Monograph on Fungal Diseases of Fish A guide for postgraduate students PART 1 One of the scenes showing livestock and fishing activities, DIANABUJA'S BLOG - WordPress.com By Mohamed Kamal Refai Sherif Marouf, Nermeen Abuelala, Rasha Hamza Sayed El-Ahl November, 2016 1 Refai et al. (2016). Monograph on Fungal Diseases of Fish. A guide for postgraduate students https://www.academia.edu/21679188/ or http://scholar.cu.edu.eg/?q=hanem/book/ or https://www.researchgate.net/publication Prof. Dr. Mohamed K Refai, Department of Microbiology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt Ass. Prof. Dr. Sherif Marouf, Department of Microbiology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt Ass. Prof. Dr. Nermeen Abuelala, Department of Fish Diseases, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt Ass. Prof. Dr. Rasha Hamza Sayed El-Ahl, Department of Mycology and Mycotoxins, Animal Health Research Institute, Dokki 2 Preface The first paper I published on fungi in fish was concerned with the isolation of Aspergillus niger and Penicillium funiculosum from imported smoked herring, which was published in Mykosen. 1968 Jan 1;11(1):83-6, and the last paper was a part of MS thesis of my student Sheimaa, entitled: [Refai, M.K., Laila, A. Mohamed, Amany, M. Kenawy, Shimaa, El-S.M.A. The Assessment Of Mycotic Settlement Of Freshwater Fishes In Egypt. Journal of American Science 2010;6(11):823-831]. (ISSN: 1545-1003). I was asked several times by my postgraduate students to prepare a review on fungal infections of fish, but I was postponing this act, because I realized the great efforts I have to do to fulfil this job, the uncertain classification of most fungi affecting fish and my limited expertise with the Oomycetes, however, I felt it is my duty to invade this field and try to get the basic knowledges as guide for the post-graduate students. This monograph is dedicated to the Egyptian pioneers in the field of fish diseases, who were the first to teach fish diseases in faculties of Veterinary Medicine, Cairo (late Professors Mohy Elsaied Eissa& Mahmoud Essam Hatem and Prof. Mohamed Marzouk), Zagazig (late prof. Noor Eldeen Amin), Alexandria (Prof. Mohamed Faisal) and Suez Canal (Prof. Ismail Eissa) Universities. I was lucky to join them as a co-author in some publication:  Easa, M., Hatem, M., Sakr, E. and Refai, M. : Phoma herbarum as a mycotic fish pathogen in Clarias lazera. Vet. Med. J. 92, 257-267 (1984)  Faisal, M., Refai, M. and Peter, G. : Augenmykosen bei Zuchtfischen. Pilzdialog 3, 56 (1986  Faisal, M., Popp, W. and Refai, M. : Hohe Mortalitaet der Nil-Tilapia Oreochromis niloticus verursacht durch Providencia rettgeri. Berl. Muench. Tieraerztl. Wschr. 100, 238-240 (1987),  Refai M, Abdel halim MM, Afify MMH, Youssef H, Marzouk KM. Studies on aspergillomycosis in catfish (Clarias Lazera). Allgemeine Pathologic and pathologische Anatomic. Tagung der Deutachen Veterinar - Medizinischen Gesellschaft& der Europaeischen Gesellschaft fur. Vet Pathol 1987; 63:1-12  Salem AA, Refai MK, Eissa IAM, Marzouk M, Bakir A, Moustafa M, Manal Adel. Some studies on aspergillomycosis in Tilapia nilotica. Zagazig Vet J 1989; 17(3):315-328. Noor El Deen Amin Mohamed Marzouk Mohy Elsaied Eissa Mahmoud Essam Hatem Ismail Eissa Mohamed Faisal Prof. Dr. Mohamed Refai Cairo, November 15, 2016 3 Contents Introduction 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. PART 1 Saprolegnia Achlya Aphanomyces Branchiomyces Dermocystidium PART 2 Sphaerothecum Ichthyophonus Lagendium Haliphthoros Halioticida Halocrusticida Atkinsiella 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 4 Aquastella Pythium Aspergillus Fusarium Exophiala LCD Ochroconis Purpureocillium Phoma Miscellaneous fungi Yeasts Mycotoxins Introduction Fish in Ancient Egypt 1. Fish as popular food Fish was enjoyed by all classes of Egyptian society, both the poor and the wealthy, and was part of most Egyptians' daily diet. Fish was often the first food a child ate after weaning. Fish was fried, smoked, boiled, salted, sun-dried, boiled, pickled, or used in soups. Food in the form of Meat, fish and poultry was flavoured with salt, pepper, cumin, coriander, sesame, dill and fennel. Meat, fish and poultry that was not eaten quickly was preserved by salting or drying. Mullet was particularly favored, and the roe was considered to be a delicacy. Favorite recipes called for the meat to be shredded and mixed with bread and spices into a fish cake, or marinating the fish in wine, beer, or oil with onions, then sprinkling it with pepper or coriander. A condiment made of preserved fish in brine was similar to the Chinese forerunner of soy sauce. 5 Ancient Egyptian Daily life (Food) .ittatoursegypt.blogspot.com The Egyptians - Food | HistoryOnTheNet 6 Bread, quails, tilapia and carp fish, figs, raisins, and wine are awaiting preparation in this ancient kitchen scene. Tomb of Minna. 18th dynasty, ancient Egypt. A man filleting fish The River Nile held a variety of fish, including Nile perch, tilapia, mullet, puffer fish, moonfish, mullets, carp, eels, elephant fish, catfish, and others. One of these catfish actually swam upside-down and was appropriately called, “the upside-down catfish.” At present, it is only found in the Nile below the Aswan dam. Another catfish that was well known to the Ancient Egyptians was the electric catfish. 2. Fish species illustrated on the walls of Ancient tombs:       carp, mullet, catfish, upside-down catfish, elephant fish, Tilapia, 7 Egyptian      Nile perch, puffer fish, Egyptian eel, moonfish, electric catfish. Ti was a supervisor of the pyramids (2474 BC). In this scene, he is out with his men on a hippopotamus hunt. Note the many fish species below the papyrus boat that include: upside-down catfish, elephant fishes, Tilapia, puffer fish, Egyptian eel, moonfish, catfish, and electric catfish. Fish species in this scene include: carp, mullet, catfish, upside-down catfish, elephant fish, tilapia, Nile perch, and eel. On the left side of the panel 8 Mereruka was a vizier to the Pharaoh Teti (2323 BC). Fish species include: Tilapia, eels, puffer fish, catfish, elephant fish, mullet, carp, Nile perch, upside-down catfish, and moonfish. Idut was a queen and the daughter of King Josser (2323 BC). This photo shows a crocodile and elephant nose fish It’s incredible that the records in their tombs have lasted almost 5,000 years. Even more amazing is the fact that many of the same species of fish thrive in the waters of the Nile – today! :: 9 All these fish species are found in Lake Nasser and Red Sea and are becoming tourist attractive hoppy for fishing. Lake Nasser, the largest manmade lake on earth is located in Egypt. The lake is famous for not just its length of 310 miles but also for fishing. Here people come from all over to enjoy fishing by the lake and there are special fishing holidays conducted by tour operators in Egypt to help visitors indulge in this do-not-miss opportunity while in Egypt Enjoy amazing Fishing Tour in Hurghada by Boat, Fishing at Hurghada 10 Carp , Mullet Grey Mullet - Fishing by Rigz 11 Nile perch Tilapia 12 catfish Jeremy Wade with another Nile catfish that he caught at night. The catfish's barbelAnimal Planet upside-down catfish Adult female upside-down catfish lighten up more than the males Aqualand Pets Plus 13 puffer fish Stellate puffer (Arothron stellatus), Red Sea, Makadi Bay, Hurghada, Egypt Eel Travel To Egypt - Dahab Eel Garden 14 moonfish, The Electric Catfish (and the First Pharaoh) ferrebeekeeper - WordPress.com 15 elephant fish Pinterest 3. Fishing in Ancient Egypt As mentioned by Dr. Kenneth J. Stein, we can only imagine that fishing was a popular pastime – it provided an opportunity for either solitude or camaraderie. Like today, some fished by themselves and others, in groups. Many did it for sport and/or food. Others made their living from it. They fished from banks and in boats or rafts that were made from papyrus and other reeds. The Ancient Egyptian anglers also used a variety of techniques, including baited hooks, hand nets, drag-nets, fish baskets or weir traps, and harpoons. Hooks were carved from pieces of bone, wood, shell or ivory. Based on the results from archaeological finds, fishhooks averaged 1/3” – 7” in length. Eventually, the Egyptians evolved and began crafting their hooks from copper and bronze. When this happened is a source of conflict. Most sources place metal fishhooks in later dynasties (Dynasty XII; 1991-1778 BC); however, a famous Egyptologist by the name of Sir William Flinders Petrie dated one specimen of a barbed, copper fishhook at 2500 BC. This latter scenario seems probable as the period was well into the Bronze Age, which began in 3300 BC. In any case, the Egyptians gave barbed metal fishhooks to the world. Fishing line was made from the fibers of flax or linen. The Egyptians did not use a loose mass of fibers but a group of individually twisted threads. Certainly, the diameter and “Lb. test” of the line would be related to the number of linen threads. Sportsmen and recreational fishermen would use one or more hooks on a single line, and those who depended on fishing as a livelihood used multiple lines to improve their catches. The fishing lines were initially weighted with clay, but the Egyptians 16 eventually upgraded to lead sinkers by 1200 BC. The British Museum of Natural History has one of these sinkers in its collection. The fishermen baited their hooks with various items such as stale bread, dates, meat, small fish, and undoubtedly, insects. In addition, they used ground bait, something that was sprinkled on top of the water to attract fish. It is interesting that they never used a small fish to target a larger fish of the same species – they may have considered it sacrilegious. The tombs do not reveal the use of fishing rods or floats in the Old Kingdoms. Both of these came into existence sometime in later dynasties. Given that they knew how to fish the bottom then, it isn’t much of a stretch to consider that they could also devise floats to carry the bait closer to the surface – and alert the angler to a strike. Accordingly, there exist reports of cork floats used by Egyptians but it remains unclear when this practice came about. Perhaps, one day, archaeologists will uncover evidence of this. Fishing with nets was common in Ancient Egypt. These were made from linen and constructed with knots that have been passed down from generation to generation. In fact, these knots (reef, mesh, and half) are universal among net fishermen today. However, fishnets were a costly item that many fishermen could not afford, and it was for this reason that the less affluent fishermen were restricted to fishing on the bank with lines. Drag-net fishing involved more than a couple of fishermen. These nets were weighted with lumps of clay at the bottom and buoyed at the top with wooden floats. Fishermen would wade through the water and encircle a group of fish with their drag-net. Upon trapping the fish, they would strike them with clubs or kill them with harpoons. During later dynasties, drag-nets made use of lead weights and cork floats. Fish traps, or weir baskets, were made from the branches of willow trees. These wickerwork basket-traps were conical in shape and used in one of two ways: For the first way, the Egyptians strategically placed these in the paths of migrating fish; for example, fish swimming upstream. The trap had the effect of corralling fish as they swam with the current. The second way involved placing the traps in water that was adjacent to submerged vegetation. People would walk into the vegetation and scatter the fish away from the shoreline and into the trap. Once captured, the fish were either clubbed or harpooned. Even today, fishermen use weir traps in various places throughout the world. When fishermen caught these in nets, the fish produced sufficient electricity that shocked the fishermen. The volleys of electricity were strong enough to cause the fishermen to release their grip on the nets, allowing the electric catfish and all the other fish to escape! This species is still found in the Nile today. Finally, during the Greco-Roman period in Egypt, some fish, such as the Nile perch and the elephant fish, were considered sacred. There were prohibitions against keeping and eating these fish. Fishermen took great care while removing these fish from their nets to ensure their survival – and to avoid severe punishments! Both of these are doing fine in Egypt today. 17 It is easy to see that the Ancient Egyptians were the early innovators of modern day fishing and most likely, not any different from us. If you have the chance to read any of their translations, you will find that they valued being in the outdoors and away from everyday life. They seemed to have a good time – like all anglers! The Tomb of Userhat (TT56), Usherhat fishing with a harpoon Tour Egypt Fishing and hunting on the Nile - Q-files Encyclopedia, Q-files The Ancient Egyptians fished from a papyrus boat or a papyrus raft, a multi-hooked lines; landing nets. 18 Kagemni was a vizier during the time of King Djoser (2649 BC). Men fish from a papyrus boat. One man fishes with a multi-hooked line; another holds a landing net. Kagemni was a vizier during the time of King Djoser (2649 BC). The two men fish from a papyrus raft. Note the men’s outstretched index fingers that indicate they are fishing. 19 Idut was a queen and the daughter of King Djoser (2323 BC). The man fishes from a papyrus boat with a fishing line in one hand and a club in the other. Note the basket of fish. 4. Fish trade Fish bones were made into beads, needles, and awls. Wages and taxes were sometimes paid in baskets of fish. Fish was also used as payment in international trade - in the report of Wenamun, 35 baskets of dried fish were destined as partial payment for a shipment of Syrian cedar Fish obtained by Hatshepsut ‘s trade expedition to Punt From the time of the old kingdom onward, Egyptians had launched expeditions to the land of Punt, a kingdom rich in gold, frankincense, myrrh, and exotic timber. Hapshepsut's task force was organized, launched and proceeded to meet their goals in bringing the trade goods of Punt to Egypt without the need for trade through middle men. 20 Bas-relief from the temple of Hatshepsut Copy from a relief recording Queen Hatshepsut's expedition to the Land of Punt The artists documenting the Egyptian expedition to Punt under Queen Hatshepsut depicted a number of sea creatures living in the Red Sea, among them:          rays, a swordfish, a unicorn fish, a flatfish, a spiny lobster, a triggerfish, a surgeonfish, a wrasse, and a Oligo squid. 21 Ray fish TripAdvisor Egypt Excursions Online - Day Tours: Swordfish Tackle Direct Blacktip Daytime Unicorn fish, Naso brevirostris, Ras Mohammed, Sinai, Red Sea, Egypt 22 Flat Fish Camouflage Stock Photos ...Alamy A moses sole swimming close to the sea bottom, Marsa Alam, Egypt, Red Sinai Underwater Richard Seaman Picasso triggerfish 23 Dreamstime.com Sohal surgeonfish (Acanthurus sohal) with coral reef Red Sea Egypt AlamyHumphead Wrasse or Napoleon fish Red Sea Egypt AlamyReef squid Sepiotheuthis lessoniana Red Sea Egypt - Stock Image 5. The electric catfish (King Catfish) Malapterurus electricus was well known to the ancient Egyptians. One of the earliest artifacts to utilize hieroglyphs, the extraordinary Palette of Narmer, depicts the electric catfish in a central location on both sides. The dense siltstone palette dates from 3100 BC and it depicts Egypt’s first pharaoh, King Narmer. On the front of the palette, King Narmer is shown wearing the white crown of Upper Egypt–the desert fastnesses to the south. On the palette’s back he is portrayed walking among beheaded enemies and wearing the red crown of Lower Egypt–the rich delta land of swamps and fertile black earth. The object was found in Neches, a community which had been inhabited for thousands of years before King Narmer united the two kingdoms. Neches later became a major center for the worship of Horace, the god of the pharaohs. 24 Why is the catfish in such a prominent place on the palette? King Narmer’s name was an elision of two hieroglyphs “n’r” and “mr”. N’r stands for catfish, and mr stands for chisel. So the first god king of Egypt was literally named “Catfish-chisel” which is exactly what the symbol on the palette consists of. Here is a longer account of the history and milieu of King Catfish from an Egyptian website (the site calls Neches by its Greek name of Hierakonpolis). A Drawing of the Front of the Palette of Narmernm, back of the Palette of Narmer https://ferrebeekeeper.wordpress.com/2010/08/03/electric-catfish-and-the-firstpharaoh/ 6. Fish in hieroglyphs 25 Penn Museum Blog | Something's Fishy in the Palace of Merneptah ... 7. Fish in the Ancient Egyptian art In Egypt, fish depicted in artworks and tomb carvings often serve as symbols of fertility and rejuvenation, life and death, and ultimately reincarnation. Tilapia was commonly farmed in the Nile river: the symbolism inherent in the perpetual birth, harvesting (i.e. death), and return of the fish must not be lost on us Fish paintings Fish paintings are located in the Mortuary Temple of Queen Hatshepsut, Nile Tilapia on the walls of Hatshepsut’s Temple Painting of fisherman at work in the tomb of Anchtifi. Anchtifi was a nomarch of Hierakonpolis. In his own autobiography, the nobleman described his time as one of famine and economic hardship. During these strenuous times, fish yielded by the farmers must have been a treasure 26 BBC , A wall painting of fish from the tomb of Minna at Thebes Fish being speared, captured in limestone. This piece was located in Thebes, but is now on display at the Cleveland Museum of Art, and was created back in the late 25th to early 26th dynasty, roughly 600-660 BCE Photo Sources (top to bottom): Marcus Cyron, Anonymous, Elke Wetzig, Elke Wetzig, Marcus Cyron, Daderot Fish carving 27 Carved wooden fish showing the cartouche of Intef VII (Nub-Kheper-Ra) from the 17th Dynasty c.1580-1550 BC. Cosmetic dishes are often in the form of the Nile fish Tilapia Nilotica, which is easily distinguished by its arrangement of fins as well as its general shape. In some examples the dish had a hinged lid. Fish carving from the Mortuary Temple of Queen Hatshepsut 28 Ancient Egyptian bas-relief carving of a fish. From the temple at Karnack Scorpionfish from within the mortuary complex of Hatshepsut, the first female Pharaoh (1500 BC). 29 Contemporary Sculpture - "ANCIENT EGYPTIAN RELEIF" Fish made from ceramic and metals Ancient Egyptian Faience Fish Amulet - 1550 BC - Egyptian - Cultures - Shop www.artancient.com 30 Egyptian Inspired Abbie Rose Collections Lotus Fish plate. This ceramic plate has been inspired by an 18th Dynasty Egyptian Faience Bowl 31 Vessel in the Form of a Fish, Egypt, 18th Dynasty, 1550–1307 BC, Los Angeles County Museum of Art, gift of the 1999 Collectors Committee This vessel represents a (Nile perch), a silver-blue fish depicted here in delicately formed Egyptian faience. It dates to the 18th dynasty, a period known for its outstanding design and fine craftsmanship, making this elegant form a desirable addition to LACMA's small collection of Egyptian art. Ancient Egyptian Art | Bowl with design of fish and lotus | F1909.71,Freer and Sackler Galleries Smithsonian 32 Ancient Egyptian Fish Art | Fish FAQs An ancient Egyptian (c.3000 BC) slate cosmetics dish in the form of a tilapia fish, a symbol of fertility and resurrection. (Kunsthistorisches Museum Wien) 33 Toilet Dish in Two Parts in the Form of a Fish A container for cosmetics or possibly medical ointment, this covered dish represents a tilapia fish. The tilapia symbolized fertility and was believed to promote abundance on earth and a renewed life after death. This container’s lid swivels open at the tail. Medium: Greywacke, inlay of shell and black paste Place Made: Saqqara, Egypt Dates: ca. 3000-2800 B.C.E. Dynasty: I Dynasty Period: early Dynastic Period Ancient Egyptian Gold Amulet of Fish Pinterest , Egyptian Gold Amulet of a Fish. This and more ancient jewelry 34 Gold amulet from a Twelfth Dynasty child’s tomb at Haraga. According to ancient Egyptian belief, the image of a fish worn in the hair gave protection against drowning. Egyptian gold fish pendant, Middle Kingdom, Late Dynasty, 1878-1640 BC (courtesy National Museums Scotland) 8. Fish in Ancient Egyptian Religion/Spirituality. Certain species of fish were associated with various deities and therefore considered to be sacred. The symbol of the patron goddess of fishermen, Hat-Mehit, was the Lepidotus Fish. The goddess Neith was associated with the Nile Perch, the Nile Carp was associated with Osiris, and the gods Ra and Atum with the eel. The Chromis and Abdju fish served as pilots for Ra's solar barque, warning him of the approach of the serpent Apophis as the barque traveled through the Duat. The Nile Mormyrid was a symbol of the evil god Set, and therefore reviled (in some instances it was held as sacred because it was thought to carry some of Osiris' own flesh.) The Bolti fish was regarded as a symbol of rebirth because it carries its eggs in its mouth. The Tilapia was believed to have multiple lives, and to be self-created. The image of a fish with lotus flowers issuing from its mouth was a symbol of resurrection. Amulets of both were buried with the dead. The heart was likened to a "red fish swimming in a pond." During the harrowing journey through the Duat, the deceased at one point changed into a fish. The possession of a fish amulet was believed to help with this transformation. Sometimes an actual mummified fish was included in the mummy wrappings. On one Egyptian coffin dating to 330 B.C.E., a fish takes the place of the Ba-bird hovering protectively over the mummy. 35 Fish were also placed in tombs to serve as food for the dead. According to the Coffin Texts, the deceased wishes to become like the crocodile god Sobek, who "lives on fish." Considered a food pleasing to the gods, Ramses III gave to the Temple of Amun some 474,640 fish, both fresh and dried. Fishing scenes were popular in tomb paintings. The wealthy had pools stocked with fish for pleasure. Fish were also purchased as gifts to feed sacred animals, especially cats. The boulti fish was associated with the sun and his image carried protective properties for ancient Egyptians. He was later linked to the cult of Hathor at Dendera. For all these reasons, it is shown on amulets, pledges of protection and charms that could be worn as jewellery by the living or even for the dead to protect them from potential pitfalls to be encountered in the afterlife. New Kingdom? Between 1150 and 1069 BC, Precious Metal.Precious Stone . This amulet was made from a fine gold leaf and engraved; it represents a fish swimming towards the left with a long dorsal fin, two small ventral fins and a tail spreading out like a fan. The gills and eye are marked by small protrusions. On the body, a rock with a bluish-turquoise tone (green feldspar?) in an almond shape is set using a thin gold band. The general form of this inlaying is reminiscent of an eye, in particular, the Udjat eye, the symbol of integrity for ancient Egyptians. This is, without doubt, a representation of the Tilapia nilotica, or Boulti as it is called today in Egypt. 36 One of the most famous tales from the Westcar Papyrus told by Bauefre, called “Tale of the rowers”, tells a story which took place at the court of King Snefru. The tale is centred on a fish-shaped amulet of turquoise which was lost in a lake during a royal rowing trip. It testifies that such fish shaped jewelers were worn by women, and beyond the miracle performed by the chief lector Djadjaemankh who folds aside the water to retrieve the lost amulet from the bottom of the deep lake, it shows the devotion towards such an adornment. Fish amulets, like this nekhau, were given to young girls to wear as a charm against drowning. Some scholars suggest they functioned as “reminders of a watery environment,” to give the owner security, according to the catalog notes, “but it is much more likely that the amulet allowed the wearer to acquire the abilities of a fish, and therefore survival, if she happened to fall into the water.” Artistic expression greatly expanded in Egypt during the Middle Kingdom. The exhibition shows how styles evolved and culture and religion transformed. Many of the motifs in protective amulets and magical objects we associate with Ancient Egypt were introduced during the Middle Kingdom. Some, like the fish, were believed to shield young girls, others were designed to protect children and pregnant women. Egyptian men were just as likely to be bejeweled – at least royal ones, like this pharaoh. 9. Fish mummies Fsh were a staple item in ancient Egyptian diets and were also mummified as represen tatives of various gods, e.g.Lates niloticus (Latidae) was worshipped as a form of the goddess Neith at Esna, giving rise to the town’s Greekname of Latopolis and Schilbe mystus (Schilbeidae) was the fetish of the delta nome of Mendes, whose localgoddess was called Hatmehyt (“foremost of the fishes”) who is usually depicted with a Schilbe on her head. Fish-eating was banned in areas were certain fish species were venerated 37 Academic Dictionaries and Encyclopedias Egyptian electric catfish mummy in the Rosicrucian Egyptian Museum in San Jose, California. Mummified fish on Pinterest 38 2800 B.C. year Egyptian mummified fish! Keep in mind that this original mummy likely dates from the Early Dynastic Period... 39 Ancient Resource Ancient Egypt. Ptolemaic period, c. 3rd – 1st Century BC. Fantastic Egyptian mummified catfish! Wrapped within linen strips with details added in pigment on ... 10.Aquaculture in Ancient Egypt Aquaculture has been known in Egypt since the beginning of written history; tomb friezes date back to 2500 B.C. and illustrate the harvest of tilapia from ponds (Bardach et al, 1972). A traditional form of aquaculture, known as “hosha”, has been commonly practiced for many centuries (Eisawy and El-Bolok, 1975) in the Northern Delta Lakes Region until a few decades ago. ca. 1350 BCE. Nebamun’s Tomb Thebes; Funerary Painted Pool in the Central Garden contains tilapia, mullet and ducks. Nilotic cultures built gardens with central swimming pools. BM.Less Bread, quails, 40 tilapia and carp fish, figs, raisins, and wine are awaiting preparation in this ancient kitchen scene. La tombe de Menna. Tilapia fingerlings and tilapia breeding colonies for sale, Tilapia farming at home A 3400 year old picture of a tilapia garden on the tomb of Nebamun. Fish of the Dead - Egyptian Tomb Paintings of Fish | Fish FAQs 11. Sham el-Nessim Sham el-Nessim is an Egyptian national holiday marking the beginning of spring. It always falls on the day after the Eastern Christian Easter (following the custom of the largest Christian denomination in the country, the Coptic Orthodox Church). Despite the Christian-related date, the holiday is celebrated by Egyptians regardless of religion. The name of the holiday is derived from the Egyptian name of the Harvest Season, known as Shemu, which means a day of creation. According to annals written by Plutarch during the 1st century AD, the Ancient Egyptians used to offer salted fish, lettuce, and onions to their deities on this day. After the Christianization of Egypt, the festival became associated with the other Christian spring festival, Easter. Over time, Shemu morphed into its current form and its current date, and by the time of the Islamic entry in Egypt, the holiday was settled on Easter Monday. The Islamic calendar being lunar and thus unfixed relative to the 41 solar year, the date of Sham el-Nessim remained on the Christian-linked date. In his book, Manners and Customs of the Modern Egyptians, Edward William Lane wrote in 1834: A custom termed 'Shemm en-Nessem' (or the Smelling of the Zephyr) is observed on the first day of the Khamaseen. Early in the morning of this day, many persons, especially women, break an onion, and smell it; and in the course of the forenoon many of the citizens of Cairo ride or walk a little way into the country, or go in boats, generally northward, to take the air, or, as they term it, smell the air, which on that day they believe to have a wonderfully beneficial effect. The greater number dine in the country or on the river. Egyptian feasts. Egyptian feasts celebrated Sham el-Nessim since 2700 BC by all Egyptians regardless of their religion, beliefs, and social status, the name Sham el-Nessim (Inhaling the breeze) is derived from the Coptic language that, in turn, is derived from the ancient Egyptian language. Originally pronounced Tshom Ni Sime, with tshom meaning “gardens” and ni sime meaning “meadows.” Like most ancient Egyptian feasts, Sham el-Nessim was also affiliated with astronomy and nature. It marks the beginning of the spring festival, which is the time they believed day and night are equal, (when the sun is in the Aries zodiac) hence marking the beginning of creation. They confirm the exact date annually by sighting 42 the sun in relation to the great pyramid. Ancient Egyptians named it The Feast of Shmo (the revival of life). At the crack of dawn Egyptians usually leave their homes to have a picnic with their families in meadows and gardens to enjoy the breeze. On this national holiday the traditional Sham El-Nasim meal consists of fish, onions and eggs. Fish was highly respected in ancient Egyptian beliefs. Salted mullet fish (known as fesikh), was offered to the gods in Esna in Upper Egypt to the extent that Esna’s ancient name was Lathpolis, which was the name of the original fish before it is salted. As for colouring eggs, it’s a custom mentioned in the pharaoh’s famous Book of the Dead and in Akhenaton’s chants, “God is one, he created life from the inanimate and he created chicks from eggs.” Hence, the egg was a symbol of life to ancient Egyptians Ancient Egyptians would boil eggs on Sham El-Nasim eve, decorate and colour them in various patterns, then write their wishes on these eggs, tuck them in baskets made of palm fronds and hang them on trees or the roof of their houses in hopes that the gods would answer their wishes by dawn. The habit of eating onions on that day is equally ancient. According to Egyptian legends, one of the pharaoh’s daughters had an incurable disease. Doctors were clueless until a high priest started giving her a few drops of onion juice. Her condition improved and her father, thrilled, named that day “the onion coalition day.” That day people would roam the city of Menf and offer onions to their dead. As for flowers and plants, ancient Egyptians considered them holy and the lotus flower was actually the symbol of the country in ancient times. In ancient Egypt, families would combine all of these: they would gather on the eve of Sham El-Nasim to colour the boiled eggs, prepare the fesikh and onion, some hanging the onions on their door steps to ward off evil spirits and putting them under their grandchildren’s pillows that night to summon the god Sukar. Before dawn, people would head to meadows, gardens and the Nile river bank to watch the sunrise while carrying food and flowers. They spend their day out in the open air, joyfully singing away the hours. Little has changed since the time of the Pharaohs, apparently. Traditional Sham el Nessim Recipes and Food Menu The main feature of the celebration is preparing and eating of traditional foods. The dishes eaten on this day consist mainly of green onions or scallions, Fiseekh which is a smelly salted fish, boiled colored eggs, lettuce and termis or Lupini Beans. 43 Significance of Sham el Nessim Recipes. Green onions have a special significance on the occasion. It has been found in ancient times and has been an important part of the festivities owing to the belief that it helped cure the son of one of the pharaohs from a mysterious illness. In modern times, scallions are believed to keep the evil eye away and prevent envy. Salted Fish, which is also an important Sham el Nessim food has been a symbol of welfare to Egyptians. It was believed that offerings of fish were made to the ancient gods to ensure a good harvest. Salted Fish symbolizes fertility and prosperity. Boiled eggs are another must-have for the day. They are a symbol of new life and luck. Before anyone steps out of the house, the eggs are colored. 44 Fesikh is eaten during the Sham el-Nessim festival, which is a spring celebration from ancient times in Egypt. The traditional process of preparing Fesikh is to dry the fish in the sun before preserving it in salt. ... The occupation has a special name in Egypt, fasakhani. 12.Modern aquaculture in Egypt As reviewed by Naglaa F. Soliman and Dalia M. M. Yacout (2016), modern aquaculture began in the mid-1930s following the introduction of the common carp at two research farms; from then until the early 1960s, the carp was kept purely for research purposes. However, the present aquaculture system began to expand noticeably from 1960s to 1970s when a scientific base for aquaculture development was practiced (FAO 2010). In the late 1970s, an aquaculture development plan was proposed to boost the development of the sector. By the end of the plan in the mid1980s, annual aquaculture production had jumped from a mere 17,000 t to about 45,000 t. Until the mid-1980s, aquaculture activities were confined to the Eastern and Northern Delta Regions. All production derived from either semi-extensive or semi-intensive pond systems using fresh and low saline brackish water. Traditional, privately owned aquaculture farms, producing mostly tilapia and mullet, operated using large shallow ponds of up to 25 ha with production as low as (250–400 kg/ha). This type of production depends mainly on enhanced natural productivity through the addition of natural fertilizers (manure) and limited use of artificial feeds (usually rice bran; FAO 2010). The beginning of marine species culture, such as Dicentrarchus labrax, Sparus aurata, Solea vulgaris, Argyrosomus 45 regius and Penaeus semisulcatus, was in the 1990s (Dighiesh 2014). In the mid1990s, intensive pond aquaculture was introduced with the aim of replacing the semi-intensive and traditional farms. Intensive farming has been expanding as a result of the high returns on investments (FAO 2010). Tilapia, carp and mullet comprise more than 95 % of Egyptian aquaculture production indicating a narrow production basket. In 2012, the share of tilapia had increased to 768,752 t. Other major species are mullet (129,651 t), carp (67,065 t), and to a lesser extent also sea bass (13,798), seabream (14,806 t) and shrimp (1109 t). Finally since 2008, production of meager started and reached 8319 t in 2012 (CAPMAS 2012; Table 2; Fig. 2). When taking a closer look at tilapia as the most important farmed species, Egypt ranks second behind only China with a total value of about 900,000 USD (Rothuis et al. 2013). African catfish farming in the country is promising and gaining importance in recent years. While production of tilapia is steadily increasing, production of carps is declining due to less consumer preferences (Tables 2, 3, 4, 5, 6, 7, 8). 46 From small levels of production in the early 1990s, fish farming has expanded rapidly while capture fishing has remained fairly constant, even declining somewhat after peaking at the beginning of the twenty-first century. Fish farming in Egypt currently represents the largest part of production, where it reached up to 74 % of the total production, and the private sector share of this production is more than 99 % (Shaheen et al. 2013; Fig. 1; Table 1). The area being farmed increased from 42,000 ha in 1999 (El-Sayed 1999) to 120,000 ha in 2012 (GAFRD 2013). Annual total fisheries and aquaculture production (GAFRD 2012 Until the mid-1980s, aquaculture activities were confined to the Eastern and Northern Delta Regions. All production derived from either semi-extensive or semi-intensive pond systems using fresh and low salinity brackish water. Traditional, privatelyowned aquaculture, producing mostly tilapia and mullet, operated using large shallow ponds of up to 25 hectares. Production per unit area was low (250–400 kilograms/hectare). This type of production depends mainly on enhanced natural productivity through the addition of natural fertilisers (manure) and limited use of artificial feeds (usually rice bran). Semi-intensive aquaculture was more commonly carried out in farms operated by the Government where poly-culture was practiced in smaller ponds of between 2–6 47 hectares with the use of fertilisers and supplementary feeding. The average annual production from these semi-intensive systems was in the range of 1.5–2.5 tons per hectare. The farming of marine species such as European seabass, gilthead seabream, sole, meagre and penaeid shrimp began in the late 1980s and early 1990s. The majority of farming of marine fish still depends on the collection of seed from the wild. Marine aquaculture in Egypt is still far from being as successful as freshwater farming. In the mid-1990s, intensive pond aquaculture was introduced with the aim of replacing the semi-intensive and traditional farms. Intensive farming is expanding as a result of the high returns on investments. These systems use smaller and deeper ponds, stocking densities are higher and intensive feeding and aeration is provided. Average annual production attained is in the range of 17.5–30 tons per hectare. Integrated desert agriculture-aquaculture activities started in the late 1990s generally in the form of intensive tank aquaculture. This form of aquaculture is also expanding rapidly particularly in the western desert region. Total aquaculture production in 2009 in Egypt reached 705 490 tons with a total market value of USD 1 354.646 million (1 USD = 5.55 Egyptian pounds) (GAFRD, 2010). Human resources Distribution of the main aquaculture production sites (Google Egypt map, 2010). 48 Currently, tilapia are the main cultured species mainly Oreochromis niloticus, O. aureus and Sarotherodon galilaeus. Although green tilapia, Tilapia zilli has been identified undesirable, they enter the fish pond adventitiously in large numbers especially wherever the control systems for water inlets and outlets are not appropriate. Mullets especially grey mullet, Mugil cephalus are among the major cultured fish species in the early practices of aquaculture. That is mainly because of their high marketability. In addition, fry could reach marketable size in one growing season. However, there was normally shortage in their natural supply required for aquaculture. In that respect, Mugil capito which are considered the following 49 important species in the mullet group were normally collected in numbers enough to make up the shortage of grey mullet fry. However, a nursing season was required to grow them up to market size. As aquaculture sector grew and the area under culture has doubled, the demand for the mullet fry especially grey mullet began to exceed their supply. Similarly, as management practices are continuously developing, other species started to contribute more in culture systems; tilapias began to have a major role in Egyptian aquaculture (El-Gamal, 1997). Accidental entry of some other fish species cannot be ignored. African catfish, Clarias lazera and Nile perch, Lates niloticus is an examples for that group. Because their predatory behaviour, these species may have an impact on fish production in fish ponds especially when they exist in high density and/or large size. However, there is a preliminary interest in culturing both fish species. Because of the high marketability of some marine fish species, especially for exportation, they started to attract more attention. European seabass, Dicentrachus labrax and gilthead seabream, Sparus auratus are the most important species. Fry and/or fingerlings of both species collected from nature are still the major source of their culture. Expansion of their culture will be dependent on their seed production via hatcheries which still at the experimental stage. On 1934 common carp, Cyprinus carpio were introduced from Indonesia, their mirror carp strain were introduced in 1949 from France. Their natural spawning was practiced in few research stations. Along with the construction of fish hatcheries starting from 1970s, more carp species, in addition to common carp were introduced. Grass carp, ctenopharyngodon idella were first introduced from Holland on 1977 for weed control where they were successfully reproduced. Although silver carp, Hypophthalmichthys molitrix and bighead carp, Aristichthys nobilis were previously introduced from Thailand and Japan on 1954 and 1962 respectively. However, their contribution to aquaculture was not significant until fish hatcheries have been constructed and more introduction took place on early 1980s. Currently, carp species are contributing significantly in aquaculture especially in governmental fish farms. Fingerlings of common carp are the species stocked in rice fields. Fingerlings of grass carp are annually stocked in canals and drains starting from 1990 for weed control. Red tilapia hybrids were also introduced targeting better utilization of saline waters (El-Gamal, 1997). The last carp species introduced on 1993 was the snail carp, Mylopharyngodon piceus for snail control in fish culture habitat. Studies are currently conducted at the Central Laboratory for Aquaculture Research at Abbassa to investigate their food habits and their possible effects on the ecosystem and also Garrah (2001) studied factors affecting seed production of black carp. Fresh water prawn, Macrobrachium rosenbergii were also introduced from Malaysia and from Thailand in 1980s where their hatching was practiced and their seed became available for grow-out either in monoculture or in polyculture with some fish species. 50 Egyptian fish resources are Depending on Marine Fisheries, lakes, Fresh water and Aquacultures. Marine Fisheries: Marine Fisheries included Mediterranean and Red Sea, Suez Canal and Aquaba Gulf. Their area together is estimated at about 11.2 million faddans or 85.2% at the natural fisheries. Aquaculture is one of the most common commercial activities in Egypt. It includes ponds fish farms, cages, rice fish farms and also intensive aquaculture. Area of fish farms is about 360.2 thousand faddans, which is equal to 3% of Egyptian fisheries. The National Project for Fish Farming in the Canal Zone The authorityof the Suez Canal Zone has started establishing 460 fish farming ponds, out of a total of 3,828 ponds. Spanish companies are participating in the project, with the fish imported from abroad, and that the project will depend on Egyptian experts. The project is important, as it provides a source of protein in light of the high prices of meat and chicken. It was emphasized that if the fish production is sufficient, it may lead to self-sufficiency and stopping importation. Moreover, fish farming can significantly aid in the development of the economy, and can produce a multitude of species that are harvested in ideal conditions through the control of water temperature, salinity and pH levels, as well as the percentage of oxygen. The project will reduce transportation costs and the rate of fish spoilage. Establishing the project in this region gives it advantages as it is close to the major ports of Suez, Port Said and Damietta. Additionally, the project is close to the local market to distribute fish in Greater Cairo (Cairo, Giza and Qaliubiya) and other governorates. The project will be implemented through many phases to reach 80 million fry annually. It includes fodder factories, fry hatching and a comprehensive production complex, canning and packaging through many stages. So, the number of basins will reach 3800 at the end of the project. There are many studies with international Spanish and Korean parties to implement the most major project for fish culture in the Middle East which will establish on the Canal banks with a length of 120 km. Suez Canal Region Constitutes an Adequate Environment for Establishing Fish Culture Projects This project aims to produce a safe seafood product by 100%. In this regard, on 25 June 2015,500 fish basins out of 1380 were completed during the 1st phase of the fish culture project. The project provides fish and bridging the food gap in Egypt, where the initial production will be ranged from 10,000 to 15,000 tons. The production is targeted to reach 50,000 tons initially. 51 UAE participate with Egyptian authorities to create the largest fish farm in the world, cementuae.com, The most important species of fish that can grow in fish farms are sea bass, sea bream, croakers and shrimps.” Sea Bass (Morone chrysops) All-Fish-Seafood-Recipes.com Seafood Training Academy. Sizes range from300g to 1kg, though they tend to average at around 450g making Sea Bream a great fish for serving whole. 52 Croakers | | FreshFishHouse.com Shrimps 53 1.Saprolegnia          Saprolegnia is often called "cotton moulds" because of the characteristic white or grey fibrous patches they form. Current taxonomy puts Saprolegnia as a genus of the heterokonts in the order Saprolegniales. Saprolegnia, like most oomycetes, is both a saprotroph and necrotroph. Saprolegnia is tolerant to a wide range of temperature, 3°C to 33°C, but is more prevalent in lower temperatures. While it is found most frequently in freshwater, it will also tolerate brackish water and even moist soil. Saprolegnia filaments (hyphae) are long with rounded ends, containing the zoospores. Saprolegnia generally travels in colonies consisting of one or more species. Colonies are generally white in colour, though they may turn grey under the precesence of bacteria or other debris which has become caught in the fibrous mass. Saprolegnia has a diploid life cycle with both sexual and asexual reproduction. In the asexual phase, a spore of Saprolegnia releases zoospores. Within a few minutes, this zoospore will encyst, germinate and release another zoospore. This second zoospore has a longer cycle during which most dispersal happens; it will continue to encyst and release a new spore in a process called polyplanetism until it finds a suitable substrate. When a suitable medium is located, the hairs surrounding the spore will lock onto the substrate so that the sexual reproduction phase can start. Saprolegnia organisms are capable of causing infection; the most pathogenic species have tiny hooks at the end of their hairs to enhance their infectious ability. Once firmly attached, sexual reproduction begins with the production of male and female gametangium, antheridia and oogonium respectively. These unite and fuse together via fertilization tubes. The zygote produced is named an oospore. Saprolegnia is generally a secondary pathogen, though in the right circumstances, it can act as primary. Saprolegnia most frequently targets fish, both in the wild and in tank environments. Through necrosis of the skin, Saprolegnia will spread across the surface of its host as a cotton-like film. Though it often stays in the epidermal layers, the mould does not appear to be tissue specific. Saprolegnia infection is usually fatal, eventually causing hemodilution, though the time to death varies depending on the initial site of the infection, rate of growth and the ability of the organism to withstand the stress of the infection. Historicals: The first references to any of the Saprolegniaceae appear to have been those of Ledermüller (1760), of Wrisberg ( 1765 ) and of Spallanzani (1777). For a long time they were regarded as algae and were described by most of the 54  workers under the generic name Conferva, which included, in its Linnaean application, the filamentous aquatic plants generally. The earliest binomials appear to be those of Flora Danica (1780), Byssus aquatica , and of Schränk (1789), Conferva piscium . Heinrich August Wrisberg (1736–1808) Lazzaro Spallanzani 1729 – 1799), Franz von Paula Schrank (1747, 1835)  The earliest figures of these fungi are those of Flora Danica (1780), of Dillwyn (1809) and of Lyngbye (1819). 55 Byssus aquatica O.F.Muel. Flora Danica [G.C. Oeder et al], fasicle 15, t. 896 (1761-1883) 56 Franz von Paula Gruithuisen Daniel Nees von Esenbeck ,    Gruithuisen (1821) described a fungus on the remains of a dead snail and figured the escaping zoospores for the first time. Carus (1823) described a fungus on Salamander larvae with spores collected in the form of a globe at the mouth of a sporangium and called it Hydronema . Nees von Esenbeck (1823) separated the water moulds into the genera Saprolegnia (Gruithuisen’s fungus) and Achlya (Carus’s fungus) on the distinctive difference in the escape of the zoospores, which are recognized as their salient features even today. Friedrich Traugott Kützing ,     Carl Gustav Carus Nathanael Pringsheim Kützing (1843) created the family name Saprolegniaceae and described Saprolegnia ferax (Gruith.) Thuret (1850) described the warming spores that he called S. ferax Kütz. And he figured for the first time the oogonia. Pringsheim (1857) defined the family Saprolegniaceae and contributed to the literature by the description of S. monoica with excellent figures; other forms of Saprolegnia, those without antheridial branches, he considered as belonging to S. ferax. de Bary (1881) united S. monoica, S. torulosa, and S. Thureti (S. ferax) into a group which he called the ferax group. In his description of S. monoica, he 57                        mentioned that the antheridial branches arise from hyphae remote from the oo gonium, i.e. are not androgynous. de Bary (1881) distinguished S. torulosa by the presence of oogonia in chains , S. Thureti (S. ferax) by single large round oogonia, with almost none of whic h have antheridia, and S. monoica by the constant presence of androgynous ant heridia de Bary (1883) described S. mixta and S. hypogyna (Pringsh.) de Bary (1888), jn his last paper published after his death, included in the fera x group S. Thureti, S. hypogyna, S. monoica, S. mixta, S. torulosa, S. dioica and S. anisospora. He also described Saprolegnia monilifera Humphrey (1892) limited the "ferax" group to S.ferax, S.mixta, and S.monoic a Humphrey (1893) Saprolegnia diclina Anton de Bary Vincenzo Maurizio Ernst Gäumann Maurizio (1899) described Saprolegnia furcata and Saprolegnia crustosa Gäumann (1918) described Saprolegnia lapponica and Saprolegnia turfosa Coker (1923) described Saprolegnia delica, Saprolegnia litoralis, Saprolegnia megasperma and Saprolegnia parasitica Emoto (1923) described Saprolegnia tokugawana Apinis (1930) described Saprolegnia latvica A. Lund (1934) described Saprolegnia pseudocrustosa and Saprolegnia glomerata (Tiesenh.) J.V. Harv. (1942) described Saprolegnia bernardensis Ant.-Murg. (1947) described Saprolegnia exigua Johannes (1950) described Saprolegnia uliginosa R.F. Elliott (1968) described Saprolegnia australis Dissmann (1970) described Saprolegnia subterranea R.L. Seym. (1970) described Saprolegnia luxurians P.G. Richt. (1970) Saprolegnia richteri R.L. Seym. (1970) described Saprolegnia unispora and Saprolegnia eccentrica T.W. Johnson & R.L. Seym. (1975) Saprolegnia irregularis Hatai, Egusa & Awakura (1977) described Saprolegnia shikotsuensis Prasher (1998) described Saprolegnia chandigarhensis Willoughby (1998) described Saprolegnia polymorpha 58 Mortada M. A. Hussein Bernard Paul                Hussein & Hatai (1999) described Saprolegnia salmonis S. Inaba & Tokum. (2002) described Saprolegnia semihypogyna Steciow (2001) described Saprolegnia longicaulis Steciow (2002) described Saprolegnia milnae Steciow (2003) described Saprolegnia oliviae Gandhe & Kurne (2003) described Saprolegnia anomala B. Paul & Steciow (2004) described Saprolegnia multispora R.L Seym. (2005) described Saprolegnia truncata . Steciow (2007) described Saprolegnia bulbosa A. Lund 1934 described Saprolegnia glomerata (Tiesenh.) R. F. Elliott 1968 described Saprolegnia australis Shigeki Inaba · Seiji Tokumasu, 2002 described Saprolegnia semihypogyna X.L. Ke, J.G. Wang, Ze M. Gu, Ming Li & X.N. Gong (2009) described Saprolegnia brachydanis Sandoval-Sierra and Diéguez-Uribeondo, 2015 described Saprolegnia racemose and Saprolegnia aenigmatica SARAH CRISTINA OLIVEIRA ROCHA et al. 2016 described Saprolegnia milanezii sp. nov. Diéguez-Uribeondo Sarah Cristina Oliveira Rocha Life Cycle of Saprolegnia The members of saprolegniaceae typically exhibit the complete life cycle, i.e.  the presence of vegetative reproduction by production of gemmae, 59    asexual reproduction by formation of zoosporangia producing bifl agellate zoospores under favourable conditions and sexual reproduction by formation of male (antheridia) and female (oögonia) gametangia producing respective gametes under unfavourable conditions. general concepts regarding sexual reproduction in Saprolegniaceae o Particular inorganic salts in a growth medium favour the development of sexual apparatus. o A distinct correlation exists between the mass (weight) of the vegetative growth and abundance of male and female gametangial production. o Carbon-nitrogen ratio (C/N ratio) and certain combination of its sources stimulate production of male gametangia in excess production of female gametangia while others are antagonistic to the male gametangial production. o The variation in the concentration of constituents in a medium affects the structural changes in the sexual apparatus. o The production of female gametangia may not be stimulated by addition of inorganic salts to a nutrient-rich medium. o The sexual apparatus appears more frequently to develop on older hyphae than on younger hyphae. baladesnaturalistes.hautetfort.com Asexual reproduction    The organism produces specialized sporangia from which emerge zoospores with an anterior tinsel flagellum and a recurrent whiplash flagellum which may be produced in succession A primary zoospore with both flagella originates at the anterior end and a secondary zoospore with the two flagella originate laterally. Zoospores then germinate to produce a body with cellulose and glucan walls 60    The filaments (hyphae) are coarse, and non-septate. Nuclear division is closed, with a persistent nucleolus and an intranuclear spindle with poles near pairs of centrioles oriented at 180o to each other. In the vegetative state, they are diploid organisms with gametic meiosis. Saprolegnia zoosporangial release www.youtube.com commons.wikimedia.org Scanning electron micrograph (SEM) of primary zoospore of Saprolegnia parasitica isolate (TP41). Spore width = 5 mm. (A.W. Burr and G.W. Beakes, University of Newcastle, unpublished material.) Scanning electron micrograph (SEM) of secondary zoospore of isolate TP41. The mastigoneme hairs on the anterior flagellum appear as bobbles. (A.W. Burr and G.W. Beakes, unpublished material.) Cryofixed scanning electron micrograph (SEM) of a germinating encysted zoospore of Saprolegnia parasitica (A.W. Beakes, unpublished material). Scanning electron micrograph (SEM) of germinating cyst, showing the appearance of long boat-hook bundles. The individual spines cannot be resolved but bundles (hoops) are clearly resolved. (A.W. Burr and G.W. Beakes, unpublished material.) 61 Sexual reproduction    begins with the apposition of two hyphae that are cut off by septa at their tips and migration of nuclei through channels from one (differentiated as an antheridium) to the other (differentiated as an oogonium). After fertilization, the zygote develops into a thick-walled oospore (resting stages) The oospores germinate by the production of a diploid vegetative filament thick-walled oospore (resting stages) of Saprolegnia www.questionstudy.com Saprolegniosis (Saprolegniasis)            Saprolegniosis is a disease of freshwater and brackish water species caused by Saprolegnia Saprolegniosis typically is seen as cottony white, gray, brown, red, or greenish masses on skin or gills of freshwater or brackish fish. Initial affected areas may be small; however, the fungi can spread rapidly and cover most of the body. Occurrence may be acute, but duration is often chronic. Saprolegnia causes superficial disease with a mild inflammatory response . Lesions rarely go beyond superficial musculature. Generally, saprolegnia infections are associated with the integument and can cause destruction of the epidermis, thus depriving the fish of the protection of the mucus. Saprolegnia infections of fish are frequently associated with the wounds and lesions and also handling fish may predispose them to infection. Saprolegnia organisms are not tissue specific and are capable of attacking virtually any tissue. Saprolegniosis is mainly a secondary infection seen after damage to the fish integument. Water pollution and overcrowding like other predisposing factors were also included. Saprolegnia can act as a primary pathogen infecting fish that have not shown signs of previous damage. 62   This disease attacks are temperature-dependant (temperature ranging from 32° to 95°F but seem to prefer 59° to 86°F) usually occurring at low temperatures. Symptoms: o Infections take many shapes, symptoms include white fluff and fur on the body and any part of the eternal anatomy, to slimy patches and cloudy eyes. Fins will begin to recede in many cases, gills also , are extremely vulnerable o Fish fungus appears as gray or white patches on the skin/gills. o Fish fungus becomes brown/green (later stage) as they trap sediment. o Patches of skin in serious infections may fall way exposing reddish wounds and bare flesh. o Fungal infections can promote colour changes in most fish, from patches through to complete blanching of colour on the entire body. o Some fish live with peripheral infections all their lives while other can succumb within days, suffering all the effects of necrosis and septacaemia, as the fungus may aid the invasion of various bacterial strains into the body. o Flexibacter columnaris is often associated with fungal infections, and it may in some cases be prudent to treat the fish for both conditions. It is very difficult for the inexperienced fishkeeper to differentiate between saprolegnia and columnaris infections Predisposing factors include: o environmental stresses (temperature drops or poor water quality, handling or transport); o aggression; o sexual maturity and o reproductive readiness; o excessive use of chemotherapeutics such as formalin or potassium permanganate; and o bacterial, parasitic, or viral diseases. Saprolegniosis in fish eggs is a significant problem in the culture of many freshwater and brackish water species. o Fungi normally do not penetrate or infect normal, healthy fertilized eggs. o Infertile eggs are very susceptible to fungal infection, however, o fungi that begin their growth on infertile eggs may rapidly spread and smother adjacent fertile eggs, preventing adequate oxygenation and gas exchange. Diagnosis  Tentative identification of Saprolegnia and related species can be made by microscopic examination of wet mounts of infected tissues and observation of 63  branching, filamentous, aseptate (not separated by cross-walls, known as septa) fungal hyphae comprising the vegetative mass known as a mycelium. Definitive diagnosis requires culture, visualization, and identification of sexual or asexual reproductive stages. Treatment and control                  Treatment of infected populations can be difficult. Anatomic location of initial lesions and severity seem to determine likelihood for survival, as do ability to determine and eliminate predisposing factors after onset. Fish that are infected primarily on the distal areas of fins have a better chance than those that are infected on the main trunk. Less severely infected fish can recover if provided with good water quality. Increasing salinity to freshwater fish assists with osmoregulatory balance and appears to affect fungi to some degree. Most freshwater species of fish can tolerate 1 to 3 g/L of salt indefinitely; some species can tolerate even more. Unfortunately, as much as 10 to 30 g/L may be required to achieve significant inhibition and control. Once Saprolegnia has taken hold of a population, however, prognosis can be poor. Other chemotherapeutics have demonstrated limited efficacy when used as bath treatments for large populations. Malachite green is considered most effective agent, although its use in food fish is prohibited because of carcinogenicity and teratogenicity. Malachite green alone, or in conjunction with formalin, can be highly toxic to some fish species. Other agents with some promise include hydrogen peroxide [11] and formalin, labeled by the US Food and Drug Administration for use as an antifungal. For aquarium (non-food) fish, more aggressive therapy, including use of combination antibacterial/ antifungal ointments, can increase survival significantly if only one or a small number of fish are affected. Anesthesia, followed by daily application of ointment to the affected areas for approximately 30 seconds has proven effective, provided that predisposing factors have been eliminated or reduced. Increasing salinity to upper tolerance levels for the fish species also is recommended in these instances. For small-scale, aquarium fish operations or hobbyist-level production, control can include manual removal of infected eggs to reduce spread. For commercial operations, antifungal agents used for fish eggs are similar to those used for fish. For a short period after fertilization and before hatch, the chorion (outer layer) of the egg is protective and allows the egg to withstand higher concentrations than those used for fish. Formalin (1000 to 2000 ppm for 15 minutes or up to 1500 ppm for Acipenseriformes, FDA-CVM), Hydrogen peroxide (250 to 500 mg/L flush for channel catfish eggs, U.S. Food and Drug Administration-Center for Veterinary Medicine FDA-CVM), Iodophors including buffered iodine (100 ppm for 10 minutes, FDA-CVM), and high water flow [8] at a rate that allows rolling of eggs are methods frequently used to prevent infection. 