ORE Open Research Exeter
TITLE
Halioticida noduliformans infection in eggs of lobster (Homarus gammarus) reveals its generalist
parasitic strategy in marine invertebrates
AUTHORS
Holt, C; Foster, R; Daniels, CL; et al.
JOURNAL
Journal of Invertebrate Pathology
DEPOSITED IN ORE
21 May 2018
This version available at
http://hdl.handle.net/10871/32942
COPYRIGHT AND REUSE
Open Research Exeter makes this work available in accordance with publisher policies.
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The version presented here may differ from the published version. If citing, you are advised to consult the published version for pagination, volume/issue and date of
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Journal of Invertebrate Pathology 154 (2018) 109–116
Contents lists available at ScienceDirect
Journal of Invertebrate Pathology
journal homepage: www.elsevier.com/locate/jip
Halioticida noduliformans infection in eggs of lobster (Homarus gammarus)
reveals its generalist parasitic strategy in marine invertebrates
T
Corey Holta,b,c,⁎, Rachel Fosterd, Carly L. Danielsc, Mark van der Giezenb, Stephen W. Feista,
Grant D. Stentiforda, David Bassa,d
a
Pathology and Microbial Systematics, Centre for Environment, Fisheries and Aquaculture Science (Cefas), Barrack Road, Weymouth, Dorset DT4 8UB, United Kingdom
Biosciences, University of Exeter, Stocker Road, Exeter EX4 4QD, United Kingdom
c
The National Lobster Hatchery, South Quay, Padstow PL28 9BL, United Kingdom
d
The Natural History Museum, Cromwell Road, Kensington, London SW7 5BD, United Kingdom
b
A R T I C LE I N FO
A B S T R A C T
Keywords:
Halioticida noduliformans
Homarus gammarus
Haliphthoros
Oomycete
18S rRNA gene
A parasite exhibiting Oomycete-like morphology and pathogenesis was isolated from discoloured eggs of the
European lobster (Homarus gammarus) and later found in gill tissues of adults. Group-specific Oomycete primers
were designed to amplify the 18S ribosomal small subunit (SSU), which initially identified the organism as the
same as the ‘Haliphthoros’ sp. NJM 0034 strain (AB178865.1) previously isolated from abalone (imported from
South Australia to Japan). However, in accordance with other published SSU-based phylogenies, the NJM 0034
isolate did not group with other known Haliphthoros species in our Maximum Likelihood and Bayesian phylogenies. Instead, the strain formed an orphan lineage, diverging before the separation of the Saprolegniales and
Pythiales. Based upon 28S large subunit (LSU) phylogeny, our own isolate and the previously unidentified 0034
strain are both identical to the abalone pathogen Halioticida noduliformans. The genus shares morphological
similarities with Haliphthoros and Halocrusticida and forms a clade with these in LSU phylogenies. Here, we
confirm the first recorded occurrence of H. noduliformans in European lobsters and associate its presence with
pathology of the egg mass, likely leading to reduced fecundity.
1. Introduction
The Oomycetes are parasitic or saprotrophic eukaryotes that group
within the Stramenopile clade (Phillips et al., 2008). They include numerous taxa which infect and cause disease in aquatic invertebrates
(Noga, 1990). Several Oomycete genera are known pathogens of lobsters and Crustacea in general. Lagenidium, has been identified as a
mortality driver in larval American lobster (Homarus americanus)
(Nilson et al., 1976) and other members of the genus have been detected in several commercially significant shrimp and crab species
(Armstrong et al., 1976; Bian et al., 1979; Bland and Amerson, 1973;
Lightner and Fontaine, 1973). Species belonging to the genera Saprolegnia and Aphanomyces are also notable pathogens of freshwater
crayfish (Alderman et al., 1984; Diéguez-Uribeondo et al., 1994); often
associated with catastrophic mortalities in natural stocks in Europe
(Holdich et al., 2009).
The genus Haliphthoros comprises three species; H. milfordensis, H.
philippinensis and H. sabahensis. These typically infect eggs and early life
stage marine invertebrates. Infection has been described in spiny rock
⁎
lobster (Jasus edwardsii) (Diggles, 2001), blue crab (Portunus pelagicus)
(Nakamura and Hatai, 1995a, 1995b), mud crab (Scylla serrata, S.
tranquebarica) (Leano, 2002; Lee et al., 2017), American lobster (Homarus americanus) (Fisher et al., 1975), white shrimp (Penaeus setiferus)
(Tharp and Bland, 1977), black tiger prawn larvae (Penaeus monodon)
(Chukanhom et al., 2003), and abalone (Haliotis spp.) (Hatai, 1982;
Sekimoto et al., 2007). Experimental challenges have also demonstrated
the susceptibility of pea crab eggs (Pinnotheres sp.) (Ganaros, 1957;
Vishniac, 1958) the European lobster (Homarus gammarus) (Fisher
et al., 1975), ova of the blue crab (Callinectes sapidus) (Tharp and Bland,
1977), adult pink shrimp (Penaeus duoraram) (Tharp and Bland, 1977)
and, the ova and larvae of brine shrimp (Artemia salina) (Tharp and
Bland, 1977). Furthermore, Haliphthoros has also been isolated from the
surfaces of several algae which may give an indication of its lifecycle
outside of an invertebrate host infection (Fuller et al., 1964). With the
exception of H. sabahensis in mud crab (Lee et al., 2017), all of these
descriptions were solely based on the morphological characteristics of
cultures isolated from the site of infection. The infection occurring in
black tiger prawn (Chukanhom et al., 2003), however, was later
Corresponding author at: Biosciences, University of Exeter, Stocker Road, Exeter EX4 4QD, United Kingdom.
