are modified strains of pathogenic forms, and their presence stimulates the host to produce
un-

Fig. 14.3 Schematic model for phytoalexin synthesis by host cells near the infected cell. Adapted
from Sequeira ( 1984).

known compounds that inhibit the replication of the pathogenic viruses. The resistance may spread
from an infected host cell to other uninfected cells. Cross-protection has economic applications.
Tomato and citrus plants can be protected from disease-causing viruses by inoculating them with
less virulent strains of the same virus. For example, a common practice used to protect tomatoes
against the tobacco mosaic virus (TMV) is to infect seedlings with a mutant form of TMV. The
mutant virus slows the growth of the plants somewhat but does not cause the severe symptoms of
the normal virus, such as leaf mosaic, growth inhibition, and fruit discoloration. The infected
seedlings produce plants that are disease resistant. This method is useful for protecting plants
grown in greenhouses, but scientists are hesitant to apply the technique to field crops. Under
natural conditions the modified viruses might spread to different plants, causing new diseases, or
they might also mutate back to the virulent strains.

14.4 PRODUCTS OF COEVOLUTIONARY

SYMBIOSES
Plants are attacked by viruses, bacteria, fungi, nematodes, insects, and grazing animals, and many
have developed adaptations to protect themselves from predatory and parasitic organisms.
Morphological features such as spines and thorns and harmful chemical substances are examples of
such adaptations ( Huxley and Cutler, 1991; Bernays, 1992).

The primary chemical substances of plants are carbohydrates, lipids, and proteins. Secondary plant
chemicals include phenylpropanes, acetogenins, terpenoids, steroids, and alkaloids. These
secondary chemicals protect plants from being eaten by herbivores. Some insects, however, have
evolved mechanisms to detoxify poisonous plant metabolites.

Passiflora adenopoda protects itself by trapping plant-eating insects by means of hooked hairs on
its outer surface. Many species of Passiflora also have structures that resemble the eggs of
Heliconius butterflies. These structures deter female butterflies from laying their eggs on the plant.

Ants have evolved mutualistic associations with a variety of flowering plants ( Keeler, 1989). In
plants such as Acacia the ants live in hollow areas located at the base of thorns ( fig. 14.4 ). The
ants are attracted to the host plant by secretions from glandular nectaries at the base of the
leaves. The ants protect the plants from other insects and disperse the plant's seeds, and their
excrement pro-

Fig. 14.4 Pseudomyrmex ants living inside the hollow cavity of thorns of the acacia plant protect
the plant from insect predators. The ants feed on glycogen-rich nodules produced by the plant.
Adapted from Perry (1983

vides nutrient to the host plant. Plants that are fed by ants include species of the genera
Hydrophton and Myrmecodia in the family Rubiaceae from Southeast Asia and northeastern
Australia. The plants typically produce swollen organs that the ants live in, and the mutualistic
interaction is known as myrmecotrophy ( Beattie, 1989).

Many evergreen plant species produce steroids that simulate the molting and juvenile hormones of
insects. These steroids accelerate larval development and may cause premature death of an insect.
Balsam fir (Abies balsamea) has evolved an analog of the insect juvenile hormone that arrests
larval development and prevents the formation of normal adults. Analogs of insect hormones are
the products of fine-tuned coevolutions between plants and insects.

Hypericin is a toxic metabolite secreted from glands of the flowering plant genus Hypericum. The
chemical, when ingested by animals, produces skin irritation and blindness and leads to starvation;
consequently, the plant is generally avoided by most grazing animals. Beetles of the genus
Chrysolina, however, can detoxify hypericin and thus have gained access to a food supply that is
generally unavailable to other animals.

All members of the mustard family, Cruciferae, produce oils that are harmful to animals and are
also effective as antibiotics. Insects such as the cabbage butterfly, mustard beetle, and cabbage
aphid have evolved mechanisms to detoxify the active ingredients of these oils, but in the process
they have become dependent on the substances for their growth and development.
14.5 ECOLOGICAL AND EVOLUTIONARY
PERSPECTIVES ON SYMBIOSIS
Ecosystems and biological communities have historical dimensions. A diverse biological community
consists of many interacting organisms where the organismic characteristics reflect a unique
evolutionary history. Plants, animals, fungi, protozoans, and microbial organisms have lived
together in communities that reflect mutually reciprocal coevolutionary adaptations.

The Symbiotic Continuum

During the past decade, the terms symbiosis and mutualism have assumed a much broader
significance. Throughout this text we have examined the mechanisms of symbiosis between
interacting species. Evolutionary ecologists, however, have a different perspective of symbiosis and
have emphasized the outcomes of symbiotic interactions, which they measure in terms of potential
fitness. For ecologists, symbiosis includes not only intimate associations but also interspecific
interactions in which the symbionts are not physically connected, for example, a diffuse mutualism
such as the relationship between flowering plants and their animal pollinators.

Mortimer Starr and others have developed a classification they call the symbiotic continuum (
Starr, 1975). This classification of symbiotic categories is based on the potential fitness, or
reproductive ability, of the symbionts. Competition and mutualism are at opposite ends of the
continuum, and neutralism is in the center ( fig. 14.5 ). Competition between interacting species
produces detrimental outcomes to both species, whereas mutualistic relationships increase the
potential fitness of the symbionts. Thus, symbiosis means that two interacting organisms can
influence each other's rates of survival and reproduction. Mutualisms are interorganismic
interactions that result in improved survival or reproduction of the partners and may involve aids to
dispersal, provision of nutrients, energy, shelter, or fertilization. Other continua in Starr's
classification deal with the duration of the symbiosis, from transient to permanent; the physical
contact between the symbionts, from incidental to close; and nutrition, from saprobic to biotrophic.