64  Elimination of most, if not all, of the agent should occur shortly before hatch, through volatilization (formalin) or water changes. Classification of Saprolegnia Chromista, Oomycota, Oomycetes, Saprolegniales, Saprolegniaceae 1. CBS-KNAW Fungal Biodiversity Centre Saprolegnia Nees, Nova Acta Phys.-Med. Acad. Caes. Leop.-Carol. Nat. Cur.: 513 (1823) Associated records S. anisospora, S. asterophora, S. crustosa, S. delica, S. diclina, S. eccentrica, S.ferax, S. furcata, S. hypogyna, S. lapponica,S. litoralis, S. rma, S.mixta, S, monilifera, S. monoica, S. parasitica, S. subterranea, S. terrestris, S. turfosa, S. unispora, S. dioica, S. polymorpha, S. semihypogyna, S.torulosa, S. debaryi,S. schachtii, S. androgyna, S. paradoxa, S. floccosa, S. bhargavae, S.glomerata, S. bernardensis, S. shikotsuensis, S. terrestris, S. australis, S.molluscorum, S. chandigarhensis, S.invadans, S. salmonis, S. milnae, S. longicaulis, S. esocina, S. dioica, S. papillosa,S. hypogna, S. intermedia, S. heterandra, S. bodanica, S. rhaetica,S. semidioica, S. paradoxa, S.retorta, S. tokugawana, S. latvica, S. pseudocrustosa, S. uliginosa, S. exigua, S. irregularis, S. richteri, S. luxurians, S. blelhamensis, S. subeccentrica, S.divisa, S. elongata, Saprolegnia thureti, Saprolegnia variabilis, Saprolegnia kauffmanniana, Saprolegnia lactea, Saprolegnia capitulifera,Saprolegnia spiralis, S. siliquaeformis, Saprolegnia stagnalis, Saprolegnia philomukes, Saprolegnia declina,S.libertiae, Saprolegnia variabilis, Saprolegnia treleaseana, Saprolegnia bhargavii, S.invaderis,Saprolegnia anomala, Saprolegnia oliviae, Saprolegnia multispora, Saprolegnia itoana, Saprolegnia truncata,Saprolegnia curvata, Saprolegnia siliquiformis, S.thuretii, S. bulbosa, S. brachydanionis,Saprolegnia anomalies, Saprolegnia brachydanis, Saprolegnia candida, Saprolegnia minor, Saprolegnia saccata,Saprolegnia tenuis, Saprolegnia xylophila, Saprolegnia var. dioica 2. Species Fungorum Accepted Saprolegnis species 1. 2. 3. 4. 5. 6. 7. 8. 9. Saprolegnia anisospora de Bary (1888) Saprolegnia anomala Gandhe & Kurne (2003) Saprolegnia australis R.F. Elliott (1968) Saprolegnia bernardensis J.V. Harv. (1942) Saprolegnia bulbosa Steciow (2007) Saprolegnia chandigarhensis Prasher (1998) Saprolegnia crustosa Maurizio (1899) Saprolegnia delica Coker (1923) Saprolegnia diclina Humphrey (1893) 65 10. Saprolegnia eccentrica (Coker) R.L. Seym. (1970) 11. Saprolegnia exigua Ant.-Murg. (1947) 12. Saprolegnia ferax (Gruith.) Kütz. (1843) 13. Saprolegnia furcata Maurizio (1899) 14. Saprolegnia glomerata (Tiesenh.) A. Lund (1934) 15. Saprolegnia hypogyna (Pringsh.) de Bary (1883) 16. Saprolegnia irregularis T.W. Johnson & R.L. Seym. (1975) 17. Saprolegnia lapponica Gäum. (1918) 18. Saprolegnia latvica Apinis (1930) 19. Saprolegnia litoralis Coker (1923) 20. Saprolegnia longicaulis Steciow (2001) 21. Saprolegnia luxurians (Bhargava & G.C. Srivast.) R.L. Seym. (1970) 22. Saprolegnia megasperma Coker (1923) 23. Saprolegnia milnae Steciow (2002) 24. Saprolegnia mixta de Bary (1883) 25. Saprolegnia monilifera de Bary (1888) 26. Saprolegnia monoica Pringsh. (1858) 27. Saprolegnia multispora B. Paul & Steciow (2004) 28. Saprolegnia oliviae Steciow (2003) 29. Saprolegnia parasitica Coker (1923) 30. Saprolegnia polymorpha Willoughby (1998) 31. Saprolegnia pseudocrustosa A. Lund (1934) 32. Saprolegnia richteri P.G. Richt. ex R.L. Seym. (1970) 33. Saprolegnia salmonis Hussein & Hatai (1999) 34. Saprolegnia semihypogyna S. Inaba & Tokum. (2002) 35. Saprolegnia shikotsuensis Hatai, Egusa & Awakura (1977) 36. Saprolegnia subterranea Dissmann (1970) 37. Saprolegnia tokugawana Emoto (1923) 38. Saprolegnia truncata R.L. Seym. (2005) 39. Saprolegnia turfosa (Minden) Gäum. (1918) 40. Saprolegnia uliginosa Johannes (1950) 41. Saprolegnia unispora (Coker & Couch) R.L. Seym. (1970) 42. Saprolegnia variabilis Minden (1912) Species given other names 1. 2. 3. 4. 5. 6. 7. 8. Saprolegnia Saprolegnia Saprolegnia Saprolegnia Saprolegnia Saprolegnia Saprolegnia Saprolegnia androgyna W. Archer (1867), (= Achlya androgyna) asterophora de Bary (1860), (= Scoliolegnia asterophora) blelhamensis (M.W. Dick) Milko (1979), (= Scoliolegnia blelhamensis) crustosa var. crustosa Maurizio (1899), (= Saprolegnia crustosa) crustosa var. punctulata Cejp (1959), (= Saprolegnia crustosa) crustosa var. similis Cejp (1959), (= Saprolegnia crustosa) curvata Minden (1916), (= Isoachlya curvata) diclina var. diclina Humphrey (1893), (= Saprolegnia diclina) 9. Saprolegnia diclina var. minima Cejp (1959), (= Saprolegnia diclina) 10. Saprolegnia diclina var. numerosa Cejp (1959), (= Saprolegnia diclina); 66 11. Saprolegnia 12. Saprolegnia 13. Saprolegnia 14. Saprolegnia 15. Saprolegnia 16. Saprolegnia 17. Saprolegnia 18. Saprolegnia esocina Maurizio (1896), (= Saprolegnia ferax) ferax var. esocina (Maurizio) Cejp (1959), (= Saprolegnia ferax) ferax var. ferax Kütz. (1843), (= Saprolegnia ferax) ferax var. hypogyna Pringsh. (1874), (= Saprolegnia hypogyna) ferax var. lapponica (Gäum.) Cejp (1959), (= Saprolegnia lapponica) floccosa Maurizio (1899), (= Saprolegnia monoica) hypogyna var. coregoni Maurizio (1899), (= Saprolegnia hypogyna) hypogyna var. hypogyna (Pringsh.) de Bary (1883), (= Saprolegnia hypogyna) 19. Saprolegnia 20. Saprolegnia 21. Saprolegnia 22. Saprolegnia 23. Saprolegnia 24. Saprolegnia 25. Saprolegnia 26. Saprolegnia 27. Saprolegnia 28. Saprolegnia 29. Saprolegnia 30. Saprolegnia 31. Saprolegnia mixta var. asplundii Gäum. (1918), (= Saprolegnia mixta) mixta var. mixta de Bary (1883), (= Saprolegnia mixta) molluscorum Nees (1823), (= Saprolegnia ferax) monoica var. acidamica S. Suzuki (1961), (= Saprolegnia monoica) monoica var. floccosa (Maurizio) Cejp (1959), (= Saprolegniamonoica) monoica var. glomerata Tiesenh. (1912), (= Saprolegnia glomerata) monoica var. monoica Pringsh. (1858), (= Saprolegnia monoica) monoica var. ocellata Schkorb. (1923), (= Saprolegnia monoica) monoica var. tortipes Schkorb. (1923), (= Saprolegnia monoica) monoica var. turfosa Minden (1912), (= Saprolegnia turfosa) monoica var. vexans Pieters (1915), (= Saprolegnia monoica) papillosa (Humphrey) Apinis (1930), (= Achlya papillosa) parasitica var. kochharii Chaudhuri & Kochhar (1935), (= Saprolegnia parasitica) 32. Saprolegnia 33. Saprolegnia 34. Saprolegnia 35. Saprolegnia 36. Saprolegnia 37. Saprolegnia 38. Saprolegnia 39. Saprolegnia 40. Saprolegnia parasitica var. parasitica Coker (1923), (= Saprolegnia parasitica) rhaetica Maurizio (1894), (= Isoachlya rhaetica) siliquiformis Reinsch (1878), (= Gonapodya siliquiformis) spiralis Cornu (1872), (= Cladolegnia spiralis) stagnalis Tiesenh. (1912), (= Cladolegnia stagnalis) subeccentrica (M.W. Dick) Milko (1979), (= Scoliolegniasubeccentrica) torulosa de Bary (1883), (= Isoachlya torulosa) treleaseana Humphrey (1893), (= Achlya androgyna) variabilis var. charkoviensis Schkorb. (1923), (= Saprolegnia variabilis) 41. Saprolegnia variabilis var. variabilis Minden (1912), (= Saprolegnia variabilis)  3. EOL Saprolegnia classification Eukaryota + o Stramenopiles +  Oomycetes +  Saprolegniales +  Saprolegniaceae +  Saprolegnia 1. Saprolegnia anisospora 2. Saprolegnia anomalies 3. Saprolegnia asterophora 4. Saprolegnia australis 5. Saprolegnia bulbosa 6. Saprolegnia cf. ferax 7. Saprolegnia delica 67 8. Saprolegnia diclina + 9. Saprolegnia eccentrica 10. Saprolegnia ferax 11. Saprolegnia furcata 12. Saprolegnia hypogyna 13. Saprolegnia lapponica 14. Saprolegnia litoralis 15. Saprolegnia longicaulis 16. Saprolegnia megasperma 17. Saprolegnia mixta 18. Saprolegnia monilifera 19. Saprolegnia monoica 20. Saprolegnia multispora 21. Saprolegnia oliviae 22. Saprolegnia parasitica + 23. Saprolegnia polymorpha 24. Saprolegnia salmonis 25. Saprolegnia semihypogyna 26. Saprolegnia subterranea 27. Saprolegnia terrestris 28. Saprolegnia torulosa 29. Saprolegnia turfosa 30. Saprolegnia unispora 31. Unclassified Saprolegnia (130 species) Differentiation of Saprolegnis species Groupings of Saprolegnia species recognized by Johnson et al. 2002 based on predominant features [SAPgroups.doc] David E. Padgett 8 Sept 04 type species S. ferax (Gruith.) Thuret 68 69 70 Cultural characteristics 71 Morphological characteristics of Saprolegnia parasitica . Cotton like colony (A) and branched aseptic hyphae (B). Bar = 50 μ m Morphological characteristics of Saprolegnia. Species are listed in order of their appearance on the 18S phenogram (Fig. 8). Data Morphological characteristics of Saprolegnia. Species. Data from Coker (1923) and Seymour (1970). (Molina et al,. 1995) Ultrastructural characteristics and %GC of Saprolegnia (Molina et al,. 1995) 72 Molecular analysis of the genus Saprolegnia The lack of a robust taxonomy in the genus Saprolegnia (Oomycetes) is leading to the presence of incorrectly named isolates in culture collections and of an increasing number of misassigned named sequences in DNA databases. Accurate species delimitation is critical for most biological disciplines. A recently proposed approach to solve species delimitation (taxon diagnosis system) of difficult organisms is the definition of molecular operational taxonomic units (MOTUs) using molecular data. 73 In this study, we have analyzed 961 sequences of internal transcribed spacer from main culture collection of Saprolegniales (461 sequences) and GenBank (500 sequences). For this purpose, we have used two phylogenetic analyses, i.e, Maximum Parsimony and Bayesian inference, and also a clustering optimization analysis using arbitrary options regarding the distance threshold values and the clustering algorithm. Thus, we have identified 29 DNA-based MOTUs in agreement with phylogenetic analyses of species. The molecular clusters support the validity of 18 species of Saprolegnia and identify 11 potential new species. Based on this system, we have listed a number of incorrectly named isolates from culture collections, misassigned species names to GenBank sequences, and type sequences for species. We conclude that GenBank represents the main source of errors for identifying species since it possesses a high number of misassigned sequences, and the presence of sequences with sequencing errors. The presented taxonomic diagnosis system might help setting the basis for a suitable identification of species in this economically important genus (Sandoval-Sierra et al., 2014) 74 Schematic tree of Bayesian inference analysis based on ITS nrDNA sequences obtained from pure cultures of Saprolegnia species and related genera of culture collections (ATCC, CBS, and RJB-CSIC). The cluster numbers represent the Saprolegnia species based on MOTUs obtained from clustering optimization analysis. The numbers above the branches represent the pp (>0.90) values and bs support (> 75) based on Bayesian inference and MP analyses respectively and performed under Mafft alignment. The numbers include in brackets represent the pp (>0.95) values and bs support (> 75) for phylogenetic considerations. Species names below each cluster name indicate sequences obtained from pure culture of the culture collections CBS and ATCC with misassigned names, i.e., names that do not correspond to the MOTU, i.e., species, found with these analyses. Sandoval-Sierra et al. (2014) 75 Schematic tree of Bayesian inference analysis based on ITS nrDNA sequences obtained from pure cultures of Saprolegnia species and related genera of culture collections (ATCC, CBS, and RJB-CSIC) 76 and combined with GenBank sequences. The cluster numbers represent the Saprolegnia species based on MOTUs obtained from clustering optimization analysis. The numbers above the branches represent the pp (>0.90) values and bs support > 75) based on Bayesian inference and MP Sandoval-Sierra et al. (2014) Description of Saprolegnis species 1. Saprolegnia aenigmatica, Sandoval-Sierra and Diéguez-Uribeondo, 2015 Hyphae of the vegetative mycelium are slender, delicate, aseptate, smooth, moderately branched. Gemmae, when present, generally branched, simple, catenulate; spherical, pyriform, irregular; often terminal. Sporangia clavate, rarely filiform; 164.3±59.0 × 21.4±3.4 μm (overall range 59.3–340.6 × 12.2–32.9 μm); mostly terminal, renewal usually by internal proliferation, with an apical papilla before zoospore discharge. Zoospore discharge saprolegnoid. Primary zoospores pyriform and apical biflagellate. Primary cysts sphaerical, 12.0±0.8 μm. (overall range 10.4– 15.4 μm) in diameter. Secondary zoospores reniform and lateral biflagellate. Secondary cysts morphologically identical to primary cysts. Oogonia abundant when are present, globose, subglobose, pyriform and sometimes forming short moniliform chains, 88.6±29.6 × 63.4±15.7 μm (overall range 44.0–219.0 × 32.3–112.3 μm), the length/breadth ratio averaged 1.4±0.3 μm (overall range 1.0–2.9 μm). Oogonial wall unpitted, except under points of attachment of antheridial cells. Oogonia axillary, terminal, and intercalary. Oospores mostly filling the oogonium, but in some case do not filling the oogonium, globose to subglobose, 1–33 (9.0±6.2) in number; 25.6±2.3 μm (overall range 17.3–34.2 μm) in diameter. The internal structures of alive oospores are centric. Antheridial hyphae well developed, persistent, highly branched, of variable origin, always diclinous. Antheridia numerous, often encircling the oogonia, tubular to ampullaceous, sometimes branched, attached by foot-like projections. 77 Specific morphological features of asexual and sexual structures of Saprolegnia aenigmatica sp. nov.Light (a, b, c, d, e, f, g, and h) and electronic micrographs (i, j, and k) of: (a) sporangia clavate, terminal, with internal proliferation (arrows) and apical papilla (arrowhead) (RJBCC0020); (b) oogonia pyriform, diclinous antheridial hyphae (arrows) well developed, branched, ampullaceous (arrowhead), and centric oospores (RJBCC0028); (c) oogonial intercalary, antheridial hyphae ampullaceous (arrowhead) and centric oospores (RJBCC0038); (d) oogonia subglobose, diclinous antheridial hyphae (arrows) well developed and from variable origin, tubular (arrowhead), and centric oospores (RJBCC0039); (e) oogonia pyriform, diclinous antheridial hyphae (arrows) from variable origin, encircling the oogonia, and centric oospores that do not fill the oogonium (RJBCC0024); (f) immature oogonia moniliform, and diclinous antheridial hyphae (arrows) from variable origin (RJBCC0027); (g) oogonia moniliform, and diclinous antheridial hyphae (arrows) from variable origin, encircling the oogonia (RJBCC0026); (h) oogonia pyriform, diclinous antheridial hyphae (arrows) well developed, attached by foot-like projections (arrowhead), and centric oospores (RJBCC0039); (i) oogonia pyriform, antheridial hyphae (arrows) well developed, encircling the oogonia, and antheridial attached (arrowhead) (RJBCC0024); (j) oogonial intercalary, antheridial hyphae attached (arrowhead) (RJBCC0028); (k) antheridial hyphae (arrows) well developed, encircling the oogonia, and antheridial attached (arrowhead) (RJBCC0028). Bar = 20 μm. Sandoval- Sierra,J. V., J. Diéguez-Uribeondo, 2015 2. Saprolegnia australis R. F. Elliott 1968 78 Mycelium dense, diffuse; hyphae slender or stout. Sporangia cylindrical; renewed internally, primary ones 250 × 32 µm; secondary ones usually shorter, but up to 600 µm long. Spores dimorphic; discharge and behavior generally saprolegnoid; primary spore cysts 11.1µm in diameter. Gemmae abundant; clavate; terminal or intercalary, usually single. Oogonia generally terminal, lateral or intercalary, when intercalary 5980 µm in diameter. Oogonial wall pitted, smooth. Oogonial stalks in length; straight, curved, twisted, or irregular; unbranched. Oospores may or may not mature, or may abort; when mature, subcentric; spherical to subspherical; 4-12 per oogonium, but usually not filling it; 22-27 µm in diameter; germination not observed. Antheridial branches, predominantly diclinous, monoclinous or androgynous. Antheridial cells simple or branched, persisting; tubular or attached in a digitated fashion; fertilization tubes present or absent, not persisting. Remarks. S. australis is a monoecious species and can be distinguished by its subcentric oospores (types I and III) with the refractive droplets surrounding the plasma or only partially so. The oospheres may or may not mature, or the oosphere may develop and then abort. S. australis and S. diclina have pitted and obpyriform oogonial wall, but in S. australis are primarily terminal and in S. diclina are these are predominantly lateral. S. australisis is distributed in Canada, Japan, New Zealand and USA (Johnson et al., 2002); this species is cited for the first time in Mexico. A), Saprolegnia australis . 1. Obpyriform oogonium; oospores subcentric; antheridia lacking. 2. Obpyriform oogonium and clasping/wrapping diclinous antheridial branch (Johnson et al., 2002). B), S. australis . Lateral spherical oogonium with more than 6 oospores subcentric, diclinous antheridial branch. Barr= 20 μm Vega-Ramírez et al. (2013) 3. Saprolegnia brachydanis X.L. Ke, J.G. Wang, Ze M. Gu, Ming Li & X.N. Gong (2009) The distinctive characteristics of S. brachydanis are the production of glomerulate oogonia wrapped around by predominantly monoclinous antheridia which can be up to eight in one oogonium. The oogonial stalks are short, straight, or curved and the antheridia, twisted, can enwind one or more oogonia. The oospores cannot mature or easily abort. Morphological features of the oomycete and the ITS sequence of its rDNA as well as the comparison with related species are discussed in this article. 79 Left: Saprolegnia brachydanis asexual reproduction. (a) Hyphae emerging out of hemp seeds in water culture, (b) matured zoosporangia, (c) sporangial renewal by internal proliferation, (d) saprolegnoid discharge of zoospores, (e) emptied zoosporangia, (f) primary and secondary zoospores, (g) encysted and germinating zoospores. a, c, d, e Scale bar = 50 μm; b bar = 100 μm; and f, g bar = 10 μm Right: Saprolegnia brachydanis sexual reproduction. (a) Portion of hyphae bearing intercalary or terminal oogonia with attached monoclinous or diclinous antheridial hyphae ((i), (ii) lateral oogonia with diclinous antheridia (black Arrowheads); (iii) lateral oogonia with monoclinous antheridia; (iv) intercalary oogonia with monoclinous antheridia (white arrowheads)), (b) mature zoospore and lateral oogonia, (c) terminal oogonia with twist stalk, (d) intercalary oogonia wrapped around with two monoclinous antheridia (white arrowheads point to monoclinous antheridia). a, b, c, d Scale bar = 50 μm Ke et al. (2009) Schematic drawing of oogonia with monoclinous antheridia in S. brachydanis. (a) Lateral oogonia with eight monoclinous antheridia, (b) intercalary oogonia with one monoclinous antheridium. Scale bar = 50 μm Ke et al. (2009) 4.Saprolegnia bulbosa, Steciow MM, Paul A, Bala K. (2007) 80 The oogonial stalks are usually bent, curved or once coiled; oospores are subcentric, (1) 2–15 (45) per oogonium and are variable in size. Taxonomical description of this new species, its comparison with related oomycetes of the genus and the nucleotide sequences of the internal transcribed region (spacers ITS1, ITS2 and the gene 5.8S) of its rRNA gene are given. Left: (a–h) Saprolegnia bulbosa. (a) Details of a naviculate zoosporangium containing zoospores. (b–d) Detail of zoospores' discharge; zoosporangium renewal by internal proliferation leads to the development of a new one inside. (e–h) Immature oogonia with curved or once coiled oogonial stalk and some of them with a characteristic bulbous oogonial stalk and antheridial branches. Scale bar: a–e, h=66.6 μm; f– g=210 μm. Right: (a–l) Saprolegnia bulbosa. (a, b, g) Characteristic monoclinous antheridial branches on immature oogonia. (c–d) Diclinous antheridial branches on mature oogonia. (e) Androgynous antheridial branch arising from a bulbous oogonial stalk. (f, h) Oogonia with smooth oogonial wall and antheridial cell laterally appressed. (i) Aspect of mycelium with numerous Steciow et al. (2007) 5.Saprolegnia diclina Humphrey 1892 Mycelium sparingly to moderately branched. Sporangia cylindrical, clavate, fusiform; straight or slightly curved; renewed internally or basipetalous succession; 75-1050 × 20-80 µm. Spores dimorphic; discharge and behavior saprolegnoid; encysted spores 9-12 µm in diameter. Gemmae, when present, pyriform, cylindrical, clavate, or irregular; terminal or intercalary, single or catenulate. Oogonia sparse to moderately abundant, and often appearing in culture only after prolonged incubation; terminal, lateral, or intercalary, single or catenulate; spherical, or subspherical when intercalary; 81 spherical ones (30-) 50-70 (-130) µm in diameter, subspherical or obpyriform ones 54-146 × 18-72 µm. Oogonial wall unpitted, pitted, or with pits only under the region of attachment of antheridial cells; pits sometimes inconspicuous; smooth. Oospores centric or subcentric, both types occurring in some oogonia; spherical (1-) 8-16 (>100) per oogonium, and may or may not fill it; (12-)18-26 (-44) µm in diameter; at germination forming a slender, irregular germ tube terminating in a small, cylindrical or clavate sporangium. Antheridial branches diclinous. Antheridial cells simple, very rarely compound; fertilization tubes, when present, persisting or deliquescing. Remarks. S. diclina is a monoecious species and can be distinguished by the predominance of diclinous antheridial branches, these are often very abundant and may indeed enclose the oogonia partially or fully. LikeS. ferax, S. diclina produces both centric and subcentric oospores, and the 2 types may occur in the same oogonia. Milanez (cited by Johnson et al., 2002) observed subcentric oospores in some oogonia of his specimens of Humphrey's species. Willoughby et al. (1984) recognized 3 types of Saprolegnia diclina (parasitic forms from salmonids and perch, and strictly saprophytic ones) based upon the ratio of oogonium length to diameter. S. diclina is distributed in Belgium, British Isles, Canada, Czechoslovakia, Denmark, France, Finland, Germany, Iceland, India, Iraq, Japan, Latvia, Middle Europe, Nepal, Poland, Portugal, Republic of China, Rumania, South America, Switzerland, USA and West Indies (Johnson et al., 2002); this species is cited for the first time in Mexico. (a–f) Characteristic spore morphology of Saprolegnia diclina. (a) Light micrograph of an asexual sporangium; (b) phase contrast light micrograph of a secondary cyst with no visible hairs on its surface; (c) oogonium, being fecundated by the antheridia; (d) fecundation by tubular diclinous antheridium (antheridium and oogonium in different hyphae); (e) unpitted wall of the oogonium; (f) spherical centric oospores. Fernández-Benéitez et al., 2008 82 6.Saprolegnia ferax (Gruith.) Kütz 1843 Mycelium stout, hyphae moderately to sparingly branched. Sporangia cylindrical, or slightly irregular, sometimes nearly spherical; renewed internally with secondary ones nesting inside discharged primary ones, or partially emerged through orifices of empty sporangia and forming bead-like chains or cylindrical segments, or emerging fully through orifices of previously emptied sporangia; rarely renewed in a basipetalous or cymose manner; 31-624 × 18-67 µm. Spores dimorphic; discharge and behavior saprolegnoid; primary spore cysts 9-12 µm in diameter. Gemmae variable in shape and position. Oogonia lateral, terminal, or intercalary, infrequently occurring in emptied sporangia or sessile; (28-) 60-80 (-194) µm in diameter. Oogonial wall generally conspicuously and abundantly pitted, rarely unpitted; smooth or rarely with 1 or 2 short, papilliform evaginations, or apiculate. Oospores centric or subcentric, spherical or ellipsoidal; (1-) 10-18 (-54) per oogonium and nearly filling it, (12-) 2228 (-44) µm in diameter; germinating by a slender germ hypha that may or may not bear a small, apical, clavate sporangium. Antheridial branches sometimes absent; when present, monoclinous or androgynous, rarely diclinous; slender, may vary slightly to prominently irregular, unbranched or very sparingly branched; persisting. Antheridial cells simple; generally tubular or clavate, occasionally irregular, infrequently once-branched; usually persisting; laterally oppressed, very rarely attached apically; fertilization tubes present, not persisting. Remarks. S. ferax is a monoecious species and can be distinguished by its oospores centric or subcentric, spherical or ellipsoidal; 10-18 per oogonium and nearly filling it, 22-28 µm in diameter and gemmae variable in shape and position. Generally, it is most easily recognized by reliance on a combination of predominating characters: large, conspicuously or sparsely pitted oogonia, centric and subcentric oospores (sometimes in the same oogonium), occasional development of oogonia in discharged sporangia, and a preponderance of androgynous or monoclinous antheridial branches (when these filaments are present at all). Saprolegnia feraxalso is associated with ulcerative dermal necrosis. S. ferax is distributed in Asia, Australia, Austria, Belgium, British Isles, Canada, Czechoslovakia, Denmark, France, Germany, India, Iraq, Japan, Lapland, Latvia, Middle Europe, Nepal, Netherlands, Poland, Republic of China, Romania, South America, Switzerland, USA and USRR (Johnson et al., 2002). This species is cited for the first time in the State of México. 83 Saprolegnia ferax . Obpyriform, pitted oogonium, centric or slightly oospores subcentric (Johnson et al., 2002). B), S. ferax. Obpyriform oogonium, oospores centric. Barr= 20 μm . Vega-Ramírez et al. (2013) Oogonia of Saprolegnia ferax (100x) Shahbazian et al., 2010 7.Saprolegnia glomerata (Tiesenh.) A. Lund 1934 Mycelium delicate; some principal hyphae stout and provided with numerous short, scattered or clustered lateral, often twig-like branches. Sporangia abundant or sparse; cylindrical, fusiform, clavate, or irregular; renewed internally; 40-220 × 18-28 µm. Spores dimorphic; discharge and behavior saprolegnoid, rarely aplanoid; primary spore cysts 10-14 µm in diameter. Gemmae very sparse; clavate or obpyriform; terminal, single. Oogonia spherical, obpyriform, napiform, or subspherical; lateral, occasionally terminal, rarely intercalary; (32-) 46-60 (-107) µm in diameter. Oogonial wall pitted or unpitted; smooth; stout; straight, curved, or bent; unbranched or with 1 or more short, lateral branches. Oospores centric; spherical, often nearly filling the oogonium; (1-) 6-16 (-28) per oogonium; (18-) 23-26 (-30) µm in diameter; at germination producing a germ hypha. Antheridial branches androgynous or monoclinous; slender, usually contorted, twisted, or irregular and sparingly branched; persisting. Antheridial cells simple, generally tubular, occasionally clavate; fertilization tubes present, not persisting. S. glomerata is readily recognizable by the short, branched or unbranched, contorted, lateral (and often clustered) hyphal extensions. Remarks. S. glomerata is a monoecious species and can be distinguished by its centric oospores; spherical, often nearly filling the oogonium, 6-16 per oogonium; 23-26 µm in diameter; at germination producing a germ hypha and gemmae variable very sparse; clavate or obpyriform; terminal, single. Saprolegnia glomerata is readily recognizable by the short, branched or unbranched, contorted, lateral (and often clustered) hyphal extensions. Secondary characters of recognition are the contorted, 84 branched antheridial filamentsand the short, lateral evaginations on many of the oogonial stalks. In S. litoralis, the hypha immediately below a terminal oogonium, may bear short, lateral branches as does S. glomerata, but in the former, the hypha is usually swollen at its juncture with the oogonial septum. In any case, the oospores in S. litoralis are occasionally subcentric, a condition not known to occur in S. glomerata. S. glomeratais distributed in British Isles, Czechoslovakia, Denmark, Germany, Iceland, India, Japan, Latvia, Poland, Switzerland and USA (Johnson et al., 2002); this species is cited for the first time in Mexico. S. glomerata . Mature oogonia; short, contorted, branched, or peg-like hyphal elements; oospores centric (Johnson et al., 2002). D), S. glomerata . Terminal oogonium, antheridial diclinous branch. Barr= 20 μm. OC: oospores centric; OS: oospores subcentric; AC: antheridial cells; PI: pitted. Vega- Ramírez et al. (2013) 8. Saprolegnia hypogyna, (Pringsh.) de Bary (1883) This species, which has been studied by Pringsheim and DeBary differs from all other known Saprolegniacece in producing antheridia without special antheridial branches. A second portion of the oogonial branch is cut off just below the oogonium and consti tutes the antheridium. Its upper wall, the basal wall of the oogonium, grows up, as a fe rtilization tube, into the cavity of the latter. The oogonia show, in their form, pitting of their walls, and in the structure and number of their oospores, near relations with the f erax group, but the species is at once recognizable by the peculiarities above mentione d. 85 86 www.researchgate.net737 × 1235Search by image, Microscopic observations of stonefly nymph, Saprolegnia infected insects, salmon eggs and frog embryos. A) Mycelia of Saprolegnia hypogyna 9.Saprolegnia longicaulis, STECIOW, M. M. (2001) Saprolegnia longicaulis differs from S. australis in having Very long Oogonial Stalks, mature ooospores, and in lacking androgynous antheridial branches; the ZoosPorangia are Very much longer; the oogonia and the oospores are larger. Saprolegnia longicaulis. A-D, Mycelium with immature and some mature oogonia showing distinctive long oogonial stalks, and a few oospores; E-F, immature oogonia with diclinous antheridial branches and stout oogonial stalk tapering toward the end. Scale bar =100 pm. STECIOW, M. M. (2001) 87 Fig. 2 Saprolegnia longicaulis. A-B, Mycelium with gemmae and abundant oogonia with the smooth wall; C-F, Detail of zoosporangia filiform, cylindrical and clavate, renewal by internal proliferation, developa new one or a gemmae, inside; G-H, mature oogonia showing, in detail, the subcentric oospores, type I. Scale bar: A-G = 100 pm; H = 50 pm. STECIOW, M. M. (2001) 10.Saprolegnia milanezii, Sarah Cristina Oliveira Rocha, Jose V. SandovalSierra, Javier Diéguez-Uribeondo, Danilo R. Gonçalves, Gustavo H. Jerônimo, Ana L. De Jesus, Agostina V. Marano, Carmen L. A. Pires-Zottarelli, 2016 88 Saprolegnia milanezii sp. nov. was described as a new species of the genus Saprolegnia (Saprolegniales, Oomycota). It has been collected from freshwater samples at “Parque Estadual da Ilha do Cardoso”, Cananéia, São Paulo State, Brazil. This species is characterized by rare antheridia, extended stalk near the oogonial septum and subcentric oospores. Phylogenetic analysis using concatenated ITS and LSU rDNA regions confirmed the position of this new species into the genus Saprolegnia as sister group of S. furcata. 11.Saprolegnia milnae Steciow, 2002 Mycelium extensive, denser near substratum, two week old hemp seed colony, 2-4.5 cm diam.; principal hyphae stout, sparingly branched, 18—70 μm diam. at the base, with secondary branches, short, irregular and contorted. Gemmae scanty, spherical, pyriform or irregular, simple or catenulate. Zoosporangia cylindrical, filiform, clavate or irregular; 127-582 X 19-46 μm; short or long, often with one or two papillae, usually terminal, renewal usually by internal proliferation or proliferating sympodially. Zoospore discharge saprolegnoid. Encysted spores globose, 10-15 μm diam. Oogonia very abundant, terminal or lateral, rarely intercalary; pyriform, spherical or obovate, infrequently doliform orapiculate; (33)51-98(145) μm; immature oogonia frequently proliferating. Oogonial wall smooth, very rarely with one papilla; pitted, or pitted only under point of attachment of antheridial cell. Oogonial stalks frequently stout and variable in length, tapering toward the end; sinuous, bent, curved or once coiled, very rarely slender and straight; 100-900 μm long. Oospheres maturing. Oospores subcentric, type I, filling the oogonium; spherical or ellipsoid; (1)3-23(40) in number; (14)18-30(60) μm diam. Antheridia present, or very rarely absent. Antheridial branches principally androgynous (44%± 2) and diclinous (35% ± 10), occasionally monoclinous (21% ± 5), branched. Antheridial cells simple or branched; attached by projections or laterally appressed. Fertilisation tube not observed. NOTES: It is important to note that the type of oospore, antheridial branches, and the measurements of oogonia are very constant features of this species. There was little variation in type and size of zoosporangia in different temperatures; they are cylindrical, filiform, clavate or irregular, and reached a mean range in length of 127582 μm. The shape of oogonia remained constant, mainly pyriform, spherical or doliform, sometimes apiculate (smooth, rarely with one papilla). 89 Saprolegnia milnae. 1 Mycelium with immature and some mature oogonia with oospores. 2-5 Mycelium withdistinctive secondary hyphae that are profusely branched, contorted and twisted. 6-7 Zoosporangia.8 Intercalary oogonium with one papilla. 9 Detail of diclinous and monoclinous antheridial branches on immature oogonium. 10 Oogonium with an androgynous antheridial branch and oospores of different sizes.Scale bars: Fig. 1-2 = 100 μm; Fig. 3-10 = 50 μm. 90 Saprolegnia milnae 11-12 Detail of once coiled and bent oogonial stalk with androgynous antheridial branches. 13 Sinuous oogonial stalk with distinctive contorted androgynous antheridial branches. 14 Lateral oogonium with a long once-coiled stalk. 15 Terminal oogonium with a long bent stalk. 16 Aspect of subcentric oospores within a smooth and pyriform oogonium. 17-18 Two oogonia on curved oogonial stalk. Scale bars = 50 μm. 91 12. Saprolegnia mixta, de Bary, 1883 Hyphse rather slender, not long. Zoosporangia cylindric-clavate. Oogonia terminal o r rarely intercalary, on main filaments or lateral branches, globular, with numerous pi ts of varying size, but often pretty large. Antheridia cylindrical, rather shorter and sma ller than in S. monoica, of androgynous or diclinous origin, absent from a part of the o ogonia, sometimes from a large part. Oospores up to fifteen or occasionally more than twice that number, centric, their average diameter Saprolegnia mixta www.dipbot.unict.it345 × 485Search by im 13. Saprolegnia monilifera DeBary (1888). DeBary separated this species as a distinct type from the other members of the genus, although, judging from his description, it would seem to represent a further developm ent on the lines of 8. torulosa. Its oogonia are formed in somewhat more definite moni liform chains, all of whose members appear to have the same fate. Their walls are occ asionally pitted, and no antheridia are developed. A striking feature is the separation o f the oogonia from the plant and from each other, often at a very early stage, so that th ey lie free in the water and complete their development independently. The species pr esents a transitional feature leading towards Aclilya, or a reversional feature towards t he commonest condition in the family, in that many of the later sporangia are produce d by cymose branching, instead of by the usual method for Saprolegnia. 92 14. Saprolegnia monoioa Pringsh. (1857). Hyphse rather stout, often long. Zoosporangia cylindric-clavate. Oogonia terminal or r arely intercalary, usually on short lateral branches, globular, their walls abundantly an d prominently marked with large pits. Antheridia long-cylindrical, uniting with every oogonium, on rather stout branches of androgynous origin, which usually arise from t he main hypha near the oogonial branch. Oospores commonly not above ten, rarely nu merous, centric. 93 15. Saprolegnia multispora, Paul and Steciow, 2004 S. multispora refers to the presence of one to many (up to hundred) subcentric oospores inside the oogonia of this new species. The oomycete grows luxuriantly on hemp-seed halves in water and on CMA. In water, the mycelium is extensive, denser near substratum, and a two week-old colony on hemp seed measures 2–4.0 cm in diameter. The main hyphae are stout, sparingly branched, and measure 24–63 lm diameter at the base. Asexual reproduction is abundant. Zoosporangia are often fusiform, filiform, clavate or rarely naviculate, measuring 121–485 X 19–50 lm; straight or bent, usually terminal. Zoospore discharge is typically saprolegnoid. Encysted zoospores are globose and measure 9–12 lm diameter. Sporangial renewal is usually by internal proliferation. Gemmae are also formed plentifully in water cultures. These are spherical, pyriform, clavate or irregular, simple or catenulate, and function as zoosporangia, with one–several papillae of discharge. Sexual reproduction is oogamous by gametangial copulation. Both male and female gametangia are formed within one week of culture in water on the hemp-seeds. Oogonia are abundant, terminal, lateral or frequently intercalary; mainly pyriform, but also spherical, obovate, cylindrical, rarely naviculate or rarely irregular, doliform when intercalary, and measure 60–111lm in diameter. Morphologically, S. multispora has close affinities with S. diclina Humphrey and S. australis Elliott in having mainly diclinous antheridial branches, which are at times monoclinous or androgynous and subcentric oospores. However, the oospores in S. diclina are predominantly centric. The number of oospores per oogonium are higher in S. multispora 11–70 instead of only 8–12 in the case of S. diclina and 6–12 in S. australis. Moreover the oospores of S. australis may or may not mature, or theoospores may develop and then abort. The subcentric oospores of S. multispora also relates it to S. parasitica Coker. However there are many differences between these two species: the conspicuous pitted oogonial wall, origin of antheridial branches, and the presence of up to 100 oospores in S. multispora separates it from S. parasitica, which has unpitted oogonial wall, diclinous antheridial branches, and lesser number of oospores 14–23. 94 Saprolegnia multispora asexual reproduction. (a) Stout hyphae emerging out of hemp seeds in water culture, (b) mature and emptied zoosporangia, (c)–(d) formation and saprolegnoid discharge of zoospores, (e) encysted and germinating zoospores, (f) sporangial renewal by internal proliferation, (g) gemmae, (h) zoospore formation within gemmae. a,b,e,g: Scale bar = 100 μm and c,d,f,h: bar = 50 μm. Saprolegnia multispora sexual reproduction. (a) Terminal oogonia with diclinous antheridia, (b) intercalary oogonia, (c) intercalary oogonia with diclinous antheridia, (d) terminal oogonia with monoclinous antheridia, (e)–(h) oogonia wrapped around with diclinous antheridia, containing multiple oospores. (a) scale bar = 100 μm, (b)–(h) bar = 50 μm. 16. Saprolegnia oliviae M. M. Steciow (2006) Distinguishing characteristics of S. oliviae are the production of smooth oogonia (with some lateral or terminal projections) and the absence of antheridial branches on the majority of the oogonia, but when present, they are mostly diclinous, at times oogonia are supplied with androgynous and monoclinous antheridial branches. The oogonial stalks are predominantly short and straight or long and bent, curved or many times coiled; oospores are distinctive subcentric, (1–) 15–50 (–70) per oogonium. Morphological details of the new species and its comparison with other described species are discussed here. 95 1–7: S. oliviae. 1–2: Aspect of mycelium with oogonia and gemmae in water culture. 3: Gemmae. 4: Zoosporangium containing zoospores. 5: Discharged zoosporangium. 6: Zoosporangium renewal by internal proliferation. 7: Naviculate discharged zoosporangium. Scale bars: 1 = 50 µm; 2,3,5 = 100 µm; 4,6,7 = 50 µm.Th 8–15: S. oliviae. 8,9: Coiled oogonial stalk. 10: Intercalary oogonium. 11,12: Single and catenulate oogonia of variable forms lacking antheridial branches but having many oospores, one of them with lateral projection. 13: Papillate oogonium with apical projection. 14: Long oogonial stalk. 15: Oogonium with lateral projection. Scale bars: 8,9 = 100 µm; 10–15 = 50 µm. M. M. Steciow (2006) 16–23: S. oliviae. 16: Naviculate pitted oogonium with subcentric oospores 17–19: Oogonia with apical projection containing oospores. 20: Branched oogonial stalk with spherical oogonia. 21,22: Pitted spherical oospores and pyriform oogonia with subcentric oospores. 23: Obovate oogonium. Scale bars: 50 µm. 24–31: S. oliviae. 24,25: Characteristic intercalary and filiform oogonia 26,27: Diclinous antheridial branches. 28,29: Androgynous antheridial branches. 30,31: Monoclinous antheridial branches. Scale bars: 24,26,28–31 = 100 µm; 25,27 = 50 µm. M. M. Steciow (2006) 96 17. Saprolegnia parasitica Coker 1923. Gemmae abundant, size and shape very variable; often in chains, mostly terminating hyphae, but sometimes intercalary. Sporangia variable, but usually bent and irregular. At times up to 0.7 mm, long, very often proliferating from the side below as in Achlya; when growing through others sometimes discharging spores through the side wall of the old sporangium; spores dimorphic; discharge and behavior saprolegnoid, 9-11.5 µm. Sexual reproduction not observed so far in our strains and very rarely observed in others. Our isolation developed as a parasite on fish or in water, rarely on trout eggs. Remarks. S. parasitica can be distinguished by gemmae abundant, size and shape very variable; often in chains, mostly terminating hyphae; but sometimes intercalary. Sporangia usually bent and irregular, at times up to 700 µm long with rounded ends, containing the zoospores (9-11.5 µm). The isolates obtained from lesions of live rainbow trout with characteristic bundles of hairs and retracted germination pattern. Sexual reproduction not observed. S. parasitica is distributed in Japan, United Kingdom, Brazil, Netherlands and Russian Federation (Global Biodiversity Information Facility, 2012); this species is cited for the first time in Mexico. Saprolegnia parasitica . 1, mature zoosporangia; 2, release mobile zoospores; 3, formation of secondary zoosporangia; 4, primary zoospore; 5, primary cyst. 6, germinated cyst; 7, secondary zoospore; 8, secondary cyst with “boat hooks”; 9, germinated cyst; 10, hyphal growth. B), S. parasitica . B1, formation of secondary sporangia. B2, mature sporangia. B3, formation of sporangia. Barr= 75 μm. B4, catenulate gemmae. Vega-Ramírez et al. (2013) 18. Saprolegnia racemose, Uribeondo, 2015 Sandoval-Sierra and Diéguez- Hyphae of the vegetative mycelium are slender, delicate, aseptate, smooth, moderately branched. Gemmae, when are present, generally branched, catenulate when are intercalary and clavate when are terminal. Sporangia clavate, rarely filiform; 129.9±48.3 × 19.3±3.4 μm. (overall range 58.7–326.5 × 11.1–30.1 μm); mostly terminal, some times apical, renewal usually by internal proliferation, with an apical papilla before zoospore discharge. Zoospore discharge saprolegnoid. Primary 97 zoospores pyriform and apical biflagellate. Primary cysts sphaerical, 12.1±0.5 μm (overall range 11.0–13–9 μm) in diameter. Secondary zoospores reniform and lateral biflagellate. Secondary cysts morphologically identical to primary cysts. Oogonia abundant, often grouped in racemose structures. Oogonia abundant, globose, rarely obovoid, 73.0±15.9 × 71.0±15.4 μm (overall range 32.5–119.0 × 27.5–118.5 μm), the length/breadth ratio averaged 1.0±0.1 μm (overall range 0.9–1.3 μm). Oogonial wall unpitted, except under points of attachment of antheridial cells. Oogonial stalks often branched to form a group, sometimes axillary and occasionally intercalary. Oospores mostly filling the oogonium, globose to subglobose, 1–30 (6.4±4.7) in number; 31.7±4.7 μm (overall range 21.3–51.3 μm) in diameter. The internal structures of alive oospores are centric. Antheridial hyphae well developed, persistent, of variable origin, mainly diclinous, sporadically monoclinous. Antheridia numerous, often circling the oogonia, tubular to ampullaceous, attached by foot-like projections. Specific morphological features of asexual and sexual structures of Saprolegnia racemosa sp. nov. Light (a, b, c, d, e and g) and electronic micrographs (f and h) of: (a) sporangia clavate, terminal, with internal proliferation (arrows) and apical papilla (arrowhead) (RJBCC0003); (b) sporangia apical and apical papilla (arrowhead) (RJBCC0011); (c) oogonia globose, monoclinous antheridial hyphae (arrows) well developed and from variable origin, ampullaceous (arrowhead), and centric oospores (RJBCC0003); (d) oogonia globose, diclinous antheridial hyphae (arrows) well developed, circling the oogonia, tubular (arrowhead), and centric oospores (RJBCC0001); (e) oogonial intercalary, antheridia ampullaceous (arrows), attached by foot-like projections (arrowhead), and centric oospores (RJBCC0017); (f) oogonia globose, diclinous antheridial hyphae (arrows) well developed, and attached by foot-like projections (arrowhead) (RJBCC0001); (g) oogonia globose, grouped like racemosa structure, and centric oospores (RJBCC0002); (h) oogonia globose, grouped like racemosa structure, diclinous antheridial hyphae (arrows) well developed, and attached by footlike projections (arrowhead) (RJBCC0002). Bar = 20 μm. Sandoval-Sierra,J. V., J. Diéguez- Uribeondo, 2015 98 19. Saprolegnia salmonis, Hussein and Hatai, 1999 Hyphae, tenues, aseptatae, modice lateraliter ramosae, ca. 22 pm crassae. Zoosporangia, clavata vel cylindrica, recta, interdum curvata, 178-186 x 24-36 #m, orificio dimissionis terminali. Zoosporae primariae saprolegnoides dimissae, pyriformes, biflagellatae, 11 l~m diam. Zoosporae secundariae emergentes e zoospora incystata, reniformes. Germinatio oogonii directus vel interdum indirectus. Oogonia abundantia, sphaerica obovata vel pyriformia plerumque 40-90 #m diam ubi antheridio affixo, elongata 90-149 x 50-73 [tm ubi antheridio non affixo. Oogonia sphaerica generatim 66.3~ et elongata 33.8~ Paries oogonii laevis, foveatus. Stipes oogonii rectus, nonramosus, 20#m diam. Oosporae sphaericae, 20 #m diam, 4-12 (plerumque 8) inoogoniis sphaericis 16-20 (plerumque 18) inoogoniis elongatis, guttula olei generatim subcentrica interdum centrica includentes. Rami antheridiorum diclini. Cellulae antheridiorum 1-cellulares, tenues, nonramosae, 8 #m diam, lateraliter vel apicaliter adpressae. Gemmae nonobservatae. Holotypus: NJM 9851, colonia exsiccata e cultura ex musculo, Onchrhynchi nerka, Hokkaido in Japonia, 7 Maius 1998, a Hussein isolata et ea collectione culturae in Universitate Veterinarii et Scientificae Animalis Nipponensis (NJM) conservata. Hyphae delicate, non-septate, slender, moderately branched (laterally), averaging about 22~m in diam. Zoosporangia clavate or straight, cylindrical, infrequently curved, 178-186 x 24-36 ~m. Zoospores discharge saprolegnoid. Primary zoospores biflagellate, pyriform, and 11 ~m in diam. Zoospores encysted 30 rain to several h after their discharge. The encysted zoospores emerge reniform secondary zoospores. Both direct (dominant 8590~ and indirect (10-1 5~ germination types of encysted zoospores observed. Oogonia abundant, spherical, ovate, pyriform, and elongated with or without antheridial attachment. Percentage of spherical and elongated oogonia 66.3 and 33.8~, respectively. Spherical oogonia 40-90(60)#m in diam, elongated oogonia 90-149(132) x 50-73(60) #m in size. Oogonial wall smooth and conspicuously pitted not only under the point of attachement of antheridial cell but also in absence of antheridial attachment. Some spherical oogonia pitted without antheridial attachment. Oogonial stalks straight, slightly bent, not branched, 20 #m in diam. Oospores spherical, 20 #m in diam, 4-12(8) and 16-20(18) in number in spherical and elongated oo gonia, respectively. Internal structure of oospores not only centric but also subcentric. Subcentric prevalent. Antheridial branches diclinous. Antheridial cells simple, tubular, delicate, not branched, 8/~m in diam, and laterally or apically apressed (sometimes attachment by footlike projections). Gemmae not observed. Effect temperature on vegetative growth The effect of temperature on vegetative growth is summarized in Fig. 3. The optimum temperature for vegetative growth was between 15 and 20~ Growth at 30~ was abnormal and stopped at 96 h. Effect of NaCI on the vegetative growth As shown in Fig, 4, the isolate was able to tolerate up to 3.0% of NaCI. No growth was observed on the GY agar containing 3.5% NaCI. 99 Morphological characteristics of sexual organs of Saprolegnia salmonis NJM 9851 isolated from sockeye salmon. A. Elongated oogonium pitted under the point of diclinous antheridial attachment (arrow); B. Pyriform oogonium pitted under the point of diclinous antheridial attachment (arrow). C. Spherical oogonium pitted without antheridial attachment (arrow). Scale bar=50Fm. Hussein et Hatai, 1999 20. Saprolegnia semihypogyna. Shigeki Inaba · Seiji Tokumasu, 2002 Hyphae slender, delicate, moderately branched, 16– 60µm in diameter at base. Gemmae, when present, spherical, pyriform, or irregular, terminal or lateral. Zoosporangia moderately abundant, elongate, cylindrical, clavate, occasionally pyriform in small ones, occasionally curved, terminal or lateral, 125–660 34–70µm, renewed internally or sympodially. Primary zoospore discharge saprolegnoid, pyriform, biflagellate, encysted zoospores 12–14µm in diameter. Secondary zoospore emerged from encysted primary zoospore, reniform. Oogonia abundant, lateral, terminal, rarely intercalary, occasionally clustered on a hypha, spherical, pyriform or obovate, doliform when intercalary, (25–)30–45(–55) µm in diameter. Oogonial wall pitted, unpitted, or pitted only under point of attachment of antheridial cells, smooth, occasionally with one or a few papillate projections. Oogonial stalks long, straight or curved, occasionally branched, then bearing oogonia cymosely. Oospheres usually maturing. Oospores subeccentric, spherical or subspherical, one, rarely two in number, (18–)25–33(–43) µm in diameter, oospore germination not observed. Antheridial branches semihypogynous, androgynous; when androgynous, usually arising from just beneath the basal walls of the oogonia, lacking on some oogonia. Antheridial cells simple, laterally or apically appressed, fertilization tubes observed. 100 Morphological characteristics of Saprolegnia semihypogyna. A Zoosporangia. B Oogonia with or without antheridium: the oospores are subeccentric. Bars A 100µm; B 25µm Shigeki Inaba · Seiji Tokumasu, 2002 Saprolegnia semihypogyna. 2 Emergence of primary zoospores from zoosporangium. 3 Secondary zoospore (left) and empty primary cyst (right). 4 Oogonium including single subeccentric Shigeki Inaba · Seiji Tokumasu, 2002 21. Saprolegnia terrestris Cookson ex R. L. Seym.1970 Hyphae slender, sparingly branched. Sporangia fusiform, obpyriform, or clavate, frequently spherical, often irregular and contorted; renewed internally, by cymose 101 branching, or in basipetalous succession; 60-400 × 16-48 µm. Spores dimorphic; discharge and behavior saprolegnoid; primary spore cysts 6-11 µm in diameter. Gemmae, when present, fusiform or obpyriform, infrequently conspicuously irregular or branched; terminal or intercalary. Oogonia lateral or terminal, infrequently intercalary; spherical or obpyriform, sometimes oval or apiculate, (35-) 60-65 (9l) µm in diameter, inclusive of papillae, if any. Oogonial wall pitted or unpitted; smooth or occasionally very sparsely papillate. Oospores subcentric; spherical or ellipsoidal; (1-) 2-11(-18) per oogonium, and nearly filling it; (20-) 24-32 (-41) µm in diameter; germination not observed. Antheridial branches androgynous and sometimes arising close to the oogonial septum, or infrequently monoclinous; stout or delicate, irregular, infrequently branched; persisting. Antheridial cells simple; generally clavate; not persisting; apically or laterally appressed; fertilization tubes sometimes present, not persisting. Remarks. S. terrestris is a monoecious species and can be distinguished by oospores subcentric 2-11 per oogonium and 24-32 μm in diameter. Like Saprolegnia litoralis, S. terrestris commonly has androgynous antheridial branches, and the general configuration of the laterally produced sexual apparatus in both is very similar. The oospores of S. litoralis are generally slightly larger than those of S. terrestris, but the latter usually has subcentricones while those of the former are consistently centric. S. terrrestrisis distributed in Australia, British Isles, Canada, Iceland, Iraq, New Zealand and Republic of China (Johnson et al., 2002); this species is cited for the first time in Mexico. Morphologil variation in Saproiegnia terrestris. A-C-Isolate 54a with persistent anthridial hypha:: A. sporangia; B. oogonium with monoclinous anthridial hyphae; C. oogomum with androgynous antheridial hypae. D-F-young oogonia with monoclinous and androgynous antheridial hyphae 102 in isolates with non-persistent anthendlal hyphae: D. isolate 28a; E. 56a'F. 61d. A. scale a. B-F. scale b.F. 61d. A. scale a. B-F. scale b. Ruth F. Elliott (1968) S terrestris . 1-3, oogonia with attendant androgynous antheridial branches; oospores subcentric (Johnson et al., 2002). D), S. terrestris . Lateral spherical oogonium, wall pitted androgynous, monoclinous and diclinous antheridial branches. Barr= 25 μm. OC: oospores centric; AC: antheridial cells; PI: pitted. Vega-Ramírez et al. (2013) 22. Saprolegnia torulosa DeBary (1881). Hyphae rather slender. Zoosporangia from cylindric becoming clavate, fusiform, or ne arly globular, often in torulose series. Oogonia globular, ovate, pyriform, or cylindrica l, terminal or intercalary, commonly in torulose series, their walls more or less abunda ntly marked by small pits, and yellowish brown when old. The members of a series m ay form, some sporangia and others oogonia. Antheridia very rarely present. Oospores as many as twelve, or rarely more, in an oogonium, centric, their average diameter ab out 25/7.. 23. Saprolegnia uliginosa Johannes 1950 Mycelium moderately dense, extensive; hyphae slender, flaccid. Sporangia clavate, cylindrical or fusiform, sometimes irregular, renewed internally, rarely in basipetalous succession; 108-266 × 12-42 μm. Spores dimorphic; discharge and behavior saprolegnoid; primary and secondary spore cysts 9-12 μm in diameter. Gemmae sparse; spherical, obpyriform, cylindrical, fusiform, or irregular; terminal or intercalary, single or catenulate. Oogonia sparse or abundant; lateral, rarely terminal, occasionally intercalary; spherical, infrequently napiform or obpyriform; (32-) 60-68 (-91) μm in diameter. Oogonial wall pitted under the region of attachment of antheridial cells; smooth. Oospores centric, rarely subcentric; spherical; (2-) 5-7 (-25) per oogonium, and nearly filling it; (21-) 25-33(-36) μm in diameter; germination not observed. Antheridial branches predominantly monoclinous, and arising very near the oogonial stalk; infrequently androgynous; rarely diclinous; slender, slightly irregular, occasionally producing 1 or 2 short, lateral branches; persisting. Antheridial cells simple; generally tubular, straight or curved, occasionally faintly cylindro-clavate; laterally appressed, persisting; fertilization tubes present, not persisting. Remarks. S. uliginosa is a monoecious species and can be distinguished by oospores centric, 5-7 per oogonium, and nearly filling it; 25-33 μm in diameter; germination 103 not observed. Like Saprolegnia turfosa, S. uliginosausually has spherical oogonia borne laterally on short stalks and containing predominantly centric oospores. The similarity between these 2 species does not go beyond these features since the antheridial branches of S. uliginosa generally are monoclinous whereas androgynous ones are clearly predominate in S. turfosa. S. uliginosais is distributed in Germany, Iceland, India and USA (Johnson et al., 2002); this species is cited for the first time in Mexico. S. uliginosa . Oogonia with oospores centric; monoclinous antheridial branches (Johnson et al., 2002). B), S. uliginosa . Spherical oogonium with 5 centric oospores. Barr= 20 μm. Vega-Ramírez et al. (2013) 24. Saprolegnia unispora (Coker and Couch) R. L. Seym., 1970 Mycelium extensive; hyphae stout, sparingly branched. Sporangia clavate, fusiform, pyriform, cylindrical, or subspherical basally and attenuated apically; often slightly irregular or curved; renewed internally or in a cymose fashion, rarely sympodially; 88-317 × 21-127 μm. Spores dimorphic, or rarely monomorphic; discharge and behavior saprolegnoid, rarely dictyucoid; primary spore cysts 10-12 μm in diameter. Gemmae abundant, fusiform, pyriform, obpyriform, cylindrical, spherical, subspherical, or irregular; terminal or intercalary; single or catenulate. Oogonia lateral, occasionally terminal or sessile, rarely intercalary; frequently clustered on the hyphae or arranged in a sympodially branched glomerulus; spherical or obpyriform, occasionally subspherical, oval, or broadly clavate, very rarely angular, sometimes cylindrical (in old sporangia); (18-) 45-55 (-91) μm in diameter, inclusive of wall ornamentations. Oogonial wall pitted or unpitted; smooth or very rarely with 1 to a few short, inconspicuous broad papillae, or a single apiculus. Oogonial stalks (1/8-) ½ -11/4 (-3) times the diameter of the oogonium, in length; stout, straight or curved, sometimes slightly irregular; unbranched, branched, or forming a glomerulus. Oospores centric or subcentric; spherical or compressed at one side, or ellipsoidal; 1-2 (-4) per oogonium, and generally not filling it; (l6-) 32-38 (-43) μm in diameter; germination not observed. Antheridial apparatus absent. Saprolegnia unispora is recognized chiefly by its short-stalked, large, generally spherical oogonia (often in glomeruli or on once-branched stalks) usually containing a single, large, centric or subcentric oospore, but having no attendant antheridial filaments. 