E-mail address: ch499@exeter.ac.uk (C. Holt).
https://doi.org/10.1016/j.jip.2018.03.002
Received 4 December 2017; Received in revised form 15 February 2018; Accepted 4 March 2018
Available online 17 March 2018
0022-2011/ © 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).
Journal of Invertebrate Pathology 154 (2018) 109–116
C. Holt et al.
sequenced and analysed phylogenetically (Sekimoto et al., 2007). It is
therefore possible that, based on morphological descriptions alone,
several of these infections could have been misdiagnosed as ‘Haliphthoros’ and more accurate diagnostics are required (Stentiford et al.,
2014).
Halocrusticida (syn. Halodaphnea) is a closely related genus isolated
from marine Crustacea, erected to contain 6 taxa belonging to the genus
Atkinsiella (Nakamura and Hatai, 1995a). All six infect invertebrates
with A. hamanaensis, A. okinawaensis and A. panulirata originally isolated from decapods (Scylla serrata, Portunus pelagicus and Panulirus
japonica, respectively) (Bian and Egusa, 1980; Kitancharoen and Hatai,
1995; Nakamura and Hatai, 1995b). Atkinsiella dubia, a crab parasite,
was the only species not to be reclassified as a member of the Halocrusticida (Atkins, 1954; Nakamura and Hatai, 1995a; Sparrow, 1973).
Sekimoto et al. (2007) isolated an unidentified Oomycete (NJM
0034) from white nodules in the mantle of an abalone (Haliotis rubra)
imported to Japan from southern Australia. The pathogen most closely
resembled a species of Haliphthoros based on characteristic morphological features such as hyphal fragmentation by cytoplasmic restriction.
However, zoosporogenesis, which has traditionally served as the principle method of species identification to discern between Haliphthoros
and its close relatives, was not observed. Upon discovery of the unidentified NJM 0034 isolate (herein referred to as 0034), Sekimoto et al.
(2007) analysed three different gene loci; the ribosomal small subunit
(SSU), the ribosomal large subunit (LSU) and the cytochrome c oxidase
subunit II (cox2). In the SSU and cox2 phylogenies, 0034 branched just
prior to the divergence of Peronosporales and Saprolegniales, separately from the other members of Haliphthoros. In the LSU phylogeny,
0034 formed a clade with ‘Haliphthoros sp. NJM 0131’, originally isolated from black tiger prawn (Chukanhom et al., 2003; Sekimoto et al.,
2007). Muraosa et al. (2009) later described a second abalone parasite
sharing morphological characteristics with Haliphthoros and erected a
new genus to describe the species as Halioticida noduliformans. H. noduliformans was later isolated in wild Japanese mantis shrimp (Oratosquilla oratoria) from Japan and cultured abalone (Haliotis midae)
from South Africa (Atami et al., 2009; Macey et al., 2011) and found to
share 100% sequence identity to the previously sequenced 0034 in the
LSU gene region (Macey et al., 2011).
As part of an ongoing programme considering novel and emerging
pathogens of the European lobster (Homarus gammarus) in the United
Kingdom, we carried out a histopathology and molecular diagnostic
survey of lobsters displaying cloudy/discoloured eggs. We designed and
applied new Oomycete-specific SSU PCR primers to reveal the presence
of 0034 associated with the egg pathology, and generated LSU sequences from the lobster pathogen to determine whether it was the
same as that in Haliotis rubra in Japan (Sekimoto et al., 2007). We also
designed and tested 0034-specific SSU primer sets for use as molecular
diagnostic tools. Our SSU analysis confirmed that 0034 cannot belong
to the genus Haliphthoros and has no directly related SSU sequence
types.
Fig. 1. Gross pathology of infected eggs of Homarus gammarus. Pale, discoloured eggs
observed in brood clutch of berried hen. Black eggs indicate healthy, uninfected eggs.
transport to the hatchery. The remaining three were chilled and immediately transported. Animals that developed pathological signs of
infection (n = 21) had spent between 24 and 106 days within the
hatchery tank system. In order to understand the nature of the disease,
animals were anaesthetised under ice for up to one hour, depending
size. Heart, hepatopancreas (HP), gonad, gut, muscle, gill and eggs were
removed using sterile dissecting equipment and fixed for DNA extraction, histopathology, and transmission electron microscopy. Six eggs
from a subset of animals were cut in half so that histological and molecular analysis could be applied to the same individual egg. From 4th
to 9th July 2016, an additional 17 egg bearing lobsters were collected
on landing, from wholesalers in the south of Cornwall and processed in
the same manner as above. These animals did not enter any holding
tanks and are herein referred to as ‘wild’. Wild lobsters were chilled on
landing and sampled that same day.
2.1.2. Environmental sampling
Littoral marine, brackish water and sediment samples were collected from Newton’s Cove and the Fleet Lagoon (Weymouth, SW
England) by Hartikainen et al. (2014), together with agricultural soil
samples (Gosling et al., 2014), and freshwater samples from the River
Avon (Bickton) and California Lake (Berkshire) (Hartikainen et al.,
2016).