Theories of Mutualism
According to Douglas Boucher ( 1985), there are four types of models for ecological theo-

Fig. 14.5 The symbiotic continuum classification is based on the potential fitness of interacting
species
ries of mutualism: (1) individual selection model, (2) population dynamics model, (3) model of
shifts of interaction, and (4) the keystone mutualist model. In the individual selection model, which
is usually a fitness costbenefit type, symbiotic associations tend to favor mutualism because the
number of competitors benefited is less in a mutualistic association. As more needs of an
association are met by the combined contributions of the symbionts, the pressure of competition
on these symbionts from similar species will decrease.Population dynamics models begin with the
Lotka-Volterra competition theory. More than half a century ago, Alfred Lotka and Vito Volterra
used mathematics to understand competition and predator-prey relationships ( Lotka, 1932;
Volterra, 1926). They devised a model that is now called a phase-plane model and is being used as
a prototype to study mutualism. In this model, the population densities of two interacting species
are plotted along vertical and horizontal axes, thus producing a plane separated by lines that form
four regions. The regions show different population densities of the two species and correspond to
what will happen to the populations; that is, both populations may increase or decrease, or one will
increase and the other will decrease. The phase-plane model has been applied to more than 25
different case histories of mutualism with virtually identical results in the forms of the graphs. The
model predicts stable equilibrium, persistence of mutualistic interaction, attainment of higher
population densities in symbiosis for both species, and decrease of mutual benefit as the population
grows larger.Models of shifts of interaction are based on the symbiotic continuum model, which
was described above, and takes into account fluctuations from one segment of the continuum to
another, for example, from competition to mutualism. In the keystone mutualist model, removal of
a particular symbiont from an association will produce significant changes in the structure of a
community ( J. H. Brown and Heske, 1990).

Categories of Mutualism
Boucher ( 1985) argues that mutualism is a major organizing principle in nature and that
researchers should be able to test the following predictions.
1. Mutualists control survival and reproduction; for example, the number of seeds produced by a
flower depends on the activity of its mutualist pollinators.
2. Mutualisms that do not appear to have clear values may, in fact, have definite fitness values.
For example, seed dispersal by mutualists is more efficient than other means such as wind
and results in a higher survival value of the seeds.
3. When mutualistic organisms are either removed from or introduced into a community, the
relative abundance of other members of the community is significantly altered.
4. Interspecific competition in nature often produces indirect mutualisms, associations in which
species are not in physical contact but positively affect each other's fitness or growth rates;
for example, plants with small populations may mimic flowers of other plants, thereby
increasing the population size recognized by pollinators.
5. Mutualistic enviroments tend to expand because mutualisms can colonize marginal habitats.
6. Antagonistic interactions stimulate mutualisms to evolve as the antagonists develop defenses
against each other.
7. The biomass of natural as well as artificial communities may increase when mutualistic
organisms (e.g., mycorrhizal fungi) are added to the community and decreases when they are
removed.
8. Sharing of mutualists, as with pollinators, will generally produce indirect mutualisms rather
than competition for mutualism. Many new examples of mutualism in nature remain to be
discovered.

Four categories of mutualism in terrestrial ecosystems have been recognized: seed dispersal,
pollination, resource harvest, and protection.

Seed dispersal mutualism

Seed dispersal mutualisms are diffuse and involve plants that are generally woody and broad-
leaved and many different animals

( Herrera, 1995). Seed dispersal occurs in many habitats, from the Arctic to the tropics. When an
animal swallows a seed and deposits it at another site, mutualism has taken place. The potential
fitness of a seed is increased if it can germinate in a site that is more favorable for growth than
where it was produced, that is, away from the parent population. Pine seeds are dispersed by
nutcrackers and red squirrels. Nutcrackers feed on pine seeds in late summer and store many
seeds in their sublingual pouches. As the birds fly to their cache sites, they bring seeds from their
pouch and bury them in the soil. Nutcrackers recover these caches throughout fall, winter, and
early next summer and seem to remember where each group of seeds was buried ( Lanner, 1996).
Plant fruits are bait, and they protect the seeds as well as provide the mutualists with their reward.
Ants disperse seeds by carrying them and frequently burying them. The system provides dispersal
for the seeds and food for the ants. There are also nonmutualistic means of seed dispersal such as
wind, water, and explosive fruits, which forcibly disperse their seeds.

Pollination mutualism

Insects, birds, bats, some rodents, marsupials, and primates make up most of the pollinators.
Many of them form diffuse types of associations, but others are obligate mutualists. The
advantages for the pollinators include food, such as pollen and nectar, and for the plants the ability
to produce more offspring per gram of pollen produced, more viable offspring, and minimized
inbreeding ( Kearns et al., 1998). For further discussion of pollination, consult chapter 12.

Harvest mutualism

In harvest mutualisms one symbiont gathers, or harvests, nutrients that it converts into a form the
other symbiont can use. A unique feature of harvest mutualism is that one organism interacts with
a geographically restricted population of the other mutualist. Examples of this type of symbiosis
include the gut mutualism of herbivores, the fungus gardens of ants, mycorrhizal fungi, and
lichens. Alimentary canals of all animals contain complex colonies of microorganisms. Some of
these endosymbionts are mutualists or parasites; others are commensalistic. These organisms may
form diffuse or obligatory mutualisms. The host pays a low cost for maintenance of the symbionts
and receives a high rate of return when the food it ingests is converted into a usable form.
Termites and ruminants are examples of digestive mutualism. If the animal had to depend on its
ability to digest, it would assimilate less material. Thus, host animals take in large amounts of
potential food that cannot be directly assimilated and depend on their mutualistic microorganisms
to make available molecules such as vitamins, amino acids, fatty acids, and carbohydrates.

Protection mutualism

One of the best known examples of protection mutualism is the association between ants and the
thorns of Acacia plants ( Janzen, 1966). In this obligate mutualism, ants live in colonies in the
hollow chambers of thorns and feed on carbohydrates provided by extrafloral nectaries on the
plant. The ants protect the host plant from insects and other plants such as vines ( Huxley and
Cutler, 1991).