104 Remarks. S. unispora is a monoecious species and can be distinguished by oospores centric or subcentric; 1-2 per oogonium; 32-38 μm in diameter; germination not observed. Antheridial apparatus absent. Saprolegnia unispora is recognized chiefly by its short-stalked, large, generally spherical oogonia (often in glomeruli or on oncebranched stalks) usually containing a single, large, centric or subcentric oospore, but having no attendant antheridial filaments. S. unisporais distributed in Australia, British Isles, Canada, Czechoslovakia, India, Japan, Republic of China, USA, and USSR (Johnson et al., 2002); this species is cited for the first time in Mexico. S. unispora. Cluster of oogonia; antheridial components lacking; variations in oogonial stalk length (Johnson et al., 2002). D), S. unispora . Oogonium with 1 oospore, without antheridial branches . Barr= 20 μm. DA: diclinous antheridium, OC: oospores centric, AC: antheridial cells . Vega-Ramírez et al. (2013) Reports: Copland and Willoughby (1982) described the clinical signs and histopathology of Saprolegnia sp. (related to S. declina) in cultured Anguilla anguilla under an intensive production system. The main observed lesions were loss of epithelium leading to ulceration, oedema and myofibrillar degenerative changes of the muscle mass. It was suggested that the rapid extension of the lesion is due to the loss of integrity of the integument and the wide spread oedema, which altered the viability of tissue and assisted the extensive liquification in severely acute cases with obvious bilateral exophthalmia. Injected fungi were re-isolated from heart, spleen, kidney, liver, intestine, gills and eyes of the examined fish. The liver and spleen showed tissue necrosis with conidia and some germinating or septate hyphae were extending into the tissue. Moreover, fungi were demonstrated in eyes and the gills with generalized oedematous changes. Noga and Dykstra (1986) stated that species of Saprolegnia are usually implicated in fungal infections of fish and fish eggs. They also mentioned that the Oomycetes are commonly associated with skin lesions in fresh water fishes. They normally cause relatively superficial infections. Clinically, Oomycetes are characterized by the 105 production of a cotton-wool-like mycelial mass that was grossly evident on the surface of an affected fish. Shaheen (1986) reported that Saprolegnia parasitica was isolated from Nile Tilapia in naturally and experimentally infected fish. Saprolegnia infects fish especially at low water temperature, which may act as predisposing factor. Handling, injury, spawning period may also play a role in infection. The result of experiment revealed that Saprolegnia affected skin and gills as a primary pathogen. Wounded fish were highly susceptible to fungal infection. Intraperitoneal and intramuscular infection revealed negative results for Saprolegnia. Also, he described the clinical signs of Saprolegniosis among fresh water fishes due to Saprolegnia parasitica. The infected fish displayed cotton-wool-like white to dark gray growths, which started at the head and tail regions then spread over the body. Wood and Willoughby (1986) studied the colonization of clean (apparently uninfected) and dead salmonid fish by Saprolegniaceae in the laboratory and in Windermere. Colonization proceeded in two stages in water: initially there was superficial mycelium, which was easily removed, and later there was a deeper penetration into the epidermis and dermis. There was evidence that colonization can occur as an intimately mixed mosaic of different Saprolegniaceae. Live char and other salmonids were normally colonized only by the specific Saprolegnia pathogen in Windermere. However, this specificity broke down, when there was colonization of dead tissue on a live char. In post-mortem, salmonid fish killed by the specific Saprolegnia pathogen did not attract other Saprolegniaceae subsequently and explanations for this were proposed. Easa and Amin (1987) reported that Saprolegnia Parasitica was isolated from Oreochromis niloticus. Identification of species of such fungus was based on the morphological characters seen in wet mounts of specimens from lesions or from growth on Sabouraud's agar medium containing hemp seeds. They mentioned also that Saprolegnia parasitica was isolated from Oreochromis niloticus showing skin lesion at water temperature of 18-22°C. The lesions appeared as dark gray or grayish white, elevated plaques on the dorsal and pectoral fins and skin. The lesion progressed to erosion of deep ulcer formation in the skin and was surrounded with hyperaemic irregular zone. Molina et al. (1995) examined restriction fragment length polymorphisms (RFLPs) in two regions of the ribosomal DNA (rDNA) repeat unit in 33 strains representing 18 species of Saprolegnia. The Polymerase Chain Reaction (PCR) was used to separately amplify the 18S rDNA and the region spanning the two internal transcribed spacers (ITS) and the 5.8S ribosomal RNA gene. Amplified products were subjected to a battery of restriction endonucleases to generate various fingerprints. The internal transcribed spacer region exhibited more variability than the 18S rDNA and yielded distinctive profiles for most of the species examined. Most of the species showing 100% similarity for the 18S rDNA could be distinguished by 5.8S + ITS restriction polymorphisms except for S. hypogyna, S. delica, S. lapponica, and S. mixta. The 106 rDNA data indicate that S. lapponica and S. lapponica and S. mixta are conspecific with S. ferax, whereas there is no support for the proposed synonymies of S. diclina with S. delica and of S. mixta with S. monoica. Results from cluster analysis of the two data sets were very consistent and tree topologies were the same, regardless of the clustering method used. A further examination of multiple strains in the S. diclina-S. parasitica complex showed that restriction profiles are conserved across different strains of S. parasitica originating from the U.K. and Japan. HhaI and BsaI restriction polymorphisms were observed in isolates from the U.S. and India. The endonuclease BstUI was diagnostic for S. parasitica, generating identical fingerprints for all stains regardless of host and geographic origin. Except for the atypical strain ATCC 36144, restriction patterns were also largely conserved in S. diclina. Correlation of the rDNA data with morphological and ultrastructural features showed that S. diclina and S. parasitica are not conspecific. Restriction polymorphisms in PCR-amplified rDNA provide a molecular basis for the classification of Saprolegnia and will be useful for the identification of strains that fail to produce antheridia and oogonia. Phenogramfromclusteranalysisofthe 18SrDNArestriction fragment data with the UI~MA method. Scale bar- Dice similarity coefficient. Molina et al. (1995) 107 Phenogram from UPGMA cluster analysis of the 5.8S + ITS rDNA restriction fragment data. Scale bar - Dice similarity coefficient. Molina et al. (1995) Hussein and K. Hatai (1999) reported saprolegniasis in cultured sockeye salmon, Onchorhynchus nerka, raised in Hokkaido, Japan. The lesions were mainly observed in the head, peduncle region and the caudal fin. All strains isolated were morphologically classified in the genus Saprolegnia. They were identified as a new species in the genus from the characteristics of the sexual organs, and named Saprolegnia salmonis. Gross appearance of sockeye salmon fingerling with saprolegniasis. The fungal growth appears adjacent to head and peduncle region (arrows). Scale bar= 1 cm. Morphological characteristics of sexual organs of Saprolegnia salmonis. Hussein and K. Hatai (1999) Leano et al. (1999) observed mass mortality among pond cultured red drum (Sciaenops ocellatus) in Hong Kong. Affected fish were lethargic and lost their appetite but no lesions on the body surface were apparent. Patches of white to 108 brownish cottony growth on the gills of affected fish were observed and microscopic examination revealed mats of hyaline mycelia with mature zoosporangia and oogonia which were identi®ed as Saprolegnia diclina. During induced sporulation, production of primary and secondary zoospores, oogonia, and antheridia were observed. A physiological study of the growth and sporulation of a representative isolate determined its optimum growth requirements. The isolate can grow from pH 4 to 10, in distilled water, at salinities of 5±30^, and temperatures of 4±30°. Maximum growth was observed at pH 5 and 8±10, at salinities of 5±10^, and 25±30°. Production of zoosporangia only occurred in distilled water, 5 and 10^ salinities, with zoospores released in distilled water and 5^ salinity. Zoospore release was also observed from 4 to 30° with greater abundance at 25 and 30°, while oogonia and antheridia were produced in distilled water and from 5 to 30^ salinities and at 20±30°. 109 Saprolegnia diclina. Fig. 1. Mats of hyaline mycelia with some trapped debris (d) on the gills (g) of affected red drum. Fig. 2. Mature zoosporangium. Fig. 3. Zoosporangium releasing zoospores (Zp). Fig. 4. Internal proliferation of the zoosporangium. Note the two layers of spent zoosporangia (arrowheads 1, 2). Fig. 5. Pyriform primary zoospore (z) and primary cyst (c). Fig. 6. Developing secondary zoospore (arrow) inside the primary cyst (c). Fig. 7. Reniform secondary zoospore. Fig. 8. Germinating secondary cyst (gt germ tube). Fig. 9. Immature oogonium with monoclinous antheridium (arrow). Fig. 10. Immature oogonium with diclinous atheridia (arrows). Fig. 11. Mature oogonium (a¯ antheridium ; o¯ oospheres). Leano et al. (1999) 110 Xu and Rogers (1991) stated that the vegetative hyphae of Saprolegnia parasitica infection of Channel catfish Ictalurus punctatus are aseptate and have a thin wall. They contain large vacules, elongated mitochondria and nuclei oriented parallel to the longitudinal axis and in the direction of hyphal extension. Presporangium hyphae, that will form sporangia also are aseptate but have thicker walls and contain smaller vacules. They do not had the longitudinal orientation of organells seen in vegetative hyphae, but they have mitochondria, dicytosome, endoplasmic reticulum dense body vesicle and primary bars. Vegetative hyphae of S. parasitica. 1. Longitudinal section of a hypha showing distribution of organelles and large vacuoles (V) at this stage. M = mitochondrion; N - nucleus. Bar = 2.0 /itm. 2. A detailed view of the cell wall (W) comprising electron-opaque layers (arrows) on either side of a more electronlucent zone. Bar = 0.2 pin. 3. A portion of a hypha showing the elongate nature of a nucleus (N) and mitochondria (M). Bar = 2.0 jan. 4. A higher-magnification view of the hyphal tip showing the accumulation of vesicles (arrows) indicative of active growth. Bar = 1.0 /tm. Xu and Rogers (1991) Bly et al. (1992) isolated pure cultures of fungus from commercially raised channel catfish exhibiting signs of a winter syndrome locally termed "winter kill". The fungal isolates were identified as members of the genus Saprolegnia. Histopathological examination of fungal associated skin lesions from diseased fish exhibited a complete lack of bacteria or of leukocytic infiltration around the site of hyphal penetration. In order to determine if the fungus was the origin of disease or an opportunistic secondary pathogen, controlled laboratory studies were conducted, which conclusively proved that if channel catfish were immunosuppressed by a rapid decrease in environmental water temperature from 22 to 10°C, the Saprolegnia sp. isolates from infected catfish were able to cause 92% infection (skin lesions) and 67% mortality within 21 days. 111 Bly et al., 1992 112 Bly et al., 1992 113 Bly et al., 1992 Gonzalez et al. (2001) described the clinical signs and histopathological disturbances produced by Saprolegnia infections in Chondrostoma polylepis and Rutilus albugineus under environmental stress. The fish were obtained during an outbreak in a river in the southern part of the Iberian Peninsula. The main alterations observed were loss of epithelium leading to ulceration and blood disorders (blood congestion and occasional haemorrhages). Infected fish developed focal lesions because the fungus invaded the stratum spongiosum of the dermis before extending laterally over the epidermis. The onset of disease was brought about by the combination of a rapid fall in water temperature which probably induced an effect of immunosuppression, and the maintenance of low water temperature which favoured the proliferation of the fungus. 114 (A) Reproductive structure (immature sporangium) of fungus of the genus Saprolegnia, separated from the hypha by means of a septum (.), PAS × 40. (B) External surface area of the fish damaged by the fungus observed with SEM; the disposition and cottony appearance of the mycelium is observed × 250. (C) Final portion of the somatic hypha, which in this species is non-septate, PAS × 20.(D) Surface of a sporangium (reproductive structure) with SEM × 400. (E) Mature sporangium, containing a large amount of zoospores, H/VOF × 40. (F) Zoospore germinating on the surface of a branchial lamina × 7500. Gonzalez et al. (2001) STECIOW (2002) described Saprolegnia milnae from litter (floating dead twigs, leaves, and roots) in the Milna River, Ushuaia Department, Tierra del Fuego Province (Argentina). The new species is illustrated and compared with other species of the genus. Distinguishing characteristics of S. milnae are the production of smooth oogonia and predominantly contorted androgynous antheridial branches, and also monoclinous and diclinous ones. The oogonial stalks are predominantly bent, curved or once coiled; oospores are subcentric, (1—)3—23(—40) per oogonium, and are variable in size, reaching a diameter of up to 60 µm. Stueland et al. (2005) examined seventeen strains of Saprolegnia spp. for morphological and physiological characteristics, and seven were examined for their pathogenicity to Atlantic salmon, Salmo salar L. Two of the Saprolegnia strains tested caused 89 and 31% cumulative mortality in challenged salmonids and were signi115 ficantly more pathogenic than the other strains tested. The positive control (Saprolegnia parasitica ATCC 90213) caused 18% mortality, but this was not significantly higher than non-pathogenic strains (0–3% cumulative mortality). All the pathogenic Saprolegnia strains and two non-pathogenic strains had secondary cysts with long, hooked hairs, a characteristic which is claimed to be typical of S. parasitica. This characteristic is apparently necessary, but does not in itself determine the ability to cause mortality in Atlantic salmon. However, all the pathogenic Saprolegnia strains in the present study showed a significantly higher initial growth rate of cysts in sterilized tap water than did non-pathogenic strains. The results of the present study suggest that initial growth rate of germinating cysts in pure water, together with the presence of long hooked hairs on the secondary cysts, may be indicators of pathogenicity of Saprolegnia strains to Atlantic salmon. Scanning electron micrographs of secondary cysts of the Saprolegnia strains included in pathogenicity testing (bar ¼ 10 lm; except strain L: bar ¼ 5 lm). Saprolegnia strain A produced oogonia at 15–20 C, and had long spines on the cyst surface. The two light micrographs show different shapes of oogonia, and the scanning electron micrograph confirms the long spines on the surface of the secondary cyst (bar ¼ 10 lm) Stueland et al. (2005. Steciow (2006) described Saprolegnia oliviae sp. nov from litter (floating dead twigs, leaves and roots) in the Olivia River, Ushuaia Department, Tierra del Fuego Province (Argentina). The new species is illustrated and compared with other species of the genus. Distinguishing characteristics of S. oliviae are the production of smooth 116 oogonia (with some lateral or terminal projections) and the absence of antheridial branches on the majority of the oogonia, but when present, they are mostly diclinous, at times oogonia are supplied with androgynous and monoclinous antheridial branches. The oogonial stalks are predominantly short and straight or long and bent, curved or many times coiled; oospores are distinctive subcentric, (1–) 15–50 (–70) per oogonium. Morphological details of the new species and its comparison with other described species are discussed here. Steciow et al. (2007) isolated Saprolegnia bulbosa sp. nov. from floating and decaying twigs and leaves in El Gato stream, Partido de La Plata, Buenos Aires Province, Argentina. The distinctive characteristics of S. bulbosa are the product of smooth oogonia and predominantly contorted monoclinous, androgynous and diclinous antheridia. The oogonial stalks are usually bent, curved or once coiled; oospores are subcentric, (1) 2–15 (45) per oogonium and are variable in size. Taxonomical description of this new species, its comparison with related oomycetes of the genus and the nucleotide sequences of the internal transcribed region (spacers ITS1, ITS2 and the gene 5.8S) of its rRNA gene are given. ABOU EL ATTA (2008) carried out a study on 100 cultured Tilapia nilotica in floating cages with high stocking density suffered from saprolegniosis. The fish were subjected to full clinical, postmortem, bacteriological and histopathological examination, also trial for control using Bafry D50/500 and hematological examination of treated fish. Clinically, the infected fish showed loss of equilibrium, lethargy, unable to feed, hemorrhage at the base of fins may extended to cover all the body surface, the main characteristic lesions of saprolegniosis was appearance of cotton wool like tufts on the fins (dorsal, caudal, and pectoral fins); also, on eyes, the head and mouth and uni or bilateral blindness ended with death. Postmortem: the infected fish showed pale grayish gills, the intestine were free from any food particles, dark enlarged liver, distended gallbladder and spleenomegaly. The microscopical and morphological finding were characteristic for saprolgnia. Saprolegnia parasitica was isolated with higher percentage from skin, followed with fins, eyes, and mouth, but neither from liver nor kidney. Trial of using Bafry D50/500 for control of saprolegniosis in a dose of 0.75 ml/L (375ppm) for 10-12 minutes for 3 successive days as a bath gave good results in treatment of the diseased fish in aquaria, where the rate of mortality reached 16.7% without any effect on the healthy state of treated fish and also no effect on hematology and blood chemistry of treated fish with Bafry D50/500. Histopathological finding of the infected fish showed epithelial desquamation of the epidermis, erosion and ulceration of the infected area, edema, hyaline degeneration, and aggregation of M.M.C. and fragment of fungal hyphae occurred in the underling dermis of the skin, muscles; The gills of the infected fish showed complete desquamation of majority secondary lamellae, and gill arch was edematous with presence of aggregation of fungal spores and M.M.C., Histopathological finding of treated fish with Bafry D50/500:skin showed increase of M.M.C. and slight sloughing of most superficial layer of epidermis but the gills showed increasing and hyperplasia of epithelial cells covering of primary lamellae, this indicate that the infected fish was treated with BafryD50/500. 117 Hemorrhage at the base of the fins, and extended to cover all the body surface ABOU EL ATTA (2008) Cotton wool like tufts on the dorsal, tail (caudal), pectoral fins also appearance on the eye, head and mouth of fish ABOU EL ATTA (2008) Unilateral cloudy and opacity of the eye ended by blindness and emaciation, cotton wool tufts on caudal fin(tail), pale to grayish gills, serious fluid or exudates in the abdominal cavity, dark enlarged liver,distended gall bladder with bile, spleenomegaly and congested kidney ABOU EL ATTA (2008) 118 The positive colonies on (SDA)at 20ºC for 3-4 days started with cysts of long hairs with white cottony color after that became grey then black after 96 hrs. The wet preparation of skin, gills, eye, and mouth lesions showed masses of mature and immature sporangia filled with large number of sporangiospores, the hyphae appeared profusely branched and were non septated, these morphological findings were characteristic of the Saprolegnia species ABOU EL ATTA (2008) The skin of infected Tilapia, showed epithelial desquamation in the epidermis. The other epidermal cells suffered vacuolar degeneration and focal necrosis. The underling dermis was edematous and contained fragments from the fungal hyphae with focal aggregation of melanomacrophages cells. The necrotic muscles infiltrated with numerous mononuclear leukocytes and some melanomacrophages. Saprolegnia spp, appeared by H&E stain as non septated hyphae of variable size and lengths The gills of infected tilapia, showed desquamation to the majority of the secondary lamellae with few mononuclear leukocytes infiltrated the primary lamellae. The gill arch was edematous and contained the fungal spores and hyphae as well as some melanomacrophages and mononuclear cells ABOU EL ATTA (2008) 119 Skin of Tilapia nilotica (O.niloticus) treated with BafryD50/500showed increase of M.M.C and slight sloughing of most superficial layer of epidermis . Gill of Tilapia nilotica (O.niloticus) treated with Bafry D50/500 showed hyperplasia of epithelial cells covering of primary lamellae ABOU EL ATTA (2008) Hirscch et al. (2008) found crayfish specimens with a fungus-like growth which after DNA-isolation and sequencing was identified as consisting of a set of previously undescribed Saprolegnia species. Four of the isolates (b) grouped in molecular clade IV after Diéguez-Uribeondo et al. (2007), and were most likely identical to S. australis, two isolates (c) grouped in clade II, and one isolate (a) in clade III. Morphological studies partly confi rmed the molecular classifi cation; all isolates remained sterile after several months of storage on various standard agar media (V8 juice agar, oatmeal agar). On hemp seed cultures only the single isolate in clade a) (isolate ID: II-2) abundantly produced spherical, smoothwalled oogonia, measuring ca. 50–105 µm (mean 69.5 ± 13.4 µm SD), and containing ca. 5–30 (mean approx. 15) globose, centric to subcentric oospores relatively uniform in size (mean 23 ± 1.6 µm SD) 120 Oogonia and oospores of Saprolegnia sp. isolate II-2 after seven days on autoclaved hemp-seed halves in water culture. A: general view (bar = 40 µm); B–D: detailed view of small (B) and medium-sized (C–D) oogonia (bar = 20 µm). Hirscch et al. (2008) Osman et al. (2008) developed a method to induce experimental saprolegniosis in Tilapia exposed to physical stress through descaling with or without wounding, in addition to sudden or gradual drop of water temperature. Osman et al., 2008 121 Ke et al. (2009) described Saprolegnia brachydanis from zebra fish (Brachydanio rerio) in Wuhan, Hubei Province, China. The species is illustrated and compared with other species of the genus. The distinctive characteristics of S. brachydanis are the production of glomerulate oogonia wrapped around by predominantly monoclinous antheridia which can be up to eight in one oogonium. The oogonial stalks are short, straight, or curved and the antheridia, twisted, can enwind one or more oogonia. The oospores cannot mature or easily abort. Morphological features of the oomycete and the ITS sequence of its rDNA as well as the comparison with related species are discussed in this article. Zebra Fish infected with Saprolegnia. Scale bar = 0.5 cm Ke et al., 2009 Krugner-Higby et al. (2010) monitored Crayfish populations in the area of the North Temperate Lakes Long Term Ecological Research (LTER) project, Wisconsin, USA for >25 yr. In 2005, native crayfish Orconectes propinquus from Big Muskellunge Lake were found with ulcerated lesions in the cuticle. In 2006, lesions occurred in 9.5% of sampled crayfish from the lake (n = 3146). Ulcers generally occurred on the appendages of affected individuals but varied in location and severity. The prevalence of ulcers varied widely among sites, sample depths, and sampling dates, ranging from 20%. The prevalence of ulcers in crayfish increased from a minimum in early June to a maximum in late July and August. In aquarium trials, healthy crayfish representing either O. propinquus or O. rusticus co-housed with ulcerated crayfish did not develop ulcers within 4 wk of exposure. Gross and histopathologic analyses of ulcerated crayfish revealed the presence of filamentous hyphae in the lesions while hemocytic infiltrates, melanotic reactions and silver-stained sections indicated that the ulcers had an oomycete etiology. Excised samples of ulcerated crayfish cuticle grown in culture developed an oomycete that was identified as Saprolegnia australis by PCR 122 amplification and sequence analysis of 2 different DNA fragments. This is the first report of the occurrence of ulcers in wild crayfish associated with S. australis infection in the USA. The advent of the outbreak and its underlying ecological causes are still under investigation. Orconectes propinquus. Gross lesions of saprolegniasis. Ulcerative lesions on the carapace and legs of some of the more severely affected crayfish Krugner-Higby et al. (2010) Orconectes propinquus. (A) Healthy carapace showing the typical organization of the cuticle into epicuticle (Epi), exocuticle (Exo), endocuticle (Endo), and membranous layer (Mem). P: Psorospermium haekelii sporont. H&E. Scale bar = 200 μm.(B) Carapace that is severely infected, putatively by Saprolegnia australis, showing lytic disruption of the cuticle, melanoticresponse to infection (arrows), and encapsulated hyphae. H&E. Scale bar = 200 μm. Krugner-Higby et al. (2010) 123 C) Detail from the same animal as in (B) showing the melanotic response that has developed along a hypha (arrow) that has grown into the dermis (D) An ulcerative lesion that is heavily melanized (black areas) with hyphae (arrows)penetrating through the cuticle. GMS stain. Scale bar = 200 μm. KrugnerHigby et al. (2010) (E) Detail of ulceration in (D) demonstrating hyphae (H) (arrows) in the lesion and penetrating into the epidermis. GMS stain. Scale bar = 50 μm. (F) Detail of ulceration in (D) showing hyphae (arrow) in the leading edge of the lesion. The hyphae elicit an intense melanitic response. GMS stain. Scale bar = 50 μm. Light microscopy Krugner-Higby et al. (2010) 124 Parsimony bootstrap tree resulting from analysis of the ITS sequence of the Saprolegnia sp. isolated from Orconectes propinquus (bold) with sequences of other members of the Saprolegniaceae. Boostrap support values are given for clades with >50% support. Note strong support (89%) for placement of the crayfish Saprolegnia sp. in the S. australis clade. Analysis was conducted with 1000 bootstrap replicates of 100 random additions Krugner-Higby et al. (2010) Noor El- Deen et al. (2010) carried out on 300 cultured fingerlings of Nile tilapias and Mugal cephalus from earthen ponds in freshwater fish farms suffered from Saprolegniosis. Diseased fish were subjected to full clinical and postmortem examination. Artemisia cina (Sheih Baladi) and Humates (humic and fulvic acid ) were tested for the control of Saprolegniosis affecting fingerlings of Nile tilapias and Mugal cephalus. Artemisia cina L. (A.cina) was used in the form of 5% and 25% stock solutions prepared by pouring boiling water on the herb in a piece of gauze and soaked for 2 hours. The doses were 0.25, 0.5 and 1 ml/l 3 times every an hour for 3 days in fingerlings of Nile tilapias and twice for 2days in fingerlings of Mugal cephalus in earthen ponds. Humates was used as HUMAPOL-FIS dry stock solution in the rates of 5, 10 and 15 g/1000 liter in earthen ponds. Three replicates were used per each treatment and 3 earthen ponds served as control where malachite green or 125 formalin were applied for comparison. Results revealed that A. cina and humates gave the best estimates of viability percentages among the Nile tilapia and Mugal cephalus fingerlings and were safe for fingerlings in the rates of 25% for A. cina and 5 and 15 gm/1000 liter for humates. Showing cotton wool like tufts on the dorsal, tail of fingerlings of Mugal cephalus (1), small fingerling of Nile tilapia (2) and large fingerlings of Nile tilapia (3). Noor El- Deen et al. (2010) Refai et al. (2010) carried out a study on 360 freshwater fishes (240 Oreochromis species and 120 Clarias gariepinus). They were collected from different governorates and during different seasons. Naturally infected fishes showed clinical abnormalities such as skin darkening, exophthalmia, corneal opacity, abdominal distention, ulceration of the skin and cotton wool like growths on various parts of the body. Fishes were then subjected to post mortem examination which revealed many abnormalities. Mycological examination revealed the isolation of 2081 fungal isolates from 150 diseased and 210 apparently healthy fish samples (1658 mould and 423 yeast isolates), of which 1334 were isolated from Oreochromis species and 747 isolates from Clarias gariepinus. Isolated moulds belonged to the following genera: Saprolegnia (4.2%), Aspergillus (43.0%), Fusarium (14.1%), Mucor (14), Penicillium (17.2), Rhizopus (4.8%), Scopulariopsis (1.2%), Paeciliomyces (1%) and Curvularia (0.4%). Saprolegnia species were obtained from skin lesion and gills of both Oreochromis species and Clarias gariepinus. Macromorphology, colonies appeared as scanty cotton wool like growth at early stage then became dense when hemp seeds were added. Microscopically, hyphae obtained from wet mount preparation of skin scrape of affected fish were broad, branched, coenocytic and non septated (Photo14). Different stages of reproductive structures were obtained after addition of hemp seeds 126 Colony of Saprolegnia species with the characteristic cotton- wool like growth. Non-septated broad hyphae of Saprolegnia species. Refai et al., 2010 Different stages of reproductive structure of Saprolegnia species on hemp seeds. Refai et al., 2010 Shahbazian et al. (2010) isolated and identified parasitic and saprophytic fungi from affected eggs of rainbow trout at two fish hatchery in Kermanshah province. The samples were inoculated in culture media (SDA,CMA,GPA and stilled water with cotton seed culture) at room temperature (18-24°C).17 species of fungi were isolated from the fungal eggs. Five fungi species belonged to the saprolegniaceae family including Saprolegnia parasitica, Saprolegnia lapponica , Saprolegnia ferax, Saprolegnia hypogyna and Saprolegnia diclina. Saprolegnia parasitica with 26.8 percent of isolations was the most important fungal infestation of eggs in kermanshah trout hatcheries. In this study S. ferax, S. hypogyna and S. diclina were reported from Iran for the first time. Oogonia of Saprolegnia lapponica (100x), Oogonia of Saprolegnia diclina (100x), Oogonia of Saprolegnia ferax (100x) Shahbazian et al. (2010) 127 Oogonia of Saprolegnia hypogyna (100x), Pyriform zoosporangium of S. parasitica (40x) Shahbazian et al. (2010) Cao et al. (2012) characterized an oomycete water mould (strain HP) isolated from yellow catfish (Peleobagrus fulvidraco) eggs suffering from saprolegniosis both morphologically and from ITS sequence data. It was initially identified as aSaprolegnia sp. isolate based on its morphological features. The constructed phylogenetic tree using neighbour joining method indicated that the HP strain was closely related to Saprolegnia ferax strain Arg4S (GenBank accession no. GQ119935), that had previously been isolated from farming water samples in Argentina. In addition, the zoospore numbers of strain HP were markedly influenced by a variety of environmental variables including temperature, pH, formalin and dithiocyano-methane. Its zoospore formation was optimal at 20 °C and pH 7, could be well inhibited by formalin and dithiocyano-methane above 5 mg/L and 0.25 mg/L, respectively. To our knowledge, this is the first report on the S. ferax infection in the hatching yellow catfish eggs. The symptom of the yellow catfish egg suffering from saprolegniosis, arrowheads showed that the mycelium penetrated egg (10×) Cao et al. (2012) de Bruijn et al. (2012) the immune response of salmonid cells is investigated at the transcript level, by analysis of a large set of immune response genes in four different rainbow trout cell lines (RTG-2, RTGill, RTL and RTS11) upon infection with S. parasitica. Proinflammatory cytokine transcripts were induced in all four cell lines, including IL-1β1, IL-8, IL-11, TNF-α2, as well as other components of the innate defences, including COX-2, the acute phase protein serum amyloid A and C-type lectin CD209a and CD209b. However, differences between the four cell lines were found. For example, the fold change of induction was much higher in the epithelial RTL and macrophage-like RTS11 cell lines compared to the fibroblast cell lines 128 RTG-2 and RTGill. Several antimicrobial peptides (AMPs) were also up-regulated in response toSaprolegnia infection, including hepcidin and cathelicidin 1 (rtCATH1) and 2 (rtCATH2). An rtCATH2 peptide was synthesised and tested for activity and whilst it showed no killing activity for zoospores, it was able to delay sporulation of S. parasitica. These results demonstrate that particular immune genes are upregulated in response to S. parasitica infection and that AMPs may play a crucial role in the first line of defence against oomycetes in fish. de Bruijn et al. (2012) Ali et al. (2013) tested the ability of Saprolegnia to form biofilms where it can survive, reproduce and resist different chemicals used for its control. Naturally formed biofilms were obtained from laboratory aquaria. Saprolegniagrowth within these biofilms was demonstrated with light microscopy and confirmed by isolation. Isolates were identified morphologically and molecularly on the basis of ITSsequences. Two isolates were identified as Saprolegnia parasitica, a species known to be highly pathogenic for fish, while the other belonged to S. australis. SelectedSaprolegnia strains obtained from natural biofilms were then used to establish simple methods for in vitro induction of Saprolegnia biofilm. The ability of Saprolegnia isolates to form biofilms with subsequent production of infective motile zoospores within the biofilm was documented by light and confocal laser scanning microscopy. We demonstrate for the first time that isolates of S. parasitica and S. australis can form biofilm communities together with multiple microorganisms, wherein they grow and reproduce. It is therefore likely that natural biofilms constitute incessant Saprolegnia reservoirs in nature and aquaculture Dissected natural biofilm showing, mature (a) and empty (b) Saprolegnia zoosporangia, together with other microorganisms and organic matter. Development of mature Saprolegnia zoosporangia (arrows) on the generator slide in the presence of other microbes and sedimented organic matter. The composition of the induced biofilm from the generator is similar to the natural one. Ali et al. (2013) 129 CLSM images showing the diversity of microbes associated with the Saprolegnia in an induced biofilm. (A) 3D rendering of a chlamydospore associated with different bacteria – long rods (white arrows), short rods (white ring) and cocci (red arrow). (B) Single plane projection of a chlamydospore (blue cell walls) with surrounding bacteria (green). Extracellular material and debris are not showing. 3D rendering of S. parasitica chlamydospores in chains in an (induced biofilm). Ali et al. (2013) 3D rendering of developmental stages of induced Saprolegnia biofilm (S. parasitica) obtained from chambered slide and stained with calcofluor white(CLSM). (a) Single germinating cyst. (b) Asterisks illustrate original cysts and arrows the septa. (c) Arrows represent empty cysts. (d) Sporangium showing apical exit papillum arrowed of an internally proliferated sporangium (*) which has grown through the original primary sporangium. Ali et al. (2013) 130 CLSM images of induced biofilm after 3 weeks. (a) Single plane image showing formation of chlamydospores. (b) 3D rendering showing the structure of the biofilm. (c) 3D rendering at 908 angle, showing the thickness of the biofilm (60–80 mm). Ali et al. (2013) Saprolegnia in treated biofilm and control. (a1) healthy, growing Saprolegnia sporangia (L) were not affected by the treatment as they were protected by the dense content of the biofilm. Dead Saprolegnia sporangia (D) and many dead hyphae stained red (a2) as the PI stain only permeate to the dead cell (not the healthy one). Controls (b1 and b2), all the hyphae are completely destroyed by the action of the treatment, the entire field stained red with PI; this is confirming the death of all Saprolegnia hyphae by the treatments. Ali et al. (2013) 131 Cao et al. (2013) characterised an oomycete water mould (strain SC) isolated from Prussian carp [Carassius gibelio (Bloch, 1782)] eggs suffering from saprolegniosis morphologically as well as from ITS rDNA sequence data. Initially identified as a Saprolegnia sp. based on its morphological features, the constructed phylogenetic tree using the neighbour joining method further indicated that the SC strain was closely related to Saprolegnia australis R. F. Elliott 1968 strain VI05733 (GenBank accession no. HE798564), and which could form biofilm communities as virulence factors. In addition, aqueous extracts from forty Chinese herbs were screened as possible antiSaprolegnia agents. Among them, a 1 g ml1 extract from Radix sanguisorbae was the most efficacious anti-Saprolegnia agent, indicated by the minimum inhibitory concentration that was as low as 256 mg L1. Relative survival of 73 and 88% was obtained against the SC strain in fish eggs at concentrations of 256 and 1280 mg L1 , respectively. 1. Morphological characteristics of the SC strain. (a) cylindrical zoosporangium (arrow); (b) sporangial renewal by internal proliferation (arrow); (c) saprolegnoid discharge of zoospores (Arrow); (d) immature oogonium (arrow). Cao et al. (2013) Eissa et al. (2013) reported mass mortalities of angelfish eggs accompanied with very low hatchability in a private ornamental fish farm in Egypt. Examined eggs were badly damaged by water mould that was decisively confirmed as Saprolegnia species. Presumptive identification of the ten retrieved isolates was initially suggestive of Saprolegnia species. Mycological investigations have revealed that only 7 out of 10 isolates were capable of producing sexual stages. Therefore, using molecular tools 132 such as PCR coupled with partial sequencing of inter-transcribed spacer (ITS) gene was one of the most important approaches to distinguish Saprolegnia parasitica from other water moulds. The sequences of ITS gene data derived from eight isolates showed 100% similarity with S. parasitica ATCC90312 sequence and the remaining two isolates were different in one nucleotide (99.9%). The phylogenetic analysis of ITS genes grouped the ten isolates with other S. parasitica in one clad. Further, to control such fungal infection, the efficacy of povidone iodine as surface disinfectant for angelfish and their fertilized eggs were tested. By trial, it was obvious that the obtained post-rinsing results were highly suggestive for the efficacy of povidone iodine as an efficient antifungal disinfectant for both fish and eggs. Sexual reproduction of the Saprolegnia parasitica The amplified fragments of the internal transcribed spacer (ITS) gene extracted from fish eggs isolates (n = 10) and the reference strains of Saprolegnia parasitica ATCC90213 (n = 1) using two ITS gene primers: (ITS1) 5′-TCCGTAGGTGAACCTGCGG-3′ and (ITS4) 5′-TCCTCCGCTTATTGATATGC3′ Lane M, Marker; lane 1, S. parasitica ATCC90213; and lanes 2–11, angelfish eggs isolates of S. parasitica. Eissa et al. (2013) Kim et al. (2013) identified Saprolegnia isolates from wild brook lamprey on the basis of their morphological and molecular characteristics. The isolates showed aseptic hyphae and clavate zoosporagium. Zoospores discharge was typically saprolegnoid. Neither oogomia nor antheridia was observed in this study. ITS sequence obtained from the isolate was compared with other Saprolegnia spp. to analyse their phylogenetic relationships. Results showed that the isolate belongs to clade I including Saprolegnia parasitica. Based on the asexual organs, zoospore discharge manner and ITS sequence analysis, the isolate was identified as S. parasitica. Morphological characteristics of Saprolegnia parasitica. Cotton like colony (A) and branched aseptic hyphae (B). Bar = 50 μm. Kim et al. (2013) 133 Light micrographs of Saprolegnia parasitica. Pyriform zoosporagium (A) and release of zoospores (B). Bar = 50 μm Kim et al. (2013) Phylogenetic relationships among 15 saprolegnia isolates based on internal transcribed spacer (ITS) sequence homology.The scale represents 0.01 nt substitutions per position Kim et al. (2013) Mohamad et al. (2013) carried out a study on 240 Clarias gariepinus fish collected from The River Nile and El- Ibrahemia canal, Assuit city and the around cities (20 fish /month). The period of study was carried out during October 2011 till the end of September 2012. The clinical finding of naturally infected fish included erosions, ulceration of skin, skin darkening, fin rot, petechial hemorrhage at different parts of the body, necrotic foci and growth of the fungl hyphe in different sites on the skin and fins. It's colour was from white to brown.Mycological examination of collected samples resulted in isolation of 1200 isolates from 240 fish in presence of 960 isolates as mixed cases. The incidence of Saprolegnia Sp. was 5%. 134 Non septated broad hyphae of Saprolegnia sp., Characteristic cotton –wool like growth of Saprolegnia, Mohamad et al. (2013) Hussein et al. (2013) isolated S. diclina from saprolegniosis outbreaks against immature stages of Nile tilapia, Oreochromis niloticus. The cumulative mortality rates of the tested fish exposed to high zoospore concentrations of S. diclina BSN-M003 were 100%. The histopathological changes associated with saprolegniosis lesions induced by S. diclina BSN-M003, were loss of the epidermis, edema of the hypodermis, different degrees of degenerative changes in the underling musculature and the fungal elements were observed penetrating the entire musculature. 1.Moribund immature Nile tilapia, O. niloticus, showing cotton-like mycelial growth after challenging with S. diclina BSN-M 003, 2. Immature Nile tilapia, O. niloticus, died 4 d after challenging with S. diclina BSN-M 003, showing lesions on tail fin and eyes Hussein et al. (2013) light destruction of the epidermal layer of immature Nile tilapia, O. niloticus exposed only to amimoni treatment. 6. Extensive oedema of the hypodermis radiating away from the invading mycelia of S. diclina BSN-M003 resulting in marked myofibrillar degeneration Hussein et al. (2013) 135 Hussein et al. (2013) van den Berg et al. (2013) focused in a review on the unique characteristics of two aquatic Oomycetes, Saprolegnia parasitica and Saprolegnia diclina with respect to their impact on aquaculture, animal health and the surrounding environment. The species characteristics, ecology, biology, infectivity and identification methods are described and the latest research insights are discussed. van den Berg et al. (2013) Vega-Ramírez et al. (2013) isolated 10 species of the family Saprolegniaceae from the fish farm “El Zarco”, State of México, obtained from samples of influent and effluent water of the farm and from infected eggs and individual fish of rainbow trout. Two species belong to the genus Achlya and 8 to Saprolegnia. Saprolegnia ferax is recorded for the first time for the State of México. Achlya ambisexualis, A. heterosexualis, S. australis, S. diclinous, S. glomerata, S. parasitica, S. terrestris, S. uliginosa and S. unispora are cited for the first time from Mexico. 136 Culture with melon seeds and in distilled water at 4° C. A), S. diclina . Cyst presented short hooked hair. B), S. parasitica . Cyst presented long, hooked hairs in bundles; the white arrow shows the hooked hairs. 100X. Vega-Ramírez et al. (2013) Culture with melon seeds and indistilled water at 4° C. A), A. ambisexualis . Spherical oogonium with more than 6 oospores eccentric. B), A. heterosexualis . Oogonium antheridial cells with 6 oospores . C), S. australis . Lateral spherical oogonium with more than 6 oospores subcentric. D), S. diclina . Spherical oogonium, wall unpitted, oospores centric. E), S. glomerata . Terminal oogonium, antheridial diclinous branch. F), S. terrestris . Lateral spherical oogonium, wall pitted androgynous, monoclinous and diclinous antheridial branches. G), S. uliginosa . Spherical oogonium with 5 oospores centric. H), S. unispora . Oogonium with 1 oospore, without antheridial branches. I), S. parasitica . Typical zoosporangia with mature zoospores. A-C, E, G- I: Barr= 20 μm. D, F: Barr= 25 μm. I: Barr= 100 μm. AA: androgynous antheridium, DA: diclinous antheridium, OC: oospores centric, OE: oospores eccentric, OS: oospores subcentric, AC: antheridial cells, PI: pitted, Z: zoospores, ZP: zoosporangia. Vega-Ramírez et al. (2013). Zahran et al. (2013) studied the oxidative stress response regarding the saprolegniosis. Nile tilapia fish were subcutaneously abraded and divided into four 137 groups: control group, abraded but not challenged, infected group, challenged with Saprolegnia. ferax (S. ferax) zoospores 2 × 104 for one week, potassium permanganate (KMnO4) group, challenged with S. ferax zoospores 2 × 104 for one week then treated with KMnO4 and lasted for 2 weeks after, Freund’s complete adjuvant (FCA) group challenged with S. feraxzoospores 2 × 104 for one week then treated with FCA and lasted for 2 weeks after and control positive group, challenged with S. ferax zoospores 2 × 104 for 3 weeks. Higher mortalities were recovered from the challenged group, which declined upon treatment compared to the continued increased rate in the control positive group. Oxidative stress indicators Nitric Oxide (NO), glutathione (GSH) and superoxide dismutase (SOD) were measured; biochemical parameters: total protein, albumin, globulin, aspartate aminotranseferase (AST), and alanine aminoranferase (ALT) were also measured. Sodium (Na+) and potassium (K+) levels were measured as indicators of Plasma osmolality. Almost of the measured parameters showed varying reduction significant levels in treatment groups compared with the infected and the control ones. Thus, this study revealed that KMnO4 have a protective role against oxidative stress response, furthermore our data provide evidence for the role of FCA in modulating the oxidative stress response and enhancing fish immune response against infections. Nile tilapia experimentally infected with 2x10 4 of S. ferax , showing cotton wool like masses on the body. Zahran et al., 2013 Chauhan et al. (2014) conducted a study to find out the pathogenicity of three species of fungi,viz. Aspergillus niger, Aphanomyces laevis and Saprolegnia parasitica isolated from Gold fish (Carrasius auratus) collected from pet shops. Experiment was conducted for 10 days period with concentration of 8x103 conidia/ml (A.niger), 125 zoospores/ml (A.laevis) and 2x103 zoospores /ml (S.parasitica). All the tested fungi were found pathogenic to fish. Among the three species of fungi tested, A. laevis and S. parasitica showed 100% mortality in fish, while A. niger showed 75% mortality. S. parasitica was found most virulent causing mortality within six days of experiment. Histopathological examination of fishes showed inflammation of epidermis , loss of epidermis and necrotized hypodermis. Degenerative changes were observed in musculature. Ulcerated skin showed mycotic granulommas. 138 . 1. white fungoid patches of S.parasitica. 2. Wet colony of S.parasitica on Soya bean seed. Chauhan et al. (2014) Granulomas formed after penetration of hyphae of S.parasitica., Fibrillar granulomas in larger view Chauhan et al. (2014) Minor et al. (2014) carried out a study to identify suitable antigens that could help generate a fish vaccine against Saprolegnia parasitica. Unexpectedly, antibodies against S. parasitica were found in serum from healthy rainbow trout, Oncorhynchus mykiss. The antibodies detected a single band in secreted proteins that were run on a one-dimensional SDS-polyacrylamide gel, which corresponded to two protein spots on a two-dimensional gel. The proteins were analysed by liquid chromatography tandem mass spectrometry. Mascot and bioinformatic analysis resulted in the identification of a single secreted protein, SpSsp1, of 481 amino acid residues, containing a subtilisin domain. Expression analysis demonstrated that SpSsp1 is highly expressed in all tested mycelial stages of S. parasitica. Investigation of other non-infected trout from several fish farms in the United Kingdom showed similar activity in their sera towards SpSsp1. Several fish that had no visible saprolegniosis showed an antibody response towards SpSsp1 suggesting that SpSsp1 might be a useful candidate for future vaccination trial experiments. Rezinciuc et al. (2014) investigated the aetiology of chronic egg mortality events occurring in farmed brown trout, Salmo trutta. A total of 48 isolates were obtained from eggs with signs of infection as well as from water samples. A molecular analysis based on nrDNA internal transcribed spacer (ITS) operational taxonomic units indicated that the majority of the isolates correspond to Saprolegnia australis. All isolates of S. australis exhibited the same random amplified polymorphic DNA (RAPD) band patterns suggesting that a single strain is implicated in egg infections. The isolates followed Koch postulates using trout eggs and fry. Under standard concentrations of bronopol commonly used in farms, these isolates could grow, but not sporulate. However, both growth and sporulation were recovered when treatment 139 was removed. This study shows that S. australis can infect and kill salmon eggs, and helps in defining oomycetes core pathogen Sandoval-Sierra et al. (2014) analyzed 961 sequences of internal transcribed spacer from main culture collection of Saprolegniales (461 sequences) and GenBank (500 sequences). For this purpose, they used two phylogenetic analyses, i.e, Maximum Parsimony and Bayesian inference, and also a clustering optimization analysis using arbitrary options regarding the distance threshold values and the clustering algorithm. Thus, they identified 29 DNA-based MOTUs in agreement with phylogenetic analyses of species. The molecular clusters supported the validity of 18 species of Saprolegnia and identify 11 potential new species. Based on this system, they listed a number of incorrectly named isolates from culture collections, misassigned species names to GenBank sequences, and type sequences for species. They concluded that GenBank represents the main source of errors for identifying species since it possesses a high number of misassigned sequences, and the presence of sequences with sequencing errors. The presented taxonomic diagnosis system might help setting the basis for a suitable identification of species in this economically important genus Sandoval-Sierra et al. (2014) investigated the main Saprolegniaspecies involved in saprolegniosis of salmonids in Chile, and their association with specific developmental stages of the host fish. For this purpose, they studied 244 isolates of Saprolegnia-affected Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), and king salmon (Oncorhynchus tshawytscha) from the salmon farming regions, using a recently developed identification strategy based on molecular taxonomical operational units. We found that the Saprolegnia species associated with diseased salmon were Saprolegnia australis, Saprolegnia delica, Saprolegnia diclina,Saprolegnia ferax, Saprolegnia parasitica and two new Saprolegnia species observed during this study. In order to determine whether there were any specific species associations with different stages in the fish life cycle, we applied mosaic plots and correspondence analyses for categorical data. These analyses showed a strong association of S. parasitica with samples from the adult stage of the fish (χ2 = 196.29,p < 0.0001), while the species S. australis, S. diclina and Saprolegnia sp. 2 were strongly associated with embryonic stages (eggs or alevins) (χ2 = 196.29, p < 0.0001). This work represents the first detailed molecular characterization of Saprolegnia species involved in saprolegniosis in Chile, and the first study showing specific association of differentSaprolegnia species with different stages in the salmonid life cycle Sandoval-Sierra et al. (2014) Saraiva et al. (2014) described the first application of transient gene silencing in Saprolegnia parasitica, a pathogenic oomycete that infects a wide range of fish, 140 amphibians, and crustaceans. A gene encoding a putative tyrosinase from S. parasitica, SpTyr, was selected to investigate the suitability of RNA-interference (RNAi) to functionally characterize genes of this economically important pathogen. Tyrosinase is a mono-oxygenase enzyme that catalyses the O-hydroxylation of monophenols and subsequent oxidation of O-diphenols to quinines. These enzymes are widely distributed in nature, and are involved in the melanin biosynthesis. Gene silencing was obtained by delivering in vitro synthesized SpTyr dsRNA into protoplasts. Expression analysis, tyrosinase activity measurements, and melanin content analysis confirmed silencing in individual lines. Silencing of SpTyr resulted in a decrease of tyrosinase activity between 38 % and 60 %, dependent on the level of SpTyr-expression achieved. The SpTyr-silenced lines displayed less pigmentation in developing sporangia and occasionally an altered morphology. Moreover, developing sporangia from individual silenced lines possessed a less electron dense cell wall when compared to control lines, treated with GFP-dsRNA. In conclusion, the tyrosinase gene of S. parasitica is required for melanin formation and transient gene silencing can be used to functionally characterize genes in S. parasitica. 141 Saprolegnia parasitica sporangia phenotypes from different individual lines treated with dsRNA.Phenotypes of individual lines of Saprolegnia parasitica treated with SpTyr-dsRNA were observed at 8 d after dsRNA-treatment. Individual silenced lines showed different phenotypes when compared with the control lines. Panels A and B (control lines) represent the normal morphology and colouration of young sporangia of S. parasitica. Panels C–F show some of the phenotypes observed in different SpTyr-dsRNA-treated lines, whereby some young sporangia have less pigmentation (C), or are smaller (D), or are elongated with less pigments (E) or sporangia that are pigmented only at the tip (F). Scale bar represents 100 μm. Saraiva et al. (2014) Transmission electron microscopy studies of dsRNA treated lines derived from wild-type strain CBS223.65. Panel A: A young sporangium of a gfp-dsRNA-treated line containing several nuclei (N) and fingerprint vesicles (FV), Scale bar represents 2 μm. Panel B: A young sporangium of a SpTyrdsRNA-treated line containing several nuclei (N), mitochondria (M) and fingerprint vesicles (FV) Scale bar represents 2 μm. Saraiva et al. (2014) Effect of SpTyr-silencing on Saprolegnia parasitica cell wall. The gene expression level for all individual lines was obtained by qPCR analysis using exogenous β-tubulin as housekeeping gene. The level of silencing achieved by the putatively silenced lines was transformed into percentage of remainingSpTyr-expression after silencing (in graph referred to as ‘SpTyr-expression’) having the minimal gene expression level obtained for the control lines (GFP-dsRNA) as 100 % non-silenced (upper left graph). A range of putatively SpTyr-silenced lines (panel A to D) as well as control lines (panel *) were prepared and analysed using TEM. This procedure revealed an electron dense layer in the cell wall (CW) of sporangia of the control lines (*) and a non-silenced line (D). The electron dense layer decreased in the cell wall of the sporangium with decreased levels of SpTyr-expression (images A–C). Saraiva et al. (2014) 142 Yahya et al. (2014) investigated causes of mass mortality in Cyprinus carpio eggs during peak- breeding season between April to July 2012 and 2013 at El-Abbassa Fish Hatchery, Sharkia Governorate. Physicochemical and microbial characteristics of culture water were examined during the induced breeding of Cyprinus carpio besides mycological and histopathological examination of egg samples with trial of treatment of different types of disinfectant. The physicochemical parameters were within the range of Cyprinus carpio eggs rearing. The total bacterial count fluctuated between 4.2 x 1010cfu/ml after 24 hrs of fertilization and 48 x 1010 cfu/ml after 4 days of incubation till hatching.The infected fertilized egg by saprolegnia spp. appeared as tuft hairy like balls with a white cottony envelope that surround it which focally invaded the cytoplasm resulted in loss of the cytoplasm content and destructed envelops. Treatment by sodium chloride at1.5 g L-1 for 60 min. daily for 4 days was showed significantly higher hatching and survival rates. Map showing the study area. Yahya et al. (2014) 143 Saprolegniosis in Cyprinus carpio fertilized egg: A. The fertilized eggs in the hatching containers (hapas) were surrounded by a cloudy white cottony envelope. B. All affected eggs appear as hairy balls with tuft hair like outgrowths. C. & D. Wet mounts of infected egg showing highly branched non septated hyphae with the presence of zoosporongia. Yahya et al. (2014) 144 Histopathological findings of Saprolegniosis infected Cyprinus carpio egg: A. Healthy egg showing 2 layers of egg envelopes (outer and inner) and eosinophilic cytoplasm, HE (Bar = 100 μm). B. Infected eggs showing numerous fungal hyphae attached to the outer surface of the whole eggs besides pores in the inner envelope, HE (Bar = 100 μm). C. Infected eggs showing fungal hyphae attached to the outer surface of the whole eggs focally invaded to the cytoplasm, HE (Bar = 100 μm). D. Infected eggs showing complete loss of the cytoplasm content and destructed envelops, HE (Bar = 100 μm). Yahya et al. (2014) Parra-Laca et al. (2015) used a preparation of hemolymph (HL) from adult female of D. coccus, to detect Saprolegnia sp. The hemolymph response, against the presence of the oomycete in vitro, was measured spectrophotometrically at 495 nm. The isolation strain of Saprolegnia sp. induced a reaction leading to the consumption of the pigment carminic acid, and was made from water and epidermic scrape of commercial fresh water fish tanks, obtaining a strain of Saprolegnia sp. identify by its´ reproductive structures and morphological characteristics. The strain of Saprolegnia sp. isolate induced reaction that leads the consumption of the pigment carminic acid, and as a consequence the formation of melanin, having the capacity to identify the presence of Saprolegnia from the amount of 5 to 282 zoospores. Seedlings at 2 weeks after sowing: showing period of insect infestation.A) sexual structures obtain from the isolated strain of Saprolegnia sp. A. Zoosporangium, (B) Primary zoospore, (C) Secondary zoospore, (D) Secondary zoospore with signs of germ tube, (E) Secondary germling cyst. ParraLaca et al. (2015) 145 Sequence response of granulocytes to Saprolegnia sp. elicitors. 1: Control granulocytes, with a typical spherical shape and carminic acid granules inside the cytoplasm. 2: Granulocyte incubated with Saprolegnia sp. with pigment consumption and degranualtion. 1: Control prove vial with the characteristically red pigmentation. 2: Prove vial with hemolymph of D.coccus and Saprolegnia sp. Spore suspension, presenting consumption of red pigment and a fibrilar aggregate precipitate. Parra-Laca et al. (2015) Sandoval-Sierra and Diéguez-Uribeondo (2015) proposed a standardized protocol for describing Saprolegnia spp. that includes good cultural practices and proper holotype preservation. In order to illustrate this new proposal, they described two species, Saprolegnia aenigmatica sp. nov. and Saprolegnia racemosa sp. nov., based on the recently described molecular operational taxonomic units (MOTUs), phylogenetic relationships, and the analyses of morphological features. They showed that they belong to two different MOTUs that are grouped into two sister clades. Morphologically, they found that S. racemosa exhibits a species-specific character, i.e., aggrupation of oogonia in racemes, while S. aenigmatica does not have any specific characters. Analyses of a combined set of characters, i.e., length and breadth of sporangia, length/breadth ratio (l/b) of oogonia, cyst and oospore diameter, and the number of oospores per oogomium, allow distinguishing these two species. To improve Saprolegnia taxonomy, they proposed to incorporate into the protologue: (i) several isolates of the new species; (ii) the rDNA sequences to compare them to databases of Saprolegnia sequences of reference; (iii) a phylogenetic analysis to check relationships with other species; (iv) to preserve holotypes in absolute ethanol and to include lyophilized material from holotype; and (v) the ex-type as a pure culture from singlespore isolates stored in at least two different collections. 