2. Material and methods
2.1. Sample collection
2.1.1. Animal sampling
From July 2015 to October 2016, 323 egg bearing female lobsters
were obtained from various fishermen and wholesale facilities around
Cornwall and the Isles of Scilly, United Kingdom, originally recruited to
take part in a larval rearing program at the National Lobster Hatchery,
Padstow (UK). The landing of egg bearing females was carried out
under authorisation granted by the Cornwall Inshore Fisheries and
Conservation Authority (IFCA). During this period, a total of 21 animals
developed abnormal egg colouration (Fig. 1) (6.5% of the total number
of animals that entered the hatchery). Eighteen of the suspect 21 animals were maintained in wholesaler tanks for up to 7 days prior to
2.2. Histology
Lobster egg samples were fixed in Davidson’s Seawater Fixative for
24–48 h before transferring tissues to 70% industrial denatured alcohol
(IDA). Cassettes were processed using a Leica Peloris Rapid Tissue
Processor and subsequently embedded in paraffin wax. Sections were
cut using a rotary microtome set at 3 µm thickness, adhered to glass
slides and stained using a standard haematoxylin and eosin protocol.
110
Journal of Invertebrate Pathology 154 (2018) 109–116
C. Holt et al.
10 min). Amplicons generated from environmental sampling were
pooled according to habitat type (soil, marine, freshwater) and cleaned
using polyethylene glycol (PEG) ethanol precipitation. Purified amplicons underwent A-tailing to improve cloning efficiency and were subsequently PEG-cleaned once more.
Clone libraries were created using the StrateClone kit (Agilent
Technologies, Santa Clara, CA, USA). 32 clones from each habitat type
were Sanger sequenced using the M13 forward primer at NHM. LSU
gene fragments were amplified using the LSU-0021 (5′-ATTACCCGCT
GAACTTAAGC-3′) and LSU-1170R (5′-GCTATCCTGAGGGAAATTT
CGG-3′) following the concentrations and conditions described by
Macey et al. (2011).
Sequences generated by the study are available in GenBank: accession numbers MH040872-MH040907 (Fig. 3).
Slides were screened for any abnormal pathologies using a Nikon
Eclipse light microscope and NIS imaging software at the Cefas
Laboratory, Weymouth.
Hyphal staining was carried out following a Grocott-Gomori methanamine silver nitrate staining protocol. Slides were de-waxed and
rinsed, followed by oxidation in 5% aqueous chromic acid for 1 h. Slides
were then washed and rinsed in 1% aqueous sodium bisulphate for
1 min to remove excess chromic acid, washed again and subsequently
placed in the incubation solution (5% sodium tetraborate, distilled
water, silver solution (5% aqueous silver nitrate, 3% aqueous methenamine)), pre-heated to 50–60 °C and covered in foil, for 10 min. Stain
development was checked after 5 min. This was followed by several
washes in distilled water, toning in 0.1% gold chloride for 3–4 min and
rinsing in distilled water. Sections were then fixed in 2% sodium thiosulphate for 2–5 min and washed under running water before counterstaining with light green dye (light green SF, acetic acid, water) for
20 s and mounting.
2.6. Phylogenetic tree construction
Sequenced amplicons were added to the collection of full-length
SSU Oomycete sequences with a Labyrinthulomycete outgroup. Distinct
OTUs were defined as having at least one nucleotide difference in two
variable regions of the gene. Those that did not satisfy this criterion
were considered duplicate sequences and grouped together. Closest
BLAST hits for each amplicon generated were included before aligning
using the multiple sequence alignment program (MAFFT Version 7;
(Katoh and Standley, 2013) and the E-INS-I iterative refinement
method. The resulting alignment was used to produce a maximum
likelihood phylogenetic tree inference using RAxML-HPC BlackBox
version 8 (Stamatakis, 2014) on the CIPRES Science Gateway (Miller
et al., 2010) using a generalised time-reversible (GTR) model with CAT
approximation (all parameters estimated from the data). A Bayesian
consensus tree was constructed using MrBayes v 3.2.5 (Ronquist et al.,
2012). Two separate MC3 runs with randomly generated starting trees
were carried out for 5 million generations, each with one cold and three
heated chains. The evolutionary model used by this study included a
GTR substitution matrix, a four-category auto-correlated gamma correction, and the covarion model. All parameters were estimated from
the data. Trees were sampled every 1000 generations. The first
1.25 million generations were discarded as burn-in (trees sampled before the likelihood plots reached stationarity) and a consensus tree was
constructed from the remaining sample.
2.3. DNA extraction
One hundred mg of lobster tissue (or one egg) was transferred to an
MPBio FastPrep (Lysing Matrix A) (MP Biomedicals, Santa Ana, CA)
tube containing 250 µL of lysis buffer (SDS, EDTA) and homogenised.
100 µg/µL of Proteinase K was added and tubes were incubated overnight at 55 °C. 75 µL of NaCl along with 42 µL of 10% CTAB/0.7 M NaCl
was added prior to further incubation at 65 °C for 10 min. DNA was
isolated through phase separation with subsequent additions of
chloroform and phenol:chloroform:isoamyl alcohol (25:24:1). DNA was
then precipitated in 2× volume of cold 100% ethanol at −20 °C for 1 h,
centrifuged to form a pellet and washed with 70% EtOH. The pellet was
air dried before elution in molecular grade water.
For water samples, up to 100 L of water was serially filtered through
55 µm and 20 µm meshes. Twenty-five L of the filtered water was later
serially passed through 3 µm and 0.45 µm filters. The filtrand was dried
and DNA extracted using the MoBio PowerSoil DNA extraction kit
(MoBio, Qiagen, Carlsbad, CA).