Distribution of Mutualism
Although the evidence is incomplete, it appears that mutualistic associations are more common in
tropical communities than elsewhere. For example, flowering plants in tropical rainforests and
tropical deciduous forests are mostly animal pollinated, whereas the vast northern forests and
temperate deciduous forests are mainly wind pollinated. Plant--ant relationships, including those of
seed dispersal and protection, are more common in the tropics, as are mutualisms involving algae
and cnidarians such as corals, cleaning symbioses, and fungus gardens of insects ( Beattie, 1985;
Boucher, 1985). The abundance of mutualisms in the tropics may be a reflection of the greater
species diversity and productivity in this part of the world. Most tropical soils and waters are poor
in nutrients, a condition that favors mutualisms between autotrophic and heterotrophic organisms.

r and K Selection: Life-History Patterns and

Symbiosis
Robert H. MacArthur and Edward O. Wilson ( 1967) proposed a concept to elucidate the connection
between the abundance of an organism and evolution. According to these sci-entists, density-
dependent selection causes the evolution of high-K traits, whereas r selection is characteristic of an
expanding population, which is density independent ( fig. 14.6 ). In environments that are
constantly changing physically, individuals with high birthrates are often favored by natural
selection because their descendants can colonize empty habitats quickly. These organisms usually
experience a high degree of mortality among juveniles, and the populations are generally below
their saturation levels with little or no competition. The individuals are short lived and are usually
small, grow fast, and mature early. Organisms such as insects and annual plants have high r
values. K-selected species inhabit relatively stable environments in which population levels are
consistently high. In these situations, fitness depends more on features that allow individuals to
compete for resources, often at the expense of their ability to produce offspring. Populations are at
the saturation level most of the time and are regulated by intense competition within and among
the species. The individuals are long lived, grow slowly, and mature late. The most obvious
examples of this strategy are found in tropical forests, coral reefs, and specialized environments
such as the termite intestine or the rumen of herbivores ( MacArthur and Wilson, 1967; Pianka,
1970).

Organisms under r selection are often described as physically controlled, and the K-selected
species are biologically accommodated. The r and K selection model offers a broad framework
within which to consider the forces of natural selection that regulate symbiotic organisms and their
hosts. Gerald Esch and colleagues ( 1977) have examined the concept of r and K selection within
the context of animal parasitism. They have shown that characteristics of morphology, physiology,
and life history of parasitic symbionts can be used to determine population density dynamics.
A biological community shows the range of the r and K continuum, the r end representing
organisms that expend most of their energy on reproduction and little on each individual. K
selection produces stabilized symbioses, with efficient use of the environment and with the
organisms saturating the environment; competition among individuals is intense. Application of the
r and K selection concept to the study of symbiosis is relatively new ( Andrews and Harris, 1986).

Evolution in a Symbiotic Environment

The evolution of a species in the absence of interspecific interactions is largely determined by
forces of natural selection. Ecologists realize, however, that a population of any given species is
embedded in the matrix of a community, and interactions with other species will produce genetic
changes resulting in an evolutionary feedback. Interspecific dynamics such as those involving
predator-prey, host--pathogen, and competitive relationships are antagonistic interactions and
therefore cause deterioration of the evolutionary environment.

Populations of mutualists will bring about an evolutionary change in other mutualists, with the
following predictable results: (1) genetic change in the mutualists will be slow, (2) there will be
tendency toward a lack of specificity between the interacting populations, and (3) there will be a
reduction in the importance of sexual reproduction. Intracellular symbioses that involve two or
more species have had a profound influence on the evolution of the symbionts as well as on the
environments in which they grow. Examples of such symbioses are those of reef-building

Fig. 14.6 Population growth curves for organisms that use r and k strategies. Adapted from Esch et
al. (1977).

corals and foraminifera, which are described in chapter 10. In both cases, symbiosis with
unicellular algae has resulted in changes in size and shape of the hosts and an increased number of
host species.

One of the unresolved mysteries of evolutionary biology is the role of selection that favors the
maintenance of sex in natural populations. Scientists have suggested that antagonistic forces in an
environment favor the evolution of sexual reproduction. Conversely, in environments where
mutualistic interactions predominate, asexual reproduction is the favored strategy because it
results in continued genetic homogeneity of the mutualists, thereby ensuring continuity of the
symbiosis.

Traditionally, scientists have considered the environment a product of geological and physico-
chemical processes to which living organisms have to adapt or perish. This view has been
challenged by James Lovelock and Lynn Margulis ( 1974), who have proposed the Gaia hypothesis.
According to this hypothesis, the composition of the atmosphere, sediments, and aquatic
environments is controlled by living organisms in their interactions with the environment. Present
levels of atmospheric oxygen, carbon dioxide, and nitrogen and temperature ranges are the
product of life mechanisms. Thus, in the broadest sense, there is a coevolution of life forms and
environmental conditions, and each depends on the other for its existence and maintenance (
Joseph, 1990; Levine, 1993; Myers, 1993; Lovelock, 1995; G. R. Williams, 1996; Volk, 1997).

Hypersea, like the Gaia hypothesis, requires an imaginative stretch. According to its authors,
hypersea represents the body fluids of land eukaryotes that are involved in symbiotic associations,
including mutualism and parasitism ( McMenamin and McMenamin 1993, 1996). Hypersea is a new
type of ocean which first became established in the Paleozoic ( Zimmer, 1991).
14.6 SYMBIOSIS IN THE ORIGIN OF
AGRICULTURE IN ANTS AND HUMAN
SOCIETIES
David Rindos ( 1984) framed the origin of agriculture within the broad scope of evolutionary
theory. He argues that the domestication of plants and animals is not merely an invention or a
historical event but rather a mutualistic relationship of varying degrees between different species
that arise under a specific set of conditions ( Rindos, 1984). Incidental domestication is the result
of human dispersal and protection of wild plants and animals in the general environment. Over
time this relationship will select for morphological changes in plants and animals for further
domestication. Specialized domestication is mediated by the environmental impact of humans in
localized areas. Agroecology is not merely a theater in which people perform their agricultural
tasks, but humans are directly interacting with plants on which they feed and with which they
establish a symbiotic relationship. Agricultural domestication is the climax of two processes within
agroecology where humans, by means of environmental manipulation, increase the rates of
evolution of domesticated plants. Weeds are opportunistic plants that parasitize the symbiosis
between people and their coevolved domesticates. Most successful weeds are those that mimic
cultivated plants in morphology and life cycle events ( Gould, 1991).