146 147 Phylogenetic relationships of Saprolegnia aenigmatica sp. nov and Saprolegnia recemosa sp. nov based on ITS rDNA. Phylogenetic tree was obtained from Bayesian inference analysis based on ITS rDNA sequences. Phylogenetic tree show the relationships among S. aenigmatica, S. racemosa and related Saprolegnia species. The numbers the branches represent the probability values (>0.95) and bootstrap support (> 75) obtained from Bayesian inference and Maximum Likelihood analyses respectively. The analyses comprise reference sequences of genus Saprolegnia [12] and all Saprolegnia isolates obtained in this study. Sandoval-Sierra and Diéguez-Uribeondo (2015) They showed that, after preservation in absolute ethanol for 12 months, oogonia stalks, number and shape of oospores, and antheridial branch origin, did not vary from those measured in fresh samples. The only differences observed were in the antheridia and lipid droplet positions in the oospores. In preserved samples, some of the antheridia attached to the oogonium walls were occasionally found to be collapsed, and oospore lipid droplets were always disrupted. Preservation of morphological characters of Saprolegnia in absolute ethanol.The figure shows that the specific morphological features of S. aenigmatica (a and b) and S. racemosa (c and d) maintained after 12 months in absolute ethanol: oogonia globose (Og), oogonia subglobose (Os), oogonia pyriform (Op), oogonial stalk terminal (Ot), oogonial stalk intercalary (Oi), antheridial hypha (arrow). Compared to fresh material the antheridia attached to the oogonium walls were sometimes collapsed (arrowhead), and lipid droplets in the oospore became disrupted (Ld) preserved saples in ethanol. Bar = 20 μm. Sandoval-Sierra,J. V., J. Diéguez-Uribeondo, 2015 Shaheen et al. (2015) isolated Saprolegnia strain from infected goldfish,Carassius auratus, fingerlings. Fish showed extensive hyphal growth on the skin, fins, gills and eyes. The isolate was molecular identified through 18S rRNA gene and internal transcript spacer, ITS, sequencing. The isolate showed 18S rRNA gene nucleotide identity, 91.6%, and ITS homology with Saprolegnia parasitica. Scanning electron microscopy was applied for evaluating the pathogenicity for the retrieved isolate; which proved its high virulence accompanied with the high mortality rate reaching 100% of the infected fingerlings 148 (a) goldfish fingerling warped with cotton wool like hyphal mats,(b) Asexual reproduction showing; branched nonseptated hyphae & sporan gia filled with large number of spherical sporangiospores (arrow) (c) sexual reproduction showing; oogoniawith centric oospores, (d) germination of oospore with formation of Gemmae. Shaheen et al. (2015) PCR products of 18S rRNA gene and ITS region; M is 1k DNA Ladder Phylogentic tree of 18S rRNA sequence of the retrieved saprolegnia isol ate (accession number in gene bank HQ384412) Shaheen et al. (2015) 149 Scanning electron microscope; A,B) a newly germinating secondary zoospores of S. parasitica with germinating tube (black arrows) and appersoria (blue arrows) C) secondary zoospores of S. parasitica with globular adhesive materials around the zoospore (blue arrows) with hair like tuft on the secondary cyst (black arrow) D) deep inclusion of the emerging tubes into the skin, note the accumulation of the adhesive materials around the germinating tube (arrow). Shaheen et al. (2015) Thoen et al. (2015) recovered 89 Saprolegnia isolates from water, eggs and salmon tissue samples that originated from salmon (Salmo salar) hatcheries along the coast of Norway. The cultures were characterized morphologically and molecularly in order to provide an overview of the species composition of Saprolegnia spp. present in Norwegian salmon hatcheries. We demonstrate that S. diclina clearly dominated and contributed to 79% of the recovered isolates. Parsimony analyses of the nuclear ribosomal internal transcribed spacer (ITS) region split these isolates into 2 strongly supported sub-clades, S. diclina sub-clade IIIA and IIIB, where sub-clade IIIB accounted for 66% of all isolates. A minor portion of the isolates constituted other taxa that were either conspecific or showed strong affinity to S. parasitica, S. ferax, S. hypogyna and Scoliolegnia asterophora. The unique sub-clade IIIB of S. diclina was most prevalent in water and salmon eggs, while S. parasitica isolates were more frequently isolated from post hatching stages. The study demonstrated that morphological criteria in many cases were insufficient for species delimitation due to lack of sexual structures or incoherent morphological expression of such features within the tested replicates. Songe et al. (2016) addressed the morphological changes of eyed eggs of Atlantic salmon, Salmo salar L. infected with Saprolegnia from a commercial hatchery and after experimental infection. Eyed eggs infected with Saprolegnia spp. from 10 Atlantic salmon females were obtained. Egg pathology was investigated by light and scanning electron microscopy. Eggs from six of ten females were infected with S. parasitica, and two females had infections with S. diclina clade IIIA; 150 two Saprolegnia isolates remained unidentified. Light microscopy showed S. diclina infection resulted in the chorion in some areas being completely destroyed, whereas eggs infected with S. parasitica had an apparently intact chorion with hyphae growing within or beneath the chorion. The same contrasting pathology was found in experimentally infected eggs. Scanning electron microscopy revealed that S. parasitica grew on the egg surface and hyphae were found penetrating the chorion of the egg, and re-emerging on the surface away from the infection site. The two Saprolegnia species employ different infection strategies when colonizing salmon eggs. Saprolegnia diclina infection results in chorion destruction, while S. parasitica penetrates intact chorion. We discuss the possibility these infection mechanisms representing a necrotrophic (S. diclina) vs. a facultative biotrophic strategy (S. parasitica). Histology of normal (a) and histopathology of infected eggs (b–f). (a) Healthy chorion separated into an outer thin layer (co; insert) and inner thicker layer (ci). The cell-rich layer inside of the chorion is likely part of the blastoderm. (b) Hyphae of S. parasitica are located inside the chorion, which shows minor changes. The hyphae are located in the mid-part and towards the yolk granules (arrows). Details of the hyphae are shown in the insert (arrow). (c) S. parasitica infection with moderate chorion changes. Numerous hyphae on the outside of the egg, and there are several pores and vacuoles seen in the chorion wall (arrows). (d) Higher magnification of (c) detailing the vacuoles and the cracks in the chorion (arrow). Note numerous hyphae. (e) S. diclina infection, with moderate-to-severe changes of 151 the chorion (and cytoplasm). Germinated cysts present below the cracked chorion and inside the egg (arrow). (f) S. diclina infection, severe chorion changes. Almost a complete wipeout of the chorion in some areas and with thinner chorion than normal in others. Chorion is also discontinuous and changes are associated with the presence of hyphae (arrow). Bars = 50 μm. Songe et al. (2016) LEFT: SEM of an infected egg. (a) Egg cracked open during processing, separating the outer and inner layer, thus exposing the inner surface. Bar = 1 mm. (b)Saprolegnia parasitica hyphae invading the inner surface of the outer layer of the chorion (arrows). Bar = 20 μm. Right: SEM of an air-dried infected egg (in the middle) Bar = 1 mm. A close-up of the areas marked a (upper) and b (lower) shows Saprolegnia parasitica cysts (arrows) and hyphae growing on the outer surface of the egg. Cysts (arrows) are also visible. Hyphae are seen penetrating into the chorion (circles). Bars a and b = 20 μm. Songe et al. (2016) (a) Saprolegnia parasitica-infected egg showing an intact or moderately disrupted chorion and with hyphae located on the outside and/or on the inside of an intact chorion and chorion changes scored as ‘mild’. Remnants of hyphae were seen on the outside of the chorion (arrowheads) without the outer membrane loosing its continuity. (b)Saprolegnia diclina infection with the outer chorion membrane disrupted and with disintegration of the inner chorion membrane. 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New records of Saprolegniaceae isolated from rainbow trout, from their eggs, and water in a fish farm from the State of México. Revista Mexicana de Biodiversidad 84: 637-649, 2013 41. Wood, S.E. and Willoughby, L.B.(1986):Ecological observations on the fungal colonization of fish by Saprolegniaceae in Windermere.J. Appl. Ecol., 23, (3), 737749. 42. Xu, D. and Rogers, W.A. (1991):Electron microscopy of infection by Saprolegnia spp in channel catfish. J. Aquat Anim. Hlth 3:63-69. 43. Yahya M.; 2 Rasha M. Reda*;3 Eletreby S. Case Study on Mass Mortality Problem of Cyprinus carpio Eggs in El- Abbassa Fish Hatchery in Egypt. International Journal of Research in Fisheries and Aquaculture 2014; 4(1): 8-13 44. Zahran E, Risha E. Protective role of adjuvant and potassium permanganate on oxidative stress response of Nile tilapia (Oreochromis niloticus) challenged with Saprolegnia ferax. SpringerPlus. 2013;2(1):94. doi:10.1186/2193-1801-2-94. 155 2. Achlya Historical                Carus (1823) described a fungus on Salamander larvae with spores collected in the form of a globe at the mouth of a sporangium and called it Hydronema. Nees von Esenbeck (1823) gave the name Achlya to the (Carus’s fungus) and separated it from Saprolegnia (Gruithuisen’s fungus) on the distinctive difference in the escape of the zoospores, which are recognized as their salient features even today. Nees described Achlya prolifera Nees (1823) Hildebrand (1867) described Achlya racemosa Pringsheim (1882) described Achlya colorata Humphrey (1893) described Achlya americana, Achlya debaryana and Achlya papillosa Maurizio (1899) described Achlya radiosa Kaufmann (1906 described Achlya treleaseana Coker (1910) described Achlya caroliniana Coker (1912) described Achlya glomerata Coker (1915) described Achlya klebsiana Coker (1923) described Achlya conspicua, Achlya dubiam Achlya flagellata, Achlya imperfecta and Achlya proliferoides Historically, the genus Achlya has been divided into different groups or subgenera (Coker 1923): o Sub-genus Centroachlya  Racemosa Group A. racemose, A. hypogyna, A. colorata, .4. radiosa o SuB-GENUS Euachlya  Prolifera Group A. Americana, A. deBaryana, .1. Orion, A. prolifera, A. proliferoides, A. flagellate, A. imperfecta, A. Klchsiana, A. caroliniana  Apicidata Group A. apicidata, A. megasperma, A. polyaiidra, A. conspicna, A. oblongata, A. rccurva o Sub-genus Glomeroachlya A. glomerata o Sub-genus Thraustoachlya A. dubia Coker & Braxton (1926) described Achlya abortiva and Achlya subterranea Coker & Couch (1927) ) described Achlya bisexualis Coker & Braxton (1927) ) described Achlya inflata 156        Coker & J. Leitner (1938) ) describedAchlya regularis Raper (1939) ) described Achlya ambisexualis F.T. Wolf (1941) ) described Achlya rodrigueziana Hamid (1942) described Achlya androcomposita Harvey (1942) described Achlya diffusa Reischer (1949) ) described Achlya sparrowii Johnson (1950) ) described Achlya spiracaulis     Whiffen (1965) described Achlya heterosexualis Dayal & J. Thakur (1969) ) described Achlya aquatica Chiou & Chang (1974) described Achlya formosana Steciow (2001) described Achlya ambispora 157       Steciow & Elíades (2002) described Achlya robusta Dick & Spencer (2002) described Achlya truncatiformis Johnson et Seymour (2005) described Achlya androgyna Johnson et Seymour (2005) described Achlya irregularis El Androusse et al (2006) described Achlya abortispora Paul & Steciow described (2008) Achlya spiralis  Beakes et al. (2014) proposed Achlyaceae fam. nov. which aggregates four genera, all with eccentric oospores in the “achlyoid/thraustothecoid clade”: o Achlya sensu stricto, o Brevilegnia, o Dictyuchus o Thraustotheca Jesus et al. (2015) described Achlya catenulate  Gordon W Beakes Mark A. Spencer Classification   Achlya has approximately 50 valid species (Johnson et al. 2002, El Androusse et al. 2006, Paul & Steciow, 2008, Kirk et al. 2008). On searching the internet, two genera of Achlya were found, one belongs to the Animalia which was is concerned with insects: Achlya, Billberg, 1820, e.g. Achlya flavicornis, Linnaeus 1758 and the other belongs to the Kingdome Protista which is concerned with the water moulds, Achlya C. G. D. Nees in Carus, 1823 Species 2000 & ITIS Catalogue of Life: April 2013 Species  Animalia + o 158  Arthropoda + Insecta +  Lepidoptera +  Drepanoidea +  Drepanidae +  Achlya , Billberg, 1820  Achlya flavicornis Linnaeus 1758  Achlya flavicornis finmarchica Schoyen, 1881      Achlya flavicornis jezoensis Matsumura, 1927 Achlya flavicornis meridionalis Wolfsberger, 1968 Achlya flavicornis scotica Tutt, 1888 Achlya kuramana Matsumura 1933 Achlya longipennis Inoue 1972 + Achlya flavicornis  o Protista + Heterokontophyta +  Oomycetes +  Saprolegniales +  Saprolegniaceae Kütz. ex Warm., 1884 +  Achlya C. G. D. Nees in Carus, 1823  Achlya ambisexualis Raper 1939  Achlya americana Humphrey 1892  Achlya apiculata de Bary 1888  Achlya aquatica Dayal & J. Thakur 1969  Achlya benekei J. S. Furtado 1965  Achlya bisexualis Coker & Couch 1927  Achlya bispora (Couch) Langsam 1986  Achlya brasiliensis A. I. Milanez 1965  Achlya caroliniana Coker 1910  Achlya colorata Pringsh. 1882 29 more... show full tree. 1. EOL Achlya ambisexualis http://eol.org/pages/19037/hierarchy_entries/36121516/names Protista +  Heterokontophyta + o  Oomycetes +  Saprolegniales +  Saprolegniaceae Kütz. ex Warm., 1884 + Achlya C. G. D. Nees in Carus, 1823 1. Achlya ambisexualis Raper 1939 2. Achlya americana Humphrey 1892 3. Achlya androgyna (W. Archer) T.W. Johnson & R.L. Seym. 2005 4. Achlya aquatica Dayal & J. Thakur 1969 5. Achlya bisexualis Coker & Couch 1927 6. Achlya bispora (Couch) Langsam 1986 7. Achlya caroliniana Coker 1910 8. Achlya colorata Pringsh. 1882 9. Achlya conspicua Coker 1923 159 10. Achlya crenulata Ziegler 1948 11. Achlya debaryana Humphrey 1893 12. Achlya dubia Coker 1923 13. Achlya flagellata Coker 1923 14. Achlya glomerata Coker 1912 15. Achlya heterosexualis Whiffen 1965 16. Achlya imperfecta Coker 1923 17. Achlya intricata Beneke 1948 18. Achlya klebsiana Pieters 1915 19. Achlya lobata Ziegler & Gilpin 1954 20. Achlya orion Coker & Couch 1920 21. Achlya oviparvula A. L. Rogers & Beneke 1962 22. Achlya papillosa Humphrey 1892 23. Achlya primoachlya (Coker & Couch) T.W. Johnson & R.L. Seym. 2005 24. Achlya prolifera Nees 1823 25. Achlya proliferoides Coker 1923 26. Achlya pseudoradiosa A. L. Rogers & Beneke 1962 27. Achlya racemosa Hildebr. 1867 28. Achlya radiosa Maurizio 1899 29. Achlya sparrowii Reischer 1949 30. Achlya turfosa Johannes 1950 2. Integrated Taxonomic Information System (ITIS) http://www.itis.gov/servlet/SingleRpt/SingleRpt Kingdom Fungi Achlya – accepted 1. Achlya ambisexualis Raper 2. Achlya americana Humphrey 3. Achlya apiculata de Bary 4. Achlya benekei J. S. Furtado 5. Achlya bisexualis Coker & Couch 6. Achlya caroliniana Coker 7. Achlya colorata Pringsh. 8. Achlya crenulata Ziegler 9. Achlya diffusa 10. Achlya echinulata 11. Achlya flagellata Coker 12. Achlya inflata Coker & Braxton 13. Achlya klebsiana Pieters 14. Achlya nowickii Racib. 15. Achlya oviparvula A. L. Rogers & Beneke 16. Achlya polyandra Hildebr. 17. Achlya prolifera Nees 18. Achlya racemosa Hildebr. 19. Achlya radiosa Maurizio 20. Achlya sparrowi Reischer 3. NCBI Taxonomy Eukaryota + http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi 160  Stramenopiles + o  Oomycetes +  Saprolegniales +  Saprolegniaceae + Achlya 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. Achlya ambisexualis Achlya americana Achlya apiculata Achlya aquatica Achlya bisexualis Achlya caroliniana Achlya colorata Achlya conspicua Achlya crenulata Achlya dubia Achlya flagellata Achlya glomerata Achlya heterosexualis Achlya hypogyna + Achlya intricata Achlya klebsiana Achlya oblongata Achlya oligacantha Achlya ornata Achlya papillosa Achlya primoachlya Achlya prolifera Achlya proliferoides Achlya racemosa Achlya radiosa Achlya recurva Achlya rodrigueziana Achlya sparrowii Achlya spinosa Achlya stellata Achlya treleaseana 4. Index Fungorum Accepted species 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Achlya abortispora B. Paul & El Andr. (2006); Saprolegniaceae Achlya abortiva Coker & Braxton (1926); Saprolegniaceae Achlya achlyoides (Coker) T.W. Johnson & R.L. Seym. (2005); Saprolegniaceae Achlya ambisexualis Raper (1939); Saprolegniaceae Achlya ambispora Steciow (2001); Saprolegniaceae Achlya americana Humphrey (1893); Saprolegniaceae Achlya androcomposita Hamid (1942); Saprolegniaceae Achlya androgyna (W. Archer) T.W. Johnson & R.L. Seym. (2005); Saprolegniaceae Achlya anomala Steciow & Marano (2008); Saprolegniaceae Achlya aquatica Dayal & J. Thakur (1969); Saprolegniaceae 161 11. 12. 13. 14. 15. 16. 17. 18. Achlya bisexualis Coker & Couch (1927); Saprolegniaceae Achlya bispora (Couch) Langsam (1986); Saprolegniaceae Achlya bonariensis Beroqui (1969); Saprolegniaceae Achlya californica J.V. Harv. (1942); Saprolegniaceae Achlya caroliniana Coker (1910); Saprolegniaceae Achlya colorata Pringsh. (1882); Saprolegniaceae Achlya conspicua Coker (1923); Saprolegniaceae Achlya crenulata Ziegler (1948); Saprolegniaceae 19. Achlya debaryana Humphrey (1893); Saprolegniaceae 20. Achlya diffusa J.V. Harv. ex T.W. Johnson (1956); Saprolegniaceae 21. Achlya dubia Coker (1923); Saprolegniaceae 22. Achlya echinulata Beroqui (1969); Saprolegniaceae 23. Achlya flagellata Coker (1923); Saprolegniaceae 24. Achlya flexuosa Nagai (1931); Saprolegniaceae 25. Achlya formosana Chiou & H.S. Chang (1976); Saprolegniaceae 26. Achlya fuegiana Steciow (2001); Saprolegniaceae 27. Achlya glomerata Coker (1912); Saprolegniaceae 28. Achlya haehneliana Cejp (1934); Saprolegniaceae 29. Achlya heteromorpha J.V. Harv. (1942); Saprolegniaceae 30. Achlya heterosexualis Whiffen (1965); Saprolegniaceae 31. Achlya imperfecta Coker (1923); Saprolegniaceae 32. Achlya inflata Coker & Braxton (1927); Saprolegniaceae 33. Achlya intricata Beneke (1948); Saprolegniaceae 34. Achlya kamatii Date (1972); Saprolegniaceae 35. Achlya kashyapia Chaudhuri & Kochhar (1935); Saprolegniaceae 36. Achlya klebsiana Pieters (1915); Saprolegniaceae 37. Achlya lobata Ziegler & Gilpin (1954); Saprolegniaceae 38. Achlya orion Coker & Couch (1920); Saprolegniaceae 39. Achlya oryzae S. Ito & Nagai (1931); Saprolegniaceae 40. Achlya pacifica J.V. Harv. (1942); Saprolegniaceae 41. Achlya papillosa Humphrey (1893); Saprolegniaceae 42. Achlya michiganensis T.W. Johnson (1950); Saprolegniaceae 43. Achlya pinnulata J.V. Harv. (1942); Saprolegniaceae 44. Achlya primoachlya (Coker & Couch) T.W. Johnson & R.L. Seym. (2005); Saprolegniaceae 45. Achlya prolifera Nees (1823); Saprolegniaceae 46. Achlya proliferoides Coker (1923); Saprolegniaceae 47. Achlya pseudoachlyoides (Beneke) T.W. Johnson & R.L. Seym. (2005); Saprolegniaceae 48. Achlya pseudoradiosa A.L. Rogers & Beneke (1962); Saprolegniaceae 49. Achlya racemosa Hildebr. (1867); Saprolegniaceae 50. Achlya radiosa Maurizio (1899); Saprolegniaceae 51. Achlya regularis Coker & J. Leitn. (1938); Saprolegniaceae 52. Achlya robusta Steciow & Elíades (2002); Saprolegniaceae 53. Achlya rodrigueziana F.T. Wolf (1941); Saprolegniaceae 54. Achlya sparrowii Reischer (1949); Saprolegniaceae 55. Achlya spiracaulis T.W. Johnson (1950); Saprolegniaceae 56. Achlya spiralis B. Paul & Steciow (2008); Saprolegniaceae 57. Achlya subterranea Coker & Braxton (1926); Saprolegniaceae 58. Achlya truncatiformis M.W. Dick & Mark A. Spencer (2002); Saprolegniaceae 59. Achlya tuberculata Ziegler (1950); Saprolegniaceae 60. Achlya turfosa Johannes (1950); Saprolegniaceae 162 Species given other names Achlya abortiva f. abortiva Coker & Braxton (1926), (= Achlya abortiva); Saprolegniaceae Achlya abortiva f. normalis Coker (1927), (= Achlya abortiva); Saprolegniaceae Achlya acadiensis C.L. Moore (1912), (= Achlya androgyna); Saprolegniaceae Achlya ambisexualis var. abjointa Raper (1939), (= Achlya ambisexualis); Saprolegniaceae Achlya ambisexualis var. ambisexualis Raper (1939), (= Achlya ambisexualis); Saprolegniaceae Achlya ambisexualis var. gracilis Raper (1939), (= Achlya ambisexualis); Saprolegniaceae Achlya americana var. americana Humphrey (1893), (= Achlya americana); Saprolegniaceae Achlya americana var. cambrica Trow, (= Achlya americana); Saprolegniaceae Achlya americana var. megasperma Crooks (1937), (= Achlya americana); Saprolegniaceae Achlya americana var. megasperma Crooks ex Cejp (1959), (= Achlya americana); Saprolegniaceae Achlya apiculata de Bary (1888), (= Newbya apiculata); Saprolegniaceae Achlya apiculata var. apiculata de Bary (1888), (= Newbya apiculata); Saprolegniaceae Achlya apiculata var. forbesiana Cejp (1959), (= Newbya apiculata); Saprolegniaceae Achlya apiculata var. prolifica Coker & Couch (1923), (= Newbya apiculata); Saprolegniaceae Achlya benekei J.S. Furtado (1965), (= Protoachlya benekei); Saprolegniaceae Achlya bisexualis var. ambisexualis (Raper) Milko (1983), (= Achlya ambisexualis); Saprolegniaceae Achlya bisexualis var. bisexualis Coker & Couch (1927), (= Achlya bisexualis); Saprolegniaceae Achlya brasiliensis A.I. Milanez (1965), (= Newbya brasiliensis); Saprolegniaceae Achlya braunii Reinsch (1877), (= Achlya androgyna); Saprolegniaceae Achlya cambrica (Trow) T.W. Johnson (1956), (= Achlya americana); Saprolegniaceae Achlya curvicollis Beroqui (1969), (= Newbya curvicollis); Saprolegniaceae Achlya debaryana var. americana (Humphrey) Minden (1912), (= Achlya americana); Saprolegniaceae Achlya debaryana var. debaryana Humphrey (1893), (= Achlya debaryana); Saprolegniaceae Achlya debaryana var. intermedia Minden (1912), (= Achlya debaryana); Saprolegniaceae Achlya diffusa J.V. Harv. (1942); Saprolegniaceae Achlya dubia var. dubia Coker (1923), (= Achlya dubia); Saprolegniaceae Achlya dubia var. pigmenta Chaudhuri & Kochhar (1935), (= Achlya dubia); Saprolegniaceae Achlya flagellata var. flagellata Coker (1923), (= Achlya flagellata); Saprolegniaceae Achlya flagellata var. yezoensis S. Ito & Nagai (1931), (= Achlya flagellata); Saprolegniaceae Achlya klebsiana var. indica Chaudhuri & Kochhar (1936), (= Achlya klebsiana); Saprolegniaceae Achlya klebsiana var. klebsiana Pieters (1915), (= Achlya klebsiana); Saprolegniaceae Achlya lignicola Hildebr. (1867), (= Achlya racemosa); Saprolegniaceae Achlya megasperma Humphrey (1893), (= Newbya megasperma); Saprolegniaceae Achlya mucronata Ziegler (1958), (= Protoachlya mucronata); Saprolegniaceae Achlya oblongata de Bary (1888), (= Newbya oblongata); Saprolegniaceae Achlya oblongata var. gigantea E.J. Forbes (1935), (= Newbya oblongata); Saprolegniaceae Achlya oblongata var. globosa Humphrey (1892), (= Newbya oblongata); Saprolegniaceae Achlya oblongata var. oblongata de Bary (1888), (= Newbya oblongata); Saprolegniaceae 163 Achlya oligocantha de Bary (1888), (= Newbya oligocantha); Saprolegniaceae Achlya oligocantha var. brevispina Schkorb. (1923), (= Newbya oligocantha); Saprolegniaceae Achlya oligocantha var. oligocantha de Bary (1888), (= Newbya oligocantha); Saprolegniaceae Achlya ornata T.W. Johnson & R.L. Seym. (2005), (= Newbya pascuicola); Saprolegniaceae Achlya paradoxa Coker (1914), (= Isoachlya paradoxa); Saprolegniaceae Achlya polyandra sensu de Bary, (= Achlya debaryana); Saprolegniaceae Achlya polyandra Hildebr. (1867), (= Newbya polyandra); Saprolegniaceae Achlya racemosa f. maxima Minden, (= Achlya racemosa); Saprolegniaceae Achlya racemosa f. polyspora Schkorb. (1923), (= Achlya racemosa); Saprolegniaceae Achlya racemosa f. racemosa Hildebr. (1867), (= Achlya racemosa); Saprolegniaceae Achlya racemosa var. lignicola (Hildebr.) Cornu (1880), (= Achlya racemosa); Saprolegniaceae Achlya racemosa var. maxima (Minden) Cejp (1959), (= Achlya racemosa); Saprolegniaceae Achlya racemosa var. racemosa Hildebr. (1867), (= Achlya racemosa); Saprolegniaceae Achlya racemosa var. spinosa (de Bary) Cornu (1880), (= Newbya spinosa); Saprolegniaceae Achlya racemosa var. stelligera Cornu (1880), (= Achlya racemosa); Saprolegniaceae Achlya recurva Cornu (1880), (= Newbya recurva); Saprolegniaceae Achlya spinosa de Bary (1882), (= Newbya spinosa); Saprolegniaceae Achlya stellata de Bary (1888), (= Newbya stellata); Saprolegniaceae Achlya stellata var. multispora J.N. Rai & J.K. Misra (1977), (= Protoachlya mucronata); Saprolegniaceae Achlya stellata var. stellata de Bary (1888), (= Newbya stellata); Saprolegniaceae Achlya treleaseana (Humphrey) Kauffman (1906), (= Achlya androgyna); Saprolegniaceae  Biological characteristics of Achlya species      Achlya isolates are able to grow at a temperature range of 5–35ºC, presenting optimum temperatures of 25–35ºC and rapid, maximal growth at 30ºC. Achlya isolates show maximal growth in GY agar without NaCl (0%) and were able to grow at concentrations up to 1.5% NaCl. Achlya isolates grow in GY broth over a wide range of pH levels from 4.0– 11.0, displaying an optimal pH of 6.0–8.0. Zoospores of isolates Achlya isolates are able to germinate in GY broth over a wide range of pH levels from 4.0–11.0 after 24 h of incubation. Histopathology: Microscopic examination of the skin lesions of infected tilapia show cellular debris and necrosis of epidermal cells associated with the invasion of aseptate hyphae. These hyphae are thick with a diameter of approximately 11.7–30.2 µm. The epidermal tissues are covered by the hyphae and some hyphae penetrate into the epidermis. Hyphae are not easily visible following H & E staining, but are clearly recognizable using PAS. The following species, were found to have a parasitic mode of life on fish: o Achlya americana (cf. Scott and Warren 1964), o Achlya dubia (cf. Bhargava et al. 1971), o Achlya klebsiana (cf. Vishniac and Nigrelli 1957), 164 o Achlya prolifera (cf. Srivastava 1976, Srivastava and Srivastava 1977). o Studies have revealed that these zoosporic fungus species grow on the eggs of numerous freshwater fish species .  Achlya ambisexualis, A. americana, A. caroliniana, A. crenulata, A. debaryana, A. diffusa, A. dubia, A. intricata, A. klebsiana, A. oblongata, A. oligacantha, A. orion, A. polyandra, A. prolifera, A. proliferoides, A. radioasa, A. rodrigueziana, A. treleaseana, Description of Achlya species 1. Achlya abortispora El Androusse et al., 2006 Distinguishing characteristics of A. abortispora are the production o long fusiform sporangia with achlyoid and aplanoid discharge of zoospores; smooth-walled spherical to club-shaped oogonia, which are usually lateral, but at times intercalary, containing 1 to 20 oospheres. The oogonia can also bear 1 to 5 appendages, which may indicate oogonial proliferation. Most of the oospheres do not mature and are thus abortive. The antheridial branches supplying the oogonia are predominantly diclinous, but at times these may be monoclinous and androgynous. Antheridial branches coil and wrap around the oogonia. 1. (A, B) Sporangia with spores clustering at the orifice in a spherical mass (achlyoid discharge). Scale bar = 50 lM for 1A and 200 lM for 1B. (C) Catenulate gemmae. (D) Terminal oogonia. Scale 165 bar = 50 lM. Scale bar = 200 lM (D-E) Terminal oogonia, (F) Lateral oogonia, (G) Different shapes of lateral oogonia. Scale bar = 50 lM. g. 2. (A, B) Oogonia supplied with appendages. (C) Oogonia and androgynous antheridia. (D) Lateral oogonia containing one oosphere and diclinous antheridia. (E) Lateral oogonia with diclinous and monoclinous antheridia. Scale bar for all = 50 lM. El Androusse et al., 2006 3. (A, B) Achlyoid discharge of zoospores. Scale bar = 200 lM. (C, D) Sporangia with spores germinating in situ (aplanoid discharge). Scale bar = 200 lM for 3C and 80 lM for 3D. (E-G) Catenulate to irregular gemmae. Scale bar 200 lM for 3E and 80 lM for 3F-G. (H) Zoospore encysting and germinating within sporangia. Scale bar = 25 lM. 4. (A, B) Terminal oogonia with two appendages. Scale bar = 200 lM for 4A and 50 lM for 4B. (C) Intercalary oogonia. Scale bar = 200 lM. (D) Terminal oogonia containing one oosphere, the antheridia are clasping the oogonium in a complicated knot. Scale bar = 80 lM. (E) Lateral oogonia with diclinous and monoclinous antheridia. Scale bar = 200 lM. (F) Immature oogonia wrapped around with diclinous antheridia. Scale bar = 80 lM. (G) Terminal oogonia completely wrapped by antheridia. Scale bar = 80 lM. (H) Aborting oospheres not filling the oogonium. Scale bar = 50 lM El Androusse et al.,. 2006 2. Achlya ambisexualis Raper 1939 Synonyms: Achlya Achlya Achlya Achlya ambisexualis var. abjointa Raper, 1939 ambisexualis var. ambisexualis Raper, 1939 ambisexualis var. gracilis Raper, 1939 bisexualis var. ambisexualis (Raper) Milko, 1983 Mycellium of oogonial tallus dense, extensive; hyphae strout, branched. Sporangia clavate, renewed sympodially; 210-500 × 21-48 μm. Gemmae abundant cylindrical, occasionally irregular; terminal or intercalary, single or catenulate. Mycelium of antheridial thallus diffuse; hyphae moderately stout, branched. Sporangia sparse, 166 cylindrical, fusiform; renewed sympodially; 160-590 × 16-41 µm. Gemmae cylindrical. Spores monomorphic in both thalli; discharge and behavior achlyoid; primary spore cysts 9-14 µm in diameter; spore cluster persisting at exit orifice or disintegrating in part. Oogonia lateral or terminal; spherical or obpyriform,(33-) 50-85 (-110) µm in diameter. Oogonial wall pitted under the region of antheridial cell attachment; smooth. Oospores almost always maturing; eccentric; spherical; (1-) l0-l8 (-36) per oogonium, and generally filling it; (14-) 18-24 (-38) µm in diameter; germination not observed. Antheridial branches arising from one hypha ("male"); long, slender, irregular, and abundantly branched; often wrapping about the oogonium and its attendant hypha; persisting. Antheridial cells compound; tubular, branched or unbranched; persisting; attached in a digitate fashion or laterally; fertilization tubes not observed. Remarks. A. ambisexualis is a dioecious species and can be distinguished by its oospheres predominantly maturing, oospores 18-24 µm in diameter, generally 1-18 per oogonium and gemmae cylindrical in both antheridial and oogonial mycelia. A. bisexualisis is also a dioecious species, and is differentiated by its oospheres infrequently to rarely maturing, oospores 22-26 µm in diameter, generally 5-10 per oogonium, and gemmae in oogonial mycelia spherical to shortcylindrical and spherical to cylindrical in antheridial mycelia. A. heterosexualis can be a dioecious or monoecious species and it is separated from both A. ambisexualis and A. bisexualis by producing monoclinous and androgynous as well as, diclinous antheridial branches. A. ambisexualisis is distributed in Africa, China, India, British Isles, South America, Canada and USA (Johnson et al., 2002); therefore this species is cited for the first time in Mexico. A) Achlya ambisexualis. Mature oogonium; wall pitting pattern; digitate antheridial cell attachment; oospores eccentric B), A. ambisexualis. Oogonium with antheridials cells with 6 oospores. Barr= 20 μm Vega-Ramírez et al., 2013 3. Achlya ambispora Steciow, New Zealand Journal of Botany, 2001, Vol. 39: 277-283 167 Mycelium densum, cultura in seminibus Cannabis sativae, 1-3 cm diam. Hyphae ramosae, pleraque 24-106 pm late in base. Sporangia copiosa in culturis juvenilibus, filiform vel naviculata, 250- 650(-995) pm longa et 15-44 pm lata, sympodia vel basipeta. Ejecto sporarum pro genus typica, spori globosi 10-12 pm. Gemmae frequentis. Oogonia copiosa, sphaerica, pyriform, doliiform, rarissimo apiculata, (30-)48-80(-110) pm diam ramulus lateralibus, intercalaribus vel terminalibus provenientia, 12-114 pm diam. Paries oogoni sine projectionibus, oospori 1-6(-22) per oogonium, centrici (80%) ad subcentrici (20%), (16-)20-27(- 29) pm diam. Ramulus antheridialis, ramosus, plerumque origine androgyna (60%) sed interdum monoclina (30%) et diclina (10%). Fig. 1-6 Achlya ambispora. Fig. 1 Mycelium with oogonia and monoclinous and androgynous antheridial branches, PCM. Fig. 2 Terminal and intercalary gemmae, PCM. Fig. 3 Oogonia with diclinous and androgynous antheridial branches, PCM. Fig. 4 Oogonia proliferating in chains, PCM. Fig. 5 Zoosporangia, PCM. Fig. 6 Intercalary oogonia with monoclinous antheridial branch. Scale bars: Fig. 1-5 = 100 pin; Fig. 6 = 50 pin. Fig. 7-14 Achlya ambispora. Fig. 7 Detail of androgynous and inonoclinous antheridial branches. Fig. 8-10 Androgynous antheridial branches; antheridia apically appressed. Centric-subcentric oospores. Fig. 11 Lateral apiculate oogonia with diclinous antheridial branch. Fig. 12 lntercalar apiculate oogonia with monoclinous antheridial branch. Fig. 13-14 Oogonia with 1-several centric-subcentric oospores inside. Scale bars: Fig. 7, 14 = 50 pm; Fig. 8-13 = 10 pin. Steciow, 2001 168 Fig. 15-22 Achlya umbisporu. Fig. 15-17 Detail of filifonn zoosporangia with characteristic discharge achlyoid; spore cluster persistent, remaining as an irregular clump. Fig. 18 Oogonium with centric oospores. Fig. 19-22 Details of oogonia with subcentric oospores. Scale bars: Fig. 15 = 100 pm; Fig. 1619 = 50 pin; Fig. 20-22 = IO pin. Steciow, 2001 4. Achlya americana Humphrey, Transactions of the American Philosophical Society 17: 116 (1892) 169 Synonyms: Achlya americana var. americana Humphrey, 1893 Achlya americana var. cambrica Trow Achlya americana var. megasperma Crooks ex Cejp, 1959 Achlya americana var. megasperma Crooks, 1937 Achlya cambrica (Trow) T.W. Johnson, 1956 Achlya debaryana var. americana (Humphrey) Minden, 1912 Mycelium extensive diffuse; principal hyphae stout, 75-150 μ in diameter at base. Gemmae abundant；filiform or fusiform, irregular; terminal single, intercalary or catenulate; zoosporangia aboundant; navivulate , filiform or fusiform; straight or curved; 200-980 μ long by 15-60 μ in diameter, renewed sympodially. Zoospore discharge achlyoid; spore cluster persistent at exit pore; encysted spores 9-10 μ. Oogonia abundant; lateral, occasionally terminal; spheriacal, infrequently pyriform, oval; 35-97 μ in diamenter, predominantly 55-70 μ. Oogonial wall pitted, smooth. Oogonial stalks 1/2-2 times the diameter of the oogonia; straight, rarely curved or bent. Antheridial branches monoclinous, rarely diclinus; almost arising near stalk; simple; attached by projections, rarely laterally appressed; fertilization tubes present. Oospores eccentric; spherical; filling the oogonium;1-38 in number, generally 6-14; 20-44 μ in diameter, generally 20-24 μ. 170 Achlya americana,coker, The Saprolegniaceae, 1923 5. Achlya androgyna (W. Archer) T.W. Johnson & R.L. Seym., 2005 Synonyms: Achlya androgyna (W. Archer) T.W. Johnson & R.L. Seym., 2005 Achlya acadiensis C.L. Moore, 1912 Achlya braunii Reinsch, 1877 Achlya treleaseana (Humphrey) Kauffman, 1906 Aplanes androgynus (W. Archer) Humphrey, 1893 Aplanes androgynus var. androgynus (W. Archer) Humphrey, 1893 Aplanes androgynus var. mindenii (Schkorb.) Cejp, 1959 Aplanes braunii (Reinsch) de Bary, 1888 Aplanes braunii var. braunii (Reinsch) de Bary, 1888 Aplanes braunii var. mindenii Schkorb., 1923 Aplanes treleaseanus (Humphrey) Coker, 1923 Saprolegnia androgyna W. Archer, 1867 Saprolegnia treleaseana Humphrey, 1893 Zygothrix brauniana Reinsch ex Rabenh., 1866 171 Mycelium extensive, diffuse. Zoosporangia fusiform or cylindrical, renewed sympodially, 200–800 x 28–34 µ. Zoospores dis charge achlyoid or rarely aplanoid. Zoospores 10-12 µ in diam. Gemmae abundant, fusiform, pyriform or cylindrical, terminal and intercalary, single and catenulate. Oogonia terminal, intercalary or lateral, predominantly catenulate, fusiform, naviculate or cylindrical, 300-400 × 50-70(90) µ. Oogonial wall pitted, smooth or rarely with some papillae. Terminal oogonia often with conical, spine-like projection Oospores subcentric or centric (20-)28-34 µ in number, filling the oogonium. Antheridial branches androgynous, semi attached by projections or laterally appressed. Fertiliza tion tubes not observed Achlya androgyna: a &ndash; zoosporangia with zoospores and oogonia, b &ndash; hyphae bearing oogonia with subcentric oospores and androgynous antheridial branches. Markovskaja S., 2007 6. Achlya apiculata deBary. Bot. Zeit. 46: 635.1888 Vcgetative growth ample and abundant, but not as stout as in A. oblongata or in the Prolifera group. The main filaments mostly about 4(>-6oµ thick, tips rounded; breaking up soon after maturity into segments with little or no change in the appearance of the threads, each segment becoming a gemma and resting indefinitely until the conditions change, then forming spores like sporangia. Sporangia moderately plentiful, long or short, usually somewhat larger than the threads and gradually pointed towards the end, emptying as usual for an Achlya, or often remaining closed and emptying as in Dictyuchus. Spores ciliated on emerging and capable of swimming under certain conditions, 12.5-14.5µ in diameter or at times larger. 172 Oogonia not formed regularly or aljundantly except at low temperatures, racemosely borne on the tips of short or rather long branches whichare usually bent and sometimes make a complete turn, rarely intercalary, ovate, short pyriform or spherical, at low temperatures very rarely formed within empty sporangia (as in Saprolegnia ferax), typically with (but often without) a more or less prominent apiculus; 60 µ thick, walls thin, smooth, unpitted. Eggs few, large, very dark, subcentric, 1-5, usually 2 or 3 (rarely 10), 25-40µ thick, sometimes larger. Antheridial branches usually androgynous, but often diclinous, arising from the main hyphae or from the oogonial branches, soon becoming inconspicuous. Antheridia small, tuberous or cylindrical, usually one or more to each oogonium. Fig. 167. I. Fig. 2. X Cylindrical and spherical oogonia. A dictiosporangium, with one of the spores sprouting into a filament. 447. X Fig. 3. Habit of oogonia and antheridia. 102. Fig. 4. Sporangia, several in serial arrangement Fig. 5. Spore in same hypha. emerging from Fig. 6. A Fig. 7. Gemmae. X mature egg. X X X 100. cyst. X 720. 447. 107. A small dictiosporangium. X 167. Fig. 9. Peculiar case in which an oogonial initial was halted and sent ofT an Fig. 8. antheridial branch. Fig. 10. Oogonia. X X 167. 247. Fig. II. Sporangia, one on left a dictiosporangium with the spores emerged but not swimming; one in center containing four sprouting spores. Fig. 12. An intercalary oogonium. X 60. X 247. Fig. 13. Sporangia and oogonium with androgynous antheridium. X 167. Fig. 14. papilla. An intercalary oogonium X 247. with a Achlya apiculate, Coker, The Saprolegniaceae, 1923 7. Achlya caroliniana Coker. Bot. Gaz. 50: 381. 1910. Hyphae rather stout, about 48µ thick at the base and 20µnear the tip, in strong cultures reaching a length of 1.5 cm. Sporangia irregularly cylindrical, about 20-30µ. in diameter, often discharging by several openings, sometimes remaining closed and emptying as in Dictyuchus, ciliated on emerging but behaving as in other Achlyas. Spores 11-12µ in diameter. Oogonia abundant, very small, 24-55µ. thick, most about 30-37µ., spherical when terminal, wall smooth, or not rarely with one or two papillae or angles, thin, not pitted, light yellow in age, terminating short or moderately long, slender branches, which are racemosely borne on the strong main hyphae, or rather rarely intercalary and elongated, at times filiform with several elongated eggs in a row. Oogonial branches generally simple, but often giving off near the base, or sometimes near the oogonia, one or two branches which also terminate in oogonia, and, as a rule, are curved downward. Eggs generally 1-2, not rarely 4, eccentric, with a large oil globule, 18.5-23µ in diameter, averaging about 22µ, often elongated by 173 pressure. Antheridia absent. A papilla, thick-walled and soon empty, often grows into the oogonium through the basal partition exactly as in other members of the Prolijera group and in A. hypogyna. Fig. I. Oogonia with papillae. X 503. Fig. 2. Young oogonia and sporangia. X 188. Fig. 3. Gemma and three sporangia which were gemmae. X 122. Fig. 4. Habit of oogonia. X 18S. Fig. 5. Sporangium with spores clustered at tip. X 251. Fig. 6. Oogonium with an ingrowth from below. X 503. Fig. 7. Two odd-shaped intercalarjoogonia containing ripe eggs. X 503. Fig. 8. Empty sporangia, gemmae and young oogonia. X 188. Fig. 9. Sporangium partly filled with spores and odd-shaped oogonia. X li Achlya caroliniana. Coker, The Saprolegniaceae, 1923 8. Achlya catenulate, Jesus et al. (2015) Colony without special pattern and limited growth. Growth of the isolates after 96 h at 21 ºC: (i) CCIBt 4029: on MP5 without salt: 2.2 cm; on MP5 with 0.5 % of salt: 2.5 cm; on MP5 with 1.0 % of salt: 1.4 cm; on MP5 with 1.5, 2.0, 2.5 and 3.0 % of salt: no growth was observed; (ii) CCIBt 4030: on MP5 without salt: 2.0 cm; on MP5 with 0.5 % of salt: 2.6 cm; on MP5 with 1.0 % of salt: 1.2 cm; on MP5 with 1.5, 2.0, 2.5 and 3.0 % of salt: no growth was observed. Monoecious. Mycelium dense; hyphae slender, sparingly branched. Sporangia abundant; fusiform or clavate; straight; renewed sympodially; 230–460 × 20–30 μm. Spores discharge and behavior achlyoid; spore cluster persisting at exit orifice; encysted primary cysts 7.5–12.5 μm diam. Gemmae present, some catenulate. Oogonia abundant, terminal or lateral; catenulate, chains of up to 11 oogonia, sometimes simple; obpyriform, 35.0–82.5 × 27.5–57.5 μm. Oogonial wall pitted under region of attachment of the antheridial cells; smooth. Oogonial stalks straight, 1–2½ times the diameter of the oogonium in length. Oospheres generally not maturing. Oospores eccentric; spherical; 1–3 (–5) per oogonium, and never filling it; 15–35 μm diam.; germination not observed. Antheridial branches diclinous; slender; frequently branched; persisting. Antheridial cell tubular or clavate; simple or branched; attached apically, laterally or by projections; fertilization tube present, persisting. 174 Achlya catenulata. A. Achlyoid discharge of the zoosporangia. B. Sympodial renewal of the zoosporangia. C. Catenulate oogonia and diclinous antheridia. D–E. Oogonia, failed to mature and eccentric oospores and antheridia. Bars: 10 μm. Jesus et al. (2015) 9. Achlya colorata Pringsh. Sitzungsber. der Akad. der Wissensch. Zu Berlin, 1882 Hyphae stout, 25-50 μm in diameter at base. Sporangia long, almost cylindrical, or slightly tapering toward the end, very little or not at all larger than the hyphae bearing them. Spores ii[x in diameter, emerging and behaving as in A. racemosa. In neither species is any spontaneous movement shown before encystment. Oogonia varying greatly in size, 4i-90 μm in diameter, rarely as much as loyix, commonly 5566[a,racemosely borne on short lateral branches and also at times on the tips of main branches; the yellow walls producing short, blunt outgrowths in varying number or rarely almost smooth. Eggs mostly 1-4, rather rarely 5 and very rarely 6, 26-39 μm in diameter, mostly about 30-37 μm, centric, the wall very thick. Antheridial branches short, arising from the oogonial branches near the basal wall of the oogonium, and, as in the typical A. racemosa, often from the neck-shaped base of the oogonium itself, rarely from the main hyphae. Antheridia 1-4 on each oogonium, commonly 2, shortclavate, usually bent and applying their tips to the oogonium. Gemmae formed at the maturity of the culture in large numbers. They are scarcely enlarged sections of hyphae arranged in rows of rarely over 5, one erid often projecting to one side below the partition and somewhat thickened. They do not form all the way to the substratum, 175 but only near the ends of the hyphae. When brought into fresh water they sprout by tubes or become sporangia. Fig. I. Habit of fruiting. X II5Fig. 2. Sporangia, one with spores sprouting as in A planes (aplanosporangium). X II5Fig. 3. Oogonium with four antheridia. X 503. Fig. 4. Oogonium with antheridia arising from the oogonial wall. X 503. Fig. 5. Gemmae sprouting to filaments. X 1 13. Fig. 6. Enlarged tip of sporangium showing spores shrunken about lO'tX away from wall before escaping. Showing that there is pressure from gelatinization of inner part of sporangial wall. X 810. Fig. 7. Oogonium with ripe eggs and two antheridia, one arising from the oogonial wall. X 503- 10. Achlya debaryana HUMPHREY in Trans. Amer. Phil. Soc. (n. s.) 17: 117. 1893 Synonym Achlya debaryana var. debaryana Humphrey, 1893 Achlya debaryana var. intermedia Minden, 1912 Achlya polyandra sensu de Bary; fide Saccardo (Saccardo's Syll. fung. XXI: 854, 1912) Myceliu m limited, sparse to somewhat dense, 4-week old colonies on hemp seeds 1.5—2 cm in diameter, primary hyphae 85—170 pm wide at the base but becoming progressively narrow towards the apices (40—60 pm), stout, sparsely branched at the base, but branches becoming more profuse towards the periphery, thin but firmwalled. — Gemma e rather uncom mon, usually sub-cylindrical, 140—700 X 60— 150 |xm, terminal or intercalary, single or occasionally in chains. — Zoosporangi a fusiform to naviculate, (120) 280—900 X 25—60 p.m, renewed sympodially or basipetally; zoospor e discharge achlyoid; spore cluster at the mouth of zoosporangium not persistent; zoospore cysts 1—12 jim in diameter, smooth, thinwalled. — Oogon i a abundant, lateral, usually forming a raceme, spherical, 55—100 fim in diameter, the •walls thin, smooth, unpitted or very rarely pitted at the point of contact with antheridia; oogonial stalk 50—200 X 10—12 |.im, straight, cylindrical, sometimes slightly curved, thin but firm-walled. — Oospore s eccentric, 5—40 per 176 oogonium, usually not filling the oogonial cavity, spherical, 18—28 |j.m in diameter, thin-walled, smooth, germination not observed. The oogonia and oospores were brownish then turned yellow at maturity. — Antheridia l branche s monoclinous, abundant, usually simple or occasionally branched, branches somewhat irregular, thin-walled, persistent. Antheridial cells tubular to occasionally clavate, laterally appressed, fertilization tubes present and pronounced. Oogonium with monoclinous antheridial branches, Couch JN. 1931; Johnson TW Jr. 1956. 11. Achlya dubia, Coker, 1923 in Saprolegniaceae, 135. 1923 Synonyms Achlya dubia var. dubia Coker, 1923 Achlya dubia var. pigmenta Chaudhuri & Kochhar, 1935 Mycelium limited, usually dense, 3-week old colonies on hemp seeds 1—1.5 cm in diameter; primary hyphae 40—100 urn wide at the base, stout, moderately branched, especially towards the periphery. — Gemma e not observed. — Zoosporangi a abundant, fusiform to sub-cylindrical, 150—• 300 X 14—30 p.m., renewed sympodially; zoospore discharge thraustothecoid in primary zoosporangia but achlyoid to aplanoid in secondary or later formed zoosporangia; spore cluster in achlyoid zoosporangia not persistent; zoospore cysts 10—12 urn in diameter. — Oogoni a less common to common, sometimes abundant, spherical to subspherical, 40—70 |xm in diameter, the walls thin, smooth, unpitted or with occasional pits at the point of contact with antheridia; oogonial stalk 60—140 X 10—18 urn, straight, thin but firm-walled. — Oospore s eccentric, 2—8 per oogonium, usually not filling the oogonial cavity, spherical, 14—25 urn in diameter, thin-walled, smooth, germination not seen. — Antheridia l branche s diclinous to monoclinous, sparsely branched, 5— 10 |im wide, thin-walled, persistent. Antheridial cells tubular, laterally appressed, fertilization tubes not observed. 177 A. dubia (18—21): 18. Oogonia showing eccentric oospores and diclinous antheridia. — 19. Zoosporangia with achlyoid zoospores discharge. — 20, 21. Thraustothecoid zoosporangia, (bar = 20 |xm). T. M. MUHSIN, 1984 12. Achlya flagellata, Coker (1923) Growth stout and moderately dense, reaching a length of about 1cm. on a mushroom grub or ant larva. Hyphae branching, tapering outward, up to 150µ thick near the base, more or less crowded and uneven, the tips hyaline and often dying and renewed from one side below as in all members of this group. Sporangia plentiful, subcylindrical, very variable in size, often bent and at times with more than one opening, scattered or clustered. Spores often falling to the bottom in an open cluster on emerging, about 11-11.5 µ thick. Gemmae abundant, usually in rows from the segmentation of the distal parts of hyphae, short or long, usually more or less cylindrical, but often pear-shaped or ten-pin-shaped or at times very irregular; usually becoming sporangia on change of medium and discharging through an elongated papilla at either end. Oogonia abundant, typically spherical, but not rarely irregular by abnormal growth on one side, and one or two papillate projections may be seen rarely; usually about 48-75 µ thick, rarely up to 100; racemosely borne on short, slender stalks about as long usually as the diameter of the oogonia or a little shorter, rarely on longer stalks and quite rarely intercalary; wall hyaline, not thick (about 1.5 µ); pits very variable, perhaps more often absent, but again numerous and rather easily seen, about 5.5 µ. wide. Eggs spherical, eccentric with a large oil drop, 1-10 (rarely 20) in an oogonium, mostly 2-6, diameter 26-35 µ, most about 28 µ, rarely small ones may be mixed with theothers. Antheridial branches abundant, usually much branched and irregular, often so much so as to make an intricate network like a group of rhizoids, originating laterally and apically from hyphae which may or may not bear oogonia and applying themselves to oogonia on the same or on other threads or to both; more often diclinous than androgynous, perhaps about three times as often usually, but varying in this respect; the antheridial branches never arising from the stalks of the oogonia. Antheridia on nearly all oogonia, one or several, elongated with the side on the oogonium, frequently touching the oogonium with foot-like projections; antheridial tubes easily observed. 178 Fig. I. Habit, showing androgynous antheridia. X 41. Fig. 2. Angular oogonium. X 447. Fig. 3. Antheridial branches on tip of hypha curling back to oogonia. X 167. Pig. 4. Laterally elongated oogonium with blunt papilla and ingrowth from below. X 250. Fig. 5. Oogonia with diclinous antheridia. X 167. Fig. 6. Empty sporangia, sporangium with sprouting spores, gemmae, and abortive oogonium. X 108. Fig. 7. Gnarled gemmae. X 60. Fig. 8. Laterally elongated oogonium with an ingrowth from below. X 250. Fig. 9. Habit, showing diclinous antheridia. X 60. Fig. 10. Part of a dictiosporangium. X 720. Fig. II. Oogonium with ripe eggs. X 447Fig. 12. Sporangium emptied in corn meal agar. X 41Achlya flagellata, Coker, The Saprolegniaceae, 1923 13. Achlya formosana, Chiou & Chang, 1974 Principal hyphae stout, branched, 60-110? in diameter at base; numerous, slender and profusely branched. Secondary hyphae intermingled with primary ones. Gemmae abundant; filiform or subfusiform, occasionally spherical; single or catenulate, functioning as zoosporangia. Zoosporangia abundant, clavate, fusiform, occasionally moniliform; 150-700 × 20-45 ? in basipetalous succession. Zoospore discharge achlyoid. Spore cluster not persistent at exit pore; encysted spore 9-11 ? in diameter. Oogonia prolifera- tion not observed. Oogonial wall smooth, pitted only under the point of antheridial cells. Oo- gonial stalk 1-4 times the diameter of the oogonium in length, stout, straight, infrequently bent or curved. Antheridial branches diclinous, sometimes monoclinous; usually coiling about hyphae which may or may not bear oogonia, not losing this feature as colony ages; irregular, frequently branched; laterally appressed or attached by projections; persistent. Antheridia up to 25 ? in width. Fertilization tube not observed. Oosphere usually not maturing. Oospores eccentric; spherical, not filling the oogonium; 2-17 in umber, generally 5-9; 17.5-25 ? in diameter, predominantly 20-23 ?. Germination not observed. 179 Achlya formosana. A. Basipetal branching sporangia. × 200. B. Sympodial branching sporangia. × 200. C. Antheridial branches wrapping about the hyphae. × 200. D & E. Catenulating gemmae. × 200. F & G. Monoclinous and diclinous antheridial branches wrapping about the oogonium. × 800. H. Oogonium with many immaturing and a maturing oospore. × 800., Yu, Gonzalo, 2011 14. Achlya heterosexualis Whiffen 1965 Mycelium diffuse; hyphae moderately stout, moderately branched; capable of self- or interspecific conjugation. Sporangia fusiform; renewed sympodially; sometimes with a lateral exit orifice; 142-309 × 23-42 µm. Spores monomorphic; discharge and behavior achlyoid; primary spore cysts 9-11µm in diameter. Gemmae abundant; fusiform, cylindrical; often disarticulating; terminal or intercalary, single or catenulate. Oogonia lateral or terminal, obpyriform, or subglobose; (66-) 80-130 (152) µm in diameter. Oogonial wall pitted; smooth. Oogonial stalks unbranched. Oospores not always maturing, but eccentric when mature; spherical; 3-18 per oogonium, and filling it or not; (18-) 20-26 (-39) µm in diameter; at germination forming a germ hypha. Antheridial branches in self-conjugating thallus diclinous or androgynous; persisting. Antheridial cells simple; fertilization tubes unknown.Remarks. A. heterosexualis is a dioecious or monoecius species and can be distinguished by its oospheres not always maturing, but eccentric when mature, oospores 20-26 µm in diameter, generally 3-18 per oogonium and gemmae abundant; 180 cylindrical; often disarticulating; terminal or intercalary, single or catenulate and produced only monoclinous and androgynous antheridial branches. In A. heterosexualis only the antheridial branches are cross-induced. The oogonia are selfinduced, but function either with cross- or self- induced antheridial filaments. A. ambisexualis and A. bisexualisare are close to A. heterosexualis and the differences are presented in the remarks of the first species. A. heterosexualisis is distributed in USA (Johnson et al., 2002), and is recorded for the first time in Mexico. A. heterosexualis. Spherical oogonium, antheridial cells attached laterally, wall unpitted, oospores centric. Barr= 20 μm. DA: diclinous antheridium, OE: oospores eccentric; OS: oospores subcentric; AC: antheridial cells; PI: pitted. Vega-Ramírez et al., 2013 15. Achlya hypogyna Coker and Pemberton. Bot. Gaz. 45: 194, 1-6. 1908. Hyphae slender, tapering gradually toward the apex, at base about 35µ. in diameter, at or near tip about 8 µ. , in vigorous cultures reaching a length of i cm. Sporangia rather plentiful or few, nearly cylindrical, a little larger at the rounded and papillate distal end, usually curved, somewhat like those of Protoachlya paradoxa; dictiosporangia common, sometimes more abundant than the typical sort; spores on emergmg ciliated, a part usually dropping to the bottom and showing a little motion from the sluggish cilia. Gemmae at times abundant, again few, pyriform or flask-shaped, less often spherical, often in chains of two, three or four; long, rod-shaped gemmae are also formed by segmentation of the hyphae. Oogonia generally borne on short branches, racemosely arranged on the main hyphae, but occasionally terminating a main hypha, and very rarely intercalary; globular or rarely oblong, the walls not pitted, more or less abundantly producing short or long rounded outgrowths, or a varying proportion smooth; yellow when old; diameter 26-83(x without the papillae which are up to 30 µ.. long, the longest at times on the smallest oogonia. Eggs 1-7 (commonly 3-5), centric, diameter 20-36 µ.., averaging 27-28 µ..; not rarely elliptic and then up to 45 X 57 µ. Antheridia cut off from oogonial branches justbelow the oogonia, very rarely absent; simple antheridial branches with one ormore branched, tuberous, antheridia also present at times and arising from the suboogonial cell or below it or even from the main hypha; in the latter case rarely tliclinous. Fertilizing tubes arising through the common septa from the suboogonial cell and penetrating the oogonia from below (hypogynous), also from the other antheridia when present. 181 [Type a quote from the document or the summary of an interesting point. You can position the text box anywhere in the document. Use the Drawing Tools tab to change the formatting of the pull quote text box.] Achlya hypogyna, Coker, The Saprolegniaceae, 1923 16. Achlya irregularis (Coker et M. W. Ward) Johnson et Seymour, Mycotaxon, 92: 14. 2005. Mycelium dense, Mycelium dense, hyphae slender. Zoosporangia cylindrical and clavate,fusiform and irregular, renewed sympodially, 200–400 × 20-30 µ. Zoospores discharge achlyoid or rarely thraustothecoid. Zoospores 10–14 ìm in diam. Gemmae abun dant, variable in shape and size. Oogonia obpyriform or spherical (50–)80–86(– 100) ìm in diam., lateral or ter minal (in our material they are larger than reported by JOHNSON et al. (2005), description – (20–)40–55(–72) ìm) Oogonial wall pitted, smooth. Oospores spherical, aborting or oospheres not maturing, mature oospores eccentric, 23–26 ìm in diam., (6–)8–10 in number, usually not filling the oogonium. Antheridial branches diclinous, long and slender, branched, clasping the oogonium (1–) 2–4 in number, laterally appressed. Fertilization tubes not observed 182 Achlya irregularis: a – zoosporangia and zoospores, b – hyphae bearing oogonia with not maturing or eccentric oospores and diclinous antheridial branches, Markovskaja S., 2007 17. Achlya klebsiana Pieters, 1915 Synonyms Achlya klebsiana var. indica Chaudhuri & Kochhar, 1936 Achlya klebsiana var. klebsiana Pieters, 1915 M y c e l i um unlimited, sparse to sometimes dense, 3-week old colonies on hemp seed 1—2 cm in diameter; primary hyphae 40—90 jim wide at the base, sparsely branched but branches become more profuse near the periphery, thin but firm-walled, usually the primary hyphae become tinted brown near the base. •— Gemmae abundant, variable in shape and may be spherical to oval or cylindrical, sometimes irregular, single or in chains, terminal or sometimes intercalary, germinating to produce zoosporangia. —•Z o o s p o r a n g i a usually abundant, cylindrical to fusiform, 140—770 X 25—55 (70) urn, renewed sympodially or sometimes basipetalous; z o o s p o r e discharge achlyoid; spore cluster at the mouth persistent; zoospore cysts 10—12 urn in diameter, thin-walled, smooth. — O o g o n i a abundant, usually lateral, spherical to subspherical, 45—90 |.im in diameter, the walls thin (1.5—3 urn), smooth, unpitted or usually pitted at the point of contact with antheridial cells; oogonial stalks straight, cylindrical, 50—240 X 14—30 urn, thin but firm-walled. — O o s p o r e s eccentric, 1—12 per oogonium, generally not filling the oogonial cavity, spherical, 16—29 |xm in diameter, smooth, germination not seen. — A n t h e r i d i a l b r a n c h e s diclinous (75%) to monoclinous, sparsely to profusely branched, branches somewhat irregular and uneven, thin-walled, 8—22 (im wide, 183 persistent. Antheridial cells tubular, often encircling the oogonia, fertilization tubes not seen Achlya kelbsiana A. Oogonia with monoclinous and diclinous antheridial branches. × 200. B. Oospores filling the oogonium with antheridial cell myco.biota.biodiv.tw Achlya klebsian, www.diark.org 184 18. Achlya megasperma Humphrey. Trans. Amer. Phil. Soc. 17: 74-77. 1892 Mycelium slenderer than in most Achlyas. Sporangia very abundant, of the typical Achlya type, borne singly or in clusters (often as many as eight) on the ends of hyphae, varying much in shape from the long, slender, tapering sporangia of Achlya apiciilata to a club-shaped form swollen at the distal end; 100-1000µ. long, most between 300 and 400µ. Spores 11µ. in diameter. Gemmae developed in considerable abundance, either single and shaped like a sporangium with pointed tip or very elaborately branched ; when solitary often separating from the hypha and falling to the bottom. Oogonia racemosely borne on branches which are about as long as or shorter than the thickness of the oogonia; rarely the oogonial branches may be longer. Oogonia without an apiculus, usually spherical, occasionally oblong, rarely cylindrical, 60-119µ thick, usually between 70 and 8oµ, oogonial wall thickened and without pits except for thin places under the antheridia. Eggs 1-10 or rarely more, usually 2-5, almost or entirely filling the oogonium, often elliptic from pressure; 3966µ thick, usually between 42 and 52µ (in an oogonium in which there were 9 eggs the average size was 44.I1µ; structure subcentric and exactly as in A. apiculata; walls 3-4. µ thick. Antheridial branches diclinous or androgynous but never arising from the oogonial stalk, usually diclinous, often much branched and not applied to oogonia; long and very slender, becoming barely visible after the eggs are formed. Antheridia tuberous and fairly conspicuous; usually one or two on each oogonium; not rarely absent. Gemmae, one of which has become a sporangium. X 103. Fi?s. 2, :v 4. , Fig. 5. Normal sporangium. X Fig. 6. Habit sketch. Habit sketch. Fig. 7. Fig. 8. 