2.4. Primer design
Universal Oomycete primers were designed by manually inspecting
an alignment of 215 Stramenopile sequences that spanned the 18S
rRNA gene: Oom278F (5′-CTATCAGCTTTGGATGGTAGGA-3′) and
Oom1024R (5′-CTCATACGGTGCTGACAAGG-3′), producing an amplicon of around 750–800 bp. The 0034-specific primers were also designed using the Stramenopile alignment with added sequence data
generated from infected lobster tissue: Hali_312_F2 (5′-TGGTTCGCCC
ATGAGTGC-3′) and Hali_415_R1 (5′-CACAGTAAACGATGCAAGTCCA
TTA-3′) giving a product of ∼100 bp.
2.7. In-situ hybridisation (ISH)
One hundred µl of hybridisation probes were generated using 20 µL
of Promega 5× Green GoTaq Flexi Buffer, 10 µL of MgCl2, 2 µL of each
primer (Oom278F and Oom1024R), 10 µL of DIG-labelled dNTPs, 1 µL
of GoTaq DNA Polymerase, 49 µL of molecular grade water and 6 µL of
template DNA. Amplification was performed using the previously
mentioned thermal cycler settings.
Slides mounted with suspect wax sections were de-waxed as above
and air-dried. De-waxed slides were then treated with 100 µg/ml
Proteinase K in H2O for 15 min at 37 °C in an opaque box soaked in 5×
saline-sodium citrate (SSC) buffer (trisodium citrate, NaCl, water). The
slides were then incubated in 100% IDA for 5 min and subsequently
rinsed in 2× washing buffer (20× SSC, Urea, BSA). Gene Frames
(Thermo Fisher Scientific) were mounted on to each slide and 300 µL of
probe in a 1 in 2 dilution with hybridisation buffer (100% formamide,
50% dextran sulphate, 20× SSC, 10 mg/mL yeast tRNA, 50×
Denhardt’s solution) was added. Slides were then denatured at 95 °C for
5 min and hybridised overnight at 40 °C. Gene Frames were removed
and slides were washed with 2× washing buffer, preheated to 40 °C, for
15 min. Hybridisation was blocked with a one hour incubation using
6% skimmed milk powder in Tris buffer. Slides were then incubated
with an Anti-Digoxigenin antibody diluted in Tris buffer (1/300 dilution) for one hour at room temperature. Antibody was removed and
slides were washed before staining with nitroblue tetrazolium and 5-
2.5. PCR and sequencing
PCR amplification was performed in 50 µL volumes using 10 µL of
Promega 5× Green GoTaq Flexi Buffer, 5 µL of MgCl2, 0.5 µL of each
primer (1 µM), 0.5 µL of DNTPs, 0.25 µL of GoTaq DNA Polymerase,
32.25 µL of molecular grade water and 2.5 µL of template DNA. Initial
denaturation was carried out at 94 °C for 2 min. This was followed by
30 PCR cycles: denaturation at 94 °C for 1 min, annealing at 64.5 °C
(Oomycete) and 67 °C (0034) for 1 min and extension at 72 °C for
1.5 min (Oomycete) and 10 s (0034), followed by a final extension at
72 °C for 5 min before being held at 4 °C.
PCR products from gill and egg tissues were directly sequenced.
Amplification of the environmental samples were conducted in 20 µL
final volumes with 1 µL of template DNA and was completed at the
Natural History Museum, UK. The thermal cycler program was adjusted
(95 °C for 5 min, 30 cycles of 95 °C for 1 min, 55 °C for 1 min and extension of 1 min 15 s at 72 °C, with a final extension at 72 °C for
111
Journal of Invertebrate Pathology 154 (2018) 109–116
C. Holt et al.
Fig. 2. Histological sectioning of infected tissues. Light microscopy images of 3 µm tissue sections. A – Hyphae protruding from the surface of the egg. Scale bar = 50 µm. B – Silver
staining of the hyphal cell walls within egg tissue. Scale bar = 50 µm. C – Low magnification image of infected gill tissue showing loss of structure and replacement with inflammatory
cells and melanisation. Scale bar = 500 µm. D – Melanised lesion showing multinucleate nature of the hyphae ramifying through gill tissue. Scale bar = 500 µm. E – Silver staining of
hyphal cell walls within the melanised lesion of the gill. Scale bar = 50 µm. F – In-situ hybridisation labelling of H. noduliformans using universal-oomycete SSU probes. Scale bar = 50 µm.
individuals) were each bisected; one half used for histological analysis,
the other for molecular analysis. All the eggs containing hyphal structures were PCR-positive using Oomycete-specific primers. In some
cases, histology-negative samples produced a positive but weaker PCR
product. All amplicons were sequenced and all but one of the histology
positive samples produced a sequence identical to the 0034 sequence.
The remaining egg, (sample 5.3) along with two histology negative
samples produced PCR products which, when sequenced, showed
98–99% sequence identity with Lagenidium callinectes (AB284571)
(Fig. 3).
Several of the histology and Oomycete PCR-positive samples were
tested using the 0034-specific primer set and produced a positive amplicon of around 100 bp. Additionally, the Lagenidium-positive egg,
sample 5.3, produced a positive, but very weak PCR product with the
0034-specific primers. Sequence data from the 0034-specific primer set
confirmed the additional presence of this lineage.
bromo-4-chloro-3-indolyphosphate (NBT/BCIP) solution. Stained slides
were then washed and counter-stained with Nuclear Fast Red before
mounting and examination under light microscopy.
3. Results
3.1. Clinical signs
Infected eggs often appeared white, pink or grey relative to uninfected eggs (Fig. 1). Upon dissection, necrotic lesions were also commonly observed within the gill chambers of infected animals. Copepod
parasitism within the gill chamber was observed in all animals.