In a recent study on the evolution of agriculture in ants, which originated some 50 million years
ago, there are some striking parallels with human agriculture. Agricultural ants of the tribe Attini
(Formicidae), which includes the famous leaf cutters, show extensive coevolutionary symbiosis
between the "attine" ant farmers and their fungal cultivars. All agricultural ants grow their fungal
crops asexually as clones. Through molecular genetic analysis it has been shown that
domestication occurred on a number of occasions and that new cultivars are acquired in an ongoing
evolutionary process. In some cases, a single ant species may farm a diversity of fungi, and at the
same time a single cultivar is grown by ants of distinct lineages. A given ant nest may contain a
single crop, but different ant nests of the same ant species alternate between as many as eight
different cultivars. Horizontal transfer, like crop sharing, is practiced by ant species that borrow a
crop from another species' nest. Biochemical modifications of fungal cultivars are matched with
olfactory and gustatory adaptations of the ants ( Mueller et al., 1998).

Although human farmers have domesticated only 14 significant species of large mammalian
herbivores, they have successfully domesticated hundreds of crop plants ranging from cereals and
legumes to root crops and nut trees. Sharing of domesticates such as maize and potatoes followed
the European expansion into the New World. Evidence shows that human farmers were also
responsible for the "domestication" of specialized human pathogens that cause diseases such as
tuberculosis, measles, and flu, which evolved from pathogens of cattle and pigs ( Diamond, 1997,
1998).

14.7 SYMBIOSIS AND HUMAN AFFAIRS

According to the late Rene Dubos, the renowned microbiologist, all living things are mutually
interdependent ( Dubos, 1959). Earlier, the Russian philosopher Peter Kropotkin wrote a series of
essays, Mutual Aid: A Factor of Evolution ( 1902), which provided an alternative explanation to the
"struggle for existence" and "survival of the fittest" concepts. Kropotkin believed that cooperation
and mutual help, rather than struggle, were the principal forces of evolutionary change.
Cooperation resulted from an instinct of preservation which gave to species and to individuals a
better chance of survival in stressful environments. Cooperation and symbiosis are an integral part
of our biological heritage. Charles Darwin himself was aware of the role of interorganismic
associations as he eloquently explores the "entangled bank" and the "grandeur in this view of life."
In The Origin of Species ( 1859), Darwin wrote:

It is interesting to contemplate an entangled bank, clothed with many plants of many kinds, with
birds singing on the bushes, with various insects flitting about, and with worms crawling through
the damp earth, and to reflect that these elaborated constructed forms, so different from each
other, and dependent on each other in so complex a manner, have all been produced by laws
acting around us. . . . Thus from the war of nature, from famine and death, the most exalted
object which we are capable of conceiving, namely, the production of the higher animals, directly
follows. There is grandeur in this view of life, with its powers, having been originally breathed into
a few forms or into one; and that, whilst this planet has gone cycling on according to the fixed laws
of gravity, from so simple a beginning endless forms most beautiful and most wonderful have
been, and are being evolved. (pp. 489490)

The germ theory of disease, developed during the height of the social Darwinian revolution,
provided supportive evidence for the struggle for survival. With much determination, civilization
declared war against microbial infections. Ecological equilibrium between host and microbes was
not then a popular view among scientists. As the twenty-first century approaches, humans have
become a dominant force on earth, but they still struggle with symbionts that cause disease in
people, crops, and domestic animals. Human diseases such as leprosy, the black plague of the
Middle Ages, typhus, malaria, and yellow fever have had consequences of historic proportions, as
have plant disease epidemics such as the potato blight in Ireland ( Nikiforuk, 1991).

Tremendous progress has been made in curbing catastrophic diseases. But mass-scale applications
of pesticides have polluted our soils, water, and air, and at the same time the pest organisms with
millions of years of evolutionary history have successfully resisted man-made challenges to their
survival. We have learned several lessons in the past three decades: (1) a symbiont can rapidly
develop tolerance to man-made poisons; (2) instability in the ecological equilibrium of the
interacting species is an important factor in an outbreak of disease epidemics; (3) genetic
uniformity in crop plants and domestic animals is an invitation for epidemics; and (4) a
multifaceted approach to pest management is the best means of pest control.

With the advent of air travel, symbionts can move across geographical barriers with ease.
Containing epidemics such as AIDS or Mediterranean fruit fly infestations of citrus fruit challenges
the resourcefulness of the scientific community.

In agriculture, concepts of symbiosis have been applied to the biological control of insects,
increasing nitrogen fixation by symbiotic cyanobacteria, and applications of mycorrhizal symbionts
to forest seedlings in order to promote their growth and thus stop soil erosion. Symbiosis provides
the conceptual tool for the management of an ecosystem. Its alternative has been the application
of chemicals that eliminate symbioses dependent on natural biological interactions. Ever since it
became known that Rhizobium fixes atmospheric nitrogen within legume root nodules, the
application of this symbiosis has become part of modern agricultural practices. Legume crops such
as alfalfa have been used as green manure, in crop rotation, and in mixed cropping. Artificial
inoculation of legume seeds with strains of Rhizobium is a regular practice in many countries. The
Azolla--Anabaena symbiosis is most active in flooded rice fields, where populations of the symbiotic
fern can fix up to 3 kg of atmospheric nitrogen per hectare per day. In rice paddies of China and
Vietnam, Azolla has been used for centuries as a source of nitrogen, with the result that Asian
farmers can harvest up to 2 tons of rice per hectare without using chemical fertilizers. Modern rice
varieties need up to 20 kg of nitrogen per hectare for each ton of rice produced. Scientists are
exploring the possibility of improving the use of this symbiosis by manipulating cultural practices.

Black locust, alder, and Casuarina are trees with nitrogen-fixing Frankia symbionts. These trees
have been used successfully in the reforestation of barren land.

Almost all higher plants form mycorrhizal associations. Mycorrhizas are increasingly being
recognized as an important factor in the uptake of nutrients, especially phosphorus, by plants.
Donald Marx has demonstrated that pine seedlings inoculated with ectomycorrhizal fungi grow
faster and survive better than uninoculated seedlings ( Marx, 1991). Commercial application of
vesicular arbuscular mycorrhizas has yet to become establisbed. These mycorrhizas are also known
to increase nitrogen fixation in nodulated legume plants.