247. A Fig. 9. An cylindrical gemma. X Fig. 10. view stalk. X 4.1.V Oogonium with peculiar 247. Oogonium showing Fig. 13. Large ten eggs. 185 X 103. section of eggs. Achlya megasperma, Coker, The Saprolegniaceae, 1923 103. oogonium. Oogonium showing surface X Fig. 12. 103. 55- elaborately branched of eggs. Fig. II. X X X X optical 43.v oogonium containing 247. 19. Achlya orion Coker and Couch. Journ. E. Mitchell Sci. Soc. 36: 100. 1920. Hyphal threads long, reaching a length of 1.5 cm. on house-flies, more slender than in most Achlyas, from 10-40µ thick close to base, rarely up to 85 µ thick, often wavy; usually little branched and pointed at tips when young; becoming considerably branched with age. Sporangia abundant, cylindrical, usually borne singly on the tips of the main hyphae in young cultures, renewed by cymose branching, often forming several clusters at regular intervals on the same hypha, irregular and wavy in old cultures, 12-37 x 36-600 µ, (rarely up to 900 µ). Spores 9-10 µ thick, emerging as usual in Achlya, but often falling to the bottom in an open group instead of forming a sphere at the sporangium mouth. Oogonia abundant on flies, grubs, and vegetable media, spread over the entire culture from the bases of hyphae to tips, giving the culture a lacy interwoven or net-work appearance; the diameter 30-60 µ, commonly 32-48 µ; usually borne singly on long, crooked, recurved stalks which arise racemosely from main hyphae and which vary in length from 2-10 times the diameter of the oogonia; often oogonial stalks may branch bearing two oogonia, and rarely oogonia may be borne on a stalk which arises directly from another oogonial wall; very rarely intercalary; oogonial wall usually without pits (except where the antheridial tubes enter) when grown on flies or grubs, but as a rule with pits when grown on boiled corn. Eggs 1-8, usually 1 or 2 in each oogonium; 25-45 µ in diameter, most 33-36 µ, eccentric when ripe, with one large oil drop; usually spherical, but often elliptical from pressure. Antheridial branches almost always anrdrogynous, usually arising from the ooponial stalk itself, less often from the main hypha; rarely diclinous; antheridia on about 75% of the oogonia, one or two on an oogonium, tuberous; antheridial tubes obvious, penetrating the oogonia and reaching the eggs. 186 I and 2. Oogonia with single egg and unbranched antheridia. X 233. 3. Oogonium with branched antheridia. 4. Oogonial stalk arising from the wall of another oogonium. X 233. 5. Eggs showing a late stage in maturation with several oil droplets not yet united into one large drop. X 387. 6. Oogonium with a typical long stalk. 7. Cluster of oogonia, one of which is barrel-shaped with the eggs in distal end and a perforated wall partly separating the two ends. Grown on a bit of boiled corn grain in dist. water at room temperature 8. Oogonia on a very much distorted oogonial stalk, as typical when cultivated three days in electric oven with temperature of 36° centigrade. X 233. 9. Oogonium with diclinous antheridium and ripe egg, showing oil drop 10. Oogonium with antheridia arising from oogonial stalk and main hyphae also . II. Habit sketches to show appearance of oogonia and antheridia and occasional behavior of spores. X 97. 12 and 13. Habit of sporangia. X 97. 14. Spores emerging from cysts. X 720. 15. Habit of sporangia. X 97Achlya orion, Coker, The Saprolegniaceae, 1923 20. Achlya prolifera NEES in Nova Acta Acad. Leop.-Carol. 11: 514. 1823. Mycelium limited, sparse to dense, 3-week old colonies on hemp seeds 1—1.5 cm in diameter; primary hyphae stout, 60—145 pm wide at the base, sparsely branched, thin but firm walled. — Gemma e abundant, variable in shape, globose to ovoid or irregular, single or in chains, intercalary or sometimes terminal, germinating to produce zoosporangia. — Zoosporangi a rare to abundant in some isolates, filiform to fusiform, sometimes naviculate, 140—700 X 14—50 }im, renewed sympodially; zoospor e discharge achlyoid, spore cluster at the mouth of zoosporangium, persistent; zoospore cysts 8—11 pm in diameter, thin-walled, smooth. The encysted primary zoospore cysts 8—11 pm in diameter, thin-walled, smooth. The encysted primary zoospores in spore cluster were often seen to germinate by germ tubes. — O o g o n i a abundant, but less common in some isolates, usually lateral, sometimes terminal or rarely intercalary, spherical, subspherical or ovoid, 98—125 X 62—95 pm, the walls thin, smooth, pitted, oogonial stalk 30—300 X 12—28 pm, straight, thin but firmwalled, cylindrical. — Oospor e s eccentric, 3—31 per oogonium, usually filling the oogonial cavity, spherical, 17—28 urn in diameter, thin-walled, smooth, germination not seen. — Antheridia l branche s diclinous, branched but irregular, wrapping around the oogonial stalk, 7—18 pm wide, thin-walled, persistent. Antheridial cells tubular, encircling completely the oogonia, sometimes laterally appressed, fertilization tubes not seen. 187 21. Achlya proliferoides, Coker, 1923 Growth moderately dense and strong, reaching a length of about I cm. on a mushroom grub. Hyphae moderately branched, variable in size, usually wavy and irregular, the tips hyaline and dying back here and there as in A. imperfecta and A. flagellata. Sporangia subcylindrical, usually bent, often with several openings; about 35-45µ. thick as a rule, short or long, at times up to 1425 µ long. Spores 11-12 µ thick, double ones not rare, often falling to the bottom in an open group on emerging. Oogonia abundant, spherical, smooth, 40-55(1 in diameter, racemosely borne on stalks that are 188 about 1-1 2/3 times as long as the diameter of the oogonia; wall hyaline, not thick; pits numerous (usually), but not very conspicuous. Eggs eccentric, with a large oil drop, about 18-24 µ in diameter, often elliptic, the great majority always going to pieces before maturity on ordinary media. Antheridial branches numerous, diclinous (inostly) or androgynous, usually long, contorted and much-branched, in many cases coiling themselves about certain selected hyphae which may or may not bear oogonia. Antheridia, one or several, on every oogonium, elongated, applying their sides to the oogonium or touching it by several blunt, foot-like processes. 1. Hypha with antheridial branches entwined around it. X 167. 2. Oogonium with ripe eggs. X 447. 3. Habit of cogonia and antheridia. X 167. 4. Spores sprouting in sporangium and below on same hypha two gemmae. X 167. 5. Oogonium growing from an abortive one. X 247. 6. Spiral-shaped oogonial stalk (culture on corn grain). X 167. 7. Oogonium. X 247. 8. Contorted antheridial branches. X 167. 9. Gemmae. X 108. 10. Hyphae showing pointed tip and atrophied tips renewed from below. X 108. Achlya proliferoides, Coker, the genus Saprolegnia, 1923 22. Achlya racemosa Hildebrand. Jahrb. f. wiss. Bot. 6:249, 1867. Synonyms: Achlya lignicola Hildebr Achlya racemosa f. racemosa Hildebr., 1867 Achlya racemosa var. lignicola (Hildebr.) Cornu, 1880 Achlya racemosa var. stelligera Cornu, 1880 Achlya racemosa var. maxima (Minden) Cejp, 1959 Achlya racemosa f. maxima Minden Achlya racemosa f. polyspora Schkorb., 1923 189 Hyphae stout, usually 25-36µ thick at base. Sporangia long, almost cylindrical, rounded or tapering at the tips, about the size of the hyphae bearing them or sometimes slightly larger, sometimes twisted like a corkscrew. Spores 9-1 µ in diameter; on emerging forming an irregular cluster or imperfect sphere which slowly expands as if embedded in jelly so that the spores become more or less separated singly or in groups. Gemmae usually few, formed by the distal parts of hyphae becoming divided into joints after being densely filled with protoplasm. Oogonia racemosely borne on short lateral branches, rarely intercalary, plentifully developed in all cultures, rather small, 40-70 µ in diameter; wall distinctly yellowish at maturity, smooth and unpitted except where antheridia touch. Eggs variable in size, 16.6-27.7 µ in diameter, most about 22 µ, centric, 1-8 in an oogonium (Humphrey says 1-10), in most cases 2-5, centric, the wall thick (about 3.5 µ). Antheridial branches short, arising from oogonial branches near the basal walls of the oogonia, or as often from the neck-shaped base of the oogonium or even from its curved surface, rarely from the main hyphae. Antheridia one or two, sometimes more, to each oogonium; shortclavate, usually bent and applied by their tips to the oogonia. Achlya racemosa Picture modified from Walter Emil Friedrich August Migula - Pilze Band III. Teil 1. Myxomycetes, Phycomycetes, Basidiomycetes (Ordn. Ustilagineae und Uredineae). (1910) - Permission granted to use under GFDL by Kurt Stueber. Source: www.biolib.de - Permission is granted to copy, distribute and/or modify this image under the terms of the GNU Free Documentation License, Version 1.3 or any later version published by the Free Software Foundation; with no Invariant Sections, no Front-Cover Texts, and no BackCover Texts. A copy of the license is included in the section entitled GNU Free Documentation License. Dimensioni del file: 52335 bytes Foto visualizzata 60 volte 190 23. Achlya radiosa, Maurizio, 1899 Achlya radiosa is slow growing. A three-week-old colony is only about 1 .5-2 cm in diameter. Main hyphae up to 25 µ thick at the base, stout and branched ; sex organs present in such an abundance as to make the entire colony appear to be composed of ornamented oogonia after some weeks. Oogonia usually lateral, at times terminal, stalked, heavily ornamented with pointed to mammiform projections which may be bifurcated. Double mammiform projections are occasionally present. Oogonia between 40 and 100 µm in diameter, including the oogonial projections. The projections themselves about 10-20 Am long and 10-13 µm wide at the base . Oogonial stalks straight, stout, the length of these stalks 0 .5-2 times diameter of oogonia. Wall unpitted. Antheridial branches short, stout, 1-2 per oogonium, androgynous arising some 15µm from the oogonium. Antheridial cells simple and laterally applied to the oogonium . Oospores spherical, usually aplerotic, sometimes plerotic, subcentric, usually one per oogonium, rarely 2-5. When single they are 25-60 µm in diameter, otherwise 7 .5-28 µm. Oospore wall about 2 .5 µm thick. 1 . Oogonia with antheridium . 2 . Unisporus Oogonium . 3 . Oogonium with two Oospores . 4 . Multisporus Oogonium, Paul Bernard, 1984 191 24. Achlya robusta Steciow & Elíades, Microbiol. Res. 157(3): 178 (2002) Mycelium densum, cultura in seminibus Cannabis sativae 2–5 cm diam. Hyphae ramosa, pleraque 45–158 µm late diam. ad basim. Sporangia in culturis juvenilibus, fusiformia, filiformia, cylindrica, vel clavata, (135) 229–500 (945) µm larga et 19–72 µm lata. Ejecto sporarum pro genus typica, zoospori incystatis globosi 5–10 (15) µm. Gemmae frequentis. Oogonia copiosa, sphaerica, subglobosa et pyriformia, (29) 66– 117 (148) µm diam. Paries oogonia foveatus, laevis vel papillatus, ramulus lateralibus provenientia, 200–1000 µm. Oospori (1) 4–17 (30) per oogonium, eccentrici, (10) 16– 40 (48) µm diam. Ramulus antheridiales, ramosus, monoclina (49%), diclina (30%) et androgina (21%). Mycelium extensive, denser near substratum, two hy week-old hemp seed colony, 2–5 cm diam.; principal hyphae stout, sparingly branched, 45–158 µm diam. at the base. Gemmae abundant, cylindrical, pyriform or irregular, single or often catenulate, functioning as zoosporangia. Zoosporangia fusiform, filiform, cylindrical or claviform; (135) 229–500 (970) X 19–72 µm; usually terminal, renewed usually by distinctive basipetalous or cymose succession. Zoospore discharge achlyoid. Encysted spores globose, 5–10 (15) µm diam. Oogonia very abundant, lateral or terminal; spherical or subglobose, rarely pyriform; (29) 66–117 (148) µm. Oogonial wall smooth or with ornamentation, papillate, tuberculate or bullate; pitted or pitted only under attachment point of antheridial cell; yellowish at maturity. Inner wall surface irregular, refringent and of varying thickness. Oogonial stalks usually slender, frequently short, straight or frequently bent, rarely curved; 15 to 200 µm long, sometimes branched. Oospheres often not maturing. Oospores eccentric, not filling the oogonium; spherical or ellipsoid; (1) 4–17 (30) in number; (10) 16–40 (48) µm diam. Antheridia always present. Antheridial branches slender, principally monoclinous (49%), occasionally diclinous (30%), rarely androgynous (21%), branched. Antheridial cells simple or branched; attached by projections or laterally appressed. Fertilisation tube not observed. 192 Achlya robusta. 1. Detail of filiform zoosporangium with a characteristic discharge achlyoid; spore cluster persistent 2–3. Zoosporangium renewed in distinctive basipetalous succession 4. Two characteristic monoclinous antheridial branches and one androgynous in water culture 6.7. Detail of smooth oogonia with immature oospheres 8. Oogonium with detail of two eccentric oospores. Scale bars, Fig. 1–5 = 100 µm; 6–8 = 50 µm Steciow & Elíades, 2002 193 Achlya robusta. 9–12. Spherical or subglobose oogonia with distinctive monoclinous antheridial branches with immature oospheres inside, developed on slender and short stalk 13–14. Androgynous antheridial branches in oogonia developed on bent oogonial stalk. Scale bars, Fig. 9–14 = 50 µm. 15– 16. Detail of ornamentated oogonia with the oogonial wall pitted and irregular inner surface 17–18. Tuberculate and papillate oogonia with aborted oospheres 19–20. detail of papillate and bullate oogonia. Scale bars, Fig. 15–20 = 50 µm Steciow & Elíades, 2002 25. Achlya spiralis, Paul and Steciow (2008) Mycelium of A. spiralis is moderately extensive, denser near the substratum and a 2week-old hemp seed colony measures up to 1.4–3 cm in diameter. Principal hyphae are branched, slender to stout, 19–72 (exceptionally up to 121) μm at the base and are profusely branched into secondary hyphae near the tips. Gemmae are sparsely produced, and are cylindrical, fusiform, pyriform, simple or catenulate. Zoosporangia are moderately abundant in young colonies, and are filiform, fusiform, to clavate, straight and tapering towards the end and frequently furnished with one to several lateral discharge pores or tubes in addition to the terminal orifice, straight or curved at the tips. The zoosporangia measure 145–750 (exceptionally up to 970) μm × (19-) 29–50 μm and are renewed sympodially, or at times in basipetalous or cymose succession. Zoospore discharge is mostly achlyoid, sometimes aplanoid and the spore clusters are not persistent at the exit pore. Encysted spores are globose, 6–12 (rarely up to 14) μm in diameter. Oogonia are formed abundantly and are lateral, occasionally terminal or intercalary, spherical, subglobose, pyriform, oval, irregular, or dolioform, rarely proliferating. These usually measure between 25 and 78 (exceptionally up to 102) μm in diameter. Oogonial walls are slender, smooth, pitted or pitted only under the attachment point of the antheridial cell. The oogonia are generally borne on bent, curved or even coiled stalks. Sometimes the oogonial stalks are straight. The length of the stalks can vary from 48 to 300 μm or longer when coiled. Antheridia always present. Antheridial branches are slender, principally monoclinous, sometimes androgynous and or diclinous, frequently branched, at times twisted or coiled. Some nonfunctional ‘antheridial’ branches also emerge towards the oogonia. Antheridial cells are simple and laterally appressed. Fertilization tubes arise as peg-like projections from long laterally applied antheridial cells. Oospheres are frequently aborting or maturing after a long period of incubation.Oospores are eccentric, filling or not filling the oogonium, spherical, ellipsoid or irregular, usually 194 2–8 in number (exceptionally 1 and up to 18). The oospores usually measure between 19 and 30 (exceptionally 12–36) μm in diameter. Achlya spiralis. (a) Basipetalous zoosporangial renewal. (b) Filiform zoosporangium. (c) Cymose zoosporangial renewal. (d) Achlyoid zoospore discharge. (a, c) Scale bar=50 μm; (b, d) scale bar=20 μm. (a) Sympodial zoosporangial renewal. (b) Aplanoid zoospore discharge. (a) Scale bar=25 μm; (b) scale bar=15 μm. Paul and Steciow (2008) (a–d) Smooth-walled oogonia on curved oogonial stalks with monoclinous, androgynous and diclinous antheridial branches. (a–d) Scale bar=20 μ . Mycelium with characteristic oogonia on straight, bent or curved oogonial stalks and monoclinous, androgynous or diclinous antheridial branches. Scale bar=50 μm Paul and Steciow (2008) (a, b) Smooth oogonia with monoclinous antheridial branches. (c) Oogonium on a typical coiled oogonial stalk. (d) Diclinous antheridial branch on pitted oogonium. (a–d) Scale bar=15 μm. (a, b) 195 Oogonia containing one to several eccentric oospores. (c) Oval oospore. (d) Spherical oospore. (a) Scale bar=10 μm; (b–d) scale bar=5 μm. Paul and Steciow (2008) 26. Achlya treleaseana (Humphry) Kaufmann in Ann. Rept. Mich. Acad. Sei. Arts Letters 8: 26. 1906. Mycelium limited, usually dense, 3-week old colonies 1—1.3cm in diameter; primary hyphae (30) 50—110 jun wide at the base, somewhat stout, sparsely, branches more common towards the periphery of the colony, thinwalled. — Gemma e rather scanty, terminal or intercalary, single or in chains, usually cylindrical to irregular, 140—900 X 40—60 jim, germinating to produce small zoosporangia. — Zoosporangi a rather scanty, fairly common during the first two weeks but disappear later on, (90) 280— 900 X 30—62 urn, cylindrical to subcylindrical, renewed sympodially; zoospor e discharge achlyoid or occasionally dictyoid; spore cluster at the mouth of achlyoid zooporangia not persistent; zoospore cysts 11—13 (xm in diameter, thin-walled, smooth. — O o g o n i a abundant, lateral to occasionally terminal, spherical, 56—112 urn in diameter, predominantly 62—98 um in diameter, the walls thin, rarely smooth but usually marked with parse papillate projections on the outer surface, unpitted or sometimes pitted at the point of contact with antheridia, oogonial stalk 30—140 X 14—21 um, cylindrical, straight, thin but firm-walled. — Oospore s subcentric, 1—30 per oogonium, usually filling the oogonial cavity, spherical, 18—42 urn in diameter, thinwalled, smooth, germination not seen. — Antheridia l branche s monoclinous to sometimes hypogynous or rarely diclinous, 10—15 (j.m wide, simple to sparsely branched, usually not persistent. Antheridial cells tubular, laterally appressed, fertilization tubes not seen. A. treleaseana (5—7): 5. Oogonium with subcentric oospores and monoclinous antheridia. — 6. Dictyoid zoosporangium. — 7. Achlyoid zoosporangia. T. M. MUHSIN, 1984 27. Achlya truncatiformis M. W. Dick & M. A. Spencer, 2002 Zoosporangia establishment cymosae ; large , fusiform , 80 ± 150¬20 ± 30 lm . Zoospore casting typical race . gems lights . Oogonium spherical , usually 30 ± 90 lm 43 lm ( bay nipples ). The wall terminal breasts Tenis or even eadern thick. Oosporae eccentric, usually 18 ± 1 4 ± 8 19 ± 26 lm origin androgyna but usually 23. antheridial Sometimes diclina , applying lateral . 196 Achlya aff. truncatiformis Uniovulate oogonia with eccentric oospores; stout truncate papillae with thin walled distal portion Bar =10µ Reports: TIFFNEY and WOLF (1937) stated that Achlya flagellate was found in 1935 attacking Triturus viridescens, in association with Saprolegnia parasitica in a small pond near Lexington, Massachusetts, and was further isolated alone from 12 out of 70 fish (Lebistes reticulatus[Poecilia reticulata]) which were kept for experimental purposes in an aquarium tank at Cambridge, Massachusetts; all the infected fish died eventually. The organism isolated from the latter was then shown experimentally to be pathogenic to other fish (Fundulus heteroclitus), slightly injured by the removal of a few scales, 9 out of 25 individuals in the infected tank dying. During the early summer of 1936 Achyla flagellata was responsible for the death of about 50 % of fingerlings of the brook trout (Salvelinus fontinalis) in Bayfield Hatchery, Wisconsin, but fingerlings of the brown trout (Salmo fario) and rainbow trout (S. irideus) in the same hatchery appeared to be almost immune from it. It appears, therefore, that under Wisconsin conditions A. flagellata may at times become a destructive parasite of fish. Barksdale and Lasure (1974) initiated sexual morphogenesis in Achlya by two hormones. Hormone A, or antheridiol, is a C-29 steroid. Four stereoisomers of antheridiol have been synthesized. The natural one (antheridiol 22S 23R) and its 7deoxy 7-dihydro form, when added to an aerated culture of hermaphroditic Achlya heterosexualis, stimulated this mold to secrete twice as much hormone B as the untreated control. An unnatural stereoisomer (antheridiol 22R 23S) and fucosterol, however, did not stimulate strain 8-6 in the same way. Methods by which hormone B can be produced in sufficient yield for isolation and characterization are described. Srivastava (1978) extended host range of Achlya caroliniana Coker to include Puntius Sophore, P. conchonius, P. ticto, Colisa fasciata, Chanda ranga, Labeo rohita (fingerlings), L. bata (fingerlings), Notopterus notopterus, Anabas testudineus, and Channa punctatus by artificial inoculation studies under controlled laboratory conditions. Khulbe et al. (1994) reported Achyla debaryana (Saprolegniales, Oomycetes) for the first time as a fish pathogen, causing an epizootic of mycosis in a catfish, Mastacembelus armatus, in Nanak Sugar, a huge artificial reservoir and recognized fish production centre in Naini Tal district, Uttar Pradesh, India. The incidence of infection was found to be influenced by the physicochemical characteristics of water. The maximum disease severity (52 and 47.4%) occurred in March with moderate water temperature (22-24 degrees C), high dissolved oxygen content (9.5 mg l-1) and 197 pH 8.6, while the lowest level of infection (3 and 1.8%) was recorded at high temperature (29-32 degrees C) during June. Kitancharoen et al. (1995) reported the first record of the discovery in Myanmar of Achlya klebsiana, belonging to the family Saprolegniaceae. Czeczuga et al. (2002) incubated muscles of four fish species (monkey goby, Neogobius fluviatilis; racer goby, N. gymnotrachelus; Chinese sleeper, Perccottus glenii; and stone moroko, Pseudorasbora parva caught in the drainage area of the Bug River) in water taken from 6 different places. A total of 59 fungus species were found to grow on fish muscles studied: Achlya ambisexualis, A. americana, A. caroliniana, A. crenulata, A. debaryana, A. diffusa, A. dubia, A. intricata, A. klebsiana, A. oblongata, A. oligacantha, A. orion, A. polyandra, A. prolifera, A. proliferoides, A. radioasa, A. rodrigueziana, A. treleaseana, Aphanomyces irregularis, A. stellatus, Blastocladiella britannica, Blastocladiopsis parva, Catenaria verrucasa, Cladolegnia unispora, Dictyuchus monosporus, Isoachlya monilifera, Leptolegnia caudata, Leptomitus lacteus, Phlyctochytrium aureliae, Pythium afertile, P. aquatile, P. arrhenomanes, P. butleri, P. dissotocum, P. hemmianum, P. intermedium, P. myriotylum, P. ostracodes, P. periplocum, P. tenue, Rheosporangium aphamidermatus, Rhizophlyctis hirsutus, Rhizophydium laterale, R. macrosporum, Saprolegnia asterophora, S. diclina, S. eccentrica, S. ferax, S. hypogyna, S. litoralis, S. mixta, S. monoica, S. parasitica, S. pseudocrustosa, S. shikotsuensis, S. torulosa, S. uliginosa, Thraustotheca clavata, Zoophagus insidians. El Androusse et al. (2006) found Achlya abortispora sp. nov. in water and floating organic matter taken form a dam near Rabat, Morocco. The new species is described and compared with other species of the genus. Distinguishing characteristics of A. abortispora are the production of long fusiform sporangia with achlyoid and aplanoid discharge of zoospores; smooth-walled spherical to club-shaped oogonia, which are usually lateral, but at times intercalary, containing 1 to 20 oospheres. The oogonia can also bear 1 to 5 appendages, which may indicate oogonial proliferation. Most of the oospheres do not mature and are thus abortive. The antheridial branches supplying the oogonia are predominantly diclinous, but at times these may be monoclinous and androgynous. Antheridial branches coil and wrap around the oogonia. Morphologic features of the oomycete and the sequence of the ITS region of its rDNA, as well as their comparison with related species, are discussed. This is the first report of the occurrence of a saprolegniaceous oomycete from Morocco. Walker et al. (2006) compared F-actin patterns in invasive and non-invasive oomycete hyphae. In Achlya bisexualis an F-actin depleted zone is present in 70% of invasive but only 9% of non-invasive hyphae. In Phytophthora cinnamomi these figures are 74 and 20%, respectively. Thus, the F-actin depleted zone appears to be associated with invasive growth. TEM images indicate that it is unlikely to represent areas of vesicle accumulation. Measurements of turgor indicate no significant increase under invasive conditions (0.65 MPa (invasive) and 0.63 MPa (non-invasive)). Similarly we found no difference in burst pressures (1.04 MPa (invasive) and 1.06 MPa (non-invasive)), although surrounding agarose may lead to overestimates of invasive tip strength. An F-actin depleted zone has the potential, along with wall softening, to increase protrusive force in the absence of turgor increases. Staining of 198 F-actin in hyphae under hyperosmotic conditions suggests that decreases in F-actin at growing tips may also enable non-invasive growth at very low turgor. Kales et al. (2007) explored the response of fish macrophage to members of the Saprolegniales using the rainbow trout monocyte/macrophage cell line, RTS11. After 48 h in co-culture, RTS11 demonstrated chemotaxis, adherence and homotypic aggregation to both live and heat-killed fungal spores and mycelia. This aggregation was enhanced when using conditioned media from co-cultured RTS11 andAchlya, suggesting the presence of synergistic effectors of aggregation. Although fungal toxins were not evident, as cells remained viable throughout fungal overgrowth, phagocytosis was inhibited due to large fungal spore size, allowing these molds to evade macrophage defenses. Although class I MH and other viral response genes showed no significant change in expression, calreticulin and interleukin-8 were moderately up-regulated implicating calcium modulation and chemotactic response, respectively. Cyclooxygenase (COX-2) and the cytokines IL-1beta and TNFalpha were strongly up-regulated in the presence of Achlya, while gene expression of the class II major histocompatibility (MH II) receptor and associated molecules appeared down-regulated, suggesting fungal interference of immune function. Previous studies have shown an increased dependence of macrophage in immune function at low temperatures; based upon data presented here, this reduction of macrophage MH II receptor expression and inability to phagocytose spores may limit host response thereby providing increased susceptibility to these opportunistic pathogens. RTS11 adherence to Achlya bisexualis and Saprolegnia parasitica. RTS11 cells were incubated with appropriate spore inoculum at ambient temperature for 48 h. In panel (A), macrophage adhere to Achlya chlamydospores. (B) Macrophage adherence to Achlya hypha. (C) Macrophage attachment above the plane of original adherent cells suggesting chemotropism and/or attachment by cells originally in suspension. (D) Non-uniform adherence of macrophage to Achlya hypha. In panels (E and F), macrophage adhere to hyphae of Saprolegnia parasitica. Magnification is indicated at the bottom right in each panel.Kales et al. (2007) Calcofluor staining of fungal hyphae in trout macrophage co-culture. In panel (A), bright-field microscopy illustrates macrophage adherence to Achlya hypha. (B) Merged bright-field and fluorescence imaging of hyphal filament at the site of macrophage aggregation, where staining was performed following 48 h co-culture. Bright-field (C) and corresponding fluorescence imaging (D) of macrophage aggregation 48 h after inoculation using pre-stained spores. Fungal spore germination was not inhibited by calcofluor pre-treatment. Fluorescence along growing hyphae indicated reutilization of spore coat cellulose. Kales et al. (2007) 199 RTS11 response to heat-killed Achlya mycelium. Panels (A and B) illustrate magnified view of macrophage adherence to heatkilled Achlya hypha. (C and D) Macrophage demonstrate spreading morphology in the presence of heat-killed Achlya spores. Magnification indicated at the bottom right Kales et al. (2007) Adherence and aggregation is cell-type and substrate specific. (A)RTS11 cultured 48 h in the presence of sterile cotton fibres showed no evidence of chemotropism or adherence towards the cellulose fibres indicating that adherence is specific to factors other than cellulose, an Achlya cellwall component. (B) RTG-2, a rainbow trout gonadal fibroblast cell line, cultured 48 h in the presence of live Achlya culture, demonstrated no evidence of chemotropism or adherence, indicating that these responses to the fungus are cell-type specific. The Achlya hyphae could be seen readily penetrating the confluent layer of fibroblasts Kales et al. (2007). RTS11 demonstrates homotypic aggregation in the presence of Achlya. Top panels illustrate 48 h cultures of RTS11 alone, Achlya alone and RTS11 with live Achlya. Media from each of the above cultures was filtered and diluted 1:1 with fresh media to serve as conditioned media for freshly plated cells. Middle panels illustrate macrophage following 6 days in conditioned media collected from top panel cultures. Homotypic aggregation was greatest in cultures grown in conditioned media from coculture (middle right panel) suggesting synergistic effectors. Bottom panels illustrate RTS11 after 10 days alone (bottom left panel) or in the presence of heat-killed spore inoculum (bottom right panel). Cells in lower panels were plated at highest density to enhance the density-dependent homotypic aggregation. Magnification 100×. Kales et al. (2007). 200 RT PCR analysis of trout macrophage gene expression in the presence of live Achlya. Target cDNAs are indicated on the left from RTS11 grown alone for 48 h (lane R). Achlya grown alone shows no amplification of target trout cDNAs (A). In lane R/A, cDNA template was derived from RTS11 grown for equal time in the presence of live Achlya culture. Genes are grouped by function as indicated on the left. PCR reactions includedwater in place of cDNAtemplate to serve as negative controls, indicated with a minus sign (−). Equal amounts of total RNA, determined by absorbance at 260 nm, were used in cDNA synthesis reactions. Band intensity serves as a measure of original transcript abundance relative to the internal standard, eEF1_ as previously described by Hansen and Strassburger (2000). Kales et al. (2007). Western blot analysis of major histocompatibility (MH) cellular protein steady-state levels in the presence of live cultures of Achlya, Saprolegnia and heat-killed Achlya inoculum. Previously developed affinity-purified polyclonal antibodies, raised against recombinant forms of troutMHsubunits, were employed against RTS11 lysates following treatments indicated above. Lane R is lysate of RTS11 grown alone. R/A and R/S are lysates of RTS11 grown for 48 h in the presence of Achlya and Saprolegnia, respectively. R and R/H are lysates from 10-day cultures of RTS11 grown alone or in the presence of heatkilled Achlya inoculum, respectively. Ponceau S staining (STD) of membrane, prior to blocking and subsequent probing, indicates protein transfer and serves as a measure of equal loading among lysates. Kales et al. (2007). RT PCR analysis of trout macrophage gene expression in the presence of heat-killed Achlya. Target cDNAs are indicated on the left from 48 h cultures of RTS11 alone (R) or grown the presence of heatkilled Achlya mycelium and spores (R + H). PCR reactions included water in place of cDNA template to serve as negative controls, indicated with a minus sign (−). cDNA template was derived from RTS11 grown for equal time in the presence of heat-killed Achlya mycelium and spore preparations. Equal amounts of total RNA, determined by absorbance at 260 nm, were used in cDNA synthesis reactions. Band intensity serves as a measure of original transcript abundance relative to the internal standard, eEF1_ as previously described by Hansen and Strassburger (2000). Kales et al. (2007). Sosa et al. (2007) investigated the role of Achlya bisexualis dimorphosporum in the etiology of ulcerative mycosis (UM) in striped mullet Mugil cephalus. They injected healthy striped mullet subcutaneously with secondary zoospores of four oomycete isolates: two concentrations (50 and 115 zoospores/mL) of SJR (an endemic isolate of Aphanomyces invadans in American shad Alosa sapidissima from the St. Johns River); two concentrations each of CAL (25 and 65 zoospores/mL) and ACH (1,400 201 and 2,000 zoospores/mL; endemic isolates of Aphanomyces invadans and Achlyva bisexualis, respectively, in striped mullet from the Caloosahatchee River); and two concentrations of the ascomycete culture MTZ (2,500 and 3,500 zoospores/mL; endemic isolate of P. dimorphosporum from whirligig mullet M. gyrans in the Matanzas Inlet). All fish injected with either concentration of SJR developed granulomatous ulcers after 8 d and died within 21 d. Eighty percent (8/10) of fish injected with the high dose of CAL developed ulcers after 13 d and died within 28 d, but only 30% (3/10) of fish injected with the low dose of CAL developed ulcers. Four of the ulcerated fish died within 28 d, and the remaining fish were terminated after 32 d. Fish injected with zoospores of Aphanomyces invadans developed ulcers that were grossly and histologically similar to those observed in naturally infected striped mullet with UM from several estuaries or rivers in Florida. These hemorrhagic skin ulcers were characterized by myonecrosis and the presence of mycotic granulomas. None of the fish injected with ACH, MTZ, or sterile water developed ulcers. This study fulfilled Koch's postulates and demonstrated that ulcers could be experimentally induced in striped mullet after exposure via injection to secondary zoospores of an endemic Florida strain of Aphanomyces invadans. Paul and Steciow (2008) isolated Achlya spiralis sp. nov. from water samples collected in the river Tille in the Burgundian region of France. The new oomycete is described, illustrated and compared with related species of the genus Achlya. It is characterized by the presence of smooth-walled oogonia that are usually borne on bent or twisted oogonial stalks; mainly monoclinous, androgynous and diclinous antheridial branches and eccentric oospores which generally do not mature or mature after a long period of time. The internal transcribed spacer (ITS) region of its rRNA is comprised of 671 bases. The taxonomic description of this new species, its comparison with related oomycetes and the sequence of the ITS region of its rRNA are discussed here. Abking et al. (2012) isolated water moulds in the genera Achlya and Saprolegnia from eggs of the Mekong giant catfi sh (Pangasianodon gigas) and from the water in the hatching tank at the Inland Aquaculture Research Institute, Phra Nakhon Sriayutthaya province, from 2008 to 2010. The optimal temperature of almost all isolates was 30 °C. The Achlya spp. and Saprolegnia spp. could tolerate an NaCl medium at 10 and 25 parts per trillion (ppt), respectively. An exception was Saprolegnia sp. (E3/52-P2) which could tolerate NaCl up to 30 ppt. The isolates could grow in broth at pH 4–11, while the optimal pH for Achlya spp. and Saprolegnia spp. was pH 5 and pH6, respectively. The study on pathogenicity of the water moulds isolated in the laboratory showed that the isolates Achlya spp. (T.MCF1-02, E.MCF 2-001 and E4/52-10) and Saprolegnia sp. (E1/53-12) were pathogenic to the catfi sh eggs 202 Arrow showing zoosporangium development after zoospore released (scale bar = 50 μm): (A) Achlya spp.; and (B) Saprolegnia spp. Abking et al. (2012) Arrow showing the invasion by water mould mycelia (dark lines) in eggs of the Mekong giant catfish, from cell membrane into egg yolk (light gray color) resulting in gap formation. (Staining with GrocottGomori methenamine-silver stain; power at 400×; scale bar = 50 μm.) Abking et al. (2012) Chauhan et al. (2012) conducted an investigation on fungal infected Indian Major Carps viz. Catla catla, Cirrhinus mrigala and Labeo rohita collected from Sarangpani Lake. During the study period from July 2011 to December 2011, total seven species of fungi have been isolated from the lesions and affected muscles of infected fishes. These fungal isolates were cultured under laboratory conditions and identified as A. americana, A. klebsiana, A. orion, A. prolifera, Saprolegnia diclina, S. ferax and S.parasitica. Pathogenicity of all the seven species of fungi was tested on different major carps. All the isolates were found pathogenic to fishes but A. prolifera and S. parasitica were found to be most virulent showed 100% mortality of the experimental fishes. Percentage prevelance of infection have also been found out. Maximum percentages of infections (47.4%) were recorded in Catla catla in the month of December and minimum (8.9%) in Cirrhinus mrigala in August 203 Czeczuga et al. (2012) investigated the occurrence of aquatic zoosporic fungi in the water of three water bodies of varied trophicity. Dead specimens of fairy shrimpStreptocephalus dichotomus (Crustacea), fish feed in aquacultures on the Indian subcontinent, were used as a bait. Thirty-five zoosporic fungus species, including 20 known as parasites or necrotrophs of many fish species were identified. They included Achlya intricata, Achlya prolifera, Achlya orion, Aphanomyces laevis,Dictyuchus monosporus, Leptolegnia caudata, Saprolegnia delica, Saprolegnia ferax,Saprolegnia monoica and Saprolegnia parasitica, all known as a frequent cause of mycotic diseases in economically important fish populations. Dead specimens ofStreptocephalus dichotomus should thus be regarded as vectors of aquatic fungi which induce mycosis in fish. Lecanu et al. (2012) used 22R-hydroxycholesterol as a sub-structure to screen natural compound databases, wherby they identified a naturally occurring steroid (sc-7) with a 16-acetoxy-22R-hydroxycholesterol moiety, in which the hydroxyl groups in positions 3 and 22 are esterified by an acetoxy group and in which the carbon in position 26 carries a functional diacetylamino. sc-7 is an analog of the sex steroids dehydro-oogoniol and antheridiol, can be isolated from the water mold Achlya heterosexualis, and promoted neurogenesis in vitro and in vivo. Mouse embryonic teratocarcinoma P19 cells exposed to sc-7 for 2days followed by a 5-day wash-out differentiated into cholinergic neurons that expressed specific neuronal markers and displayed axonal formation. Axons continued growing up to 28days after treatment. In vivo, infusion of sc-7 for 2weeks into the left ventricle of the rat brain followed by a 3-week wash-out induced bromodeoxyuridine uptake by cells of the ependymal layer and subventricular zone that co-localized with doublecortin and glial fibrillary acidic protein immunostaining, demonstrating induction of proliferation and differentiation of neuronal progenitors. Migrating neuroblasts were also observed in the corpus callosum. Thus, under these experimental conditions, adult ependymal cells resumed proliferation and differentiation. Taken together, these results suggest that sc-7 is an interesting molecule for stimulating in situ neurogenesis from resident neuronal progenitors as part of neuron replacement therapy. sc-7 did not bind to nuclear steroid receptors and was not metabolized as a steroid, supporting our hypothesis that the neurogenic effect of sc-7 is not likely due to a steroid-like effect. 204 sc-7 Promotes long-term differentiation of P19 cells into neurons. After 2 days of treatment and 5 days of washout, sc-7 triggered sprouting in P19 cells (B) and induced the expression of bIII-tubulin (B), MAP2 (D), and ChAT (F). No neuronal marker expression was observed in control cells (A, C, E and G). The differentiating P19 cells expressed the neuroblast marker DCX (H), which was not found in control cells (G). To assess the irreversibility of the differentiation process induced by sc-7, P19 cells were treated for 2 days and washed out for 14 or 28 days before immunolabeling for neuronal markers. After 14 days of wash-out (I), the processes had dramatically elongated, as shown by bIII tubulin staining (DyLight 594, red). Synaptophysin labeling (DyLight 488, green) showed that the newly formed neurons established synaptic connections. After 28 days of wash-out (J), neuronal processes had dramatically elongated as shown by increased bIII tubulin (DyLight 594, red) and synaptophysin immunofluorescent labeling (DyLight 488, green) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.). Lecanu et al. (2012) Proliferation and differentiation of neural progenitor cells in the ependymal layer and SVZ of sc-7-treated rats. sc7 (375 lM) Was administered by the intracerebroventricular route for 2 weeks, corresponding to a total quantity of 530 nmol per rat. Control animals were infused with vehicle. Rats were euthanized 3 weeks after the end of sc-7 infusion. The animals received a daily injection of BrdU (100 mg/kg, i.p.) starting the day following the surgery and ending the day of euthanasia. In the first set of experiments, brain sections were immunostained for BrdU (A, control and B, sc-7-treated rats). In the second set of experiments, brain sections were immunostained for BrdU (C), DCX (D), and GFAP (E). Co-localization of marker expression was visible when the three channels were merged (F). BrdU, FITC, or DyLight 594; DCX, DyLight 488; GFAP, aminomethylcoumarin acetate. F, merge. Scale bar = 50 lm. Lecanu et al. (2012) Cells coexpressing DCX and vimentin in the SVZ and corpus callosum of sc-7-treated rats. DCX-positive cells (A–D), vimentin-positive cells (E–H), and DAPI counterstaining (I–L) in the SVZ (A, B, E, F, I and J) and corpus callosum (C, D, G, H, K and L) of control (A, C, E, G, I and K) and sc-7-treated (B, D, F, H, J and L) rats. sc-7 (375 lM) was administered by the intracerebroventricular route for 2 weeks, corresponding to a total quantity of 530 nmol per rat. Control animals were infused with vehicle. Rats were euthanized 3 weeks after the end of sc-7 infusion. DCX, DyLight 488; Vimentin, RRX. M-P, merge. Scale bar = 50 lm. Lecanu et al. (2012) 205 Cao et al. (2013) isolated an oomycete water mold (strain YC) from yellow catfish (Peleobagrus fulvidraco) eggs suffering from saprolegniosis and characterized morphologically and by its internal transcribed spacer (ITS) gene sequence. Based on its morphological features, the mold was initially identified as an Achlya sp. isolate. Using the neighborjoining method to construct a phylogenetic tree, the YC strain was closely related to A. klebsiana strain CBS101.49 (GenBank accession no. AF119579), previously recorded as infecting plant roots as well as fish. Aqueous extracts from 31 Chinese herbs were screened as possible anti-Achlya agents. Of these, a 10 mg/ml extract from Rhizoma coptidis was the most efficacious. Significant protective efficacy of 66.34% and 92.20% was obtained against the YC strain in fish eggs by the R. coptidis extract provided at concentrations of 256 mg/l and 1280 mg/l, respectively. Yellow catfish eggs suffering from saprolegniosis (arrows). Cao et al. (2013) Zoosporangia and their renewal in the YC strain of Achlya: (a) a cylindrical zoosporangium; (b) a fusiform zoosporangium; (c) sporangial renewal in basipetalous succession; (d) sporangial renewal in sympodial arrangement. Discharge of zoospores (arrows) in the YC strain of Achlya. Cao et al. (2013) 206 Sexual reproduction characteristics of the YC strain of Achlya: (a) a mature oogonium, (b) an antheridial branch diclinous with the oogonium, (c) an antheridial branch monoclinous with the oogonium, and (d) a laterally formed oil globule in the oospore. Cao et al. (2013) Chained gemmae (arrows) of the YC strain Achlya, produced on branches of the main hyphae: (a) two catenulate gemmae, (b) a gemma catenulate with an oogonium, (c) three catenulate gemmae, (d) thirteen catenulate gemmae. Cao et al. (2013) Phylogenetic tree showing 100% similarity between the YC strain and Achlya klebsiana, constructed using the neighbor-joining method. Cao et al. (2013) Chauhan et al. (2013) examined 126 diseased fishes for the isolation of associated aquatic fungi. Fourty two isolates of Achlya were obtained from various species of fishes. These isolates were categorized in five different species of water mold, Achlya (A.americana, A.flagellata, A.hypogyana, A.klebsiana and A.prolifera.) Fifteen species of fishes viz. Catla catla, Channa punctatus, Channa striatus, Cirrhinus mrigala, Clarias batrachus, Labeo rohita, Mastacembalus armatus, Mystus cavasius, Mystus seenghala, Nandus nandus, Notopterus notopterus, Poecilia sphenops , Puntius sarana, Puntius ticto, Trichogaster fasciatus found infected with Achlya spp. and maximum host range was found in A.prolifera ,found associated with 10 species of fish. Maximum percentage of isolates was recorded from M.cavasius (21%). Most of the isolates of A.americana and A.prolifera were found in combination. Among 207 fungal isolates, maximum (37%) belong to A.prolifera. Histopathological finding of the most infected fish, M.cavasius showed epithelial desquamation of the epidermis, erosion and ulceration of the infected area, edema, hyaline degeneration, fragment of fungal hyphae occurred in the underling dermis of the skin, muscles and formation of granulomas 1. Hyphae of A.prolifera on head region of Mystus seenghala, 2. M.seenghala showing ulcer with hyphal tufts of A.americana. 3. A.prolifera hyphae on head and tail region of M.cavasius , 4. A.prolifera colony on soyabean seed. 5. Six days old A.prolifera culture on Corn Meal Agar , 6. The skin of infected M.cavasius , showed epithelial desquamation in theepidermis. The other epidermal cells suffered vacuolar degeneration and focal necrosis. 7. Epidermis is completely lost. The underlying dermis is necrotized, 8. The underling dermis was necrotized and contained fragments from the fungal hyphae with focal aggregation of melanomacrophages cells. The necrotic muscles infiltrated with numerous mononuclear leukocytes and some melanomacrophages , 9. Necrotized muscular tissue, cells are distended and lost their cytoplasm and nuclei.Numerous granulomas are formed encircled fungal hyphae Chauhan et al. (2013) Hussein et al. (2013) conducted an experimental study to evaluate the pathogenicity and pathology of Achlya proliferoides BSN-M005 isolated from saprolegniosis outbreaks against immature stages of Nile tilapia, Oreochromis niloticus. The cumulative mortality rateof the tested fish groups that exposed to high zoospore concentrations of A. proliferoides BSN-M005 was 60 %. The histopathological changes associated with saprolegniosis lesions induced by A. proliferoides BSNM005 showed mats of hyphae attached the surface of epidermis, necrosis, degenerative changes close to hyphae and sometimes fungal elements were observed penetrate into dermal layer but never reach the underling musculature. It is clear from our results that S. diclina BSN-M003 is highly pathogenic than A. proliferoides BSNM005 to Nile tilapia 208 Moribund immature Nile Tilapia, O. niloticus showing cotton-like mycelial growth scattered on the head and body surface, 6 days after challenging with A. proliferoides, Note superficial penetration of the fungal elements to the epidermal layer Hussein et al. (2013) Panchai et al. (2014) obtained 34 water mould isolates from the water of the Nam Phong River, Khon Kaen Province, Thailand. All isolates belonged to the genus Achlya and were identified as Achlya bisexualis, A. diffusa, A. klebsiana, A. prolifera and unidentified species of Achlya. Isolates of A. bisexualis and A. diffusa were the most abundant (35%), followed by the unidentified species of Achlya (18%) and then, A. klebsiana and A. prolifera (6% each). The ITS1-5.8S-ITS2 region of the unidentified isolates was sequenced for phylogenetic analysis. Three out of 6 isolates were indicated to be A. dubia (BKKU1005), A. bisexualis (BKKU1009 and BKKU1134), and other 3 out of 6 isolates (BKKU1117, BKKU 1118 and BKKU1127) will be an as-yet unidentified species of Achlya. The biological characteristics of the isolates showed optimum temperatures for vegetative growth of 25–35ºC. All of the Achlya isolates were able to grow under up to 1.5% sodium chloride. The isolates grew well, and their zoospores were able to germinate at pH 4.0–11.0. Microscopic examination of the skin lesions of the infected tilapia revealed bacteria and hyphae. Some of the hyphae penetrated into the epidermis, and numerous small blood vessels and scattered macrophages could be observed throughout the infected area 209 A - Cross-section of Nile tilapia skin showing aseptate hyphae (arrows) (Uvitex 2B–H&E); B numerous small blood vessels (and scattered macrophages (*) in dermis (Giemsa); C - germinated and fragmented hyphae (arrows) found in the disintegrated dermis (Giemsa); D - the underlying musculature tissue degenerated, enclosed by inflammatory cells and scattered melanomacrophages (PAS); E - scattered rod-shaped bacteria ( ) among hyphae (arrows) (Giemsa). Panchai et al. (2014) Hatai et al. (2015) performed an experimental infection of Nile tilapia (Oreochromis niloticus) fry using 6 Achlya isolates from cultured Nile tilapia with water mold infections. The experimental fish were exposed to 1.0 x 102 and 1.0 x 104 zoospores mL-1 of each Achlya isolate after ami-momi treatment. The cumulative mortality rates of fish exposed to 1.0 x 104 zoospores mL-1 of A. klebsiana BKKU1003, and A. diffusa BKKU1012 were 88.8 and 77.7%, respectively. A. klebsiana BKKU1003 was more pathogenic than the other isolates. Histopathological examination of the skin of Nile tilapia fry exposed to 1.0 x 102 zoospores mL-1 of A. klebsiana BKKU1003 showed numerous hyphae grew on the skin surface and some areas of skin were sloughed. The fish exposed to 1.0 x 104 zoospores mL-1 of A. klebsiana BKKU1003 showed massive accumulated hyphae on skin lesions with necrosis of the epidermal cells and the hyphae penetrated from the epidermis to the musculature without granulomatous response surrounding the hyphae. We found that it is possible to infect tilapia fry by exposing them to zoospores of Achlya after the ami-momi treatment. 210 The positions of skin section: head (A), trunk (B), caudal regions (C) of Nile tilapia fry. Hatai et al. (2015) Gross morphology of Nile tilapia fry after net shaking. A: the control group; B: the treatment group shaking for 2 minutes. . Hatai et al. (2015) 211 Cross section of skin from the head (A, D, G), trunk (B, E, H) and caudal (C, F, I) regions of Nile tilapia fry after the ami-momi treatment showing epidermis (E), scale (s), dermis (D), hypodermis (H), muscle (M) (H & E). A-C: the control group; D-F: the AM group for 2 minutes; G-I: the AM group for 5 minutes (arrow heads = mucous cells). . Hatai et al. (2015) A: Nile tilapia fry exposed to 1.0 x 102 zoospores mL-1 of A. klebsiana BKKU1003 for 3 days. Note the hyphae on the head, dorsal and ventral fins and caudal peduncle. B: Histopathology of skin of moribund tilapia fry showing hyphae with pink color (arrows) attached on epidermis (PAS). C: Serial section of B showing positive Uvitex 2B-H&E stain of hyphae with blue-white fluorescence (arrows). . Hatai et al. (2015) 212 A: Nile tilapia fry exposed to 1.0 x 104 zoospores mL-1 of A. klebsiana BKKU1003 for 4 days. Note the hyphae on the head, dorsal fin and caudal peduncle. B: Histopathology of skin of moribund tilapia fry showing aseptate hyphae with pink color (arrows) invaded musculature (PAS). C: Serial section of B showing positive Uvitex 2B-H&E stain of hyphae with blue-white fluorescence (arrows). . Hatai et al. (2015) 213 Photomicrographs of sexual apparatus in Achlya diffusa (After Prabhuji 2010 ). ( a ) Oögonium with many antheridial branches attached. ( b ) Oögonium with eggs (oöspheres) and fi nger-shaped branched antheridium. ( c ) Antheridium encircling it. ( d ) Several coiled antheridial branches. ( e ) Coiled monoclinous antheridial branches and one hypogynous antheridium. ( f ) Oögonium with distinct wall pitting. ( g ) Eccentric oöspores. ( h ) Abortive oögonia Jesus et al. (2015) collected Achlya catenulata sp. nov. from water samples in a mangrove swamp of the “Parque Estadual da Ilha do Cardoso”, São Paulo State, Brazil. This new species is characterized by the presence of achlyoid type of zoospore discharge from both primary and secondary sporangia, catenulate smooth-walled oogonia in chains of up to 11 oogonia, diclinous antheridial branches and eccentric oospores, which generally failed to mature. Maximum likelihood phylogenetic analyses based on the ITS and LSU regions (rDNA) placed this species within the Achlya sensu stricto clade. Maximum likelihood tree inferred from ITS rDNA sequences of isolates of Achlya. Numbers next to branches indicate bootstrap support (%) and the bar shows the number of substitutions per site. Jesus et al. (2015) 214 Saraswathi et al. (2015) carried out a study to assess the presence Achlya species and their effect in different aquatic systems in Pudukkottai District of Tamil Nadu. Of the total 314 fishes chosen, a total of 63 species were found with mycotic infections. The percentage of infection was found to vary from 15.3 to 38.09% examination of carps showed they had multiple infections. Among the nine species of fish examined, Channa striatus was the most prone to infection of Achlya species. Panchai et al. (2016) carried out study to determine the oomycetecidal effect of copper sulfate on both vegetative and zoosporic stages of water molds, Achlya spp., in in vitro tests and to evaluate the efficacy toxicity on Nile tilapia (Oreochromis niloticus) fry. The results show that copper sulfate at 100 mgL-1 killed both the vegetative stage of five selected Achlya spp. and the zoosporic stage of A. diffusa BKKU1012, A. prolifera BKKU1125 and Achlya sp. BKKU1127. Additionally, 25 mgL-1 copper sulfate solution could kill the zoosporic stage of A. klebsiana BKKU1003 and Achlya sp. BKKU1117 and also inhibited zoospore germination of all selected Achlya spp. with 30 minutes treatment. In addition, 6.25 and 12.5 mgL-1 copper sulfate solution had no toxic effect (0% mortality) on the tilapia fry. In contrast, 25, 50 and 100 mgL-1 copper sulfate solutions had strong toxicity to the fish (100% mortality) with 6 hours, 2 hours and 30 minutes treatment, respectively. Thus, this study revealed that it is possible to use copper sulfate to kill the aquatic oomycetes, Achlya spp., if it is given 30 minutes treatment References 1. Abking, N., Wichukarn Fuangsawat and Ong-ard Lawhavinit. Pathogenicity to Mekong Giant Catfish Eggs of Water Moulds Isolated in the Laboratory from Mekong Giant Catfish Eggs and Rearing Water. Kasetsart J. (Nat. Sci.) 46 : 91 - 97 (2012) 2. Barksdale AW, Lasure LL. Production of hormone B by Achlya heterosexualis. Appl Microbiol. 1974 Oct;28(4):544-6. 215 3. Cao, H,, Renjian Ou , Shan He , Xianle Yang. Identification of an Achlya klebsiana Isolate as the Causal Agent of Saprolegniosis in Eggs of Yellow Catfish (Pelteobagrus fuvidraco) and Control with Herbal Extracts IJA_65.2013.851, 9 pages 4. Coker, W. C. (1923) The Saprolegniaceae, with Notes on other Water Molds. University of North Carolina Press, Chapel Hill. 5. Chauhan.R, P.Kaur and S.sharma (2012) Pathogenicity of some species of Achlya and Saprolegnia on Indian Major Carps viz.Catla catla, Cirrhinus mrigala and Labeo Rohita ,J.of environ.sci.comp.sc. engg.and technology,vol1.no3,422-428. 6. Chauhan, R.,Shakaut Lone and Hussian Beigh, Gazala Tabbsum. MYCOTIC STUDIES OF SOME FRESH WATER FISHES WITH EMPHASIS ON ACHLYA SPP. International Journal of Research in Fisheries and Aquaculture, 2013; 3(4): 165169 7. Czeczuga B., Kiziewicz B., Danilkiewicz Z., 2002. Zoosporic fungi growing on the specimens of certain fish species recently introduced to Polish waters. Acta Ichthyol. Piscat. 32(2): 117–125. 8. Czeczuga, B.; Semeniuk, A.; Godlewska, A.; Czeczuga-Semeniuk, E.;Velu, C. S. Dead specimens of fairy shrimp Streptocephalus dichotomus (Crustacea) as vectors of mycosis-inducing fungi in fish aquacultures.Current Trends in Ecology, Volume 3 2012 pp. 53-60 9. El Androusse Amal, El Aissami Aïcha, Paul Bernard/Achlya abortispora, a new oomycete isolated from water samples taken from a water reservoir in Morocco. Curr Microbiol 2006 Jul 29;53(1):60-7. Epub 2006 May 29. 10. Hatai, Kishio , Panchai, Kwanprasert , Hanjavanit, Chutima , Rujinanont, Nilubon , Wada, Shinpei , Kurata, Osamu , (2015) Experimental pathogenicity of Achlya species from cultured Nile tilapia to Nile tilapia fry in Thailand. AACL Bioflux, 8 (1). pp. 70-81. 11. Hussein, MMA, Walid H. Hassan and Maha A. Mahmoud . Pathogenicity of Achlya proliferoides and Saprolegnia diclina (Saprolegniaceae) Associated with Saprolegniosis Outbreaks in Cultured Nile Tilapia (Oreochromis niloticus), World Journal of Fish and Marine Sciences 5 (2): 188-193, 2013 12. Jesus,A. L., Agostina Virginia Marano, José Ivanildo de Souza, Timothy Young James, Gustavo Henrique Jerônimo, Sarah Cristina Oliveira Rocha, Danilo Reis Gonçalves, Marcela Castilho Boro, Carmen Lidia Amorim Pires-Zottarelli. Achlya catenulata sp. nov., a new Saprolegniales (Oomycetes, Straminipila) from Brazilian mangrove swamp. Phytotaxa 212 (3): 221–228, 2015 13. Kales SC, DeWitte-Orr SJ, Bols NC, Dixon B. Response of the rainbow trout monocyte/macrophage cell line, RTS11 to the water molds Achlya and Saprolegnia. Mol Immunol. 2007 Mar;44(9):2303-14. Epub 2007 Jan 3. 14. Khulbe RD, Bisht GS, Joshi C. Epizootic infection due to Achlya debaryana in a catfish. Mycoses. 1994 Jan-Feb;37(1-2):61-3. 15. Lecanu L, Hashim AI, McCourty A, Papadopoulos V. A steroid isolated from the water mold Achlya heterosexualis induces neurogenesis in vitro and in vivo. Steroids. 2012 Feb;77(3):224-32. 16. Muhsin , T. M. , S. S. Rattan & A. L. S. Ismail. Aquatic fungi of Iraq: Species of Achlya Sydowia, Vol. XXXVII, 1984.224-237 17. Nilubol Kitancharoen , Kishio Hatai 1, Rina Ogihara 1, Daw Nwe Ni Aye . A new record of Achlya klebsiana from snakehead, Channa striatus, with fungal infection in Myanmar. Mycoscience Volume 36, Issue 2, July 1995, Pages 235-238 18. Osman, A (2010). "Genotoxicity of two pathogenic strains of zoosporic fungi (Achlya klebsiana and Aphanomyces laevis) on erythrocytes of Nile tilapia 216 Oreochromis niloticus niloticus.". Ecotoxicology and Environmental Safety. 73 (1): 24–31. 19. Panchai,K. Chutima Hanjavanit, Nilubon Rujinanont, 3Shinpei Wada, Osamu Kurata, Kishio Hatai. Freshwater oomycete isolated from net cage cultures of Oreochromis niloticus with water mold infection in the Nam Phong River, Khon Kaen Province, Thailand. AACL Bioflux, 2014, Volume 7, Issue 6. http://www.bioflux.com.ro/aacl 20. Panchai, K., Chutima Hanjavanit, Narumon Sangpradub, Kishio Hatai. Antioomycetic effect of copper sulfate in vitro on Achlya spp. isolated from infected Nile tilapia (Oreochromis niloticus) . AACL Bioflux, 2016, Volume 9, Issue 2, 414-421 21. Paul,B., Monica M Steciow. Achlya spiralis, a new aquatic oomycete with bent oogonial stalks, isolated from the Burgundian region of France. FEMS Microbiol Lett 2008 Jul;284(1):120-5 22. Saraswathi , R., P. Sumithra1 and R. Sivakami Studies on the effect of Achlya species in various fish species cultured in different freshwater systems of Pudukkottai District, Tamilnadu, India. Int.J.Curr.Microbiol.App.Sci (2015) 4(12): 670-674 23. Sosa ER, Landsberg JH, Kiryu Y, Stephenson CM, Cody TT, Dukeman AK, Wolfe HP, Vandersea MW, Litaker RW. Pathogenicity studies with the fungi Aphanomyces invadans, Achlya bisexualis, and Phialemonium dimorphosporum: induction of skin ulcers in striped mullet. J Aquat Anim Health. 