3.2. Histopathology
Egg samples from 8 out of the original 21 animals showed abnormal
pathology (38% of animals in total) (Fig. 2A and B). Eggs showed a
reduction or complete lack of egg yolk protein and were instead filled
with large, hyphal structures. In some cases, thalli made up the entire
egg mass and structures were seen protruding out of the egg membrane,
potentially representing zoospore discharge tubes (Fig. 2A). Hyphae
were irregular in shape and multinucleated.
Gill samples from 5 out of the 21 (24%) animals showed similar
thallic structures (Fig. 2C–F). Nine of the 21 gills showed evidence of an
immune response characterised by the presence of haemocyte aggregation (not shown) and melanisation (Fig. 2C and D). Hyphal cell
walls were stained with silver (Fig. 2E). In situ hybridisation with
general Oomycete SSU probes demonstrated the localisation of the gene
target in infected tissues (Fig. 2F).
3.4. Environmental sequencing using oomycete-specific primers
To test the specificity of the Oomycete primers we used them to
amplify DNA extracted from a range of environmental samples: 16
samples from filtered coastal sea and brackish water, 24 samples from
agricultural soil, and 48 samples from filtered freshwater. Eighty of the
88 environmental samples (90.9%) amplified using the Oomycete primers: 16/16 of the marine water samples, 22/24 of the soil samples and
42/48 of the freshwater samples. These sequences clustered into 71
operational taxonomic units (OTUs), which branched across the full
range of Oomycete diversity as shown in Fig. 3. Thirty seven of these
were identical or very similar to GenBank sequences using the same
grouping criterion as described in the methods. The other 34 OTUs
were novel and are indicated by FW, Soil, and Marine prefixes in Fig. 3.
Twenty six of the OTUs generated in this study grouped within the
Peronosporales (12 FW, 10 Soil and two in both FW and Soil), 7 in the
Saprolegniales (6 FW, one Soil) and one Soil sample (Soil 24) branched
prior to the divergence of these two orders (Fig. 3). All the sequences
generated from marine water samples also branched before the radiation of the Peronosporales and Saprolegniales. Three out of the 31 OTUs
generated from marine sampling branched outside of the Oomycete
3.3. Molecular characterisation of the 18S ribosomal SSU in infected eggs
Oomycete-specific PCRs on all but three of the 13 eggs from the
initial group produced positive PCR products (∼800 bp). Sequences
obtained from excised positive bands were 99–100% identical to the
Haliphthoros sp. NJM 0034 GenBank entry (AB178865.1). Both positive
control DNA samples, Aphanomyces invadans and Saprolegnia parasitica,
also amplified. Further individual egg samples (30 eggs from 5
112
Journal of Invertebrate Pathology 154 (2018) 109–116
C. Holt et al.
Pythium scleroteichum AY598680
Pythium rhizo oryzae HQ643757
Uncultured freshwater eukaryote AB721049
Lagenidium myophilum AB284577
Pythium deliense AY598674
Pythium aphanidermatum AY742755
FW 32 MH040872
.95/- FW 15 MH040873
.93/- Pythium adhaerens HQ384422
Lagenidium sp. KJ716873
Pythium monospermum HQ643697
FW 14 MH040874
Soil 4 MH040875
Pythium grandisporangium AY598692
FW 28 MH040876
FW 11 MH040877
Lagenidium callinectes AB284571 (Lobster egg sample 5.3)
Lagenidium thermophilum AB284572
Peronophythora litchii AY742750
Phytophthora pinifolia JN635272
.82/Phytophthora capsici MF784270
Phytophthora cinnamomi JN635269
Phytophthora alticola JN635262
.83/77
Soil 31 MH040878
Soil 22 MH040879
.96/Plasmophara viticola AY742754
Hyaloperonospora parasitica AY742752
FW 7 MH040880
Pythium salinum KF853244 x13
Pythium spiculum KF853242
.91/91
Pythium polare KJ716858
.96/Uncultured eukaryote EF100343
Soil 7 MH040881
1.0/88
Soil 3 MH040882
Uncultured eukaryote AB534496
Uncultured eukaryote AB902023
FW 10 MH040883
FW 21 MH040884
FW 26 MH040885
Uncultured eukaryote KC315824
.96/92
FW 1 MH040886
.92/65
Lagenidiales GU067950
FW 29 MH040887
Uncultured freshwater eukaryote AB771874
Sapromyces elongatus AB548399
FW 24 MH040888
.99/86
FW 18 MH040889
Uncultured Saprolegniales GU479948
1.0/93 Pythiopsis cymosa AJ238657
Pythiopsis terrestris KP098379
.86/Apianopsis terrestris AJ238658
Saprolegnia parasitica XR001099850
1.0/Aphanomyces cfrepetans HQ384411
.93/FW 12 MH040890
Aphanomyces sp. FJ794897
.95/93
Leptolegnia chapmanii AJ238660
Saprolegnia s.p SAP1 FJ794914
Atkinsiella dubia AB284575
.74/30
.96/Blastulidium paedophthorum KR869808
FW 27 MH040891
.91/40
Soil 24 MH040892