The alimentary canals of all animals are unique ecosystems that contain large numbers of
interdependent microbial species. Ruminants, with their modified stomachs, have successfully
exploited the energy of cellulose and lignin by fermentation symbioses.

Social scientists have found symbiosis to be a useful explanation for a variety of phenomena. For
example, Peter Corning has applied the general principles of mutualistic symbiosis to the evolution
of human social dominance. According to his synergistic hypothesis, cooperation enhances survival
and reproduction of humans ( Corning, 1983). Similarly, psychologists are using symbiosis as a
conceptual framework to study interactions between parents and their offspring.

The increasing ability of humans to manipulate natural symbiotic associations for their own
advantage and create artificial symbioses through genetic engineering will have far-reaching
consequences within the next few decades. Corn and soybeans are being used to manufacture
human antibodies ("plantibodies") that have shown promise as drugs against human diseases such
as cancer ( Gibbs, 1997). Disease-resistance genes have been introduced into rice plants ( Ronald,
1997). Whether all consequences will be positive is difficult to predict. Parasitic symbionts have a
long history of survival and despite our best efforts may still invent ways to circumvent our
attempts to eradicate them.

14.8 SUMMARY
The concepts of symbiosis and coevolution have attracted much attention from evolutionary
biologists and ecologists who are not satisfied with the classical Darwinian approaches to organic
evolution. Pairwise coevolution occurs between two species, whereas diffuse coevolution involves
larger numbers of species. Coevolution involves reciprocal genetic changes in the interacting
populations and includes coadaptation as well as cospeciation. Genetic conflicts have been
responsible for a number of evolutionary phenomena, including the evolution of sex. Ultra-selfish
genes are self-promoting genetic elements. Mitochondria are involved in apoptosis.

Gene-for-gene relationships between parasites and hosts have been found in both plant and animal
symbioses. Plants may have clusters of genes that confer resistance to more than one pathogen. In
some host--parasite interactions the expression of disease by one parasite depends on the
presence of another parasite.

Parasitic symbioses are thought to be the principal causes of genetic polymorphism within a
population. Resistance to pathogenic symbionts by vertebrate hosts involves immune mechanisms,
cross-protection, and sequestration of the parasites. New advances in the molecular biology of
immune responses are offering unique opportunities for combating infectious illnesses. Some
parasites have evolved unique ways to protect themselves from the host's immune systems.

Plant responses to parasites include hypersensitivity, cross-protection, and the production of

phytoalexins. Plants have evolved morphological and chemical adaptations to protect them from
parasitic organisms and herbivores. Some insects have evolved mechanisms to detoxify poisonous
compounds produced by plants. The immune system helps to regulate the interactions between a
host and its symbiont.

By measuring the potential fitness of a species, ecologists are reexamining the role of competition
and mutualism in the coevolution of interacting species. The symbiotic continuum is a classification
based on the potential fitness of the partners of a symbiotic association. Competition between
interacting species produces decreased fitness, whereas mutualisms result in enhanced potential
fitness of both partners.

There are four main models of mutualism: individual selection model, population dynamics model,
shifts of interaction model, and keystone mutualist model. Some scientists believe that mutualisms
are an important organizing principle in nature and have a significant influence on the environment
and community. Mutualisms result in the evolution of stable ecosystems with increased parasexual
forms of reproduction. Mutualism in terrestrial ecosystems includes seed dispersal, pollination,
resource harvest, and protection.

Although the phenomenon of mutualism has been recognized for more than 100 years, it has not
been part of the development of modern ecology, despite its prevalence in many biological
communities. Only recently have ecologists recognized that the subject of mutualism is worthy of
serious study. Such interest is welcome, since ecologists, with their theoretical perspectives, bring
a new dimension to a subject that has long fascinated descriptive biologists.
Symbiosis can be viewed in terms of r and K selection. Most parasites demonstrate the rselection
strategy; K-selected species are those that occur in stable or specialized environments such as
those of tropical forests or the rumen of herbivores.

According to the Gaia hypothesis, the earth's environment is a product of interactions between life
and nonlife molecules, and its fragile equilibrium is ultimately controlled by the dynamics of living
organisms. Hypersea is a new type of ocean which consists of the body fluids of eukaryotes
involved in symbiotic association. Gaia and Hypersea are ultimate manifestations of symbiotic
interactions. Agricultural ants grow fungi for food. The evolution of agriculture in ants such as the
leaf-cutters shows striking similarities to human agriculture.

The concept of symbiosis has been applied to human affairs in which cooperation and mutual aid
have been the principal forces of evolutionary change. The importance of studying symbiosis is
underscored by our increased ability to control pathogenic organisms by understanding the
molecular mechanisms of biological interactions. In agriculture, our understanding of symbiotic
systems has been of great value in the biological control of insect pests, the increase of nitrogen
production by natural means, and the artificial inoculation of forest trees and crop plants with

APPENDIX 1
mycorrhizae and nitrogen-fixing symbionts.

Heinrich Anton de Bary (1831-1888)

a phenomenon in which dissimilar organisms live together, or symbiosis

Die Erscheinung der Symbiose ( 1879)

In an address to the 1878 meeting of German naturalists, Professor Heinrich Anton de Bary of the
University of Strassbourg proposed the term "symbiosis" to designate phenomena such as
parasitism and mutualism that were found in the plant kingdom. 1 The need for such clarification
arose because earlier that year Professor Pierre-Joseph van Beneden, a renowned authority on
tapeworms, had published a book titled Commensalism and Parasites. Professor de Bary felt that
plant associations had unique properties and therefore required new definitions. He distinguished
between two main categories of symbiosis: "antagonistic," which involved a struggle between the
symbionts, and "mutualistic," in which the participating organisms benefited from the association.
Thus began the birth of a concept which this book adopts--namely, the broad definition of the word
symbiosis. During the twentieth century, symbiosis was a popular subject in biology, as well as in
society at large. Curiously, for many people symbiosis became synonymous with mutualism. When
he coined the term "symbiosis," de Bary was studying lichens, and his original paper on symbiosis
used lichens as common examples.