2007 Mar;19(1):41-8 24. Srivastava RC. Host range of Achlya caroliniana Coker on certain fresh water fishes. Mykosen. 1978 Oct;21(10):355-8. 25. Tiffney, W. N. ; Wolf, F. T. Achlya flagellata as a fish parasite.Journal of the Elisha Mitchell Scientific Society 1937 Vol.53 No.2 pp.298-300 26. Walker, S.A., K Kenny Chitcholtan, YuPing Yu, Gabrielle M Christenhusz, Ashley Garrill. Invasive hyphal growth: an F-actin depleted zone is associated with invasive hyphae of the oomycetes Achlya bisexualis and Phytophthora cinnamomi. Fungal Genet Biol 2006 May 6;43(5):357-65 3.Aphanomyces (de Bary, 1860) genus Historical   Aphanomyces genus was described firstly in 1860 by de Bary and included initially four species: o A. laevis, o A. phycophilus, o A. scaber and o A. stellatus. Other species from this genus have been described by Drechsler (1929) and Scott (1961). o according to Index Fungorum, Aphanomyces genus comprises 45 taxa and 40 species (David and Kirk, 1997), o according to Ballesteros et al. (2006)- 30 species o according to Dieguez- Uribeondo et al. (2009)- 35-40 species. 217    Dick (2001) classified Aphanomyces together with Leptolegnia and Plectospyra to Leptolegniaceae family. Some species specialize in plant or animal parasitism, other ones are saprotrophic growing on decaying plant and animals debris o Plant parasitic species include:  A. cochlioides- pathogen of roots of sugar beet  P. euteiches which parasitize on Fabaceae species o Fish parasitic species include :  A. astaci is a parasite of a freshwater crayfish  A. invadans and A. frigidophilus (A. piscicida) devastate both freshwater and estuarine fishes laevis, A. stellatus and A. helicoides belong to opportunistic pathogens Classification 1. Species 2000 & ITIS Catalogue of Life: April 2013 Chromista +  Oomycota + o Oomycetes +  Saprolegniales +  Leptolegniaceae +  Aphanomyces  Aphanomyces astaci Schikora 1906  Aphanomyces brassicae S. L. Singh & Pavgi 1977  Aphanomyces cladogamus Drechsler 1929  Aphanomyces cochlioides Drechsler 1929  Aphanomyces euteiches Drechsler 1925  Aphanomyces frigidophilus Kitanch. & Hatai 1997  Aphanomyces helicoides Minden 1912  Aphanomyces iridis Ichit. & Tak. Kodama 1986  Aphanomyces irregularis W. W. Scott 1961  Aphanomyces laevis de Bary 1860  Aphanomyces ovidestruens Gickelh. 1923  Aphanomyces parasiticus Coker 1923  Aphanomyces phycophilus de Bary 1860  Aphanomyces raphani J. B. Kendr. 1927  Aphanomyces scaber de Bary 1860  Aphanomyces stellatus de Bary 1860 2. NCBI Taxonomy Cellular organisms +  o Eukaryota + Stramenopiles +  Oomycetes +  Saprolegniales +  Saprolegniaceae + 218                                                Aphanomyces Achlya spiralis Aphanomyces astaci Aphanomyces cf. repetans Aphanomyces cladogamus Aphanomyces cochlioides Aphanomyces euteiches + Aphanomyces frigidophilus Aphanomyces helicoides Aphanomyces invadans Aphanomyces iridis Aphanomyces laevis Aphanomyces piscicida Aphanomyces repetans Aphanomyces salsuginosus Aphanomyces sinensis Aphanomyces sp. WLL-2012 Aphanomyces stellatus 3. Index Fungorum Aphanomyces acinetophagus A.F. Bartsch & F.T. Wolf 1938 Aphanomyces americanus (A. F. Bartsch & F. T. Wolf) W. W. Scott 1961 Aphanomyces amphigynus Cutter 1941 Aphanomyces apophysii Lacy 1950 Aphanomyces astaci Schikora 1906 Aphanomyces balboensis J. V. Harv. 1942 Aphanomyces bosminae W. W. Scott 1961 Aphanomyces brassicae S. L. Singh & Pavgi 1977 Aphanomyces camptostylus Drechsler 1929 Aphanomyces cladogamus Drechsler 1929 Aphanomyces cochlioides Drechsler 1929 Aphanomyces coniger H.E. Petersen 1910 Aphanomyces daphniae Prowse 1954 Aphanomyces euteiches Drechsler 1925 Aphanomyces euteiches f. sp. phaseoli W. F. Pfender & D. J. Hagedorn 1982 Aphanomyces euteiches f. sp. pisi W. F. Pfender & D. J. Hagedorn 1982 Aphanomyces euteiches f.sp. euteiches W.F. Pfender & D.J. Hagedorn 1982 Aphanomyces exoparasiticus Coker & Couch 1926 Aphanomyces frigidophilus Kitanch. & Hatai 1997 Aphanomyces gordejevi Skvortsov 1925 Aphanomyces helicoides Minden 1912 Aphanomyces hydatinae Valkanov 1931 Aphanomyces invadans Willoughby, R. J. Roberts & Chinabut 1995 Aphanomyces iridis Ichit. & Tak. Kodama 1986 Aphanomyces irregularis W. W. Scott 1961 Aphanomyces keratinophilus (M. Ôkubo & Kobayasi) R. L. Seym. & T. W. Johnson 1974 Aphanomyces laevis de Bary 1860 Aphanomyces laevis f. keratinophilus M. Ôkubo & Kobayasi 1955 Aphanomyces laevis f. laevis de Bary 1860 219                 Aphanomyces laevis var. helicoides (Minden) Cejp 1959 Aphanomyces laevis var. laevis de Bary 1860 Aphanomyces magnusii Schikora 1922 Aphanomyces norvegicus Wille 1899 Aphanomyces ovidestruens Gickelh. 1923 Aphanomyces parasiticus Coker 1923 Aphanomyces patersonii W. W. Scott 1956 Aphanomyces phycophilus de Bary 1860 Aphanomyces pisci R. C. Srivast. 1979 Aphanomyces piscicida Hatai 1980 Aphanomyces polysporus Milovtz. 1936 Aphanomyces raphani J. B. Kendr. 1927 Aphanomyces scaber de Bary 1860 Aphanomyces sparrowii Cutter 1941 Aphanomyces stellatus de Bary 1860 Aphanomyces volgensis Domashova 1974 Diseases caused by Aphanomyces 1. Epizootic Ulcerative Syndrome (EUS)     Synonyms: Mycotic Granulomatoses (MG ) Red Spot Disease (RSD Ulcerative Mycosis (UM) Epizootic Granulomatous Aphanomycosis (EGA) Definition Epizootic ulcerative syndrome (EUS) is a fungal disease of freshwater and brackish fish affecting more than 100 fish species. It is caused by the fungal species, Aphanomyces invadans. The organism requires a specific combination of factors in order to germinate within the dermis of the fish. The disease causes lesions in both the skin and visceral organs. Historical An epizootic ulcerative condition in fish, known as mycotic granulomatosis, was first described in:  Japan in 1971 (Egusa & Masuda 1971) o Australia (Queensland – 1972, o New South Wales – 1989, o Northern Territory – 1990 and Western Australia – 1994); o Papua New Guinea (1975 –1976?; 1982–1983?; 1986); o Indonesia (1980?; 1993–1994); o Singapore (1977?); o Malaysia (1979?; 1980); o Thailand (1981); o Myanmar, Lao People’s Dem. Rep. and Cambodia (1983 or 1984); o Viet Nam (1983?); 220 o United States of America (North Carolina, Florida and Connecticut – 1984); o China (1982?; 1987–1988?; 1989?); o China, Hong Kong SAR (1988?); o Philippines (1985); o Sri Lanka (1987); o Bangladesh (1988); o India (1988); o Bhutan and Nepal (1989); o Philippines (1995), o Pakistan (1996); o Botswana (2006?; 2007); o Namibia (2006?; 2007); o Zambia (2007?; 2008, 2009). Distribution Disease is present in parts of the Asia-Pacific region and Australia. A similar disease has also been reported in the eastern USA. Map showing the current global distribution epizootic ulcerative syndrome (1971 to 2008) Signalment   Over 100 species have been confirmed to be affected by EUS. Infections have been reported in a variety of freshwater and brackish water species, including: • barbs (Africa), • bream (Africa), • catfishes (Africa), • cichlids (Africa), • churchill (Africa), • eels (Asia), • gobies (Asia), • gouramies (Asia), • Indian carps (catia, mrigal, rohu) (Asia), • ayu (Japan), • menhaden (United States of America), • mullets (Asia), • perches (Asia, Africa), • seabass (Asia), • seabream (Asia), • snakehead (Asia) and • tilapias (Africa). 221   EUS occurs commonly during periods of low temperature and heavy rainfall in tropical and sub-tropical waters. These conditions favour sporulation and cold temperatures delay the inflammatory response of the fish to infection. Some species have been shown to be resistant including Chinese carps, milkfish and tilapia. Aetiology     Aphanomyces invadans Aphanomyces invaderis Aphanomyces piscicida EUS related Aphanomyces (ERA) Pathogenesis        Spread of A. invadans is via zoospores in the water. Secondary zoospores enter the skin following a breach of the epidermal barrier by physical or environmental causes (Baldock et al. 2005; Lilly & Roberts 1997). The zoospores germinate and hyphae invade the skin and musculature, causing a focal necrotising granulomatous dermatitis and myositis. Hyphae may invade more deeply, causing granulomatous inflammation in the internal organs (Baldock et al. 2005). The tissue necrosis in early lesions is associated with an intense inflammatory reaction that is observed as a characteristic focal reddening and swelling of the skin, hence the name ‘red spot disease’ used in some countries (Anon 2008). More advanced lesions are characterised by large and deep ulcers surrounded by a raised rim of inflamed tissue. The descriptive name epizootic ulcerative syndrome (EUS) has become the internationally accepted term for infection with A. invadans. Predisposing factors include    lower temperatures (less than 25C, 77F), reduced salinity in brackish water systems [17,19], presumably suppressing the immune system or primary defenses (mucus production, epithelial integrity) against invasion Susceptibility varies among species.   Tilapia (Orechromis niloticus) are considered relatively immune, snakehead (Channa species) and some barb species (Puntius species) are considered very susceptible. Clinical signs  In early disease, red spots or small haemorrhagic lesions are found on the surface of the fish.  These progress to ulcers and eventually large necrotic erosions. Fungal mycelium is often visible on the surface of ulcers.  Death then follows rapidly due to visceral granulomata, septicaemia and failure of osmoregulatory balance. 222 Barbus paludinosus – Straightfin barb, Typical severe mycotic granulomas (black arrows) from muscle section of EUS fish (snakehead from the Philippines) (haematoxylin and eosin [H&E]), FAO, 2009. Mugil sp. – Mullet, Aphanomyces sporangium , FAO, 2009 Plecoglatus altivelis – Ayu Barbus poechii – Dashtail barb Clarias gariepinus – Sharptooth catfish, FAO, 2009 Brycinus lateralis – Striped robber FAO, 2009 Sargochromis codringtonii – Green bream EUS susceptible fish Serranochromis robustus FAO, 2009 223 Channa striata – Snakehead FAO, 2009 Aphanomyces sporangium, Japanese isolate Aphanomyces sporangia, Botswana isolates, FAO, 2009 Histopathology of EUS-infected dashtail barb (Botswana) showing typical mycotic granulomas surrounding the invasive fungal hyphae (stained black, black arrows) in the skin layer (Grocott’s silver stain) Typical severe mycotic granulomas (black arrows) from muscle section of EUS fish (barb from Namibia) (H&E), FAO, 2009 Diagnosis 224  Analysis of histological sections demonstrating mycotic granulomas In early lesions:    acute spongiosis epithelial cell loss. Degenerative changes progress through the dermis with hyperaemia, haemorrhages and inflammatory infiltration. In advanced stages,   sarcolysis is also obvious.  mycotic granulation spreads through the infected muscle and internal organs.   Fungal hyphae are enclosed by a well defined epithelioid cell layer Muscle fibres eventually disappear altogether and are replaced by fibrosis, inflammatory cells and new blood vessels. These distinct features of EUS ulcers make histological analysis enough for a definitive diagnosis.  Squash preparations of skeletal muscle from beneath an ulcer will also demonstrate septate fungal hyphae for a rapid provisional diagnosis.  Isolation of the causal fungus.   Moderate, pale, raised lesions are best for fungal isolation. Muscle should be exposed in a sterile manner by removing scales and searing the skin with a red hot spatula before dissecting with a sterile scalpel.  2mm samples should be placed in a petri dish containing Czapex Dox agar with penicillin G and oxolinic acid.  They should be incubated at room temperature and examined daily so that emerging hyphal tips can be transferred onto fresh plates to produce contaminant free cultures.  The fungus can then be identified by inducing sporogenesis and demonstrating its typical asexual characteristics.  Injection of spore containing innoculum into susceptible fish at 20⁰C and demonstration of histological growth after 7 days and granulomas in muscle after 14 days is also diagnostic. Treatment    Keeping diseased fish in good quality, clean water may allow recovery, but only if lesions are not too extensive and dark scars are often left behind on healing. There is no effective treatment for advanced disease. Control of EUS in natural waters (e.g. rivers) is impossible. o Fish farmers whose farmed fish have been affected with EUS are encouraged to culture non-EUS susceptible species or avoid farming susceptible species during EUS season, i.e. rainfall period and low temperature season. o A strict ban on the movement of fish from infected waterways or river systems, especially those with lesions of EUS, to other waterways or river systems is recommended; 225        o diseased fish should not be moved from one fish farm to another. o Properly dried, salted and iced fish have not been reported as potential carriers of EUS, therefore trade of these products can be allowed to continue. A number of simple biosecurity measures can minimize or prevent the spread of EUS. These include: o All possible carriers or vectors such as freshly dead fish, birds or terrestrial animals as well as contaminated fishing gears/net and fish transport containers should be prevented from getting into water bodies or fish ponds. o In outbreaks occurring in small, closed water bodies, liming of water and improvement of water quality, together with removal of infected fish, are often effective in reducing mortality. o Increasing salinity in holding waters may also prevent outbreaks of EUS in aquaculture ponds. o During dry and cold seasons, close observation of wild fish should be made to determine the presence of EUS–diseased fish in neighbouring tanks or canals, in which case, exchange of water should be avoided. EUS infected fish should not be thrown back to the open waters and should be disposed of properly by burying them into the ground or through incineration. Additional practical aquaculture biosecurity measures include: – o good farm hygiene (e.g. handwashing between tanks, separation of nets/tanks/stocks, regular and correct disinfection procedures, etc.) o good husbandry practices o good water quality management o proper handling of fish o regular monitoring of fish health o good record keeping (gross and environmental observations and stocking records including movement records of fish in and out of aquaculture facility, etc.) Early reporting or notification to concerned authorities of a disease outbreak or suspicion of any abnormal appearance, behavior or other observations in fish stocks. Report immediately a suspected outbreak to concerned authorities (nearest fisheries or veterinary authority) and ask for guidance concerning collection of samples Take note of simple observations such as: o abnormal fish behaviour (e.g. fish swimming near the surface, sinking to the bottom, loss of balance, flashing, cork-screwing or air gulping (for non air-breathers) or any signs which deviates from normal behaviour o date and time of observed outbreaks o estimate of mortalities o species of fish affected and estimate of mortalities o pattern of mortality (small number of fish dying everyday, large number of fish dying at one time, etc.) o any unusual events in the event of a disease outbreak o Live samples if available are best for laboratory examination. 226 o The fish should be packed in double plastic bags, filled with water to one third of their capacity with the remaining 2/3 volume inflated with air/oxygen. Bags should be tightly sealed (with rubber band or tape). o If live fish which can be transported to the laboratory is not available, freshly dead or moribund fish with clinical lesions can be used. o Using a scalpel or a blade, take samples of skin/ muscle sections (<1 cm3) ), including the edge of the lesion and the surrounding tissue. Parts of internal organs may also be collected by dissecting the whole fish. o Fix the tissue samples immediately in 10 percent formalin (10 ml of formalin in 90 ml of water preferably distilled water) in a plastic or bottled container. The amount of formalin should be 10 times the volume of the tissue to be fixed. Tissues should be fixed for at least 24 hours before processing. o Fixed tissues can be wrapped into formalin-moistened tissue paper and placed into small plastic bags to prevent leakage or smell during transport. o Make sure that samples are properly labelled with the following information: date of samples, type of tissue samples (e.g. skin, gills, muscle, kidney, other internal organs), collected, locality (place of collection), species of fish (weight and length measurements if possible), name of collector, type of fixative used (10 percent formalin, etc.). o Samples can be packed into a padded envelope or container and sent by mail if no courier services exist. o Call the laboratory to inform of the kind of samples collected and when they are expected to arrive or to be delivered. 2. Crayfish plague Synonyms:      Fish plague Crayfish aphanomyciasis, La peste, Krebspest, Kraftpest. Aetiology: Aphanomyces astaci It is probably the first epidemic disease reported from a crustacean. Historical In the 1850s, the pathogen was most likely introduced to northern Italy; probably on crayfish in ballast in ships from the USA or Canada. 227      First report in Italy (1860) France, 1875, Germany, 1880, Russia, 1890, Finland, 1893 In the 1960s and 1970s, the pathogen was re-introduced to Europe through the importation of infected crayfish, Pacifastacus leniusculus and Procambarus clarkii, from the USA during attempts to revive the crayfish industry (Alderman, 1996; Holdich, 2003)     United Kingdom, 1981. Turkey, 1984 Ireland, 1987 Sweden 1988, Disease signs at the farm level      high mortality at time of initial outbreak many dead or weak crayfish floating or lying in watercourses or ponds (mortalities may go unnoticed in the wild) crayfish in open water with an unsteady and raised gait ('walking on stilts') rapid tail escape response weakens crayfish fall over, unable to right themselves (more evident out of water) Clinical signs of disease in an infected fish       fungal growth on soft, non-calcified parts of shell browning or blackening spots on the carapace, where fungal hyphae proliferate white necrotic musculature in tail instead of the pearl grey of healthy crayfish fine black lines on the soft shell underneath the tail melanised (black) shell in chronically infected individuals death, within weeks, of nonresistant European crayfish Crayfish plague. Segment with brown markings shows signs of typical infection from fungus. Segments either side indicate healthy muscle tissue, Crayfish plague, showing classic darkening at base of walking legs Source: D Alderman 228 Infected white-claw crayfish showing areas of whitening of the musculature and melanin deposits, Nicky Buller, nbuller@agric.wa.gov.au Melanin deposits on claws of white-clawed crayfish, Nicky Buller, nbuller@agric.wa.gov.au Host range             Crustaceans known to be susceptible to crayfish plague: Japanese crayfish (Cambroides japonicus) Louisiana swamp crayfish (Procambarus clarkii) noble crayfish (Astacus astacus) signal crayfish (Pasifastacus leniusculus) stone crayfish (Austropotamobius torrentium) Turkish crayfish (Astacus leptodactylus) white-clawed crayfish (Austropotamobius pallipes) Chinese mitten crab (Eriocher sinensis) freshwater crayfish (Cherax spp) giant Tasmanian crayfish (Astacopsis gouldi) Gippsland spiny crayfish (Euastacus kershawi) 229 Epidemiology            Mortalities of up to 100% have occurred in Europe, with extermination of susceptible populations. The North American crayfish is largely resistant and is a carrier of the disease agent and source of transmission among less resistant species of crayfish. All species of freshwater crayfish are believed to be susceptible to infection with crayfish plague The disease was introduced into Europe in American freshwater crayfish and has decimated European crayfish stocks (both wild and cultured). There has been no evidence of developing resistance to the disease among European species during the 100 years since its introduction. Crayfish plague can occur at any time of year, but is more likely in the summer months. Death can occur 5–50 days (or more) from initial infection, depending on water temperature and the initial number of zoospores. The fungal infestation releases motile zoospores direct to the water column when the crayfish dies, and these infect other crayfish. Transmission is horizontal through the water column. Translocation and migration of fish, birds and other wildlife can allow them to act as vectors, transporting the disease to previously unexposed waters. The fungus can be introduced to a new susceptible population on contaminated ropes, traps, fishing gear, boots, nets and other equipment. The epidemiology of the disease and its genetic diversity has been possible due to the application of random amplified polymorphic DNA (RAPD) analysis. This has allowed the description of five genotypes. Pathogenesis      The life cycle of A. astaci is simple with vegetative hyphae invading and ramifying through host tissues, eventually producing extramatrical sporangia that release amoeboid primary spores. The primary spores initially encyst, but then release a biflagellate zoospore (secondary zoospore). Biflagellate zoospores swim in the water column and, on encountering a susceptible host, attach and germinate to produce invasive vegetative hyphae. Free-swimming zoospores appear to be chemotactically attracted to crayfish cuticle and often settle on the cuticle near a wound. Zoospores are capable of repeated encystment and re-emergence, extending the period of their infective viability. 230 Susceptible host species        Almost all species of freshwater crayfish have to be considered as susceptible to infection with A. astaci. The outcome of an infection varies depending on species. All stages of European crayfish species, are highly susceptible including the o Noble crayfish (Astacus astacus) of north-west Europe, o The white clawed crayfish (Austropotamobius pallipes) of south-west and west Europe, o The related Austropotamobius torrentium (Aphanomyces astaci) o The slender clawed or Turkish crayfish (Astacus leptodactylus) of eastern Europe and Asia Minor o North American crayfish such as the signal crayfish (Pacifastacus leniusculus), Louisiana swamp crayfish (Procambarus clarkii) and Orconectes spp. are infected by A. astaci, but under normal conditions the infection does not cause clinical disease or death. All North American crayfish species have been shown to be susceptible to infection and it is therefore currently assumed that this is the case for any other North American species. The only other crustacean known to be susceptible to infection by A. astaci is the Chinese mitten crab (Eriocheir sinensis) but this was reported only under laboratory conditions. All live stages need to be considered as susceptible to infection.. Target organs and infected tissue     The tissue that becomes initially infected is the exoskeleton cuticle. Soft cuticle, as is found on the ventral abdomen and around joints, is preferentially affected. In the highly susceptible European crayfish species, the pathogen often manages to penetrate the basal lamina located underneath the epidermis cell layer. From there, A. astaci spreads throughout the body primarily by invading connective tissue and blood vessels; however, all tissues may be affected. In North American crayfish species, infection is usually restricted to the cuticle. Transmission mechanisms The main routes of spread of the pathogen are through o movement of infected crayfish o movement of spores with contaminated water or equipment, as may occur during fish movements, o colonisation of habitats, initially occupied by highly susceptible species, by North American crayfish species carrying A. astaci is likely to result in an epidemic among the highly susceptible animals. o transmission from crayfish to crayfish occurs, in short, through the release of zoospores from an infected animal and attachment of such zoospores to a naïve crayfish. o the zoospores of A. astaci swim actively in the water column and have been demonstrated to show positive chemotaxis towards crayfish. 231 o o o the main route of spread of crayfish plague in Europe between the 1960s and 2000 was through the active stocking of North American crayfish into the wild or escapes from crayfish farms. nowadays, spread mainly occurs through expanding populations of North American crayfish, accidental co-transport of specimens, and release of North American crayfish into the wild by private individuals. Fish transports may facilitate the spread of A. astaci in a number of ways, such as through  the presence of spores in the transport water,  A. astaci surviving on fish skin,  co-transport of infected crayfish specimens, or a combination of all three.  contaminated equipment (nets, boots clothing, etc.). Geographical distribution       First reports of large crayfish mortalities go back to 1860 in Italy Further reports of crayfish mortalities in the Franco-German border region in the third quarter of the 19th century. Infection spread then to o the Balkans o the Black Sea, o Russia o Finland o Sweden. The first outbreaks in Spain were reported in the 1960s In the 1980s further extensions of infection to the British Isles, Turkey, Greece and Norway. Australia has not experienced any outbreaks of crayfish plague 232 Diagnosis Nicky Buller, Animal Health Laboratories Department of Agriculture and Food Western Australia Clinical signs    Gross clinical signs are extremely variable and depend on challenge severity and water temperatures. The first sign of a crayfish plague is the presence of large numbers of dead crayfish in a river or lake In susceptible species infection will spread quickly and stretches of over 50 km may lose all their crayfish in less than 21 days from the first observed mortality. 233     Infected susceptible crayfish generally do not survive and 100% mortality is the norm. Infected crayfish of the highly susceptible crayfish species have a reduced escape reflex, and progressive paralysis. Dying crayfish are sometimes found lying on their backs. Occasionally, the infected animals can be seen trying to scratch or pinch themselves. Gross pathology     Foci of infection in crayfish may be seen by the naked eye Foci can best be seen under a low power stereo microscope and are most commonly recognisable by localised whitening of the muscle beneath the cuticle Brown colouration of cuticle and muscle may occur Hyphae may be visible in infected cuticles in the form of fine brown (melanised) tracks in the cuticle itself, cuticle between the carapace and abdomen, the joints of the pereiopods (walking legs), particularly the proximal joint and finally the gills. Histopathology  Tissue scrapings from affected areas can be smeared on a glass slide, air dried and stained with Wrights-Giemsa stain or a commercial equivalent (e.g., DiffQuick® or Hemacolor®). Examine stained air dried slides under the microscope for hyphae of A. astaci. Fungal hyphae, formation of single spores in sporangium, and encysted spores in a wet preparation. Nicky Buller, Animal Health Laboratories Department of Agriculture and Food Western Australia  The presence of distinctive aseptate, wide hyphae (5 to 10 µm in width) may be observed in tissue section (preferably containing lesions found in the 234 cuticle) that are stained with haematoxylin and eosin stain. Haemocytes that surround and encapsulate the hyphae, and become melanised can give the hyphae a knobbly appearance Grocott silver stain combined with a haematoxylin and eosin counter stain showing fungal hyphae in connective tissue. Nicky Buller, Animal Health Laboratories Department of Agriculture and Food Western Australia Grocott silver stain combined with a haematoxylin and eosin counter stain showing fungal hyphae in connective tissue around nerve cord Nicky Buller, Animal Health Laboratories Department of Agriculture and Food Western Australia  The presence of host haemocytes and melanisation closely associated with and encapsulating the hyphae give good presumptive evidence that the hyphae represent a pathogen rather than a secondary opportunist invader. 235 Electron Microscopy:  Penetration of the soft cuticle of crayfish by A. astaci zoospores begins with the lysis of the lipid surface layer of the crayfish and the formation of a germ tube (infection peg) that penetrates through the epicuticle by histolytic activity combined with mechanical penetration.  A hypha developed from the germ tube usually forms below the inner epicuticular surface and in the endocuticle.  Hyphae grow preferentially parallel to the surface, occasionally perpendicular to it. Subsequently, the hyphal tips swell and some hyphae start to penetrate through the cuticle. Culture:    Diagnosis of crayfish plague requires the isolation and characterisation of A. astaci using simple mycological media (12.0 g agar; 1.0 g yeast extract; 5.0 g glucose; 10 mg oxolinic acid; 1000 ml natural water (from river or lake); fortified with antibiotics (4 international units/ml penicillin G (sterile) added after autoclaving and cooling to 40°C) to control bacterial contamination Isolation may only be successful before or within 12 hours of the death of infected crayfish. o Excise small pieces of infected cuticle and muscle, transfer them to a Petri dish of sterile distilled water for extensive washing and further cutting into smaller pieces (1 to 2 mm²). o With sterile instruments, aseptically place the small pieces on the surface of the medium. o If no lesions are evident, sample muscle and cuticle for at least three sites in each animal (especially around the base of the walking legs close to the body and inside the thorax). o A sterile glass ring may be placed around the inoculum to force hyphae emerging from the piece of the cuticle to grow within the agar. o Incubate at 16°C for about 15 days. Note that fungi associated with crayfish may grow as contaminants in the cultures and could overwhelm A. astaci (Cerenius et al. 1988). Growth of A. astaci is almost entirely within and on the surface of the agar but with no aerial hyphae. Colonies are colourless. o the process of sporulation, where spores are produced to discharge from the hyphal tip and encyst before producing motile spores (zoospores) that swim away, is used to identify A astaci o sporangia form readily in 20 to 30 hours at 16°C and 12 to 15 hours at 20°C. o Sporangia are myceloid, terminal or intercalary, and develop from undifferentiated vegetative hyphae. o Terminal sporangia are simple, developing from new extramatrical hyphae. o Intercalary sporangia are quite complex and develop by the growth of a new lateral extramatrical branch, which forms the discharge tube of the sporangium. 236 o The cytoplasm of developing discharge tubes is noticeably dense, and slightly wider (10 to 12 µm) than ordinary vegetative hyphae. o Sporangia are delimited by a single basal septum in the case of terminal sporangia and by septa at either end of the sporangial segment in intercalary sporangia. o These septa are markedly thicker than the hyphal wall and have a high refractive index. o Primary spores (cytoplasmic units) are formed from the contents of the sporangium and are released (within 5 minutes) from the sporangial discharge tube and accumulate at this point. o The spores become round and a cyst wall develops. Most spores remain as a cluster (15-30 spores) at the sporangial tip, but some encyst away from the sporangial tip. o The number of spores in a cluster of A. astaci is generally less than that of other Aphanomyces species. The clusters are adherent and fairly resistant to physical disturbance, and spores will remain encysted for 8-12 hours. o A reniform shaped, biflagellated zoospore (8 x 12 µm, motility takes 5-20 minutes to develop) emerges from each cyst and swims away leaving the empty capsules of the encysted spore Isolation methods o Isolation medium (IM): 12.0 g agar; 1.0 g yeast extract; 5.0 g glucose; 10 mg oxolinic acid; 1000 ml river water; and 1.0 g penicillin G (sterile) added after autoclaving and cooling to 40°C. o Any superficial contamination should first be removed from the soft intersternal abdominal cuticle or any other areas from which cuticle will be excised by thoroughly wiping the cuticle with a wet (using autoclaved H2O) clean disposable paper towel. o Simple aseptic excision of infected tissues are placed as small pieces (1–2 mm3) on the surface of isolation medium plates, will normally result in successful isolation of A. astaci from moribund or recently dead (48 hours may be required in infected crayfish tissues o The PCR method Amplification of a 569 bp fragment of A. astaci DNA is performed using primers targeting the ITS region (internal transcribed spacer) of the protist: 5’-GCT-TGTGCT-GAG-GAT-GTT-CF-3’ (primer 42) and 5'-CTA-TCC-GAC-TCC-GCA-TTC-TG-3’ (primer 640). Control and prevention   Once A. astaci has been introduced into a population of highly susceptible crayfish species in the wild, the spread within the affected population cannot be controlled. To avoid the main pathways of introduction, the following measures are necessary: o Movements of potentially infected live or dead crayfish, potentially contaminated water, equipment or any other item that might carry the pathogen from an infected to an uninfected site holding susceptible species should be prevented. o Any fish movements from the site of a current epidemic of crayfish plague carries a high risk of spread and should generally be avoided. 237 o o If fish movements from a source containing North American crayfish are being planned, fish harvest methods at the source site need to ensure that:  crayfish are not accidentally co-transported;  the transport water does not carry A. astaci spores,  equipment is disinfected between use;  the consignment does not become contaminated during transport. The release of North American crayfish into the wild should be prevented. Recommendations to avoid an introduction of A. astaci onto the site: 1. Movements of potentially infected live or dead crayfish, potentially contaminated water, equipment or any other item that might carry the pathogen from an infected to an uninfected site holding susceptible species must be prevented. 2. If fish transfers are being planned, these must not come from streams or other waters that harbour potentially infected crayfish (either susceptible crayfish populations that are going through a current outbreak of crayfish plague or North American carrier crayfish). 3. North American crayfish must not be brought onto the site. 4. Fish obtained from unknown freshwater sources or from sources, where North American crayfish may be present or a current outbreak of crayfish plague may be taking place, must not be used as bait or feed for crayfish, unless they have been subject to a temperature treatment that will kill A. astaci 5. Any equipment that is brought onto site should be disinfected. Summarized from Crayfish plague (Aphanomyces astaci) 68 Manual of Diagnostic Tests for Aquatic Animals 2009 Aphanomyces species isolated from fish and shellfish, Takuma et al. 2010 238 Aphanomyces species reported in fish, Czeczuga et al., 2015 Description of Aphanomyces species reported in fish 1. Aphanomyces astaci (Schikora 1906) non-septate, branching hyphae, ca. 7-10 µm in diameter with rounded hyphal tips can be seen in the cuticle of infected crayfish. The oomycete reproduces asexually through formation of mobile zoospores. Infection of the host commences with the encystment of the zoospore in the cuticle of the crayfish. After settling on the crayfish cuticle the zoospore discards the flagella and encysts. Germination proceeds, a germ tube penetrates the cuticle and hyphae ramify the cuticle. hyphae penetrate into deeper tissues and organs. This may also happen in individuals of resistant species weakened by other infections, injuries or stress. The final phase of the infection is sporulation and release of zoospores, which occurs just prior to or soon after death, when hyphae grow outwards and give rise to sporangia. The primary spores are extruded through the hyphal tip and cluster around the sporangial ope i g to for a typi al spore all . These pri ary spores the dis harge as se o dary zoospores, develop flagella and swim off in the hunt for a new host. The zoospores remain viable only for a few days, after which they either encyst in a favourable site and germinate, 239 or, if the encystment site is not suitable, develop into a new zoospore. This process of repeated zoospore emergence can occur up to three times before the zoospore finally dies Hyphae (a) and sporangium with emerging spore ball (b) of the oomycete Aphanomyces astaci. Photo ©: Trude Vrålstad (a) and David Strand (b), Norwegian Veterinary Institute Generalized life cycle of A. astaci. Sporangia with primary spores (1) are released as a spore ball (2) from where motile zoospores (3) are released into the water. Zoospores will either encyst (4) and repeat zoospore emergence in the absence of a suitable host, or locate a suitable host with chemotaxis (5) and encysts on the host surface (6). Aided by turgor pressure and enzymes, a developing infection spike penetrates the host (7) and non-septate hyphae ramify (8-9) within the host cuticle (exoskeleton). The immune response reaction in North American crayfish species rapidly restrict the infection to invisible or minor dark spots (hyphae encapsulated in crayfish generated melanin, se Fig. 2), while in susceptible crayfish species the infection rapidly proceed into the body cavity and nerve system, leading to host death. The host disease and death triggers the production of new spore producing hyphae/sporangia (10). Crayfish mass mortalities will therefore cause a drastic spore blooming of A. astaci. From Vrålstad et al., 2006 with permission. © Trude Vrålstad 2. Aphanomyces brassicae, Singh & Pavgi 1977 240 Hyphae hyaline, delicate, 4.5-8.5/~ in width, moderately branched approximately at right angles. Zoosporangia terminal, moderately long, 4.57.5/~ in width, formed from the undifferentiated vegetative hyphae, never tapering towards the apex, of even diameter from basal to distal end. Primary zoospores spherical, 20-30 in number, encysting upon emergence above the orifice; primary zoospore cysts 5-8.5/~ in diam. Secondary zoospores ovate to reniform, laterally biflagellate. Oogonia terminal on short branches, primarily subglobose, thin-walled, measuring 24.5-35.5# in diam. Antheridia arising from a simple, short stalk, clavate, measuring 5.5-8.5 • 8-14.5/~, always single, diclinous. Oospores spherical, thick-walled with smooth outer wall, hyaline, measuring 16.5-21.5 /~ in diam. Germination either by formation of germ sporangium or by direct production of vegetative mycelium and zoosporangia. Aphanomyces brassicae, Singh & Pavgi 1977 3. Aphanomyces cladogamus Drechsler, Journal of Agricultural Research 38: 309-361, 1929. Mycelium is composed of colourless aseptate hyphae, 4-10 µm diam., sparingly branched, with a delicate appearance in water and on solid media, producing antheridia and oogonia from close or distant hyphal branches ('monoclinous' or 'diclinous'). Sporangia very long, 2-3 mm x 7-9 µm, not tapering towards the apex, with many lateral branches, 1,5-2 mm long. Primary zoospores elongate, highly abundant, encysting at the sporangium orifice on emergence. Primary cysts 7-10 µm diam., germinating to give reniform, laterally biflagellate secondary zoospores which germinate to produce hyphae. Antheridia borne on often branched subtending stalks 241 10-20 µm long, usually observed joined to an oogonium, clavate to cylindrical, 10-13 x 5-8 µm, with an apical prolongation 10 x 4 µm, and a branched extension 8-18 µm long adjacent to the delimiting septum; fertilisation tubes short. Oogonia subspherical, colourless (20-) 25 (-33) µm diam., with a wall externally smooth and internally sinuous, borne terminally on branches of variable length and winding around 2-3 antheridia. Oospores spherical, colourless, (15-) 21 (-25) diam. ('aplerotic'), wall 3 µm thick, contents granular with a large central oil globule and a small, conspicuous refractive body; germination not observed. A & B. A. cladogamus A. Inner wall of oogonium irregular. × 1000. B. Zoosporangium. C. Aphanomyceshelicoids. Antheridial branches coiling about the oogonial stalk × 1000. D. Achlya americana,with numerous maturing oospores. × 800. E. Aphanomyces parasiticus, parasitic in the hyphae of A. americana. × 1200. F. A. stellatus. Oogonium covered with bluntly conical tubercles × 1000. T. S. Chiou and H. S. Chang 4. Aphanomyces cochlioides Drechsler, Journal of Agricultural Research 38: 309-361, 1929. 242 Mycelium scanty on solid media, extensive and mat-like in submerged liquid culture, composed of colourless, aseptate hyphae 5-10 µm diam., branching at right angles infrequently, with some branches forming short diverticulate spurs 5-10 µm long, especially near the oogonia, producing antheridia and oogonia from distant hyphal branches ('diclinous'). Sporangia up to 4 mm long, variable in diam., sinuous, delimited from the vegetative mycelium by a septum. Primary zoospores elongate, (10-) 100-200 (-300) per sporangium, encysting at the sporangial orifice on emergence. Primary cysts spherical, 6-15 µm diam., with a papilla 2 µm diam. Secondary zoospores reniform, 8-13 x 7-8 µm, laterally biflagellate. Antheridia borne on a stalk which produces sterile branches, usually observed joined to an oogonium, curved-clavate, 6-10 x 9-18 µm, with apical prolongations; fertilisation tubes short. Oogonia subspherical, 22-28 µm diam., with an irregularly thickened wall having a smooth outer surface and a sinuous inner contour, borne terminally on a short lateral branch separated from the vegetative mycelium by a septum; surrounded by 1-5 antheridia. Oospores spherical, 16-24 µm diam. ('aplerotic'), colourless to bright yellow, wall 1,5-2 µm thick, with granular contents, a large central oil globule 12 µm diam., a smaller conspicuous, spherical or rectangular refractive body, and one or more layers of finely granular protoplasm. Oospores of Aphanomyces cochlioides with: A) a densely organized uniform granular appearance typical of living oospore compared to B) a loosely organized nonuniform granularappearance of a dead oospore Windels et al. 2003, Proceedings of the 2nd Inaternational Aphanomyces Workshop 5. Aphanomyces frigidophilus Kitanch. & Hatai, 1997 The isolate exhibited thin hyphae with rounded hyphal tips, and with a hyphal diameter that ranged from 5 to 7 μm. The isolate produced sporangia with a single row of primary spores. The primary spores were eventually released and encysted at the hyphal tip forming spore-balls characteristic for the genus Aphanomyces. No oogonia or antheridia were seen in either individual cultures or in co-culture with the other isolates or with representive strains of four genetic groups of A. astaci. Thus the strains appeared to be sterile and lack sexual reproduction.The encysted zoospores did not undergo repeated zoospore emergence and instead germinated. 243 Aphanomyces frigidophilus. a) Hypha with a rounded tip growing within the cuticle of the freshwater crayfish Austropotamobius pallipes. b) “Spore balls” characteristic of the genus Aphanomyces (Bars 10 –e) Chitinase assay for production of chitinase constitutively during growth: c) negative control, without fungus; d) A. frigidophilus (negative); e) A. astaci (positive). Ballesteros et al., 2006 244 6. Aphanomyces helicoides Minden, Kryptogamen-Flora der Mark Brandenburg 5: 559 (1912) Hyphae 5-9μ in diameter, delicate, branched and forming characteristic knots. Zoosporangia filamentous, long, formed from undifferentiated vegetaive hyphae, isodiametric. Primary zoospore cysts 8-11.5μ in diameter. Oogonia terminl on lateral branches of variable length, often formed in dense clusters; spherical; 21-38μ in diameter; smooth-walled; contents finely granular with a large, central oil globule. Antheridia one to five, large elongate cylindrical, closely wrapped about the oogonium. Antheridial stalk simple or branched; diclinous or monoclinous; sometimes forming helicoidal spirals wrapping about the oogonial stalk, and extensively wrapping about themselves and around adjacent hyphae. Fertilization tubes not observed. Germination on the oospore by the formation of a single germ tube. 7. Aphanomyces invadans Willoughby, R.J. Roberts & Chinabut, Journal of Fish Diseases 18 (3): 273 (1995) 245 ≡Aphanomyces invaderis Willoughby, R.J. Roberts & Chinabut (1995) Aphanomyces invadans (Saprolegniales, Oomycetes) has an aseptate fungal-like mycelia structure. This oomycete has two typical zoospore forms. The primary zoospore consists of round cells that develop inside the sporangium. The primary zoospore is released to the tip of the sporangium where it forms a spore cluster. It quickly transforms into the secondary zoospore, which is reniform with laterally biflagellate cells and can swim freely in the water. The secondary zoospore remains motile for a period that depends on the environmental conditions and presence of the fish host or substratum. Typically, the zoospore encysts and germinates to produce new hyphae, although further tertiary generations of zoospores may be released from cysts (polyplanetism) Hyphae and sporangium of Aphanomyces invadans from EUS-infected fish. J.H. Lilley 8. Aphanomyces izumoensis sp. nov. Takuma, Hatai & A. Sano, 2013 246 The vegetative mycelium was delicate, about 5μm in diameter, aseptate, smooth, slightly wavy, moderately branched. Zoosporangia were slender and isodiametric, primary zoospores were produced in a single row within zoosporangium and were encysted in a cluster at the top of the zoosporangium. Primary zoospores were about 7- μm in diameter. Secondary zoospores were reniform, laterally biflagellate. The sequence identities based on 649 base pairs to A. frigidophilus closest species to the present isolates was 97.7%. Morphological characteristics of Aphanomyces sp. NJM 0705 isolated from ice fish. a Primary zoospores, which encysted as cluster at the orifice. b Zoospores swimming away from zoosporangium in a row. Bar 30 μm Takuma, Hatai & A. Sano, 2013 Morphological characteristics of Aphanomyces sp. NJM 0705 isolated from ice fish. a Primary zoospores, which encysted as cluster at the orifice. b Zoospores swimming away from zoosporangium in a row. Bar 30 μm Hatai & A. Sano, 2013 9. Aphanomyces laevis deBary. Jahr. f. wiss. Bot. 2: 179, 1860. Hyphae saprophytic or rarely parasitic on desmids and diatoms, slender, much branched, about 5-7.5µ thick. Sporangia long and of the same size as the hyphae, often extending to the substratum. Spores 7.3-1 µ in diameter after emerging, rodshaped in the sporangium. Oogonia terminal on short lateral branches, globular or nearly so, with smooth thin walls without pits, 18-33 µ in diameter. Eggs single, 16.5247 26 µ in diameter, mostly about 19-22 µ., thickwallcd,eccentric, with one very large fat drop enclosed in the protoplasm and very near the surface on one side. Antheridial branches very abundant, sometimes twining around the oogonial branches in a knot, androgynous or diclinous. Antheridia large, abundant on all oogonia and extensively wrapping them about; antheridial tubes developed and plainly visible. Aphanomyces laevis 1. Tip of empty sporangium showing spore cluster. 2. Vegetative threads and two spores sprouting within the sporangium. 3. Oogonium with diclinous antheridia. 4. Oogonium with dicHnous antheridia and visible antheridial tube. 5. Oogonia, the one on left with both diclinous and androgynous antheridia. 6. Oogonium with mature egg showing structure. 7. Oogonium clasped by finger-like antheridia; two antheridial tubes clearly visible. All figures X 670. Aphanomyces laevis, Saprolegniaceae, 1923 10.Aphanomyces parasiticus, Coker 1923 248 Hyphae 3-7μ in diameter, intramatrical at first, later penetrating the walls of the host filament; hyline; straight and unbranched but becoming swollen and distorted with age. Zoosporangia long, filamentous, unbranched; not different from the vegetative hyphae, isodiametric. Primary zoospore cysts 9-11μ in diameter. Discharge poroid. Secondary zoospores reniform; laterally biflagellate. Oogonia terminal on short, lateral branches; spherical, almost filling the oogonium; contents finely granular with a conspicuous, central oil globule. Antheridial stalk long, unbranched, diclinous in origin. Oospore germinating not observed. 249 Scott WW. 1961; Seymour and Johnson TW Jr. 1973; Smith RL. 1940. 11.Aphanomyces salsuginosus. Takuma, Hatai & A. Sano. 2010 Mycelium aseptatum, subtile, 5–10 μm diametro, laeve, leviter undulatum, modice ramosum; zoosporangia isodiametra diam hyphae aequantia; zoosporae prope orificio emergentes et incystatae, conglobatae in globum; oogonia sphaero vel subsphaerica raro pyriformia, 21–33 μm diametro, oospore singulars, 19–27 μm diametro. A congeneribus sequentiis nucleotidi distinctus, a specie proxima A. astraci 93.5 per centum identitati regionis ITS. A colony of the isolate NJM 0801 cultured on glucose–yeast (GY) agar with 0.5% sodium chloride (NaCl) at 20°C Morphological characteristics of Aphanomyces sp. NJM 0801 isolated from ice fish. aZoospores swimming away from zoosporangium in a row. b Primary zoospores, which encysted as cluster at the orifice. c A young oogonium, which has irregular short papillated antheridia on the outer surface. d A matured oogonium with an antheridium, which has a subcentric oospore. A matured oogonium with irregular short papillate. Bars40 μm, Takuma et al., 2010 250 Morphological characteristics of Aphanomyces sp. NJM 0801 isolated from ice fish. aZoospores swimming away from zoosporangium in a row. b Primary zoospores, which encysted as cluster at the orifice. c Young oogonia, which have irregular short papillated antheridia on the outer surface. d Matured oogonia with an antheridium, which have a subcentric oospore. Bar 40 μm Takuma et al., 2010 251 12. Aphanomyces. shimanensis, Takuma, Hatai & A. Sano, 2013 The vegetative mycelium was delicate, about 5μm in diameter, aseptate, smooth, slightly wavy, moderately branched. Zoosporangia were slender and isodiametric. Primary zoospores were produced in a single row within zoosporangium and were encysted in a cluster at the top of the zoosporangium. Primary zoospores were about 6- μm in diameter. Secondary zoospores were reniform, laterally biflagellate. The sequence identities based on 651 base pairs to A. laevis closest species to the present isolates was 96.4%. Morphological characteristics of Aphanomyces sp. NJM 0912 isolated from ice fish. Primary zoospores, which encysted as cluster at the orifice. Bar μm Takuma et al., 2010 252 Comparison of colony cultured on GY agar with 0.5% NaCl at 20℃ for 4 days a NJM 0705. b NJM 0801. c NJM 0912. d NJM 0913. Takuma et al., 2010 13.Aphanomyces stellatus de Bary, 1860 Hyphae 4-7µ in diameter, delicate, hyaline, straight and sparingly branched. Zoosporangia filamentous, unbranched, formed from undifferentiated vegetative hyphae; primary zoospores elongate with rounded ends, encysting upon emergence at the orifice. Oogonia terminal on lateral branches of variable length; spherical or subspherical; 23.5-32.2µ in diameter, covered with bluntly conical homogeneous, lacking a conspicuous oil globule. Antheridia one to several, cylindrical-clavate. Antheridial stalk unbranched, diclinous or monoclinous, rarely androgynous in origin. Oospore germination by the formation of a branched germ tube. 253 Fig. I. Tip of sporangium, showing spore duster with a good many empty cysts and two spores which remainded in the sporangium. Fig. 2. Young oogonia, one showing the beginnings of the papillae. Fig. 3. Young oogonium without papillae. Fig. 4. Oogonium before formation of egg. Fig. 5. Oogonium with oval-shaped egg. Fig. 6. Oogonium without antheridium. Fig. 7. Oogonium with papillae and single egg. All figures X 503Aphanomyces scaber Fig. 8. Oogonium with ripe egg and attached hypha. Figs. 9 and 10. Oogonia with ripe eggs. All figures X 810. Aphanomyces stellatus, Saprolegniaceae, 1923 Reports : 1. Epizootic ulcerative syndrome Roberts et al. (1993) carried out a survey of fish affected with epizootic ulcerative syndrome taken from outbreaks in countries throughout South and South-East Asia, which showed that a morphologically typical fungus was consistently present within lesions. Although the majority of the fungal mycelium was dead in most lesions it proved possible to isolate a very delicate and culturally demanding Aphanomyces from such lesions in a few cases. It also proved relatively easy to isolate other members of the Saprolegniaceae including Aphanomyces from the surface of lesions, but these were considered saprophytes derived from background spore burdens in the water. Sporangium morphology of the putatively pathogenic isolates of Aphanomyces was different from that of saprophytic Aphanomyces strains and they also had a lower thermal tolerance. When a mycelium from these strains was placed below the dermis of healthy fish, it caused an inflammatory response and proceeded to migrate down into the tissues of the fish, inducing severe myonecrosis with chronic epithelial reaction. The saprophytic isolates induced a local host response followed by healing of the induced lesion, and destruction or expulsion of the mycelium. It is considered that the specific slow254 growing, thermo-labile Aphanomyces is the pathogenic fungus which causes so much tissue damage in this disease, although it may not be a primary pathogen in its own right. Callinan et al. (1995) recovered fungi morphologically consistent with class Oomycetes on primary culture from 20 of 22 ulcers on 21 fish w~th epizootic ulcerat~ve syndrome (EUS) collected from 5 sites in the Philippines. Eleven primary isolates, and the unifungal cultures derived from them, were identified as Aphanomyces spp.; the remaining 9 primary isolates were lost through contaminant overgrowth. The Aphanornyces isolates were morphologically and culturally indistingu~shable from those reported from red spot disease (RSD) in Australia. Comparison of 4 representative Aphanomyces isolates from Australian fish with RSD and 3 representative Aphanomyces isolates from Philippine fish with EUS, using SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis), revealed similar peptide banding profiles, indicative of a single Aphanornyces species These findings, combined with epizoot~ological and pathological sinlilanties between EUS and RSD, suggest the 2 syndromes are identical, and that a single Aphanomyces sp. may be the primary infectious cause. Aphanomyces sp. recovered from skeletal muscle underlying a dermal ulcer on a striped snakehead Channa stnatus from a freshwater pond, Bautista, Pangasinan Province, Central Luzon. (a) Vegetative hyphae (Lugol's iodine; x110); (b) spore cluster (long arrow) and primary spores [short arrows) within sporanglum (Lugol's iodlne; x225) Callinan et al.. 1995 Skeletal muscle underlying dermal ulcers in EUS-affected fishes from Buguey, Cagayan Province, Northern Luzon. There is severe necrotlslng myositis and early granuloma formation (short arrows) associated with invading fungal hyphae (long arrows) (Gomori rnethenamine silver and haernatoxylin and eosin; x300). (a) Striped snakehead Channa striatus from a ricefield; (b) mullet Mugil sp from a coastal lagoon Callinan et al.. 1995 255 hanomyces spp. Silver-stained SDS-PAGE polypeptide profiles of representative isolates from EUSaffected fish from the Philippines and RSD-affected fish from Australia, and type isolates of A. cochloides, A. laevis, and A. euteiches. Lane A: isolate from mullet Mugil sp., Buguey lagoon, Cagayan Province, Northern Luzon; B: isolate from striped snakehead Channa striatus, Bautista, Pangasinan Province, Central Luzon, C: isolate from striped snakehead Channa striatus, Laguna Lake, Laguna Province, Southern Luzon; D: isolate from sea mullet Mugil cephalus, Saltwater Creek, north Queensland; E: Aphanomyces cochloides (IMI 300493, International Mycological Institute, Kew, UK); F: Aphanomyces laevis (CBS 107.52; Centraalbureau voor Schimmelcultures. Baarn, The Netherlands); G: Aphanomyces euteiches (IMI 300494, International Mycological Institute, Kew, UK); H: isolate from sand whiting Sillago ciliata, Richmond River, New South Wales; I: isolate from yellowfin bream Acanthopagrus australis, Clarence River. NSW; J: isolate from sea mullet Mugil cephalus, hchmond River, NSW. Molecular weight standards are indicated on the left Callinan et al.. 1995 Diéguez-Uribeondo (1997) described the steady spread of the crayfish plague through populations of A . pallipes since 1978 and the t situation of the crayfish plague in 1997. Thus, the first diagnosis of this disease since 1978 and the first isolation of A. astaci from A. pallipes in Spain were also presented. 