Haliphthoros sp NJM 0034 AB178865 / Lobster Oomycete sequences (99 - 100% identical)
Marine 16 MH040893
Uncultured stramenopile KP685311
Marine 7 MH040894
.79/65
Marine 12 MH040895
Marine 26 MH040896
Marine 23 MH040897
Uncultured marine eukaryote AY381206
1.0/94
Olpidiopsis sp.AB363063
.30/28
.64/55
Marine 24 MH040898
.38/37
Marine 30 MH040899
Uncultured eukaryote AY789783
Marine 13 MH040900
Halodaphnea panulirata AB284574
Halocrusticida baliensis AB284578
Marine 3 MH040901
Marine 10 MH040902
Marine 17 MH040903
Haliphthoros milfordensis AB178868
.99/82
.99/88
Haliphthoros sp NJM 0440 AB284580
Haliphthoros sp NJM 0443 AB284579
Marine 1 MH040904
Haptoglossa AB425203
Haptoglossa heterospora AB425199
Eurychasma dicksonii AB368176
.86/-
.93/76
.99/99
1.0/86
.83/1.0/80
Peronosporales
Saprolegniales
Halioticida noduliformans
Unknown clade
Haliphthoros/
Halocrusticida clade
Marine 28 MH040905
Marine 18 MH040906
Marine 9 MH040907
Uncultured
m
a
r
i
n
e
eukaryote DQ103774
1.0/98
Uncultured marine eukaryote DQ103786
Bicosoeca petiolata AY520444
Cafeteria roenbergensis FJ032655
Bicosoecida sp. EF432537
Blastocystis hominis U51151
Mallomonas caudata JN991176
Chlamydomyxa labyrinthuloides AJ130893
Aureoumbra lagunensis HQ710574
Developayella elegans U37107
Haptoglossales
Eurychasmales
Outgroup
0.2
Fig. 3. SSU gene phylogeny of the Oomycete class. Bayesian phylogeny indicating the range of oomycete diversity detected using Oomycete-specific SSU primers. Shapes accompanying
tip labels indicate number of environmental samples grouped with each OTU. Circle = freshwater sample (blue), triangle = soil sample (yellow) and square = marine water sample
(green). Red tip labels indicate sequences derived from lobster tissue. Grey highlights cultured, positive control. Nodes labelled with black circles indicate Bayesian/Maximum likelihood
(%) support of over 0.95/95. With the exception to nodes surrounding the Haliphthoros/Halocrusticida clade, only support greater than 0.8/75 is annotated. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article.)
AB284579) and as a sister to the Saprolegniales but without strong
support (Bayesian PP 0.74; ML bootstrap 40%). The remaining three
sequences grouped with AB284571 (Lagenidium callinectes) (98–99%
identity) isolated from marine crustacea (unpublished). Two further
low-quality sequences were not analysed. LSU PCR amplification of
three heavily infected eggs produced an amplicon of around 1 kb in
length. Sequences generated from the isolation and purification of these
products aligned with Halioticida noduliformans sequences (GU289906,
AB506706, AB285230, AB285227) and 0034 (AB178866) with
99–100% identity. Phylogenetic analysis of the LSU region by Macey
et al. (2011) showed how this H. noduliformans sequence branches
alongside Haliphthoros and Halocrusticida species.
radiation, near the Bicosoeca.
In phylogenetic analyses of a comprehensive taxon sampling of
early-branching oomycete diversity, including 0034, Haliphthoros,
Halocrusticida, Olpidiopsis and Anisolpidium, lineages cluster to some
extent according to known host (Fig. 4). Four OTUs branch in a clade
with the brown algae parasites Anisolpidium spp., three OTUs form a
clade with the red algae parasites Olpidiopsis, and a further four OTUs in
a clade with Haliphthoros, Halocrusticida, and Haliphthoros (parasites of
marine invertebrates), including one grouping strongly with
(AB178868) Haliphthoros milfordensis.
3.5. Phylogenetic relationships of the Haliphthoros-like samples
Twenty two out of the 27 sequences generated from lobster egg
samples were 99–100% similar to Haliphthoros sp. NJM 0034
(AB178865). In ML and Bayesian phylogenetic analyses of the consensus sequence (Fig. 3), this lineage branched separately from the
three other described Haliphthoros sequences (AB178868, AB284580,
3.6. Follow-up health screen of wild lobsters
Histology of 17 wild lobster tissues did not show any abnormalities
or Oomycete-related pathology. No amplicons were generated when
Oomycete and H. noduliformans (0034) primers were applied to the eggs
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Journal of Invertebrate Pathology 154 (2018) 109–116
C. Holt et al.
.89/86
Uncultured marine eukaryote AY381206
Uncultured Stramenopile JQ781892
Marine 23 MH040897
.86/85
Marine 7 MH040894
Marine 12 MH040895
Marine 26 MH040896
.90/.99/79
Uncultured eukaryote EF100276
Anisolpidium rosenvingei KU764783
Anisolpidium ectocarpii KU764786
.85/-
Parasites of...