The exact nature of lichens was a highly contentious issue in mid-nineteenth century German
botany. Lichens were considered to be the "neglected plants" of botany until the birth of the dual
hypothesis by Simon Schwendener, a Swiss botanist. Schwendener, and to some extent de Bary,
provided evidence that lichens were associations of fungi and algae. 2 This view was bitterly
opposed by leading botanists, including William Nylander, who regarded lichens as autonomous
plants like fungi and mosses. The opponents of the dual hypothesis feared that lichens would lose
their special identity once they were recognized as fungi that imprisoned algal cells. There was an
excitement in the air. In his 1878 address to the German naturalists, de Bary described lichens as
follows:

The mass of green cells which characterize Lichens have had the most chequered fate in the history
of science. Until it was shown some ten years ago, lichens are not truly part of the plant having
fungus-like fruiting body, but they are algae which live and grow on or in certain fungi and do not
exist outside of this special association. A specific fungus and a specific alga form a specific lichen
through their association and without this association, there would be no lichen. (p. 305)

Later de Bary stated that although the algal partners of lichens could live on their own when
artificially isolated, the fungal partner died without its algae. He stated:

The host depends on the tenant in order to live. The tenant is, as a result well treated; not only its
growth not impeded but is favored in comparison with those grown independently. The algae
 APPENDIX 1
remain in harmony with the fungus. The fungus

Heinrich Anton de Bary (1831-1888)

a phenomenon in which dissimilar organisms live together, or symbiosis

Die Erscheinung der Symbiose ( 1879)

In an address to the 1878 meeting of German naturalists, Professor Heinrich Anton de Bary of the
University of Strassbourg proposed the term "symbiosis" to designate phenomena such as
parasitism and mutualism that were found in the plant kingdom. 1 The need for such clarification
arose because earlier that year Professor Pierre-Joseph van Beneden, a renowned authority on
tapeworms, had published a book titled Commensalism and Parasites. Professor de Bary felt that
plant associations had unique properties and therefore required new definitions. He distinguished
between two main categories of symbiosis: "antagonistic," which involved a struggle between the
symbionts, and "mutualistic," in which the participating organisms benefited from the association.
Thus began the birth of a concept which this book adopts--namely, the broad definition of the word
symbiosis. During the twentieth century, symbiosis was a popular subject in biology, as well as in
society at large. Curiously, for many people symbiosis became synonymous with mutualism. When
he coined the term "symbiosis," de Bary was studying lichens, and his original paper on symbiosis
used lichens as common examples.

The exact nature of lichens was a highly contentious issue in mid-nineteenth century German
botany. Lichens were considered to be the "neglected plants" of botany until the birth of the dual
hypothesis by Simon Schwendener, a Swiss botanist. Schwendener, and to some extent de Bary,
provided evidence that lichens were associations of fungi and algae. 2 This view was bitterly
opposed by leading botanists, including William Nylander, who regarded lichens as autonomous
plants like fungi and mosses. The opponents of the dual hypothesis feared that lichens would lose
their special identity once they were recognized as fungi that imprisoned algal cells. There was an
excitement in the air. In his 1878 address to the German naturalists, de Bary described lichens as
follows:

The mass of green cells which characterize Lichens have had the most chequered fate in the history
of science. Until it was shown some ten years ago, lichens are not truly part of the plant having
fungus-like fruiting body, but they are algae which live and grow on or in certain fungi and do not
exist outside of this special association. A specific fungus and a specific alga form a specific lichen
through their association and without this association, there would be no lichen. (p. 305)

Later de Bary stated that although the algal partners of lichens could live on their own when
artificially isolated, the fungal partner died without its algae. He stated:

The host depends on the tenant in order to live. The tenant is, as a result well treated; not only its
growth not impeded but is favored in comparison with those grown independently. The algae
remain in harmony with the fungus. The fungus penetrates deep into the hard rock to obtain
elements necessary to form the association.

. . . many phenomena similar to parasitism, mutualism etc occur in associations of organisms of

different species. Parasitism, mutualism, lichenism are each a special case of a tendency to
associate for which the term symbiosis is proposed as a general term. (p. 307)

As a student, de Bary was greatly influenced by Alexander Braun, one of the most celebrated mid-
nineteenth century German botanists, and studied algae, parasitic fungi, and water molds. At the
age of 24, de Bary succeeded Carl Nageli as Extraordinary Professor at Freiburg and immediately
began to attract research students worldwide. "The Professor" de Bary considered teaching
research students to be one of his most important responsibilities, and his instructional method
included teaching students to be self-reliant, to think critically, and to overcome difficulties and
errors. Posing the proper question was important. Between 1855 and 1888 de Bary trained more
than 100 scientists. Many of his students became distinguished scientists and used de Baryan
experimental approaches to study the morphology, physiology, and life history of organisms. De
Bary's most productive years ( 1872-1888) were at Strassbourg.
The spirit of de Baryan laboratory life was described in an essay, "Strassburg and its botanical
laboratory" by William Dudley in 1888. 3 De Bary and his family lived in one wing of the ground
floor of The Botanical Institute, which also housed research laboratories, instrument rooms, a
library, rooms for two assistants, a lecture room, and a museum. The new campus of Kaiser
Wilhelm University, built in 1871, consisted of several modern buildings around a quadrangle that
provided lecture halls for philosophy, literature, and mathematics on one side and the chemical,
physical, and botanical institutes and the observatory on other sides. The German model of a
research university was emulated in the United States by Johns Hopkins University ( 1876) in
Baltimore, Maryland and by Clark University ( 1887) in Worcester, Massachusetts.