256 Signal crayfish, Pacifastacus leniusculus, from river Arakil (Navarra) with melanized spots (arrows) harbouring the crayfish plague fungus, Aphanomyces astaci. Diéguez-Uribeondo (1997) Melanized hyphae of the crayfish plague fungus, Ap/ranomyces asfac/ (arrow), from a «black spot». Diéguez-Uribeondo (1997) 257 Cuticle of a native crayfish, Austropotamobius pallipes, infected with the crayfish plague fungus, Aphanomyces astaci. Diéguez-Uribeondo (1997) Sporulating hyphae of the crayfish plague fungus, Aphanomyces astaci. Arrows show typical sporangium of Aphanomyces spp, spore balls, and a row of primary spores within the hyphae. Diéguez-Uribeondo (1997) 258 First introductions of North American species of crayfish, Procambarus clarkii (*) and Pacifastacus leniusculus (#) in Spain, and the first crayfish plague outbreaks diagnosed in Spain (*).DiéguezUribeondo (1997) Kiryu et al. (2002) investigated the infectivity and role of Aphanomyces invadans in the etiology of skin ulcers in Atlantic menhaden Brevoortia tyrannus with two laboratory challenges. In the first experiment, Atlantic menhaden received subcutaneous injections with secondary zoospores from one of three cultures of Aphanomyces: WIC (an endemic isolate of A. invadans in Atlantic menhaden from the Wicomico River, Maryland), PA7 (an isolate of A. invadans from striped snakehead Channa striata (also known as chevron snakehead), infected with epizootic ulcerative syndrome from Thailand), and ATCC-62427 (an isolate from Atlantic menhaden from North Carolina). Fish were injected with 1.9 3 102 (WIC-low), 1.9 3 103 (WIC-high), 5.2 3 102 (PA7), or 6.0 3 102 (ATCC-62427) zoospores and held in static water at 23.58C (6‰ salinity) for 21 d. Both low and high doses of WIC caused incipient, granulomatous lesions after 5 d. Fish injected with the high-dose WIC died within 7 d. All fish injected with the low-dose WIC were dead after 10 d. Fish injected with zoospores of PA7 developed lesions after 9 d. Fish injected with the ATCC-62427 isolate or those that received subcutaneous injections of sterile water (controls) did not develop lesions. In the second experiment, fish were bath-exposed with zoospores of the WIC isolate after various trauma-inducing treatments. These treatments consisted of handling fish with a net (net stress, exposed for 2 h to either 70 or 700 zoospores/mL), physically removing a few scales (trauma, exposed for 1 h to 700 zoospores/mL), or acclimating fish with less handling (acclimated, untraumatized, exposed for 5.5 h to 110 zoospores/mL). Unexposed fish served as controls. Mortality ranged from 94% to 100% for net-handled and traumatized fish, with the prevalence of ulcerous lesions ranging from 70% to 79% in net-handled fish. However, mortality was 24% for the ‘‘untraumatized’’ fish and the prevalence of lesions was 32%. Fish injected with or exposed to bath challenges of zoospores developed lesions that were grossly and histologically identical to those observed in 259 naturally infected Atlantic menhaden from several estuaries and rivers along the midAtlantic coast of the USA. The deeply penetrating ulcers were characterized by dermatitis, myofibrillar degeneration, and deep, necrotizing granulomatous myositis. Experimentally induced lesions, however, exhibited invasiveness, often involving the kidney. Injected or bath-exposed fish developed incipient granulomas after 5 d, which progressed to overt lesions over 7–9 d. Gross pathology of Atlantic menhaden subcutaneously injected (a–c) or bath-exposed (d–f) to secondary zoospores of A. invadans (WIC isolate). Bar 5 1 cm. (a) Incipient lesion (arrow) at the injection site (5 d postinjection); (b) Advanced lesion exhibiting central necrosis at the injection site (9 d postinjection); (c) Control fish sham-injected with 1‰ saline water (5 d postinjection); (d) Multifocal early lesions in bath-exposed fish exhibiting reddening and striated pattern (5 d postbath exposure; netstress high dose, 700 zoospores/mL); (e) Multiple pale, striated lesions (7 d postbath exposure; netstress high dose, 700 zoospores/mL); (f) Focal advanced, deeply penetrating ulcer exposing underlying muscle (17 d postexposure; treatment 2, 110 zoospores/mL). Kiryu et al. (2002) Histopathology of Atlantic menhaden subcutaneously injected with secondary zoospores of A. invadans (WIC and PA7 isolates). (a) Moribund fish injected with high-dose WIC (1,900 zoospores/mL) at 4 d postinjection. Shown are oomycete hyphae (arrows) prominent along the myosepta and myodegenerative changes (arrowheads; Harris’ hematoxylin and eosin [H&E] staining; bar 5 50 mm). (b) Parallel section of (a), stained with Grocott’s methenamine–silver nitrate. Hyphae stained positively (arrows; bar 5 50 mm). (c) Fish injected with low-dose WIC (190 zoospores/fish) that exhibited 4 3 6 mm ulcer at 5 d postinjection. H&E staining shows well-developed granulomas (G) and intense leukocytic infiltration at injection site and extensive myodegeneration in deep tissue (bar 5 200 mm). (d) Higher magnification of (c), showing mature granulomas sequestering hyphae (arrow; H&E stained; bar 5 50 mm). (e) Fish injected with low-dose PA7 (520 zoospores/fish) that exhibited 10 3 15 mm ulcerous lesions at 9 d postinjection. H&E staining shows granulomas (G) widely distributed within the skeletal muscle, immediately overlying the injection site, with adjacent myodegeneration (arrowheads; bar 5 500 mm). (f) Control fish, sham-injected with 1‰ saline water, exhibiting resolving injection site at 5 d postinjection. Mild degeneration of skeletal muscle is observed in the absence of necrosis, granulomatous inflammation, or hyphae (H&E staining; bar 5 500 mm). Kiryu et al. (2002) 260 —Histopathology of Atlantic menhaden bath-exposed to secondary zoospores of A. invadans (highconcentration WIC, 700 zoospores/mL, net-stressed). Fish were killed 5 d postexposure. Bar 5 100 mm. (a) Unencapsulated hyphae (arrows) in the skin (hematoxylin and eosin [H&E] stain). (b) Parallel section of (a), stained with Grocott’s methenamine–silver nitrate (GMS). Hyphae (arrows) stained positively. (c) Well-developed granulomas surrounding hyphae (arrows), accompanied by necrotizing cores with strongly basophilic staining in the skeletal muscle immediately beneath the dermis (H&E stain). (d) Parallel section of (c), stained with GMS. Hyphae (arrows) stained positively. Kiryu et al. (2002) Histopathology of Atlantic menhaden bath-exposed to A. invadans zoospores. (a) Moribund fish 11 d postexposure to low-concentration WIC (70 zoospores milliliter, net-stressed). Skin erosion with exposure of underlying musculature (arrowheads) is visible, as are well-developed granulomas (G) and extensive myodegeneration in deeper muscle tissue. Kidney tissue was invaded by hyphae (hematoxylin and eosin [H&E] stain; bar 5 300 mm). (b) Moribund fish at 7 d postexposure to highconcentration WIC (700 zoospores/mL, net-stressed). Shown is kidney tissue (K) invaded by hyphae (arrows) and exhibiting extensive necrosis and vacuolation (V) of renal and hematopoietic tissues. Intense myodegeneration and necrosis were observed surrounding the kidney tissue (H&E stain; bar 5 100 mm). (c) Parallel section of (b), stained with Grocott’s methenamine–silver nitrate. Hyphae (arrows) stained positively (K 5 kidney; V 5 vacuoles; bar 5 100 mm) Kiryu et al. (2002) 261 Hawke et al. (2003) submitted eight cases of chronic ulcerative mycosis affecting populations of channel catfish Ictalurus punctatus, black bullheadAmeiurus melas, and bluegill Lepomis macrochirus cultured in recreational fishing ponds to the Louisiana Aquatic Diagnostic Laboratory between April 2000 and January 2002. Diseased clinical specimens presented with multiple foci of skin ulceration, typically overlying more extensive areas of granulomatous myositis that extended to the vertebral column in advanced cases. Lesions were predominated by fields of plump macrophages and multinucleated giant cells surrounding nonseptate, thick-walled hyphae with nonparallel cell walls suggestive of an oomycete. Because deep ulcerative mycoses in other fish species in the western Atlantic (USA), Australia, and Southeast Asia have been attributed to Aphanomyces spp., diagnostic methods were employed to isolate and identify this oomycete along with other potential pathogens. The organism was isolated from sites deep in the musculature with modified peptone−yeast−glucose medium containing 200 μg/mL streptomycin and 100 μg/mL ampicillin. Morphological features were consistent with A. invadans; the internal transcriber spacer (ITS) and 5.8S subunit regions of the rRNA gene were amplified, sequenced, and found to be identical with the ITS1 sequences from five isolates of A. invadans deposited in the GenBank database, indicating complete homology. Koch's postulates were fulfilled in juvenile (40–50-g) channel catfish. This report of ulcerative mycosis caused by A. invadans represents new host records for channel catfish and black bullheads from freshwater ponds in southeastern Louisiana Kiryu et al. (2003) conducted a dose response study. Juvenile menhaden were inoculated subcutaneously with 0, 1, 5, 10, 100, and 500 secondary zoospores per fish and monitored for 37 d post-injection (p.i.). Survival rates declined with increasing zoospore dose, with significantly different survivorship curves for the different doses. Moribund and dead fish exhibited characteristic ulcerous lesions at the injection site starting at 13 d p.i. None of the sham-injected control fish (0 zoospore treatment) died. The LD50 (lethal dose killing 50% of exposed menhaden) for inoculated fish was estimated at 9.7 zoospores; however, some fish receiving an estimated single zoospore developed infections that resulted in death. Menhaden were also challenged by aqueous exposure and confirmed that A. invadans was highly pathogenic by this more environmentally realistic route. Fish that were acclimated to culture conditions for 30 d, and presumably free of skin damage, then aqueously exposed to 100 zoospores ml–1, exhibited 14% lesion prevalence with 11% mortality. Net-handled fish that were similarly infected had a significantly higher lesion prevalence (64%) and mortality (64%). Control fish developed no lesions and did not die. Scanning electron microscopy of fish skin indicated that zoospores adhered to intact epidermis, germinated and penetrated the epithelium with a germ tube. Our results indicate that A. invadans is a primary pathogen of menhaden and is able to cause disease at very low zoospore concentrations. 262 Brevoortia tyrannus. Gross pathology: (a) Ulcerous lesion at the injection site at 14 d post-injection (p.i.) (1 zoospore per fish) (scale bar = 10 mm). (b) Cut appearance through lesion of fish from (a) (scale bar = 5 mm). (c) Multiple ulcerous lesions, 1 involving the vent (10 zoospore treatment at 26 d p.i.). Primary injection site is shown (arrow) (scale bar = 10 mm). (d) Early stage of tissue repair process exhibiting raised, shiny red, granular, and gritty appearance (10 zoospores treatment at 37 d p.i.) (scale bar = 5 mm). (e) Advanced stage of tissue repair process exhibiting concave, translucent, and smooth appearance (1 zoospore treatment at 37 d p.i.) (scale bar = 5 mm) Kiryu et al. (2003) Brevoortia tyrannus. Histological sections of menhaden injected with zoospores sampled at 22 d postinjection, moribund fish. (a) Sloughed skeletal muscle, 1 zoospore treatment (H&E; scale bar = 1500 µm). (b) Higher magnification of (a) in circled area showing granuloma (arrow) in the spinal cord and isolated macrophages with pseudopodia along with eosinophilic granular cells infiltrating between the spinal cord and neural arch (H&E; scale bar = 200 µm) Kiryu et al. (2003) 263 Brevoortia tyrannus. Histological sections of menhaden injected with zoospores sampled at 37 d postinjection exhibiting tissue repair at early (a–c) and late (d–f) stages, 100 zoospores treatment. (a) Granular appearance at the skin surface and welldeveloped granulomas (G) in a confined area (H&E; scale bar = 500 µm). (b) Well-organized capillary vascularization (arrowheads) and granular tissues (H&E; scale bar = 100 µm). (c) Higher magnification showing eosinophilic granular cells (EGCs; 264 arrowheads) and fibrocytes (arrows) filling the lesion area H&E; scale bar = 50 µm). (d) Thickening of epithelial cells and subdermal layer of the skin tissue and remodeling of the skeletal muscle with regenerated myocytes (H&E; scale bar = 200 µm). (e) Scale regeneration (arrowheads) (H&E; scale bar = 100 µm). (f) Elongated granuloma in the deep skeletal muscle, not exhibiting surrounding inflammatory cells (scale bar = 100 µm) Kiryu et al. (2003) Aphanomyces invadans. (a) Scanning electron micrograph of secondary zoospore with protruding germination tube starting to invade deeper to the menhaden skin (scale bar = 6 µm). (b) Detail of secondary zoospore with penetration of germination tube firmly into the skin epithelium (scale bar = 1.5 µm) Kiryu et al. (2003) Abbas et al. (2004) reported Aphanomycosis in about 8% of the examined Clarias gariepinus. The disease was recorded during spring and autumn. Aphanomyces fungus was found as sparsely branched-thin-long non septated hyphae in squash preparation from autolysed infected tissues. The organism was isolated on glucose peptone yeast broth using 5 steps culture technique with some modifications as addition of metalexyle, amphotricin B and fluconazol (antimycotics). Culture 265 characters on glucose peptone yeast broth and agar were studied. On sporulating media, sporangia were formed at the hyphal tips where spores encysted in clusters from which secondary zoospores released. Biochemical identification of the isolated fungus was recorded. Clinical signs and lesions on naturally and experimentally infected fish were also described. Histopathological examinations revealed presence of severe inflammatory reaction with heavy inflammatory cellular infiltration and multiple granulomas formed from aggregation of inflammatory cells mainly epitheliod cells. Muscle degeneration and necrosis were observed. Numerous multinucleated gaint cells were found and some of them showing fragmented fungal parts within their cytoplasm. Moreover, PAS positive spores and fungal hyphae were noticed. Naturally infected C. gariepinus with reddening of the entire surface focal ulcer and erosions (A); squash preparation of autolysed infected tissue showing hyphae of ahanomyces x 40 (B); fungal growth on GPY agar appeared slightly opaque and had white velvety surface (C), and at 2 weeks PI linear growth fulfill the entire plate (D);wet preparation from GPY agar culture showing sparsely branched non septated hyphae, with tapered end and contain cytoplasmic organelles (E), the hyphae at the center appeared wide, coarse and with undulating outline while those at the periphery were thin and with smooth outline (F); wet preparation from culture on tap water contained hempseed showing primary zoospore as one row linked together by a thin cytoplasmic thread (G); retained refractile encysted mature spores within the sporongia (H) and clusters ball of encysted zoospores at the hyphal tip (I). Abbas et al. (2004) 266 Experimentally infected C. gariepinus with aphanomyces sp. with reddening swelling and necrosis at the site of inoculation 6 day post infection (A); Skin of catfish (C. graiepinus) inoculated with spores of aphanomyces sp. showing inflammatory cellular infiltration mostly lymphcytes and macrophages together with necrosis of the muscles. H & E stain × 100 (B); mononuclear inflammatory cells and numerous multinucleated giant cells contained fungal element. H & E stain × 200 (C) and PAS stain × 100 (D); multiple fungal granulomas formed from aggregation of inflammatory cells particularly epitheliod cells. H & E stain × 200 (E) and positive PAS stained fungal hyphae – PAS stain × 100 (F). Abbas et al. (2004) Johnson et al. (2004) reported that along the eastern seaboard of the US, Atlantic menhaden, Brevoortia tyrannus, develop characteristic ulcerative lesions, a condition termed ulcerative mycosis. These lesions are identical to those seen across Asia in fish affected by epizootic ulcerative syndrome, a condition caused by the fungus-like oomycete Aphanomyces invadans. Young-of-the-year menhaden inhabiting estuarine environments are the primary species affected in the USA and little is known about the factors involved in the initiation of the lesions, or why menhaden are predominantly infected. Atlantic menhaden, hogchoker, Trinectus maculatus, striped killifish, Fundulus majalis, and mummichog, Fundulus heteroclitus, were inoculated with A. invadans (80 zoospores per fish) to explore species differences in infection and lesion development. All four species developed lesions. Killi fish developed frank lesions similar to those observed in menhaden but the gross lesions occurred later, approximately 5–10 days after those on menhaden. Hogchoker and mummichog did not develop gross skin ulcers; rather, their lesions appeared as reddened areas under the epidermis. Mummichogs also showed evidence of significant healing with a well developed granuloma and significant myocyte regeneration. These experiments show that species barriers as well as ecological barriers can explain some of the factors involved in the development of lesions in, and specificity of the water mould for, menhaden. 267 Gross pathology of Aphanomyces invadans infections. (a) Menhaden, approximately 5 days postinoculation showing early lesion (arrow). Striped killifish, 20 days (b) and 18 days (c) post-inoculation showing ulcerative lesions similar to those in menhaden (arrows). (d) Hogchoker, 6 days postinoculation. Reddened lesion developing at the injection site (arrow). (e) Mummichog, 20 days postinoculation showing curvature of vertebral bones (lordosis, arrow). Johnson et al. (2004) (a) Striped killifish, 14 days post-inoculation with Aphanomyces invadans. Hyphae (arrows) surrounded by a few layers of epithelioid cells, associated with myositis (H & E, bar ¼ 100 lm). (b) Striped killifish, 18 days post-inoculation. Multi-nucleate giant cells, both foreign body type (short arrow) and Langhans type (long arrow) engulfing hyphae (H & E, bar ¼ 50 lm). (c) Mummichog, 20 days post-inoculation. Granuloma within the areolar connective tissues (H & E, bar ¼ 200 lm). (d) Mummichog, 27 days postinoculation. Brown pigmented hyphae are located in the core of the granuloma (G) (H & E, bar ¼ 200 lm). (e) Higher magnification of (d). Note ceroid droplets in the core of the granuloma (H & E, bar ¼ 50 lm). (f) Mummichog, 27 days post-inoculation. Remodelling of the skeletal muscle is indicated by regenerating basophilic myocytes (arrow) (H & E, bar ¼ 100 lm). Johnson et al. (2004) Oidtmann et al. (2004) developed a diagnostic procedure, based on a polymerase chain reaction method (PCR) to detect infection of crayfish with the Oomycete Aphanomyces astaci. A set of oligonucleotide primers was designed to specifically amplify A. astaci DNA in the ITS region surrounding the 5.8S rDNA gene. The PCR amplifies a 115bp amplicon. The specificity of the primers was demonstrated by testing on 27 A. astaci strains and against 20 non-A. astaci Oomycetes and 5 fungal species. Most of the non-A. astaci Oomycete or fungal species included in the study are either known parasites of freshwater crayfish cuticle or can be found in their natural environment. Specificity was also tested against crayfish tissue and some known parasites and bacteria infecting crayfish. A protocol for the extraction of A. 268 astaci DNA from infected crayfish tissue was developed. The optimised method allows the detection of two genome equivalents of purified A. astaci genomic DNA. The method was tested on noble crayfish (Astacus astacus), artificially infected with A. astaci. Detection of A. astaci was possible at the very first time of sampling, which was 2 days after the beginning of spore exposure. Phadee et al. (2004) designed primers derived from the ITS1 and ITS2 regions of A. piscicida NJM 0204 for the detection and identification of fish-pathogenic A. piscicida, with the potential for the diagnosis of mycotic granulomatosis (MG). Polymerase chain reaction amplification showed that the primer set was specific only to fish-pathogenic A. piscicida, In addition, PCR revealed an improved sensitivity sufficient to detect A. piscicida in artificially infected goldfish Carassius auratus. Results demonstrated that the PCR method established in this study is effective for the detection and identification of A. piscicida with MG Royo et al. (2004) proposed the name Aphanomyces repetans for isolates that like A. astaci, had repeated zoospore emergence and lacked sexual reproduction. However, the isolates of A. repetans were significantly different from A. astaci in three characteristics: 1) A. repetans was not capable of killing susceptible crayfish (the Australian crayfish Cherax destructor and the European noble crayfish Astacus astacus) following standardised experimental infection, 2) it had randomly amplified polymorphic DNA - polymerase chain reaction (RAPD-PCR) and internal transcribed spacer (ITS) region sequences different from the A. astaci reference strains and 3) it did not express chitinase constitutively during growth or sporulation. Vandersea et al. (2006) developed sensitive PCR and fluorescent peptide nucleic acid in situ hybridization (FISH) assays to detect A. invadans. Laboratory-challenged killifish (Fundulus heteroclitus) were first tested to optimize and validate the assays. Skin ulcers of Atlantic menhaden (Brevoortia tyrannus) from populations found in the Pamlico and Neuse River estuaries in North Carolina were then surveyed. Results from both assays indicated that all of the lesioned menhaden (n = 50) collected in September 2004 were positive for A. invadans. Neither the FISH assay nor the PCR assay cross-reacted with other closely related oomycetes. These results provided strong evidence that A. invadans is the primary oomycete pathogen in ulcerative mycosis and demonstrated the utility of the assays. The FISH assay is the first molecular assay to provide unambiguous visual confirmation that hyphae in the ulcerated lesions were exclusively A. invadans. 269 The Aphanomyces invadans FISH probe (Ainv-FLU3) was tested for cross-reactivity by hybridization to other closely related oomycetes. Light micrographs (top) and epifluorescent micrographs (bottom) are shown of Aphanomyces invadans (A), Aphanomyces astaci (B), Saprolegnia parasitica (C), and Achlya americana (D). Note the green fluorescence of the probe (A2) versus the orange autofluorescence of S. parasitica (C2) and A. americana (D2) hyphae. The other oomycetes listed in Table Table11 were also hybridization negative (data not shown). Scale bar, 20 μm. Vandersea et al. (2006) Hybridization of the Ainv-FLU3 probe to Aphanomyces invadans secondary zoospores. A light micrograph (A) and epifluorescent micrograph (B) are shown. (C) A falsely colored epifluorescent image is superimposed on the light micrograph. Scale bar, 10 μm. Vandersea et al. (2006) Aphanomyces invadans FISH assay of UM-infected Atlantic menhaden. Light micrographs (top) and epifluorescent micrographs (bottom) are shown. (A) Hybridization of the positive control probe EuUni1. (B) Hybridization of the Ainv-FLU3 probe. (C) Hybridization of the negative control probe, PpiscFLU-1. (D) No-probe control. Arrowheads indicate A. invadans hyphae. Scale bar, 20 μm. Vandersea et al. (2006) 270 (A) The Aphanomyces invadans PCR primers were tested for cross-reactivity with genomic DNA from three strains of A. invadans, nine other related oomycetes, and two fish species. Lane 1, 100-bp ladder; lanes 2 to 4, A. invadans; lane 5, A. astaci; lane 6, Aphanomyces frigidophilus; lane 7, Aphanomyces sp. ATCC 62427; lane 8, Aphanomyces sp. ATCC 58381; lane 9, Saprolegnia ferax; lane 10, Saprolegnia diclina; lane 11, S. parasitica; lane 12, Achlya americana; lane 13, Achlya bisexualis; lane 14, Fundulus heteroclitus; lane 15, Brevoortia tyrannus; lane 16, negative DNA control. (B) Aphanomyces invadans PCR assay of ulcerated menhaden. Lane 1, 123-bp ladder; lane 2, A. invadans-positive control; lanes 3 to 14, DNA from ulcerated menhaden tissues; lane 15, negative DNA control; lane 16, blank DNA extraction control; lane 17, PCR inhibition control. Vandersea et al. (2006) Pradhan et al (2007) described the sequential inflammatory response of fingerlings of Indian major carps (IMC) to experimental infection of the fungal pathogen, Aphanomyces invadans. In all the three species of IMC, at one day of post injection (dpi), few fungal hyphae penetrating the muscle fibres were observed in the lesion area but no inflammatory response was found at the site. At 2 dpi, numbers of hyphae in the lesion increased and there was extensive infiltration of inflammatory cells. At 4 dpi, the mycotic lesion spread in the musculature at the site of injection and the lesion further extended even to the non-injected lateral side of the body. The fungal hyphae at the central part of the lesion were encapsulated by macrophages and/or epithelioid cells forming granulomata. At 6 dpi, both injected and non-injected sides and most of the internal organs revealed extensive mycotic lesions. There was extensive myonecrosis in large areas of myotome. 271 1.Traumatic damage (arrows) at the site of injection at one day post injection (H&E, X400). 2. Invasion of fungal hyphae through muscle fiber (arrow) at one day post injection in catla. Note the absense of inflammatory cells around the hyphae (Grocott-H&E, X1000). PRADHAN et al (2007) 3. Mycotic lesion area showing many number of hyphae (arrows) at 2 days post injection (GrocottH&E, X100). 4. Center of the lesion showing extensive infiltration of inflammatory cells (arrowheads) and development of inflammatory foci of macrophages around the hyphae following 2 days post injection in catla (H&E, X 400). PRADHAN et al (2007) 5. Mycotic lesion area in the injected side at 4 days post injection in catla. Note, the increased hyphae number (arrows) compared to 2 days post injection (Fig. 3) (Grocott -H&E, X 100). 6. Mycotic lesion area in the non-injected side showing many number of hyphae (arrows) and degenerated muscle fibres (arrowheads) at 4 days post injection in catla (Grocott -H&E, X 100). PRADHAN et al (2007) 7. Center of the mycotic lesion showing extensive infiltration of inflammatory cells and encapsulatory response around hyphae (arrows) at 4 days post injection in catla (H&E, X 200). 8. High magnification of the lesion area outside of the center of the lesion showing degenerated muscle fibers (arrow heads) and no inflammatory cells around the hyphae (arrow) at 4 days post injection in catla (Grocott -H&E, X 400) PRADHAN et al (2007) 272 9. Mycotic lesion area at 8 days post injection in rohu showing many fungal hyphae and large proliferative lesion (arrows) (Grocott– H&E, X 100). 10. Mycotic lesion area at 9 days post injection in mrigal showing many fungal hyphae and large proliferative lesion (arrows) (Grocott– H&E, X 100). PRADHAN et al (2007) 11. Outside of the center of the lesion, showing extensive myonecrosis (arrow heads) and no or few inflammatory cells around hyphae (arrows) at 8 days post injection in rohu (Grocott – H&E, X400). 12. Extensive mycotic lesion area at 9 days post injection in mrigal showing myonecrosis (arrow heads) and large number of fungal hyphae (arrows) (Grocott -H&E, X100). PRADHAN et al (2007) 13. Higher magnification of the centre of the lesion, showing well developed encapsulatory response by epithelioid cells around the hyphae (arrows) at 8 days post injection in rohu (Grocott – H&E, X400). 14. Higher magnification of outside of the center of the lesion, showing fungal hyphae (arrows) and extensive myonecrosis (arrowheads) at 9 days post injection in mrigal (Grocott – H&E, X 400). PRADHAN et al (2007) Sosa et al. (2007) injected healthy striped mullet subcutaneously with secondary zoospores of four oomycete isolates: two concentrations (50 and 115 zoospores/mL) of SJR (an endemic isolate of Aphanomyces invadans in American shad Alosa sapidissima from the St. Johns River); two concentrations each of CAL (25 and 65 zoospores/mL) and ACH (1,400 and 2,000 zoospores/mL; endemic isolates of Aphanomyces invadans and Achlya bisexualis, respectively, in striped mullet from the Caloosahatchee River); and two concentrations of the ascomycete culture MTZ (2,500 and 3,500 zoospores/mL; endemic isolate of P. dimorphosporum from 273 whirligig mullet M. gyrans in the Matanzas Inlet). All fish injected with either concentration of SJR developed granulomatous ulcers after 8 d and died within 21 d. Eighty percent (8/10) of fish injected with the high dose of CAL developed ulcers after 13 d and died within 28 d, but only 30% (3/10) of fish injected with the low dose of CAL developed ulcers. Four of the ulcerated fish died within 28 d, and the remaining fish were terminated after 32 d. Fish injected with zoospores of Aphanomyces invadans developed ulcers that were grossly and histologically similar to those observed in naturally infected striped mullet with UM from several estuaries or rivers in Florida. These hemorrhagic skin ulcers were characterized by myonecrosis and the presence of mycotic granulomas. None of the fish injected with ACH, MTZ, or sterile water developed ulcers. This study fulfilled Koch's postulates and demonstrated that ulcers could be experimentally induced in striped mullet after exposure via injection to secondary zoospores of an endemic Florida strain ofAphanomyces invadans Andrew et al. (2008) reported that in late 2006, diseased fish of a variety of species began to appear in the Chobe and upper Zambezi rivers in southern Africa. In April 2007, investigations showed that the levels of pesticides and heavy metals in the tissues of the fish were very low, discounting pollution as an underlying cause for the disease. However, histological evidence showed that the disease closely resembled the epizootic ulcerative syndrome caused by the oomycete Aphanomyces invadans, a serious aquatic pathogen that has been isolated from freshwater and estuarine fish in Japan, south-east Asia, Australia and the USA since the 1970s, but not previously recorded in Africa. Pradhan et al (2008) used artificial infection tests with Aphanomyces invadans, the etiological agent of epizootic ulcerative syndrome (EUS), to study the disease susceptibility and inflammatory response of advanced fingerlings of four different carp species, i.e., three species of Indian major carps (catla, rohu and mrigal) and common carp averaging 12.1±1.8, 11.4±1.1, 12.7±1.5 and 10.2±0.96 cm in body length, respectively. The findings of disease susceptibility experiments indicated that over an experimental period of 12 days, there was 100% mortality with severe gross lesion development in Indian major carps, whereas in common carp, neither any mortality nor any gross visible lesions were observed. Inflammatory response studies demonstrated that the injected zoospores were able to germinate in the muscles of all the four experimentally infected carp species. Only in Indian major carps, the germinated hyphae were able to massively proliferate and induce extensive necrotic lesions in the large areas of myotome. In the common carp, the lesion area was confined to the line of injection and with time course the lesion area appeared to be healed with regenerated muscle fibres. Thus, the findings inferred that advanced fingerlings of Indian major carps are highly susceptible to EUS. Therefore, in the EUS season, the cultured populations of Indian major carps, which are in this age group, are likely to be at high risk. 274 Mycotic lesion area in catla after 6 days of post injection (dpi) showing massive proliferation of hyphae (arrows) (Grocotts – H&E, x100). Extensive liquefaction of muscle fibers (arrow heads) and hyphae without any inflammatory cells around (arrow) in the lesion area of rohu at 8 dpi (Grocotts – H&E, x400). Pradhan et al (2008) Well developed encapsulatory response by the epithelioid cells around the hyphae (arrows) and adjacent normal muscle fibers in the mycotic lesion area of common carp (CC) at 12 dpi (Grocotts – H&E, x200). Mycotic lesion areas in CC at 4 dpi, showing extensive infiltration of inflammatory cells (H&E, x200). Pradhan et al (2008) Well developed epithelioid cell granulomata consisting of several layers of epithelioid cells in CC at 8dpi (arrows) (H&E, x 400). Lesion areas in CC at 12 dpi, appearing to be healed with well developed regenerated muscle fibers and injection site showing many fungal hyphae (arrows) (Grocotts–H&E, x40). Pradhan et al (2008) Oidtmann (2008) challenged European catfish Silurus glanis, European eel Anguilla anguilla and rainbow trout Oncorhynchus mykiss by intramuscular injection of zoospores of Aphanomyces invadans, the oomycete associated with epizootic ulcerative syndrome (EUS). The tropical three-spot gourami Trichogaster trichopterus is known to be highly susceptible and was used as a positive control. European catfish were highly susceptible and rainbow trout had moderate to low susceptibility, whereas eels appeared largely unaffected. Inflammatory host response in European catfish deviated from the effects seen in most other susceptible fish species and was 275 characterised by a more loosely arranged accumulation of macrophages, small numbers of lymphocytes and multinucleated giant cells without occurrence of EUScharacteristic mycotic granulomas. Semi-nested and single round PCR assays were developed for this study to detect A. invadans DNA in clinical samples of experimentally infected fish. The detection limit of the assays equals 1 genomic unit. Specificity was examined by testing the DNA of various oomycetes, other relevant pathogens and commensals as well as host DNA. The single round assay used was fully specific, whereas cross-reaction with the closely related Aphanomyces frigidophilus was observed using the semi-nested assay. Analysis of samples by PCR allowed detection prior to detectable histopathological lesions. Two other published PCR protocols were compared to the PCR protocols presented here. Aphanomyces invadans. Skin lesions (arrows) of fish injected with A. invadans spores: (A) three-spot gourami, (B) and (C) European catfish, and (D) rainbow trout Oidtmann (2008) 276 Aphanomyces invadans. Skin and muscle histopathology of infected fish: (A) mycotic granuloma (arrows) in a three-spot gourami, H&E, (B) multinucleated giant cell, European catfish, H&E, (C) stained hyphae, rainbow trout, Grocott and (D) mycotic granuloma (arrows), rainbow trout, H&E Oidtmann (2008) Sensitivity of the semi-nested and single round (primers BO73 and BO639) PCR using genomic Aphanomyces invadans DNA. M, 100 bp marker. (A) Results of semi-nested PCR. Lane 1, 25 ng of genomic DNA; Lane 2, 2.5 ng; Lane 3, 250 pg; Lane 4, 25 pg; Lane 5, 2.5 pg; Lane 6, 250 fg; Lane 7, 25 fg; Lane 8, 2.5 fg; Lane 9, 250 ag; N1, negative control of first round PCR; N2, negative control of semi-nested PCR. (B) Results of single round PCR. Lane 1, 2.5 ng; Lane 2, 250 pg; Lane 3, 25 pg; Lane 4, 2.5 pg; Lane 5, 250 fg; Lane 6, 25 fg; Lane 7, 2.5 fg; N, negative control Oidtmann (2008) Sensitivity of semi-nested PCR using DNA extracted from Aphanomyces invadans spores. M, 100 bp marker; N1, negative control of first round PCR; N2, negative control of semi-nested PCR; Lanes 1 to 7, serial dilution of DNA extracted from zoospores: Lane 1, 250 spores; Lane 2, 25 spores; Lane 3, 2.5 spores; Lane 4, 0.25 spores; Lane 5, 0.025 spore; Lane 6, 0.0025 spore; Lanes 7 to 11, 25 mg fish tissue spiked with spores prior to DNA extraction: Lane 7, 10 000 spores; Lane 8, 1000 spores; Lane 9, 100 spores; Lane 10, 10 spores; Lane 11, 1 spore Oidtmann (2008) Results of semi-nested PCR with Aphanomyces invadans DNA. M, 100 bp marker; N1, negative control of first round PCR; N2, negative control of semi-nested PCR; Lane 1, A. invadans NJM9701; Lane 2, A. invadans WIC; Lane 3, A. invadans PA7; Lane 4, A. invadans PA8; Lane 5, A. invadans UM3; Lane 6, A. invadans B99C; Lane 7, A. invadans T99G2 Oidtmann (2008) 277 Aphanomyces spp. and other species used to test specificity of the PCR assays. --------------------------------------------------------------------------------------------------------- Oidtmann (2008) Diéguez-Uribeondo et al. (2009) analyzed molecular phylogenetic relationships among 12 species of Aphanomyces de Bary (Oomycetes) based on 108 ITS sequences of nuclear rDNA. Sequences used in the analyses belonged to the major species currently available in pure culture and GenBank. Bayesian, maximum likelihood, and maximum parsimony analyses support that Aphanomyces constitutes a monophyletic group. Three independent lineages were found: (i) plant parasitic, (ii) animal parasitic, and (iii) saprotrophic or opportunistic parasitic. Sexual reproduction appeared to be critical in plant parasites for survival in soil environments while asexual reproduction seemed to be advantageous for exploiting specialization in animal parasitism. Repeated zoospore emergence seems to be an advantageous property for both plant and animal parasitic modes of life. Growth in unspecific media was generally faster in saprotrophs compared with parasitic species. A number of strains and GenBank sequences were found to be misidentified. It was confirmed molecularly that Aphanomyces piscicida and Aphanomyces invadans appear to be conspecific, and found that Aphanomyces iridis and Aphanomyces euteiches are closely related, if not the same, species. This study has shown a clear evolutionary separation between Aphanomyces species that are plant parasites and those that parasitize animals. Saprotrophic or opportunistic species formed a separate evolutionary lineage except Aphanomyces stellatus whose evolutionary position has not yet been resolved. 278 B Phylogenetic relationships among plant and animal parasites, and saprotrophic/opportunistic species in Aphanomyces. The figure shows the ayesian 50% majority rule consensus tree obtained from the analysis of the ITS data matrix. The topology of this tree is almost identical to the maximum likelihood and the parsimony strict consensus trees. Numbers on the branches are Bayesian Posterior Probability/Parsimony Bootstrap support. Arrows indicate the three major clades within the genus. In each clade, sequences of reference are in bold and underlined style, and sequences not matching with the main clades are listed in normal style Diéguez-Uribeondo et al. (2009) . Saylor et al. (2010) described a mass mortality event of 343 captive juvenile bullseye snakehead Channa marulius collected from freshwater canals in Miami-Dade County, 279 Florida. Clinical signs appeared within the first 2 d of captivity and included petechiae, ulceration, erratic swimming, and inappetence. Histological examination revealed hyphae invading from the skin lesions deep into the musculature and internal organs. Species identification was confirmed using a species-specific PCR assay. Despite therapeutic attempts, 100% mortality occurred. This represents the first documented case of EUS in bullseye snakehead fish collected from waters in the USA. Future investigation of the distribution and prevalence of A. invadans within the bullseye snakehead range in south Florida may give insight into this pathogen-host system. Channa marulius infected with Aphanomyces invadans. (A) Snakehead juvenile with petechiation (arrows) and ulceration (arrowhead). (B) H&E-stained section showing hyphae (arrow) invading through the skeletal muscle with granulomatous inflammation (arrowheads) that was characterized by macrophages, lymphocytes, and eosinophils. Inset is a Grocott-Gomori’s methenamine silver stain of the same section that more clearly shows the hyphae (arrows). (C) Grocott-Gomori’s methenamine silver-stained section of kidney showing hyphae (arrows); a glomerulus is noted (arrowhead) for orientation. (D) H&E-stained section of an eye showing a hyphal element (arrowhead) in the cornea. Inset is a Grocott-Gomori’s methenamine silver stain highlighting the hyphalal element in the cornea Saylor et al. (2010) 280 Gel electrophoresis of the Aphanomyces invadans PCR assay of samples collected from 4 lesioned (2 samples per fish) Channa marulius. Lanes 1 and 12: Mark IX molecular weight ladder (Roche Diagnostics); Lane 2: A. invadans positive control that incorporated 50 ng of A. invadans gDNA in the reaction mixture (234 bp); Lanes 3 to 10: bullseye snakehead tissue samples; Lane 11: negative control that substituted 1× PCR reaction buffer for DNA in the reaction mixture. The image was captured using a MultiImageTM Light Cabinet (Alpha Innotech) and UV illumination Saylor et al. (2010) Takuma et al. (2010) isolated a species of Aphanomyces was isolated from the ice fish Salangichithys microdon living in brackish water in Japan. White cotton-like growth was found on the heads and fins of the fish. Hyphae penetrated into the dermal layers, subcutaneous tissues, muscular layers, and cartilaginous tissue of the mandible and maxilla; these hyphae were associated with cellular debris and lesions in host tissue. White fluffy colonies from subcultures of these growths were isolated on glucose–yeast agar plates with 0.5% sodium chloride (NaCl). These isolates consisted of delicate, slightly wavy, and moderately branched hyphae. Zoosporangia were isodiametric with the vegetative hyphae. Oogonia were abundant and approximately 21–33 μm in diameter, with irregular short papillae. Generally they were spherical or subspherical and only rarely pyriform. Individual oogonia usually contained a single oospore, which was spherical and 19–27 μm in diameter, with a large shiny vesicle. Antheridial branches, when present, were usually androgynous; however, they were sometimes monoclinous or diclinous. The optimal growth temperature of the isolates was 20°C, and cultures grew well at low salinity (0–0.5% NaCl). Phylogenic analysis based on the internal transcribed space 1-5.8S-ITS 2 of the ribosomal RNA gene indicates that these isolates will be an as-yet unidentified species of Aphanomyces. Ice fish affected with water mold. Lesions of white, cotton-like masses of mycelia on the head, mouth, and/or fins of ice fish. Bar 1 cm Takuma et al. (2010) 281 Histopathological characteristics of diseased fish. a Mycelial growth in the rostrum, lower jaw, and oral cavity (arrow). Grocott-hematoxylin and eosin (H&E). b Hyphae penetrating deep into tissue are associated with necrosis (arrow). Grocott-H&E. cInflammatory cells surround hyphae (arrow). Grocott-H&E. d Gram-negative short-rod bacteria in the lesion (arrow). Gram stains. Barsa 300 μm; b 100 μm; c, d 30 μm Takuma et al. (2010) Baruah et al. (2012) transmitted the epizootic ulcerative syndrome (EUS) pathogen to catla (Catla catla) using two experimental infection models: intramuscular injection and cohabitation. Oomycetes, recovered from naturally infected ulcerated bata (Labeo bata), were identified as Aphanomyces invadans based on morphology and histopathology. Lesions typical of EUS were reproduced in the catla using an intramuscular injection of 1×105 zoospores/ml autoclaved water from a EUS-affected pond. Lesions were first visible six days after injection; all lesions were swollen and ulcerative 10 days after injection. In the cohabitation experiment with EUS-affected bata, apparently healthy catla exhibited lesions eight days after infection. Histopathology of the muscle and liver from experimentally-infected catla showed the presence of hyphae and granuloma. Twelve days after infection, immunological parameters (superoxide anion and nitric oxide production, leukocyte proliferation, lysozyme activity) of experimentally-infected catla were significantly higher (p<0.05) than in the control. Among the hematological parameters, red and white blood cell counts were significantly altered (p<0.05) in infected groups whereas differences in hemoglobin content and packed cell volume were not statistically significant (p>0.05). Biochemical parameters (total serum protein, serum glutamate pyruvate transaminase, serum glutamate oxaloacetate transaminase, and serum alkaline phosphatase were significantly higher (p<0.05) in intramuscularly-injected catla than in apparently healthy fish, however, in catla infected by cohabitation, only total serum 282 protein significantly differed from the control (p<0.05). Naturally-infected Labeo bata: (a) Aphanomyces invadans hyphae (arrow) penetrate the muscle, (b) elongated fungal hyphae and associated granuloma (arrow) in the liver. Baruah et al. (2012) Achlyoid cluster (arrow) of primary zoospores of Aphanomyces invadans. Baruah et al. (2012) Boys et al. (2012) sampled. in June 2010, bony herring Nematalosa erebi, golden perch Macquaria ambigua, Murray cod Maccullochella peelii and spangled perchLeiopotherapon unicolor with severe ulcers from the Murray-Darling River System (MDRS) between Bourke and Brewarrina, New South Wales Australia. Histopathology and polymerase chain reaction identified the fungus-like oomycete Aphanomyces invadans, the causative agent of EUS. Apart from one previous record in N. erebi, EUS has been recorded in the wild only from coastal drainages in Australia. This study is the first published account of A. invadans in the wild fish populations of the MDRS, and is the first confirmed record of EUS in M. 283 ambigua, M. peelii and L. unicolor. Ulcerated carp Cyprinus carpiocollected at the time of the same epizootic were not found to be infected by EUS, supporting previous accounts of resistance against the disease by this species. The lack of previous clinical evidence, the large number of new hosts (n=3), the geographic extent (200 km) of this epizootic, the severity of ulceration and apparent high pathogenicity suggest a relatively recent invasion by A. invadans. Diseased fish collected from the Barwon-Darling River between Bourke and Brewarrina weirs in June 2010.A) L. unicolor, B) M. peelii with raised lesions, C,D) M. ambigua and F) M. peelii with deep ulceration and muscle or fin necrosis, and E) N erebi showing severe ulceration and tissue necrosis exposing the peritoneal cavity and internal organs. Boys et al. (2012) 284 A) N. erebi, skin and underlying muscle. Photo micrograph of developing linear granulomas (thin arrow) surrounding faintly eosinophilic fungal hyphae (*). The overlying epithelium is ulcerated ( ) H & E. (X200). B) N. erebri, skin and underlying muscle. Photo micrograph of black staining longitudinal and cross sectional fungal hyphae (*) against green stained tissue. GMS. (X200). Boys et al. (2012) Huchzermeyer and Van der Waal (2012) reported in late 2006 an unusual ulcerative condition in wild fish was reported for the first time in Africa from the Chobe and upper Zambezi Rivers in Botswana and Namibia. Concern increased with subsistence fishermen reporting large numbers of ulcerated fish in their catches. In April 2007 the condition was confirmed as an outbreak of epizootic ulcerative syndrome (EUS), caused by Aphanomyces invadans. Ulcers followed infection of tissues by oomycete zoospores, resulting in a granulomatous inflammation associated with invading oomycete hyphae. Granulomatous tracts surrounding oomycete hyphae within the necrotic tissues characterised the diagnostic histological picture. The upper Zambezi floodplain at the confluence with the Chobe River spans the four countries of Botswana, Namibia, Zambia and Zimbabwe, making disease control a challenge. The floodplain ecosystem supports a high fish diversity of around 80 species, and is an important breeding and nursery ground. The annual cycle of flooding brings about changes in water quality that are thought to favour the infectivity of A. invadans, with diseased fish appearing soon after the plains become flooded. Since 2006 the disease has spread rapidly upstream along the upper Zambezi and its tributaries. By 2010 the disease was reported from the Okavango Delta in Botswana and in 2011 from the Western Cape Province of South Africa. EUS has the potential to disrupt floodplain ecosystems elsewhere in Africa where high fish diversity forms the basis of subsistence fisheries and local economies, and is a direct threat to freshwater fish culture. 285 Tissue necrosis associated with an intense inflammatory reaction in an early lesion in a dashtail barb (Barbus poechii) from the Kabompo River, upper Zambezi. Huchzermeyer and Van der Waal (2012) (a) Aphanomyces invadans hyphae in necrotic muscle tissue of Labeo lunatus from the Chobe River. Note granulomatous tracts surrounding the hyphae (arrow). Grocotts silver stain. (b) Deeply penetrating Aphanomyces invadans hyphae (arrow) within granulomatous tracts in the kidney of Labeo lunatus from the Chobe River. Grocotts silver stain. Huchzermeyer and Van der Waal (2012) (a) Intense inflammation accompanying a progressing epizootic ulcerative syndrome lesion inHydrocynus vittatus caught in the Okavango River near Shakawe during September 2010 Huchzermeyer and Van der Waal (2012) . 286 (b) Deep ulceration typical of an advanced epizootic ulcerative syndrome lesion in Serranochromis robustus caught in the Okavango River below Popa Falls during July 2010. Huchzermeyer and Van der Waal (2012) Songe et al. (2012) conducted a field investigation in the Sesheke District of Zambia along the Zambezi River to determine the fish species susceptible to epizootic ulcerative syndrome (EUS), a newly confirmed disease in Southern Africa. A total of 2,132 fishes were inspected for gross EUS-like lesions, of which 188 (8.82%; 95% CI=7.67-10.1%) were found with typical characteristic lesions of EUS. Of these 188 samples, 156 were found to have mycotic granulomas on histopathological analysis, representing 83.0% (95% CI=76.7-87.9%) of the initially identified in the laboratory through gross examination. The following 16 species of fish were examined and found with EUS lesions; Clarias ngamensis, Clarias gariepinus, Barbus poechii, Tilapia sparrmanii, Serranochromis angusticeps, Brycinus lateralis, Micralestes acutidens, Sargochromis carlottae, Hydrocynus vittatus, Phryngochromis acuticeps, Schilbe intermedius, Hepsetus odoe, Labeo lunatus, Oreochromis andersonii, Barbus unitaeniatus, and Barbus paludinosus. T. sparrmanii did not show any lesions, while the Clarias species were found to be the most afflicted with EUS. These results could be useful to fish farmers and organizations interested in improving aquaculture in the area. Gross lesions of EUS. aClarius gariepinus exhibiting a severe ulcerated lesion on the dorsal surface. bSerranochromis angusticeps showing an ulcerated lesion where the infection led to loss of the dorsal fin part. The ulcers (arrows) involve the skin and the underlying lateral musculature Songe et al. (2012) 287 Muscle tissue of mature C. gariepinus showing enveloped fungal hyphae (arrows) and an inflammatory focus of macrophages around the hyphae. H&E ×100 Songe et al. (2012) Afzali et al. (2013) conducted to isolate and identify fresh water fungi species from the Malaysian natural water bodies and fish farms and to examine the pathogenicity of the isolates as a confirmative identification tool for epizootic ulcerative syndrome (EUS) outbreak in Selangor state, Malaysia. For this aim, 165 water samples and 62 infected fish collected from 12 stations were tested in which 35 and 24 samples were found to be positive for fungi contamination and/or infection, respectively. The isolates were morphologically characterized; from 59 isolates, 32 were identified as Saprolegina, 21 as Achlya and 6 as Aphanomyces species. Experimental infection was carried out by intramuscularly injection of the Aphanomyces spp. isolates to the Malaysian moonlight gourami (Trichogaster Microlepis), where no mortality and no signs of EUS were observed in the fish groups. Histopathology test also revealed no signs of damage in the skin, muscles and other tissues following infection with the isolates indicating that all the Aphanomyces isolates were non-pathogenic. 288 Wet mount preparation of Aphanomyces spp. aseptate hyphae. (a) ASS1 isolated from stream (50 μm). (b) ASFT2 isolated from fish tank (50 μm). (c) ASR3 isolated from river (50 μm). (d) ASE4 isolated from estuary (50 μm). (e) ASP5 isolated from pool (50 μm). (f) ASFT6 isolated from fish tank (50 μm). Afzali et al. (2013) 289 Cultural characteristics of isolated fungi cultured on glucose-yeast (GY) media. (a) Cotton like and whitish colony of Saprolegna sp. (b) Puffy and whitish colony of Achlya sp. (c) A colony of the Aphanomyces sp. isolate ASFT6 growing on hempseed. Afzali et al. (2013) Moonlight Gourami artificially injected with saprophytic Aphanomyces isolate ASFT6. Some reddening observed in injection area which was healed after 2 days. Afzali et al. (2013) 290 Blazer et al. (2013) reported that during the summer and fall of 1997, an unusually high prevalence of skin lesions in fishes from Chesapeake Bay tributaries as well as two fish kills in the Pocomoke River stimulated significant public concern. Atlantic menhaden Brevoortia tyrannus were the most frequent target of the acute fish kills and displayed skin lesions that were attributed to the presence of the toxic dinoflagellate Pfiesteria piscicida. Hence, the penetrating skin ulcers so commonly found in this species are now widely viewed by the general public and some scientists as Pfiesteria-related and to be caused by exposure to Pfiesteria toxin. 121 menhaden with these ulcers collected from both Maryland and Virginia waters of the Chesapeake Bay in 1997 and 31 from the Pocomoke and Wicomico rivers in 1998 were examined, histologically. All of the deeply penetrating ulcers, as well as raised lesions (with or without eroded epithelium), were characterized by deeply penetrating fungal hyphae surrounded by chronic, granulomatous inflammation. These lesions had an appearance identical or similar to epizootic ulcerative syndrome (EUS), an ulcerative mycotic syndrome of fishes in other parts of the world caused by the fungal pathogen Aphanomyces invadans. They were also identical to ulcerative mycosis of menhaden previously reported along the Atlantic coast of the USA as associated with Aphanomyces spp. In 1998, using methods for isolation of A. invadans, it was possible to culture from affected menhaden an Aphanomyces sp. that by preliminary tests is similar or identical to A. invadans. It was believed that these findings sugges that factors other than Pfiesteria toxin need to be considered as the cause or initiator of these lesions. Filipová et al. (2013) investigated the prevalence of A. astaci in French signal crayfish populations to evaluate the danger they represent to local populations of native crayfish. Over 500 individuals of Pacifastacus leniusculus from 45 French populations were analysed, plus several additional individuals of other nonindigenous crayfish species Orconectes limosus, O. immunis and Procambarus clarkii. Altogether, 20% of analysed signal crayfish tested positive for Aphanomyces astaci, and the pathogen was detected in more than half of the studied populations. Local prevalence varied significantly, ranging from 0% up to 80%, but wide confidence intervals suggest that the number of populations infected by A. astaci may be even higher than our results show. Analysis of several individuals of other introduced species revealed infections among two of these, O. immunis and P. clarkii. 291 Takuma et al. (2013) isolated Aphanomyces spp. from ice fish Salangichithys microdon living in brackish water in Japan. Thirteen strains were isolated from affected ice fish from 2007 to 2009. Zoosporangia were slender with the same diameter as hyphae and isodiametric. Primary zoospores were produced in a single row within zoosporangia and encysted in a cluster at the top of the zoosporangia. Based on this mode of zoospore formation and the other morphological criteria, all strains were identified as members of the genus Aphanomyces. Aphanomyces salsuginosus was isolated from the fish in 2008. Two different Aphanomyces spp. were isolated in 2007 and 2009. Phylogenic analysis based on the internal transcribed spacer 1 and 2 including 5.8S rRNA gene region (ITS rDNA) indicated that both isolates were new species of Aphanomyces spp. They named Aphanomyces izumoensis and Aphanomyces shimanensis. Ice fish affected with the water mold. Note the cutaneous lesions with white, cotton-like masses of mycelia on the head, mouth, and/or fins Takuma et al. (2013) Afzali et al. (2014) conducted an experimental study from February to April 2012, to examine the susceptibility of Malaysia’s indigenous fish to EUS infection. In this experiment, forty apparently healthy moonlight gourami (Trichogaster microlepis) (10 ± 2 g body weight and 7.5 ± 1 cm in body length) were kept at 20 °C and challenged by intramuscular injection of zoospores (0.1 ml of 10,000 spores ml–1suspension ) of Aphanomyces invadans (isolate NJM9701). Fish were observed daily for characteristic EUS clinical signs during the 14-day trial and sampled at 1, 2, 4, 6, 7, 8, 9, 10, 11, 13, 12, 14 days post-injection. The infected skin and muscle were then sampled for histopathological examination. The results demonstrated that injected fish started to develop lesions that were histopathologically and grossly identical to those found in naturally EUS-infected fish and they died within two weeks after the infection. The profoundly penetrating ulcers had characteristics such as severe 292 dermatitis, myofibrillar degeneration, and deep necrotizing granulomatous myositis. Therefore, the result of this study proved that moonlight gourami was vulnerable to the EUS agent Showing (a) Electron microscopy of Aphanomyces invadans reisolated from injected gourami tissues showing non-septate hyphae. SEM, X400. (b) Light microscopy of Aphanomyces invadans reisolated from injected gourami tissues showing primary zoospores (65μm). Afzali et al. (2014) Longitudinal muscle section of APW injected gourami sampled at 13 days post injection. No microscopic lesion was observed as those seen in zoospore injected fish (H&E, X200). Experimentally infected gourami with A. invadans NJM 9701 at 100 spores/ml showed (A) necrotic ulcer surrounded with hyperaemic area, at day 6 post injection and (B) deep necrotizing ulcer at injection site seen at day 13, bordered with swollen hyperaemic ulcer and blanched margin. Afzali et al. (2014) Histopathological section of zoospores-injected gourami sampled at 8 days post injection, showing degeneration of muscles fibres, interspersed with severe necrotic area and infiltrated with mononucleated inflammatory cells in lacunae-like spaces . Also seen mycotic granulomas (arrow).in the necrotic areas. Note the loss of muscles fibre architecture (H&E, X200). Afzali et al. (2014) Histopathological characteristics of zoospores injected gourami sampled at 13 days post injection. mNote the presence of mycotic granulomas (arrows), interstitial oedema and inflammatory cells infiltration in epidermis and dermis (H&E, X200). Afzali et al. (2014) BARUAH et al. (2014) investigated the occurrence of epizootic ulcerative syndrome (EUS) in bata (Labeo bata) and the infectivity and role of Aphanomyces invadans in the aetiology of ulcerative condition a local fish farm in Tripura and were 293 acclimatised in laboratory tanks with proper management. Case histories, clinical and gross signs of the sampled fishes were recorded. Histopathological study of muscle and liver tissue from the ulcerated fish showed the presence of oomycete hyphae and granulomas. Oomycete isolation was done from the affected muscles and was identified as Aphanomyces invadans based on colony morphology, growth and microscopic morphology. The results suggest that the ulcerative condition in bata was due to EUS associated with A. invadans. (a) EUS-affected Labeo bata with typical dermal ulceration on different parts of the body surface, (b) Lesions causing destruction of muscle tissues near caudal peduncle with brown coloured fungus like growth. BARUAH et al. (2014) (a) Muscle section of Labeo bata showing granuloma formation (arrow) with myonecrosis around the granuloma (H&E, × 200), (b) Fungal hyphae (arrow) in muscle of section of bata (H&E, × 200), BARUAH et al. (2014) (c) Fungal hyphae (arrow) penetrating across the liver of naturally infected bata (H&E, ×200), (d). Presence of fungal hyphae encapsulated by granuloma (arrow) in the liver of bata (H&E, ×200). BARUAH et al. (2014) 294 (a) Glucose-peptone agar plate (GP-agar) with the growth of broad, non-septate, and sparsely branching oomycete hyphae (arrow), (b) Zoosporangia formation on the tip of the oomycete hyphae (arrow). BARUAH et al. (2014) Chauhan et al. (2014) described the isolation of A.laevis from fresh water tropical fish (Gray.) which is a new host record of this species of fungi and also described the experimental exposure of the isolated on some fresh water fishes to find its host range by which the virulence of given species of fungi can be found out. For the present study a total number of 28 specimens gotyla (Gray) were collected from Larpur reservoir in polythene bags and brought to the laboratory. They were kept in aquaria for further observations. Most of the collected fishes showed mycelial growth over the body. Cultures were prepared by taking small innocula from fish. Cultures were grown on 18-22 °C. From Garra gotyla the A. laevis, (de Bary).was reported first time from , it’s a new host record for and it was found pathogenic to all the fishes causing infection and mortality within six days of experiment. Garra gotyla found infected with Aphanomyces laevis Chauhan et al., 2014 295 Description of infected fishes artificially infected Aphanomyces laevis. Fig2A- 2K De scaling in epidermis in C.ranga, caudal region infection in out growth on anterior region of C.idella & L.rohita decoloration in L.bata, mycelia growth on posterior region of armatus, a large ulcer with hyphal growth in M.seenghala, N,nandu with hyphae covering the body, patch. Chauhan et al., 2014 Pradhan et al. (2014) reported that epizootic ulcerative syndrome (EUS), a disease listed by World Organisation for Animal Health (OIE) has been reported in 26 countries across 4 continents. Till date, 94 fish species have been found to be naturally infected with EUS and its host range is gradually expanding. In the year 2010–11, outbreaks resulting in heavy fish mortality were recorded in wetland districts of Uttar Pradesh, India, and EUS was confirmed as the cause of mortality on the basis of histopathology, isolation of Aphanomyces invadans, bioassay and PCR. A prevalence of ~ 69% (371/540) was recorded and 13 fish species were found to be infected. Interestingly, EUS was observed in seven new species (Aristichthys nobilis, Channa punctatus, Wallago attu, Mastacembelus armatus, Mystus cavasius, Anabas testudineus and Puntius conchonius) for the first time in natural outbreaks. Furthermore, the disease was observed even in the month of May when the mean water temperature was 31.6  0.65C. This suggests that the disease can result in severe losses even after two decades of its emergence. 