Uncultured eukaryote EF100297
Brown algae
Olpidiopsis sp AB363063
-/76
.95/84
Marine 24 MH040898
Marine 30 MH040899
.93/-
Uncultured eukaryote AY789783
.82/-
Red algae
Marine 13 MH040900
Olpidiopsis feldmanni KM210530
Olpidiopsis pyropiae KR029826
.91/83
Olpidiopsis porphyrae AB287418
Marine 16 MH040893
Uncultured stramenopile KP685311
Haliphthoros sp NJM 0034 AB178865 / Lobster Oomycete sequences (99 - 100% identical)
.98/93
Halodaphnea panulirata AB284574
Halocrusticida baliensis AB284578
Marine 17 MH040903
1.0/92
Marine invertebrates
Marine 3 MH040901
Marine 10 MH040902
.96/-
Haliphthoros milfordensis AB178868
Haliphthoros s.p NJM 0440 AB284580
.99/89
Haliphthoros sp NJM 0443 AB284579
Marine 1 MH040904
Haptoglossa AB425203
Nematode, rotifer
Brown algae
Haptoglossa heterospora AB425199
Eurychasma dicksonii AB368176
0.03
Fig. 4. SSU gene phylogeny of the lineages surrounding NJM 0034. Bayesian phylogeny of NJM 0034 and its close relatives. Accompanying (green) squares indicate number of
environmental samples grouped with that OTU. Red tip labels indicate sequences derived from lobster tissue. Nodes labelled with hollow circle indicate Bayesian/Maximum likelihood
(%) support of over 0.95/95. Nodes showing support greater than 0.8/75 are annotated. (For interpretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)
Sekimoto et al. (2007) showed the original 0034 isolate branching as a
sister to the Haliphthoros milfordensis NJM 0131 strain (AB178869). In
Macey et al.’s (2011) LSU phylogeny, 0034 is apparently identical to
Halioticida noduliformans (GU289906); an Oomycete also isolated from
nodules in the mantle of abalone and described as a causative agent of
abalone tubercle mycosis disease, which causes significant mortalities
in South Africa (Macey et al., 2011). There is no available corresponding SSU sequence belonging to H. noduliformans to allow the
comparison of both gene markers however, it is likely that 0034 is
Halioticida noduliformans, based on its LSU sequence and phylogeny and
that the Halioticida, Haliphthoros and Halocrusticida genera are mutually
related, together comprising parasites of aquatic invertebrates. LSU
amplicons from our own isolate were identical to Halioticida noduliformans sequences isolated from both shrimp and abalone along with the
0034 isolate.
and guts. However, 5 out of the 17 gill samples weakly amplified using
the Oomycete primers. Three of these tested positive for H. noduliformans using the species-specific primer set. There was no evidence of
infection by histology in any of these samples other than the copepod
parasitism of the gills as observed in the previous group of animals.
4. Discussion
4.1. Phylogenetic position of Halioticida noduliformans
In this study, we confirm the presence of the Oomycete pathogen
Halioticida noduliformans as an egg parasite of the European lobster
(Homarus gammarus). By application of improved Oomycete diagnostic
primers and, by phylogenetic analysis of the amplicon derived from
these primers applied to infected lobster eggs, we show that the parasite
is also the same as the abalone pathogen 0034 (previously described as
a Haliphthoros sp. NJM 0034) (Sekimoto et al., 2007). Isolation and
amplification of the SSU region of the parasite from a number of eggs
produced amplicons that shared 99–100% identity with 0034
(AB178865). This sequence has only previously been associated with
diseased abalone in Japan (Sekimoto et al., 2007). Despite similar
morphological characteristics, 0034 did not group with Haliphthoros
milfordensis (AB178868) from black tiger prawn (Penaeus monodon) or
Haliphthoros sp. (AB284580, AB284579) isolated from marine Crustacea
in our SSU phylogenetic analyses and in already published analyses
using the same marker gene (Beakes and Sekimoto, 2009; Sekimoto
et al., 2007). The lobster egg parasite instead branched before the radiation of the more derived Saprolegniales clade. We therefore agree
with the suggestion by Sekimoto et al. (2007) in that, although 0034
shares morphological similarities to H. milfordensis (both in terms of
their wet mount observations and our histological sectioning), the
isolate is clearly distinct from already described Haliphthoros species.
LSU rRNA gene phylogenies provide further insight into the position
of the 0034 sequence type. The LSU Maximum-Likelihood phylogeny of
4.2. Pathology of Halioticida noduliformans and its relatives
To our knowledge, this is the first report of Halioticida nofuliformans
in the European lobster or any host species from the United Kingdom
and Europe. Very few references exist in terms of the histopathological
descriptions of H. noduliformans and its closely related Oomycetes, such
as Haliphthoros. Atami et al. (2009) offered the first histological descriptions of H. noduliformans in their shrimp host. They described the
presence of hyphae in the gill filaments and base of those filaments. In
our lobster hosts, we first detected the pathogen in discoloured egg
samples. Infiltration of the egg had resulted in a mass of vegetative
hyphae and the breakdown of the egg yolk protein within. Infection in
adult tissues was similarly confined to the gills where growth was likely
halted by the surrounding areas of melanisation; a key defence mechanism of the host. It should be noted however, that gill fouling may
have contributed to the presence of necrotic tissue. No other negative
health effects were observed, however, severe melanisation and subsequent necrotic lesions may well interfere with ecdysis or compromise
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C. Holt et al.
Halioticida noduliformans. Using a combination of these primers, we
detected cases of co-infection with Lagenidium in several lobster egg
samples. It is possible that H. noduliformans infection commonly occurs
in tandem with other pathogenic Oomycetes as previously reported in
mangrove crab; where co-infection with Lagenidium callinectes, Haliphthoros milfordensis and Halocrusticida baliensis caused mortality rates
of nearly 100% in tanked larvae (Hatai et al., 2000).
respiratory function (Diggles, 2001; Fisher, 1977; Fisher et al., 1975).
Macey et al. (2011), who reported the pathogen in abalone, also conducted a histological examination. They describe large numbers of
hyphae penetrating the affected areas. However, in contrast to our own
observations, where infected lobsters showed vast areas of melanisation, they note that there was ‘very little inflammation and in most
cases no reaction zone’. Haliphthoros pathology in juvenile spiny rock
lobster (Jasus edwardsii) shows a similar histology of the gills with the
presence of multinucleate hyphae within the filaments. Hyphae and
melanised lesions were also observed within the leg musculature and
hepatopancreas (Diggles, 2001).