Asa Gray, a Harvard botanist and one of Charles Darwin's American supporters, asked his student,
William Gilson Farlow, to go to Germany and study the new experimental approaches to
morphology and development of plants under de Bary. In a letter to Farlow in January 1873, Gray
outlined what was expected of him when he returned from his studies:

We are going to have the Cryptogamic Flora of the U.S. done up, pari passu with the Phanerog.
Sullivant is engaged to do the Mosses, in a volume--to which I hope to have Austin do the
Hepaticae.I want you to come home prepared to do--
1. The Algae--one vol.
2. The Fungi--
3. --since they are only Algae & Fungi! either dwelling together--or the lamb inside of the lion--
you will have to do lichenes! Unless we can get them out of Tuckerman, in an intelligible form-
-which is doubtful. He is somewhere among your friends the Germans. 4

Farlow, on his return from Germany, established a research laboratory at Harvard University and
trained a generation of mycologists in the United States.

De Bary died on January 19, 1888 at the age of 57 but his legacy endures, and his brilliant career
as an experimental botanist encompassed several firsts in the history of life sciences. 5 At the age
of 22 he published his book, Die Brandpilze, in which he challenged the idea of spontaneous
generation and described the life cycle of the rust fungus from summer to winter spores, from
wheat to barberry hosts, and back to wheat again. With regard to the emerging cell theory, de
Bary showed that slime molds do not have cell walls. He discovered the zoospores of the potato
blight fungus, which he renamed Phytophthora infestans. In 1866, the first edition of his classic
book Morphologie und Physiologie der Pilze, Flechten und Myxomyceten was published. Marshall
Ward of Cambridge University, who studied in de Bary's laboratory, wrote the following about this
book in 1888:

He gave definiteness to the scattered knowledge (of these organisms) and enabled the scientific
world to see clearly the remarkable power of the man. His un-flinching honesty and rigorous
selfcriticism and modesty had already attracted the attention of all who came into contact with him
or his work. Now, however, was seen the marvelous grasp of details, the power of logical
generalization which he possessed, and thenceforward the name of de Bary was associated with
the leadership of the modern school of biologists, he was himself creating. 6

De Bary is regarded as a founder of modern mycology and plant pathology. De Bary, along with
Wilhem Hofmister, Hugo von Mohl, Nageli, Schwendener, and Julius Sachs were part of a mid-
nineteenth century German scientific movement that brought about a paradigm shift to botany as
an inductive science. 7 What started out as a way to describe "the lichen situation" in 1878 has
come to define our age where life is a complex web of interdependent interactions. Symbioses
range widely from those formed by viruses, as evolutionary brewmeisters, to the Gaian view of the
unity of life and global interdependence. There is a conceptual congruence of symbiosis, life,
science, and religion that de Bary could not have anticipated.

NOTES
1. De Bary's address to the German naturalists was published in 1879 ( A. de Bary, "Die
Erscheinung der Symbiose." Vortag, Gehalten and versammlung Deutscher Naturforscher and
Aerzte zu Cassel. Strassburg: Verlag von Karl J. Thubner). The French translation was
published in 1879 in Revue Internationale des Sciences, edited by J. L. DeLanessan and Tome
Troisieme ( Paris: Octave Dion). Professor David Blitz, Department of Philosophy, Central
 Connecticut State University, in New Britain, kindly provided the English translation.

2. De Bary. ( 1879). "De la symbiose." Revue Internationale des Sciences 3: 301-309.

3. William R. Dudley, "Strassburg and its botanical laboratory." Botanical Gazette, 1888, 13(
12): 305-311. J. T. Rothrock discussed the laboratory environment under Professor De Bary's
guidance in Botanical Gazette ( 1881, 1888).

4. Hilda F. Harris, "The Correspondence of William G. Farlow during his student days at
Strasbourg." Farlowia, 1945, ( 2) 1: 9-27.

5. Some significant essays on De Bary include Frederick K. Sparrow. ( 1978) "Professor Anton De
Bary," Mycologia, 1978, 70: 222252; G. Murry, "Heinrich Antone De Bary," Journal of Botany,
1988, 4: 65-67; James G. Horsfall and Stephen Wilhelm, "Heinrich Anton De Bary: Nach
Einhundertfunfzig Jahren," Annual Review of Phytopathology, 1982, 20: 27-32.

6. Marshall Ward, "Anton De Bary," Nature, 1888, 37: 297-299.

7. Eugene Cittadino, Nature as the laboratory. Darwinian Plant Ecology in the German Empire,
1880-1900 ( Cambridge: Cambridge University Press, 1990).

APPENDIX 2
HISTORICAL LANDMARKS OF SYMBIOSIS
1500 BC Papyrus Ebers describes the human intestinal parasite Ascaris lumbricoides and the
tissue parasite Dracunculus medinensis.
AD 1200 Albertus Magnus describes a parasitic symbiont of green plants--a mistletoe.
1674-81 Antonie van Leeuwenhoek describes microscopic protozoans such as Emeria and
Giardia.
1684 Francesco Redi, father of parasitology, was the first scientist to search for parasitic
symbionts of fish, birds, mammals, and humans and report the presence of these organisms
in intestines, air sacs, and swim bladders. Redi refutes the idea of the spontaneous generation
of life in 1668 by demonstrating that maggots develop from the eggs of flies.
1743 Turbevill Needham finds the nematode Anguina tritici in cockled wheat seeds.
1755 Matlieu Tillet, a pioneer experimentalist, proves that bunt disease of cereals is
contagious.
1796 Edward Jenner develops the first method of inoculation against an infectious disease
(vaccination against smallpox).
1807 Isaac-Benedict Prevost, a founding father of plant pathology, experimentally proves that
a plant disease (bunt disease of wheat) is caused by a fungus.
1835 Agostino Bassi demonstrates that a fungus, Beauveria bassiana, causes muscardine
disease of silkworm larvae.