Low levels of Halioticida noduliformans were detected by PCR in the
gills from our wild lobster health screen. However, we did not observe
any pathological evidence of an infection. It is likely that adult lobsters
in the wild are better able to combat the pathogen and infected eggs are
prematurely dispersed to make way for the next brood (Leano, 2002).
Although we do not understand its effect on the wild population, H.
noduliformans and other similar pathogenic Oomycetes are likely to
pose an increased threat to hatchery and/or aquaculture based lobsters
and other invertebrates in culture situations. Intensive culture systems/
sub optimal culture situations can cause physiological stresses which
can increase the disease susceptibility of cultured organisms (Robohm
et al., 2005). With increasing food demands and the continual growth in
the world’s aquaculture industry, it is estimated that by 2030, 62% of
consumed seafood will come from a farming environment (World Bank,
2013). It is therefore becoming increasingly important to better understand the health risks associated with such a shift and identify
simple means in which we can detect and monitor them within these
environments. That is particularly true of the Oomycetes and, more
specifically the Halioticida/Haliphthoros/Halocrusticida clade, as they
have demonstrated their ability to dramatically affect commercially
important invertebrate species.
5. Conclusions
We provide the first evidence of infection of European lobsters
(Homarus gammarus) by Halioticida noduliformans causing a destructive
pathology of the eggs. To our knowledge, this is also the first report of
the parasite in any animal collected from European waters. Potentially
due to the unavailability of Halioticida and Halocrusticida SSU sequences, the AB178865 sequence does not resolve the phylogenetic
positioning of this parasite in SSU trees. However, LSU analysis confirms its clustering within the Haliphthoraceae clade, which also contains the Haliphthoros and Halocrusticida genera.
Incidence of Halioticida noduliformans in the European lobsters not
only demonstrates its ability to impact animals outside of its known
hosts (abalone and Japanese mantis shrimp) but also, highlights the farreaching geographical distribution of the pathogen, which has not been
previously reported in Europe. This relatively newly discovered
Oomycete has proven its ability to impact commercially important
species and may pose a threat to future aquaculture efforts. Based on its
similarity and relatedness to the genus Haliphthoros, it is possible that
Halioticida noduliformans can impact a range of invertebrate species (as
does Haliphthoros milfordensis) and therefore further work is required to
highlight the extent of its host range and subsequent effects on the
hatchery and aquaculture industry.
The general Oomycete and H. noduliformans-specific primer sets we
have developed during this study should better facilitate the identification of this and other potentially problematic Oomycetes, and allow
the exploration of other susceptible host species. They have been subject to environmental testing on a range of different sample types and
have demonstrated their ability to identify a diverse spectrum of species
that span the entire Oomycete diversity.
4.3. Oomycete-specific PCR primers
The cytochrome c oxidase subunits (cox) and internal transcribed
spacers (ITS) have been suggested as DNA barcodes for the Oomycetes.
However, these loci can be problematic for phylogenetic reconstruction, which is an important element of the interpretation of amplicon
diversity (metabarcoding) data (Hartikainen et al., 2014). Choi et al.
(2015) report that the ITS regions can contain large insertions exceeding 2500 bp for some species which will introduce biases in PCR
amplification. Furthermore, together with an insufficient reference
database, cox1 amplification does not identify all known Oomycete
lineages (Choi et al., 2015). The authors demonstrate how amplification
of the sequence region spanning the cox2 gene and the hypervariable
cox2-1 spacer amplified all the lineages tested (n = 31) and therefore
suggest that the cox2 region is better suited as a gene marker. However,
these primers were not tested through means of an environmental
survey and were only applied to individual lineages belonging to the
Peronosporales.
LSU primers have also been used to analyse the molecular characteristics of the Oomycetes and, based on its ability to separate
Halioticida sequences within the Haliphthoraceae, the D1/D2 region of
the LSU has been suggested as a useful marker to discern between
members of this family (Muraosa et al., 2009). Although the cox2 and
LSU gene regions have proven beneficial in the identification of the
Haliphthoraceae and the Oomycetes in general, their reference databases are not as extensive as that of the SSU gene marker. The SSU
primers that we present here will facilitate better phylogenetic comparisons to be made as comparative gene sequences are more readily
available. Environmental testing of the primer set has indicated their
ability to detect a wide phylogenetic range of Oomycetes across all
sample types tested (freshwater, marine water and soil). Thus we were
able to detect H. milfordensis for the first time in a UK marine water
sample. We have also developed a second primer set that is specific to
Acknowledgements
We thank Ben Marshall and Adam Bates at the National Lobster
Hatchery who facilitated the transfer of samples. Further thanks to
Stuart Ross and Dr. Michelle Pond who aided in lobster dissection,
Matthew Green for histological support and advice and Dr. Kelly
Bateman and Chantelle Hooper for their guidance with the ISH protocol.
Funding
This work was conducted within the Centre for Sustainable
Aquaculture Futures, a joint initiative between the University of Exeter
and the Centre for Environment, Fisheries and Aquaculture Science
(Cefas) and funded by a Cefas-Exeter University Alliance PhD
Studentship to CH. Work was also supported through the Agri-Tech
Catalyst, Industrial Stage Awards, Lobster Grower 2 project funded by
Innovate UK (102531) and BBSRC (BB/N013891/1) and Defra contracts C6560 and C7277 to DB.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.jip.2018.03.002.
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