This was the first experimental proof that microbes cause animal disease.
1836-44 J. Schmidberger and T. Hartig coin the term "ambrosia" and recognize its fungal
nature.
1840-44 David Gruby, founder of medical mycology, experimentally shows that fungi cause
diseases in human and animals.
1842 Johann J. Steenstrup demonstrates the penetration of a snail's body by cercariae and
elucidates the life cycle of a liver fluke. His idea of alternation of generations greatly
accelerated research on the life histories of flukes, tapeworms, and nematodes.
1853 Heinrich Anton de Bary unravels the complex life cycle of wheat stem rust and lays the
foundation for the experimental study of plant diseases. In 1879, de Bary coins the term
"symbiosis."
1857-60 Louis Pasteur discovers fermentation as a microbial process and produces artificial
immunity to anthrax, chicken pox, cholera, and rabies.
1858 J. G. Kuhn writes one of the early textbooks on the theme that pathogens cause disease
(the germ theory of disease).
1858 Rudolf Virchow writes a classic book, Cellular Pathology, in which disease processes are
viewed as abnormalities at the cellular level.
1860 Claude Bernard formulates the concept of homeostasis. In later decades the internal
environment of the host is recognized as a specialized habitat for symbionts
1867 Simon Schwendener proposes the dual hypothesis to explain the nature of lichens and
states that lichens are fungi that live parasitically on algal hosts.
1875 William G. Farlow, after completing studies under de Bary, begins the study of fungi in
the United States.
1876 P. van Beneden coins the terms "commensalism" and "mutualism."
1876-84 Robert Koch proves that in anthrax disease a specific symbiont produces a specific
disease and develops criteria for establishing microbes as disease agents ( Koch's postulates).
1878 Thomas J. Burrill was the first scientist to show that bacteria cause plant disease by
demonstrating the cause of fire blight of pears.
1878 Patrick Manson observes the development of Wuchereria bancrofti in the body of female
mosquitoes and demonstrates the importance of the intermediate host in the life history of a
pathogenic symbiont.
1878 M. Woronin describes the nature of legume nodules.
1880 Alphonse Laveran describes the malarial organism Plasmodium malariae in the red blood
cells of humans; he wins a Nobel Prize in 1907.
1881-82 Rudolph Leuckart and Algernon Thomas describe the life cycle of a digenetic
trematode Fasciola hepatica.
1883 A. E. W. Schimper suggests that chloroplasts may have evolved from bluegreen
bacteria.
1884 Elie Mechnikov discovers the phenomenon of phagocytosis and pioneers studies on
cellular immunity; he wins a Nobel Prize in 1908.
1885 A. B. Frank coins the term "mycorrhiza" to describe the fungus growth around the roots
of woody plants.
1888 Hermann Hellriegel, H. Wilfarth, and Martinus W. Beijerinck discover the role of root
nodule bacteria in the legume symbiosis and their significance in the nitrogen cycle.
1888 Roland Thaxter begins his lifelong studies on the Laboulbeniales.
1890 S. Kitasato and Emil von Behring discover antibodies for diphtheria and tetanus
antitoxins.
1891 Theobald Smith successfully transmits the cattle fever organism by using ticks as
vectors.
1891-1903Dimitri Iwanovski describes the first plant virus disease, tobacco mosaic virus, and
demonstrates its filterability. He made microscopic observations and described crystal line
inclusions of the virus in diseased tobacco leaves.
1894 Richard Altmann reports on the discovery of mitochondria.
1895 David Bruce discovers the role of the tsetse fly in the transmission of African sleeping
sickness.
1897 Friedrich Loeffler and Paul Frosch discover the first animal virus, the causal agent of
foot-and-mouth disease.
1897 Ronald Ross discovers the sexual stages of the malarial organism in mosquitoes and was
awarded a Nobel Prize in 1902.
1898 Martinus W. Beijerinck experimentally isolates the tobacco mosaic virus and describes it
as contagium vivum fluidum because of its exclusively intracellular mode of reproduction.
1905 K. C. Mereschovsky proposes the bacterial origin of mitochondria.
1915 Frederick W. Twort discovers bacteriophages.
1921 Paul Buchner, a pioneer of research on mutualism, writes a classic textbook,
Endosymbiosis of Animals with Plant Microorganisms.
1921 Elias Melin synthesizes a mycorrhizal association and describes its physiological
relationships.
1923-25 Lemuel R. Cleveland explores the relationship of symbiotic intestinal flagellates with
their termite and cockroach hosts.
1924 W. Goetsch reports on the mutualistic association between hydra and unicellular algae.
1927 Ivan E. Wallin presents evidence for the bacterial nature of mitochondria and the role
such bacteria may have played in the origin of species.
1928 Frederick Griffith discovers the transformation of pneumococci from avirulent to virulent
strains following exposure to extracts of killed virulent

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which the planet Earth is a complex selfregulatory, resilient living system.

1977 A. M. Maxam and W. Gilbert discover a new method for sequencing DNA.
1981 Vernon Ahmadjian and his coworkers report separation and artificial synthesis of lichens
and introduce the concept of "controlled parasitism" to describe the nature of lichen
symbioses.
1982 W. D. Hamilton and M. Zuk suggest that defense against parasites is involved in the
evolution of bird coloration. Accordingly, the birds should choose mates with bright
ornamental plumage and complex songs because these characteristics signal resistance to
parasites.
1982 Human insulin is the first human gene product manufactured using recombinant DNA
and is licensed for therapeutic use.
1985 First genetically modified crop plants such as corn, cotton, and tobacco are produced by
using Ti plasmid of Agrobacterium tumefaciens for glyphosate-resistance to a herbicide.
1985-95 Birth of the International Symbiosis Society, journals ( Endocytobiosis and Cell
Research, Symbiosis) and international conferences to promote research on and teaching of
symbiosis.
1992 H. Mason and C. Arntzen produce hepatitis B vaccine in tobacco as a host plant by using
Agrobacterium-based vectors. Similarly, alfalfa plants are used as hosts for the vaccine
against cholera toxin, opening a new age of molecular symbiosis that spans across all
kingdoms of life.
1992 Scott O'Neill and his colleagues open a new field of reproductive symbionts of arthropods
such as Wolbachia that distort the sex ratio of the host by feminization or male killing.
1995 R. D. Fleischmann and his colleagues sequence the first complete genome sequence of
Haemophilus influenzae. Since then, complete sequences of several other prokaryotes and the
yeast Saccharomyces cerevisiae, the first eukaryotic organism, have become known.
1997 Stanley Prusiner is awarded a Nobel Prize for his research on the nature of prions as
infectious proteins.

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