296 Epizootic ulcerative syndrome (EUS)-affected fishes. a, Large-scale mortality of fishes in epidemic form. b, c, EUS-affected fishes being dried on the pondside. Pradhan et al. (2014) d–f, Deeply ulcerated lesions in Cirrhinus mrigala Pradhan et al. (2014) g, Red spots and caudal fin necrosis in Labeo rohita. h, Loss of scale and skin in Catla catla. i, Deeply ulcerated Aristichthys nobilis. Pradhan et al. (2014) j, Severely ulcerated Channa punctatus. k, Exposed of peritoneal cavity in Wallago attu. l, Ulcerated Mastacembelus armatus. Arrows indicate the EUS-affected fishes and/area of lesion. Pradhan et al. (2014) 297 Histopathology of EUS-affected fishes. a, Mycotic granulomas (arrows) replacing most of the host tissue in C. catla (400). b, Myonecrosis (arrow head) and A. invadans hyphae (arrow) in a section taken well away from the ulcer in A. nobilis (200). c, Aphanomyces invadans hyphae (arrow) penetrating across the peritoneum into kidney in C. mrigala (100). d, A. invadans hyphae (arrow) in the epidermis of L. rohita. Note the dermis and musculature are intact in large-sized L. rohita (100). Pradhan et al. (2014) Detection of A. invadans DNA in EUS-infected tissue of different fish species by PCR. Lane 1, Marker (100 bp DNA ladder, Fermentas, USA); lane 2, Positive control (NJM9701); lane 3, Negative control; lane 4, C. mrigala; lane 5, L. rohita; lane 6, C. catla; lane 7, A. nobilis; lane 8, C. punctatus; lane 9, Channa striatus; lane 10, W. attu; lane 11, Puntius conchonius; lane 12, M. armatus; lane 13, Colisa fasciata; lane 14, Glossogobius giuris; lane 15, Mystus cavasius; lane 16, Anabas testudineus. Pradhan et al.(2014) 298 Neighbour-joining phylogenetic tree for two Aphanomyces invadans isolates, INM20101 (KC137250) and INP20102 (KC137251). Numbers at branch nodes are bootstrap percentages based on 1000 resamplings; only values greater than 50% are shown. The tree was rooted with Aphanomyces astaci. Scale bar represents 0.005 substitutions per nucleotide position. GenBank accession numbers are given in parentheses. Pradhan et al. (2014) Gross and histopathological lesions of moribund L. rohita in bioassay. a, Severe swollen haemorrhagic areas following experimental infection with A. invadans at 18 dpi. b, Area of lesion showing myonecrosis (arrowheads) and A. invadans hyphae (arrows) (100). c, Higher magnification of the area of lesion showing severe myonecrosis (arrowheads) and hyphae (arrows) (400). d, A. invadans hypahe (arrows) invading the kidney tissue (400). Pradhan et al. (2014) Yadav et al. (2014) carried out experimental infection with A. invadans in one of the Indian major carps, Labeo rohita. Sequential changes in various innate immune parameters were monitored. The results indicated mthat at early stages of infection, no significant changes in any of the studied innate immune parameters were observed. However, at the advanced stages of infection from 6 to 12 days post infection (dpi), the respiratory burst and alternate complement activity were significantly higher whereas lysozyme, antiproteases and a-2 macroglobulin values were significantly lower than the control group and also from the infected group at earlier stages of infection. Since, the possibility of vaccination of fish against A. invadans appears remote due to difficulties in eliciting a specific antibody response, the information generated in the present study could be useful for developing strategies for improving resistance to A. invadans infection by stimulating the innate immunity through immunomodulation. 299 Sequential pathology of Aphanomyces invadans infected Labeo rohita . a. Normal muscle fibres of control rohu. b. Lesion area showing of fl occulent necrosis (arrow heads) and hyphae (arrows) at 3 days post infection (dpi). c, d. Mycotic lesion areas at 6 and 12 dpi respectively showing extensive myonecrosis (arrow heads) and oomycete hyphae (arrows). e. Lesion area at 18 dpi showing liquefaction (arrow heads) and mycotic granuloma (arrows). f. Lesion area showing extensive liquefaction of muscle fi bres (arrow heads) and no in fl ammatory cells around hyphae (arrows) (Grocott-H & E). Yadav et al. (2014) Afzali et al. (2015) developed a diagnostic procedure, based on a polymerase chain reaction method (PCR) to detect infection of fish with the A.invadans. A set of primers (1APM 1F and 1APM 6R) was used to specifically amplify A. invadans DNA. The PCR amplifies a 400 bp amplicon. A protocol for the extraction of A. invadans DNA from infected fish tissue and pure fungal cultures was developed. The method was tested on seven EUS-susceptible fish species (snakehead, snakeskin gourami, moonlight gourami, koi carp, catfish, gold fish, climbing perch) and one EUS-resistant fish (tilapia), artificially infected with A. invadans and pure cultures of Aphanomyces spp., Saprolegnia spp., Achlya spp., and Allomyces sp. Detection of A. invadans was possible at the early stage of sampling, which was 24 hours post injection in both EUS-susceptible and resistant fish. Resistant fish was found to be PCR300 negative after 6 days of inoculation but in susceptible fish PCR-positive results obtained even after day 28 or in dead fish. Therefore PCR may be a useful method for detection EUS infection in fish from early stage of disease onset. Agarose gel showing the PCR products, from snakehead fish tissue DNA obtained by amplification of genomic DNA of snakehead lesion infected with A.invadans NJM9701. The left margin in figure (M) indicates the position of size markers in base pairs (100-1000 bp). Lane N: negative control with no DNA template. Lanes P: positive control with genomic DNA of pure cultured A. invadans NJM 9701. Lanes 1-9: genomic DNA of intact snakehead from day 1, 2, 4, 6, 8, 10, 12, 14 and 21 post-injection. Lane 10: genomic DNA from non lesioned snakehead injected with sterilized tab water. Afzali et al. (2015) Agarose gel showing the PCR products, from moon light gourami (a) and snakeskin gourami (b) fish muscle DNA obtained by amplification of genomic DNA of gouramies lesion infected with A.invadans NJM9701. The left margin in figure (M) indicates the position of size markers in base pairs (100-1000 bp). Lane N: negative control with no DNA template. Lanes P: positive control with genomic DNA of pure cultured A. invadans NJM 9701. Lanes 1-8: genomic DNA of intact gouramies from day 1, 2, 4, 6, 8, 10, 12 and 14 post-injection. Lane 10: genomic DNA from non lesioned gouramies injected with sterilized tab water Afzali et al. (2015) 301 Agarose gel showing the PCR products, from Koi carp fish muscle DNA obtained by amplification of genomic DNA of koi carp infected with A.invadans NJM9701. The left margin in figure (M) indicates the position of size markers in base pairs (100-1000 bp). Lane N: negative control with no DNA template. Lanes P: positive control with genomic DNA of pure cultured A. invadans NJM 9701. Lanes 1-9: genomic DNA of intact koi carp from day 1, 2, 4, 6, 8, 10, 12, 14 and 18 post-injection. Lane 10: genomic DNA from non lesioned koi carp injected with sterilized tab water Afzali et al. (2015) Agarose gel showing the PCR products, from catfish muscle DNA obtained by amplification of genomic DNA of catfish infected with A.invadans NJM9701. The left margin in figure (M) indicates the position of size markers in base pairs (100-1000 bp). Lane N: negative control with no DNA template. Lanes P: positive control with genomic DNA of pure cultured A. invadans NJM 9701. Lanes 1-9: genomic DNA of intact catfish from day 1, 2, 4, 6, 8, 10, 12, 14 and 20 post-injection. Lane 10: genomic DNA from non lesioned catfish injected with sterilized tab water. Afzali et al. (2015) Agarose gel showing the PCR products, from goldfish muscle tissue DNA obtained by amplification of genomic DNA of goldfish lesion infected with A.invadans NJM9701. The left and right margins in figure (M) indicate the position of size markers in base pairs (100-1000 bp). Lane N: negative control with no DNA template. Lane P: positive control with genomic DNA of pure cultured A. invadans NJM 9701. Lanes 1-10: genomic DNA of intact goldfish from day 1, 2, 4, 6, 8, 10, 12, 14, 21 and 22 postinjection. Lane 11: genomic DNA from non lesioned goldfish injected with sterilized tab water. Afzali et al. (2015) 302 Agarose gel showing the PCR products, from climbing perch muscle tissue DNA obtained by amplification of genomic DNA of fish lesion infected with A.invadans NJM9701. The left margin in figure (M) indicates the position of size markers in base pairs (100-1000 bp). Lane N: negative control with no DNA template. Lane P: positive control with genomic DNA of pure cultured A. invadans NJM 9701. Lanes 1-10: genomic DNA of intact climbing perch from day 1, 2, 4, 6, 8, 10, 12, 14, 21 and 28 post-injection. Lane 11: genomic DNA from non lesioned climbing perch injected with sterilized tab water. Afzali et al. (2015) Agarose gel showing the PCR products, from Tilapia muscle tissue DNA obtained by amplification of genomic DNA of fish lesion infected with A.invadans NJM9701. The left margin in figure (M) indicates the position of size markers in base pairs (100-1000 bp). Lane N: negative control with no DNA template. Lane P: positive control with genomic DNA of pure cultured A. invadans NJM 9701. Lanes 1-11: genomic DNA of intact Tilapia from day 1, 2, 4, 6, 8, 10, 12, 14, 21, 28 and 35 postinjection. Lane 12: genomic DNA from non lesioned Tilapia injected with sterilized tab water. Experiments testing the potential cross-reactivity of the A. invadans-specific PCR assay were performed with genomic DNA of other oomycete species. The diagnostic tests were used on 12 Aphanomyces isolates, and 5 other fungi (two Achlya spp., two Saprolegnia spp., isolate and one Allomyces sp. isolate) and control samples (with no DNA template) which all were PCR-negative. Afzali et al. (2015) Afzali et al. (2015) challengedSnakehead, Channa striata (Bloch, 1793); snakeskin gourami, Trichopodus pectoralis (Regan, 1910); koi carp, Cyprinus carpio (Linnaeus, 1758); broadhead catfish, Clarias macrocephalus (Günther, 1864); goldfish, Carassius auratus (Linnaeus, 1758); climbing perch, Anabas testudineus (Bloch, 1792); and Nile tilapia, Oreochromis niloticus (Linnaeus, 1758) by intramuscular injection using zoospores of Aphanomyces invadans (NJM9701). The infected fish skins and muscles were examined for EUS histopathological characteristics, and the results on the severity of lesions and mortality were analyzed using SPSS program. All zoospore-injected fish were shown to be susceptible to the EUS infection except Nile tilapia. Although, the general histopathological pattern was similar in the zoospore-injected group, but there were some variation in granulomatous reaction, that is the presence or absence of giant cells, and time of mortality were detected. The result of statistical analysis showed that there was a significant difference between species, (c2=145.11 and p<0.01). It was concluded that, Gourami, koi carp, and catfish were demonstrated to be highly susceptible while goldfish and climbing perch were found to be moderately susceptible to the EUS 303 infection. These findings suggested that the cellular response of fish to mycotic infection and granulomatous reaction varied in different fish species, which could not be an indicator of susceptibility or resistant to the EUS itself, although it was shown that the granulation rate and the level of maturity or solidification (consolidation of granulomas) were higher in resistant fish. Visual description of skin lesion scoring system of examined fish is shown using epizootic ulcerative syndrome - affected snakehead lesions. (a) Score 1: Skin blanching lost of scale and epithelial cells. (b) Score 2: Red spot and marked swelling. (c) Score 3: Ulcerative lesion. (d) Score 4: Deep ulcers involving underlying muscles. Afzali et al. (2015) 304 Smear preparation of epizootic ulcerative syndrome oomycete fungus “Aphanomyces invadans” reisolated from artificially infected snakehead (a) and Snakeskin gourami (b). Typical Aphanomyces invadans non-septate hyphae are shown with cluster of encysted primary zoospores (arrows) (×100). Afzali et al. (2015) Epizootic ulcerative syndrome skin lesions (arrows) of fish injected artificially with Aphanomyces invadans spores: (a) Snakehead, (b) Snakeskin gourami, (c) Koi carp, (d) Broadhead catfish, (e) Goldfish, (f) Climbing perch. Afzali et al. (2015) 305 Histopathological characteristic of epizootic ulcerative syndrome-affected fish intramuscularly injected with Aphanomyces invadans NJM9701 zoospores. (a) Mature granuloma with necrotic center surrounded by fibroblast layers (F) in Snakehead (12 pi). Note the deposition of melanin pigments, H & E, 400×. (b) Formation of granulomata (arrow) characterized by thick fibroblast layers in Snakeskin gourami (10 pi), H & E, 200×. (c) A granuloma (arrow) surrounded by epithelioid cells in Koi carp (10 pi), H & E, 200×. (d) Foreign body type giant cells (circle) with surrounding connective tissues in broadhead catfish (14 dpi), H & E, 400×. Afzali et al. (2015) 306 Histopathological characteristic of epizootic ulcerative syndrome-affected fish intramuscularly injected with Aphanomyces invadans NJM9701 zoospores. (a) Presence of Langhans type multinucleated giant cells (circles) in Goldfish (6 pi), H & E, 400×. (b) A number of granulomata (arrow) surrounded by fibroblast layers in climbing perch (10 pi), H & E, 400×. (c) Encapsulation fungi by Foreign body type giant cells (circle) and a granulomas (arrow) surrounding by thick fibroblast layers (4 pi), H & E, 400×. (d) Non-capsulated hyphae (arrow) in necrotic areas in Koi carp (18 dpi), P.A.S, 200×. Afzali et al. (2015) Saiful Islam et al. (2015) recorded a prevalence of ~ 64% (322/500). Channa punctatus was found to be the most infected fish among the examined fishes. Petechia haemorrhages and moderate necrosis with melanomacrophages and multinucleated giant cells without fungal hyphae were pronounced in the early stages in the muscles. Moderate to severe necrosis friable tissues possessing trailing fungal hyphae associated with fungal, protozoan and bacterial infection may have caused denuding or total erosion of the affected tissues. Extensive ulcers and high mortality were prominent in the late stages of infection. Cystic granulomas associated with multinucleated giant cells often engulfing fungal hyphae were the most characteristic features at the late stages in EUS affected fish. Other observations made were muscle degeneration, surrounding perforated muscle fibres and frequent degeneration of the blood vessel walls. However, the causative link between EUS and the observed histopathological features needs to be further elucidated. (a) Lesions of Colisa fasciatus that contain the fungal pathogen (late stage) (b) Deep ulceration of an Epizootic Ulcerative Syndrome(EUS) in Anabus testudinus, (c) EUS infected Mystus vittatus with eroded scales, (d) Channa punctatus shows EUS lesions on dorso- lateral side of rhe body Saiful Islam et al. (2015) 307 (a) A section through the primary lesions of lateral muscle of Puntius ticto . HES. Bar = 100 (10 × 10). (b) A section through the primary lesion of Puntius ticto showing eroded stratum spongiosum over which the superficial fungal hyphae are present and inflammation of (10 ×10). (c) Giant cells subsutaneous tissues and muscle degeneration (md) PAS. Bar = 100 and granuloma in the muscle of early stages of Channa punctatus. Bar = 100 (10 × 10). (d) Giant cells and granuloma in the muscle of late stages of Mystus vittatus. Bar = 100 . (10 × 10). (e) A section through the granulomatous tissues of Mystus vittatus showing a multinucleated giant cell engulfing a foreign body, fungal hypha. Bar = 100 (10 × 10). (f) A granulomatous tissues of Mystus vittatus showing a multinucleated giant cell without engulfing a foreign body, surrounded by fibrous tissue and segmented skeletal muscles. Bar = 100 (10 × 10). Saiful Islam et al. (2015) YASUNARI KIRYU, JEFFREY D. SHIELDS,* WOLFGANG K. VOGELBEIN, DAVID E. ZWERNER, AND HOWARD KATOR. Induction of Skin Ulcers in Atlantic Menhaden by Injection and Aqueous Exposure to the Zoospores of Aphanomyces invadans. Journal of Aquatic Animal Health 14:11–24, 2002 Afzali SF, Hassan Hj Mohd Daud2 , Shiv Shankar3 , M Shuaib Khan4 , Samanesadat Afzali. Detecting Aphanomyces Invadans in Pure Cultures and EUS-infected Fish Lesions by Applying PCR. Malays. j. med. biol. Res. Volume 2, No 2/ 137-146, 2015, Afzali SF, Mohd Daud HH, Sharifpour I, Afsharnasab M, Shankar S. Experimental infection of Aphanomyces invadans and susceptibility in seven species of tropical fish. Veterinary World. 2015;8(9):1038-1044. doi:10.14202/vetworld.2015.1038-1044 308 Saiful Islam, M., Hamida Khanum, Asma Sultana Rimi Farhana Zaman and Shahela Alam. HISTOPATHOLOGICAL STUDIES ON EPIZOOTIC ULCERATIVE SYNDROME IN SOME FISHES FROM DEMRA, DHAKA. Bangladesh J. Zool. 43(1): 121-130, 2015 ISSN: 0304-9027 (Print) 2408-8455 (Online) DIÉGUEZ-URIBEONDO, C.TEMINO, J. , J.L. MÜZQUIZ. THE CRAYFISH PLAGUE FUNGUS (APHANOMYCES ASTACI) IN SPAIN. Bull. Fr. Pêche Piscic. (1997) 347:753-7 Detection of genomic DNA of the crayfish plague fungus Aphanomyces astaci (Oomycete) in clinical samples by PCR. Vet Microbiol. 2004 Jun Oidtmann B, Schaefers N, Cerenius L, Söderhäll K, Hoffmann RW. 3;100(3-4):269-82. Hochwimmer G, Tober R, Bibars-Reiter R, Licek E, Steinborn R. Identification of two GH18 chitinase family genes and their use as targets for detection of the crayfish-plague oomycete Aphanomyces astaci. BMC Microbiology. 2009;9:184. doi:10.1186/1471-2180-9184. Kozubikova et al. (2009) used molecular methods-species-specific amplification and sequencing of the pathogen DNA-to investigate the prevalence of individuals latently infected with A. astaci in 28 populations of two invasive American crayfish species (6 of the signal crayfish [Pacifastacus leniusculus] and 22 of the spiny-cheek crayfish [Orconectes limosus]) in the Czech Republic. The pathogen occurred in 17 investigated populations. We recorded a high variation in positive reactions, ranging from 0% to 100%, in populations of O. limosus. In P. leniusculus, however, only one individual out of 124 tested positive for the pathogen. There was a clear relationship between the water body type and pathogen prevalence in O. limosus. Infection ratios in isolated standing waters were usually low, whereas in running waters, pathogen prevalence often exceeded 50%. Other evaluated characteristics of potential plague pathogen carriers (size, sex, and the presence of melanized spots in the cuticle) seemed to be unrelated to infection. Our data suggest that in contrast to other European countries, O. limosus seems to be the primary reservoir of crayfish plague in the Czech Republic. Although all populations of alien American crayfishes may be potential sources of infections and should be managed as such, knowledge on the prevalence of the plague pathogen at various localities may allow managers to focus conservation efforts on the most directly endangered populations of native crayfishes. Cammà et al. (2010) observed in the summer of 2009, high levels of mortality among white clawed crayfish Austropotamobius pallipes in 3 watercourses of central Italy. PCR and culture methods were used to detect the causative agent of the disease. Two strains of Aphanomyces spp. were isolated and identified by PCR and DNA 309 sequencing as Aphanomyces astaci and A. repetans. This is the first crayfish plague outbreak in Italy to be confirmed by the isolation in culture of a pathogen from Austropotamobius pallipes. Austropotamobius pallipes. Histogical examination. Severe hyphal infiltration in the soft abdominal cuticle of a dead crayfish. Arrow: undamaged exoskeletal area; asterisks: paths carved out by hyphae; arrowheads: hyphae within paths. Periodic acid-Schiff stain Cammà et al. (2010) Austropotamobius pallipes. PCR testing for Aphanomyces astaci using Primers 42 and 640 (Oidtmann et al. 2006). Amplicons obtained were of the expected size of 569 bp. Lane M: PCR Marker (Sigma); Lanes 1 to 6: DNA extracted from soft abdominal cuticles of 6 dead crayfish; Lane 7: DNA extracted from soft abdominal cuticle of 1 moribund crayfish; Lane 8: PCR negative control Cammà et al. (2010) 310 Strand et al. (2010) adopted the species specific TaqMan® MGB real-time PCR assay developed for detection and quantifying A. astaci in crayfish for detection of A. astaci directly from water samples. Results from lab and field work demonstrated the possibility to detect down to one A. astaci spores directly from water samples. This provides new tools for further research and surveillance of the crayfish plague agent in aquatic environments. Strand et al. (2011) presented an approach for detecting and quantifying A. astaci directly from water samples using species-specific TaqMan minor groove binder realtime PCR. Samples of a 10-fold dilution series from approximately 10(4) to approximately 1 spore of A. astaci were repeatedly tested, and reliable detection down to 1 spore was demonstrated. Further, to simulate real-life samples from natural water bodies, water samples from lakes of various water qualities were spiked with spores. The results demonstrated that co-extracted humic acids inhibit detection significantly. However, use of bovine serum albumin or the TaqMan Environmental Master Mix largely removes this problem. The practical application of the approach was successfully demonstrated on real-life water samples from crayfish farms in Finland hosting infected North American signal crayfish Pacifastacus leniusculus. Direct monitoring of A. astaci from aquatic environments may find application in the management of wild noble crayfish Astacus astacus stocks, improved aquaculture practices and more targeted conservation actions. The approach will further facilitate studies of A. astaci spore dynamics during plague outbreaks and in carrier crayfish populations, which will broaden our knowledge of the biology of this devastating crayfish pathogen. Makkonen et al. (2012) tested the differences in A. astaci virulence under experimental conditions using both PsI- and As-genotypes with 3 Finnish noble crayfish Astacus astacus populations. We infected crayfish with adjusted quantities of A. astaci zoospores and monitored the symptoms and mortality of the crayfish. The PsI-genotype isolate caused rapid and total mortality among the tested populations, while the As-genotype isolates expressed more variable virulence. In some cases, mortality among the As-genotype-infected crayfish did not exceed the mortality level of the control group. All of the tested noble crayfish stocks showed lower mortality towards the As-genotype of A. astaci isolated from the River Kemijoki epidemic. We conclude that there are clear differences in virulence between different A. astaci genotypes and also differences in virulence within As-genotypes. Furthermore, we observed clear signs of increased resistance in different populations of noble crayfish towards some of the tested strains belonging to the As-genotype of A. astaci. Jussila et al. (2013) detected significant virulence differences among five tested (PsIPuujärvi, PsI-Pyhäjärvi, PsI-Kukkia, PsI-Saimaa I and PsI-Saimaa II) PsI-genotype crayfish plague (Aphanomyces astaci) isolates against lake Mikitänjärvi noble crayfish population. The crayfish were inoculated with a dose of 300 m·L−1 A. astaci spores in ambient water under experimental conditions. Mortalities started from four to seven days after inoculation, depending on the PsI-genotype isolate. In all the experimentally infected groups it took no more than three days for all the crayfish to die after the first mortality. The PsI-Puujärvi isolate proved to be the most virulent strain, while PsI-Kukkia was the least virulent. The average day of death for these experimental groups was fifth and ninth day, respectively. There was no correlation between the average day of death and gender or level of additional Psorospermium haeckeli infestation. The results showed that there are, from the practical point of 311 view, minor virulence differences among PsI-genotype A. astaci isolates, and that all the tested five isolates are highly virulent. The present results emphasize the necessity to prevent all further spread of highly virulent strains of A. astaci to aid and shelter successful conservations attempts of the native European crayfishes. Map of France with administrative division to regions (dark-bordered areas) and departments within them (light-bordered areas), showing the distribution of the invasive signal crayfish Pacifastacus leniusculus (small empty circles; based on approximate location of analysed populations (triangles), and the recent status of the native white-clawed crayfish Austropotamobius pallipes (green shading) and reported cases of its mass mortalities (red crosses and black dots). Filipová et al., 2013 Schrimpf et al. (2013) examined whether a new invasive crayfish of North American origin, the calico crayfish (Orconectes immunis), also carries A. astaci. Orconectes immunis is a recent invader of the Upper Rhine plain, where it seems to displace its invasive predecessor Orconectes limosus, which is a known carrier of the agent of the crayfish plague. Using real-time PCR, we identified the calico crayfish as the fourth invasive crayfish species to be a carrier of the crayfish plague pathogen in Europe and confirmed the infection with A. astaci in O. limosus. These findings supported the concern that all North American crayfish species in European waters are carriers of the crayfish plague pathogen. Such knowledge should prove useful for conservation 312 efforts, management, legislation, and public education about the spread of crayfish plague and non-indigenous crayfish species. The known distribution of Orconectes immunis in Europe as summarized in Chucholl (2012) with data from Chucholl and Dehus (2011; triangles), Collas et al. (2011; diamonds) and Gelmar et al. (2006; circles), completed with unpublished data from Chucholl (squares). Black stars indicate the sampling sites of this study. The international River Basin District Rhine (data: European Environment Agency, 2011) is colored light grey. Data of waters and national borders: GADM (2012). Schrimpf et al. (2013) 313 Orconectes immunis ♀ (top) and O. limosus ♂ (bottom) from the Rhine River. Arrows denote key characters to distinguish the two species (modified from Gelmar et al. 2006 and Chucholl et al. 2008): dn – distinct tooth followed by a notch on the dactylus of the chelipeds (only present in O. immunis); ht – hair tufts on the ventral side of the chelae joints of the 1st and 2nd pereiopod (only present in O. immunis); db – distinct dark bandage adjacent to the orange cheliped tips (only present in O. limosus); hp – hepatic spines (only present in O. limosus). Schrimpf et al. (2013) Viljamaa-Dirks et al. (2013) isolated the crayfish plague agent Aphanomyces astaci from 69 noble crayfish Astacus astacus samples in Finland between 1996 and 2006. All isolates were genotyped using randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). Altogether, 43 isolates belonged to the genotype group of Astacus strains (As), which is assumed to represent the genotype originally introduced into Europe around 1860 and into Finland in 1893. There were 26 crayfish plague isolates belonging to the group of Pacifastacus strain I (Ps1), which appeared in Europe after the stocking of the North American species signal crayfish Pacifastacus leniusculus. The geographical distribution of the 2 genotypes in Finland 314 corresponded with the stocking strategies of signal crayfish. The majority of Ps1strains (83%) were associated with a classical crayfish plague episode involving acute mortality, compared with only 33% of the As-strains. As-strains were found more often by searching for reasons for population declines or permanently weak populations, or through cage experiments in connection with reintroduction programmes. In some water bodies, isolations of the As-strains were made in successive years. This study shows that persistent crayfish plague infection is not uncommon in noble crayfish populations. The described epidemiological features suggest a difference in virulence between these 2 genotypes Gruber et al. (2014) investigated whether variation in crayfish plague resistance, the indicators of immune defence (encapsulation response, phenoloxidase and lytic activity), and the exploration behaviour among four subpopulations of noble crayfish is explained by potential local adaptation through differences in crayfish plague history, or alternatively by geographical divergence in a large watershed. We examined whether the strength of immune defence is associated with survival and exploration behaviour. Survival time after experimental crayfish plague infection and phenoloxidase activity differed among the subpopulations of the watershed but did not reveal local adaptation to the disease. Increased investment in immune defence (i.e. encapsulation response) compromised survival time after infection, suggesting the self-reactivity costs of mounting a strong immune response. Exploration behaviour was negatively associated with phenoloxidase activity before and after immune challenge Jussila et al. (2014) demonstrated that both virulence of A. astaci isolates and resistance of native European crayfish stocks vary notably. Some native European crayfish stocks latently carry crayfish plague, indicating adaptation and contemporary co-evolution between host and pathogen. The earliest introduced A. astaci genotypes have adapted to novel, susceptible native European crayfishes, likely under an evolutionary pressure to maintain a necessary host population as an essential habitat. Then, highly virulent genotypes that were introduced together with their original American hosts, have more resistant host populations present in Europe. This creates a dilemma for A. astaci: whether to increase virulence to better utilize invasive American hosts or to reduce virulence to better utilize the native European hosts. All A. astaci genotypes are potent killers, but they already show lowered virulence similarly to previous examples of virulence evolution in novel pathogens. 315 Noble crayfish (Astacus astacus) from the Kemijoki infected with As-genotype Aphanomyces astaci during the year 2006 acute Aphanomyces astaci epidemic showing melanisation and resulting exoskeleton erosion. Jussila et al. (2014) Signal crayfish (Pacifastacus leniusculus) from Lake Saimaa infected with PsI-genotype Aphanomyces astaci. (A and B) shell erosion, and (C) the melanised and eroded swimmerets. Jussila et al. (2014) Makkonen et al. (2014) compared the killing rate of different A. astaci strains in controlled infection experiments. Two separate infection experiments with three A. astaci strains (UEFT2B (As), Evira6462/06 (As) and UEF8866-2 (PsI)) were made to compare the noble crayfish populations from the Lake Viitajärvi, Tervo, (Expt I) and the Lake Mikitänjärvi, Hyrynsalmi (Expt II). In the Expt III, the Lake Koivujärvi population noble crayfish were infected with A. astaci strains UEF8866-2 (PsI) and Evira6462/06 (As) using different dosages (1, 10, 100 and 1000 spores ml1 ) of A. astaci zoospores. The results confirmed that PsI-genotype strain is highly virulent and kills all the crayfish within a few days. The tested two As-genotype strains caused the mortalities more slowly, and part of the challenged crayfish survived until the end of the follow-up period. Our results also confirmed the variance of virulence among A. astaci strains within the As-genotype and demonstrated that the mortality is dependent on the number of zoospores used in the infections. It also appeared, that some noble crayfish populations show increased resistance towards the crayfish plague, especially against the As-genotype of A. astaci. Rezinciuc et al (2014) investigated three major series of crayfish plague outbreaks in indigenous crayfish populations of Austropotamobius pallipes, located in the areas of influence of P. clarkii. All samples collected tested positive for A. astaci using a rnDNA ITS-PCR test. An AFLP-PCR analysis was also performed on 19 isolates, and it was found that all isolates belonged to genotype D. These isolates exhibited similar properties, i.e., adaptation to warm temperatures. We demonstrate, for the first time, the transmission of A. astaci genotype D to indigenous European populations of crayfish, and confirm that the properties of adaptation to warm water temperatures seem to be a specific character of genotype D. 316 Micrograph of cuticle of an indigenous European crayfish species, Austropotamobius pallipes, infected with A. astaci. The micrograph shows typical hyphae of Aphanomyces (HP) with characteristic rounded tip growing within the cuticle (arrow). Rezinciuc et al (2014) PCR products after amplification using A. astaci specific primers (42/640). Left to right: Size standard 1 Kb Plus DNA ladder (M), Lanes 1e7: new A. astaci isolates (Nv1, Nv3, Nv4, Nv7, An3, Ca2, Ca6), Lane P: pure DNA sample of A. astaci (strain Pc), and purified water as a negative control (N). Rezinciuc et al (2014) 317 RAPD-PCR profiles after amplification of DNA from representative Aphanomyces astaci strains using primer B01. Left to right: Size standard 1 Kb Plus DNA ladder (M), and strains V€a (genotype A), Pl (genotype B), Kv (genotype C), Pc (genotype D), followed by the new A. astaci isolates (An1, An2, An3, An4, An5, An6, An7, Ca1, Ca3, Ca5, Ca6, Nv1, Nv2, Nv3, Nv4, Nv5, Nv6, Nv7, Nv8) purified water as a negative control. Note the similarity between the new isolates and reference strain Pc for genotype D isolated from invasive crayfish species Procambarus clarkii. Rezinciuc et al (2014) Tilmans et al. (2014) evaluated A. astaci prevalence in Dutch populations of six alien crustaceans using species-specific quantitative PCR. These included three confirmed crayfish carriers (Orconectes limosus, Pacifastacus leniusculus, Procambarus clarkii), two recently introduced but yet unstudied crayfish (Orconectes cf. virilis, Procambarus cf. acutus), and a catadromous crab Eriocheir sinensis. Moderate levels of infection were observed in some populations of O. limosus and P. leniusculus. Positive results were also obtained for E. sinensis and two Dutch populations of O. cf. virilis. English population of the latter species was also found infected, confirming this taxon as another A. astaci carrier in European waters. In contrast, Dutch P. clarkii seem only sporadically infected, and the pathogen was not yet detected in P. cf. acutus. Our study is the first confirmation of crayfish plague infections in the Netherlands and demonstrates substantial variation in A. astaci prevalence among potential hosts within a single region, a pattern possibly linked to their introduction history and coexistence 318 Map of the Netherlands with approximate locations of analyzed populations of O. limosus (circle), O. cf. virilis (triangle), P. leniusculus (cross), P. acutus (star), P. clarkii (diamond) and E. sinensis (hexagon). Populations in which A. astaci infection was detected are indicated by black shapes, those without A. astaci detection by white shapes. In cases where sampled populations are in close vicinity to each other, only one location is marked in the map. Tilmans et al. (2014) Vrålstad et al. (2014) carried a study to investigate molecularly if A. astaci was involved in a selection of mass-mortality events in Norwegian noble crayfish populations from 1971 to 2004, and to determine the eventually involved A. astaci genotype groups both from these historical and also more recent mass-mortality events. DNA was extracted directly from presumptively infected crayfish tissues, and screened by A. astaci specific qPCR. A representative selection of positive samples was confirmed by ITS-sequencing. Finally, genotype determination was performed with microsatellite markers that distinguish all known A. astaci genotype groups. The molecular examination detected A. astaci in crayfish materials from all examined mass-mortality events. The first event in 1971-1974 was caused by the A. astaci genotype group A, presumably the first genotype group that entered Europe more than 150 years ago. All later outbreaks were caused by the A. astaci genotype group B which was introduced to Europe by importation of signal crayfish in the 1960s. The results suggested that molecular methods can verify the involvement of A. astaci in the vast majority of observed crayfish mass mortalities in Europe whenever preserved materials exist. Moreover, microsatellite genotyping can reveal at least parts of the underlying epidemiology. 319 Peiró et al. (2016) used quantitative PCR, and conventional PCR for detection of Aphanomyces astaci in feral P. clarkii populations established in southeastern Brazil. This is an alarming result because in South America, especially in Brazil, there is considerable endemic crayfish species diversity, especially in the genus Parastacus. Possible contacts between P. clarkii and the endemic crayfish could be seen as a major threat to the native crayfish, mainly because of the possibility of A. astaci transmission. Furthermore, the results indicated preliminary evidence of possible A. astaci infection, agent level A2, in two sympatric native species, namely Parastacus defossus and Parastacus pilimanus. This study provided the first overview concerning the presence of the crayfish plague pathogen, A. astaci, in South America Svoboda et al. (2016) wrote a review, in which they summarized advances in knowledge about various aspects of A. astaci biology, particularly with respect to the host range and transmission. They highlighted several aspects that have recently received particular attention, in particular newly confirmed or suspected A. astaci hosts, latent A. astaci infections in populations of European crayfish, and the relationship between A. astaci genotype groups and host taxa. Viljamaa-Dirks et al. (2016) Crayfish plague, a devastating disease of freshwater crayfish, is caused by an oomycete organism,Aphanomyces astaci. Currently five genotypes of A. astaci are known, but variable features between the strains or genotypes have not been studied extensively. This study analysed 28 isolates of the As genotype and 25 isolates of the Ps1 genotype and reveals that the radial growth rate is significantly (P < 0.001) different between these two genotypes, although highly variable inside the genotype As. Two Ps1 genotype isolates and two As genotype isolates with different radial growth rates were tested in an infection trial. Clear differences were detected in the development of mortality in the test groups. The representatives of the Ps1 genotype caused total mortality within a short time span. The As genotype isolates were much less virulent. The slow-growing As isolate showed higher virulence than the As isolate with a high growth capacity. Although slow growth could be one survival strategy of the pathogen, several other mechanisms are involved in the pathogenicity and warrant further studies. 320 References: 1. 2. 3. 4. 5. 6. 7. 8. Abbass, A. A., Adel A. Shaheen and Ashraf M. Abdel-Latif. APHANOMYCOSIS IN AFRICAN CATFISH “CLARIAS GARIEPINUS” . 1rst Ann. Confr. , FVM., Moshtohor, Sept, 2004 Afzali SF, Mohd Daud HH, Sharifpour I, Afsharnasab M, Shankar S. Experimental infection of Aphanomyces invadans and susceptibility in seven species of tropical fish. Veterinary World. 2015a;8(9):1038-1044. doi:10.14202/vetworld.2015.10381044 Afzali SF, Hassan Hj Mohd Daud , Shiv Shankar , M Shuaib Khan , Samanesadat Afzali. Detecting Aphanomyces Invadans in Pure Cultures and EUS-infected Fish Lesions by Applying PCR. Malays. J. Med. Biol. Res. Volume 2, No 2/ 137-146, 2015v, Blazer V. S., Volgelbein W. K., Densmore C. L., May E. B., Lilley J. H., Zwerner D. E. (2013).Aphanomyces as a cause of Ulcerative Skin Lesions of Menhaden from Chesapeake Bay tributaries. J. Aquat. Anim. Health. 11, 340–349 Boys, C. A., Stuart J. Rowland, Melinda Gabor, Les Gabor, Ian B. Marsh, Steven Hum, Richard B. Callinan. Emergence of Epizootic Ulcerative Syndrome in Native Fish of the Murray-Darling River System, Australia: Hosts, Distribution and Possible Vectors. PlosOne, April 25, 2012 http://dx. doi.org/10.1371/ journal.pone.0035568 Cammà, C., Nicola Ferri, Daniela Zezza, Maurilia Marcacci, Alessandra Paolini, Luciano Ricchiuti, Rossella Lelli. Confirmation of crayfish plague in Italy: detection of Aphanomyces astaci in white clawed crayfish. Dis Aquat Org. Vol. 89: 265–268, 2010 Diéguez-Uribeondo, C.Temino, J. , J.L. Müzquiz. The Crayfish Plague Fungus (Aphanomyces Astaci) In Spain. Bull. Fr. Pêche Piscic. (1997) 347:753-7 Hochwimmer G, Tober R, Bibars-Reiter R, Licek E, Steinborn R. Identification of two GH18 chitinase family genes and their use as targets for detection of the crayfishplague oomycete Aphanomyces astaci. BMC Microbiology. 2009;9:184. doi:10.1186/1471-2180-9-184. 9. Huchzermeyer, K., Van Der Waal, B.. Epizootic ulcerative syndrome: Exotic fish disease threatens Africa’s aquatic ecosystems.Journal of the South African Veterinary Association, 83, sep. 2012. Available at: <http://www.jsava.co.za/index.php/jsava/ article/view/204/936>. 10. Islam, S., M., Hamida Khanum, Asma Sultana Rimi Farhana Zaman and Shahela Alam. Histopathological studies on epizootic ulcerative syndrome in some fishes from demra, dhaka. Bangladesh J. Zool. 43(1): 121-130, 2015 ISSN: 0304-9027 (Print) 2408-8455 (Online) 11. Jenny Makkonen. The crayfish plague pathogen Aphanomyces astaci Genetic diversity and adaptation to the host species. Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences No 105, 2013 12. KIRYU, Y., JEFFREY D. SHIELDS,* WOLFGANG K. VOGELBEIN, DAVID E. ZWERNER, AND HOWARD KATOR. Induction of Skin Ulcers in Atlantic Menhaden by Injection and Aqueous Exposure to the Zoospores of Aphanomyces invadans. Journal of Aquatic Animal Health 14:11–24, 2002 13. Oidtmann B, Schaefers N, Cerenius L, Söderhäll K, Hoffmann RW. Detection of genomic DNA of the crayfish plague fungus Aphanomyces astaci (Oomycete) in clinical samples by PCR. Vet Microbiol. 2004 Jun 3;100(3-4):269-82. 321 14. Rezinciuc S, et al., AFLP-PCR and RAPD-PCR evidences of the transmission of the pathogen Aphanomyces astaci (Oomycetes) to wild populations of European crayfish from the invasive crayfish species, Procambarus clarkii, Fungal Biology,2014, 118,7,612-620 15. Roberts, R. J., Willoughby, L. G. And Chinabut, S. (1993), Mycotic aspects of epizootic ulcerative syndrome (EUS) of Asian fishes. Journal of Fish Diseases, 16: 169–183. 16. Viljamaa-Dirks, S., S Heinikainen, A-M K Virtala, H Torssonen, S Pelkone. Variation in the hyphal growth rate and the virulence of two genotypes of the crayfish plague organism Aphanomyces astaci. J.Fish Dis. Volume 39, Issue 6, June 2016 , 753–764 17. Vrålstad T1, Strand DA2, Grandjean F3, Kvellestad A4, Håstein T4, Knutsen AK4, Taugbøl T5, Skaar I.. Molecular detection and genotyping of Aphanomyces astaci directly from preserved crayfish samples uncovers the Norwegian crayfish plague disease history. Vet Microbiol. 2014 Sep 17;173(1-2):66-75. 18. Yadav, M. J., Pravata K. Pradhan , Neeraj Sood , Dharmendra K. Chaudhary , Dev K. Verma , Chandan Debnath , Lopamudra Sahoo , U.K. Chauhan , Peyush Punia , Joy K. Jena, Innate immune response of Indian major carp, Labeo rohita infected with oomycete pathogen Aphanomyces invadans. Fish & Shellfish Immunology 39 (2014) 524e531 4. Branchiomyces Branchiomyces sanguinis and Branchiomycesdemigrans are the cause of a fungal disease involving gill tissues, affecting the most species of freshwater fish. The disease is caused by Branchiomyces species It is characterized by areas of infarctive necrosis of the gills, anorexia, and marpling appearance of the gills. Causative agent:  Branchiomyces sanguinis : It grows mainly in the blood vessels of gill arches, filaments and in the gill lamellae.  Branchiomycesdemigrans: This fungal species is found in the parenchymal tissues of the gills. The species B. sanguinis and B. demingrans have been differentiated based on differences in hyphae and spore diameter and on the location of hyphae within fish gills. 322        Branchiomyces sanguinis has hyphae that are 8-30 µm in diameter, spores that are 5-9 µm in diameter, and it is found only in the blood vessels of the gills. Branchiomycesdemigrans is found in gill tissues outside of blood vessels, and has spores larger in diameter (12-17 µm) than those of B. sanguinis. It is possible that there is actually just one species and that its morphology is different in blood vessels than it is in tissues. The relationship between species and location in gill tissues has been complicated in channel catfish, where hyphae (presumably B. sanguinis) have been seen exiting blood vessels into the gill parenchyma. There is a report of simultaneous infection by both Branchiomyces spp. occurring in tench that could also be interpreted as a single Branchiomyces sp. growing in two locations. There is no DNA sequence available for Branchiomyces spp. so the relationship between the two species remains ambiguous. 323 Branchiomyces species, intravascular, located at the base of the gill arch. GA, base of gill arch. (B) Branchiomyces species; GC, gill cartilage, support structure of primary lamellae. (Courtesy of Lester Khoo, University of Pennsylvania, Philadelphia.) Classification: Species 2000 & ITIS Catalogue of Species  Chromista + Life: April 2013 o view in classification  Oomycota + Oomycetes +  Saprolegniales +  Not assigned +  Branchiomyces +   324 Branchiomyces sanguinis Plehn 1912 Branchiomyces demigrans Wundsch Index Fungorum  Sp. Chromista + view in classification o  Oomycota + Oomycetes +  Saprolegniales +  Incertae sedis +  Branchiomyces Plehn 1912 +   Branchiomyces sanguinis Plehn 1912 Branchiomyces demigrans Wundsch 1929 Geographical Range   Branchiomyces spp. are globally distributed in regions where summer water temperatures are in the range optimal for Branchiomycosis disease (above 20°C). Branchiomycosis has been reported from Europe, Asia, the Middle East, Australia, and from North America including at least 10 states scattered across the eastern half of North America. Host Species Branchiomycosis has been reported in a broad taxonomic range of fish species including                 American eel Anguilla rostrata, European eel A. anguilla, Japanese eel A. japonica, common carp Cyprinus carpio, tench Tinca tinca, Indian carps, European catfish Siluris glanis, black bullhead Ameriurus melas smallmouth bass Micropterus dolomieu, largemouth bass M. salmoides, pumpkinseed Lepomis gibbosus , bluegill L. macrochirus, rainbow trout Oncorhynchus mykiss , brown trout Salmo trutta tilapia Oreochromis niloticus, O.mossambicus, O. aureus, Northern pike Esox lucius, 325        European perch Perca fluviatilis, striped bass Morone saxatilis, burbot Lota lota, three-spined stickleback Gasterosteus aculeatus, and guppies Poecuilia reticulate. Mode of transmission:   Fungal spores are transmitted by water to gills. These spores adhere to the gills, germinate and produce hyphae. The hyphae penetrate gills epithelium or within the blood vessels of gills depending on species of fungi. Incubation period: Incubation period for disease is related to water temperature. The disease has been rapidly developed within 2-4 days under suitable conditions. Epizootiology               Branchiomyces sanguinis and B. demingrans have been reported only within the gill tissue of freshwater fish. Both species produce spores that are shed from necrotic gill tissue and probably infect new hosts through a water-borne route. Mortality may be very high when conditions favor development of the disease. Branchiomycosis is most commonly associated with temperatures above 20°C. There is a strong association between outbreaks of the disease and poor water quality, especially In wild fish populations, eutrophication is thought to be a significant factor predisposing fish to the disease. Stress factors: elevation of the water temperature low dissolved oxygen. reduced water flow. over crowded conditions. high levels of nutrients in the water and phytoplankton blooms. high ammonia levels, high organic loads, and dense plankton blooms. Prognosis: 326   Morbidity rate among fish populations with epizootics of brachiomycosis usually reach 100% depending on fish species and susceptibility. Mortality rate may each 30 to 50% of the fish population during late summer epizootics. Disease Signs Behavioral Changes Associated with the Disease  Fish with branchiomycosis may swim listlessly and exhibit signs consistent with oxygen deprivation or osmoregulatory distress. External Gross Signs   Fish with severe infections will have typical “gill rot” lesions The lesions may be similar to those associated with columnaris disease or other gill infections. Discoloration of gill filaments in a largemouth bass with branchiomycosis. Pale filaments are those in which the gill vasculature has been blocked or damaged by Branchiomyces sp. resulting in greatly reduced numbers of erythrocytes in gill lamellae. Photo by Andrew Goodwin. Figure 2. A largemouth bass with branchiomycosis. In this fish, there is multifocal necrosis of gill filaments producing a notched appearance of the gill margin. Photo by Andrew Goodwin. 1 cm 1 cm 327 Branchiomycosis (branchiomyces sanquinis) in gill of largemouth bass. www.ag.auburn.edu Mottled Gills ilovepets.co Branchiomycosis (branchiomyces sanquinis) in gill of carp. Photo Bayerische Biologische Versuchsanstalt (Adopted from Fish pathology by Reichenbach-Klinke's) 328 Histopathological Changes   Oomycete hyphae and spores can be easily seen in the gill vasculature and in extravascular gill tissues stained with special stains for fungi (periodic acid Schiff’s (PAS) or silver stains), There is little host response to hyphae within blood vessels, but extravascular hyphae provoke an inflammatory granulomatous response. Necrosis of filament tips occurs distal to regions where the gill vasculature has been damaged or blocked. A histological section from the gill of a pumkinseed with branchiomycosis. In this PASstained paraffin section, hyphae and spores stain red. On the right, a higher-magnification view of the same section shown in Figure 3. Spores can clearly be seen within hyphae growing in the central sinus of the fish gill. Photo by Fred Meyer. E. Branchiomycosis (branchiomyces sanquinis) in histological section of eel gill. www.ag.auburn.edu Diagnosis Presumptive Diagnosis It is based on gross clinical signs of typical “gill rot” lesions . 329 Confirmatory Diagnosis  The presence of the oomycetes within the gill tissue is a critical part of the diagnosis of branchiomycosis o detection of non-septate branching oomycetes with spores within gill vasculature or within other gill tissues examined microscopically in wet mounts. o histological sections and special stains (PAS or silver). Branching hyphae of Branchiomyces sp. seen in wet mounts from a largemouth bass with branchiomycosis. Arrows point to hyphae. Photo by Andrew Goodwin. Figure 6. Branching hyphae of Branchiomyces sp. seen in wet mounts from a largemouth bass with branchiomycosis. The lighting in this picture more clearly reveals spores (arrows). Photo by Andrew Goodwin. Branching hyphae of Branchiomyces sp. seen in wet mounts from a largemouth bass with branchiomycosis. Photo by Andrew Goodwin. Figure 8. Branching hyphae of Branchiomyces sp. seen in wet mounts from a largemouth bass with branchiomycosis. In this photomicrograph, hyphae and inflammation appear to be associated with a single lamella. Photo by Andrew Goodwin. Treatment and control:      Strict sanitation and disinfection are essential for disease control. Dead fishes should be collected and daily and burned or deeply buried. Ponds with enzootic branchiomycosis should be dried and treated with calcium oxide (quicklime) or 2 to 3 kg copper sulphate per hectare. Diseased fish can be treated with malachite green at 0.1mg/l for extended periods of time or 0.3mg/l for 12 hours. Transportation of infected fish areas to non-infected areas must be prevents. 330    Increase of water supply help in control of that disease. Stress factors must be avoided. Regulating the feeding rate during warm weather Reports: Meyer and Robinson (1973) reported Branchiomycosis jn North American fish Klein (1977) reported Branchiomyces sanguinis as an agent of s secondary mycosis in carps in Austria. The fungus could be detected in wet preparation and films stained with PAS of the gills of 40 out of 60 fish collected from a fish farming pond. . Gills with hyphae of Branchiomyces sanguinis 331 Chien et al. (1979) mentioned that Branchiomycosis broke out among the reared Japanese eel (Anguilla japonica) in summer, 1977, in Taiwan. The causative fungus had characteristics similar to B. sanguinis. Fungal hyphae grew in the filamental arteries and lamella capillaries in the gills. Intrahyphal spore-formation was observed in the well grown hyphae. The affected lamellae showed severe circulatory disturbance as dilatation of capillaries, hemorrhages and fibrin deposition. Metastatic lesions with hyphae and spores were observed in the epicardium, but the spleen only spores. Easa (1984) reported an outbreak of branchiomycosis at El-Abbasa fish farms in July 1977 and July 1981, with respiratory distress and 70% mortalities within 4-8 days. The disease occurred, when the water temperature was 25 - 29ºC. Pathological findings were in the form of dilatation of the branchial blood vessels and hypertrophy and oedema of the gill lamellae. Branchiomyces demigrans was identified in histological sections of the gills. Paperna and Semirnova (1997) identified Branchiomyces-like fungus from histologically examined gills of juvenile red tilapia Oreochromis nilotjcus X 0. mossambicus and green tilapia 0. niloticus X 0. aureus obtained from intensive fish farms in Israel. Hyphae embedded in the gill tissue contained undivided and sporulating stages. In one instance infection appeared to be subclinical, limited to one or a few loci in the gill filament and causing only localized tissue damage. In the second case, infection, which was severe and caused extensive tissue damage, was identified in fish from a stock suffering massive mortality which resulted in the loss of 85% of the pond fish. 332 Oreochromls spp. Fig. 1. Heav~ly proliferated gill filaments due to branchiomvcosis next to unaffected filaments (arrows: hyphae loaded with sporonts) (~150). Fig, 2. Multinucleate sporangium-like body bordering a blood vessel (~1050). Fig,3, Hyphae containing (a) a multinucleate plasmodium or (b) d~vlslon progeny of single-nucleus bodies with foamy cytoplasm (~1050). Fig. 4. Branching 'daughter' plasmodia (see drawing, Fig. 7b) (~750). cig..5.. Section through (a) non-differentiated hyphae; (b) hyphae loaded with progeny of plasmodia (sporonts) at various stages of differentiation (arrow: thinwalled sporonts, or 'daughter' plasmodia); adjoining area (c) of epithelia1 degradation pyknosis, karyorrhcxis, wlth infiltration of leucocytes (~750). Fig,6: Cross-section of hyphae loaded with softand hard-walled sporonts (fine and bold arrows, respectively) (x750) Paperna and Semirnova (1997) 333 Stages in the differentiation of the Branchiomyces-like fungus from Oreochromis spp. (a) Plasmodium. (b) Uninuclear progemy of the plasmodium. (c) 'Daughter' plasmodia extending into the surroundings of their hypha. (d, e) Single and bi-nucleate 'sporonts'. (f) Divided 'sporont' (g) 'Sporont' at final stage division, filled with spores Paperna and Semirnova (1997) 334 Oreochromls spp Fig 8 Hyphae containing (a) unlnucleate and b~nucleate progeny of plasmodia and (b) divided sporonts (xl050) Fig 9 Hypha containing thin-walled daughter plasmodla (a) and dividing bod~es (sporonts) (b) the ep~thelial tlssue contains degeneration loci (c) and the remalns of defunct hyphae (d) (x750) Hyphae (a) with sporonts at vanous stages of differentiation (b) and a few daughter plasmodia (c) Flg 11 Longitudinal section of a hypha (a) containing divided sporonts (detailed sectlon to right lower-denslty image enabling a better vlew of the dlvlded bodles) (b) a degcln~rate hypha (X 1050) Fig 12 Spores scattered and contained ivlthin a sporont (arrow) (X 1050) Flg 13 Spores contalned within phagocytic cells in thc filamental epithelium (A 1050) Fig 14 Hyphae contaming spore-loaded sporonts (see inset) emerging from the g111 filament (X 1050) Paperna and Semirnova (1997) 335 Oreochromis spp Fig 15 Concentnc proliferahon of the filamental epithelia1 tlssue around the fungal remains (x750) Fig 16 Branchiomycos~s-induced necrosls of the gill filament with (a) residues of congestion and (b) some remains of fungal sporonts (x750) Paperna and Semirnova (1997) Khoo et al. (1998) identified 4 cases (representing outbreaks in four different ponds on three farms) of branchial mycosis caused by Branchiomyces spp. in channel catfish fry during the summer of 1996. Mortalities ranged from a few hundred to several thousand fish per pond. Significant gross and histopathological findings from these four cases were limited to the gills. All fry examined had fungal mycelia that were mainly but not entirely confined to the base of the primary lamellae and the gill arches. These fungal hyphae were intravascular and occluded vessels in the gill tissues. A wet mount of a gill: the arrow indicates the branching fungal hyphae that were present in the gill arch and extending into the primary lamellae (bar = 150 mm). A representative portion of the fungal mycelium from cultures on corn meal agar (bar = 8 mm). Khoo et al. (1998) 336 A section of gill revealing the vascular location of the fungus in the gill arch as well as in the primary lamellae, as indicated by the arrows. The primary lamella in the centre is less affected, and adjacent to it there is necrosis and loss of the infarcted primary lamellae (GMS and H&E, bar = 150 mm). A higher magnification of the gill arch with sections of fungal hyphae within a vessel (i.e. the portion highlighted by the larger arrow in Fig. 3). Note the spores within one of the sections of the fungal hyphae (GMS and H&E, bar = 40 mm). Khoo et al. (1998) A branchial vessel (arrow) that is completely occluded by the fungal hyphae (H&E, bar = 40 mm). Journal of Fish Diseases 1998, 21, 423±431 L Khoo et al. Branchiomyces in channel catfish The granulomatous inflammatory response consisting of mononuclear cells and macrophages surrounding the fungus as it exits a vessel (H&E, bar = 40 mm). Khoo et al. (1998) 337 Electron micrograph of the fungus revealing the tubular configuration of the mitochondrial (arrow) cristae (bar = 0.2 mm). Khoo et al. (1998) Ibrahim (2011) diagnosed branchiomycosis by isolation and histopathological changes of examined gills in common carp fish (Cyprinus carpio) which, were obtained from fish farm in Duhok Governorate, Iraq. The infected fish were suffering from respiratory disorders; gulping air at the water surface, rapid movement of operculum and massive mortality, which resulted in the loss of 95% of fish pond. The gills appeared marbled with necrotic areas on the localized damage gills.The causal pathogen was identified as Branchiomyces sanguinis, in which the diameter of spores and non-septated hyphae were 5-7 μm and 12 – 20 μm, respectively. In histo pathological preparation, the spores and the non-septated hyphae have been shown to be embedded in the gill tissues contained undivided and sporulating stages. Cyprinus carpio infected with Branchiomyces species showing marbled appearance with the pale and necrotizing areas (arrows) Ibrahim (2011) 338 Direct examination of the gills spores of Branchiomyces spp., Culturing of Branchiomyces spp. on the Sabouraud's dextrose agar after three days from Cyprinus carpio. Ibrahim (2011) Branchiomyces spp. culture after 7 days of cultivation, the colonies shows as folded heaped, glabrous and velvety, white in color and with white –yellowish in reveries, Spores and hyphae of Branchiomyces spp. stained with Lactophenol cotton blue stain from cultivation. Ibrahim (2011) Histological section of gill Cyprinus carpio infected with Branchiomyces spp., revealing, hyphae containing (a) uninucleate and binucleate progeny of plasmodia. H & E 400 X. Histological section of gill Cyprinus carpio infected with Branchiomyces spp., revealing hypha containing (a) plasmidium (b) uninuclear progemy of plasmodium (c) adjoining area of epithelial degeneration pyknosis, keryorrhexis. H & E 1000X Ibrahim (2011) Rahemo and Taha (2013) examined samples of fish, Barbus esocinus, caught from Mosul dam lake, a fungus was revealed with incidence 5%. Depending to its characters especially its branched sporophores the fungus was diagnosed as Branchiomyces demigrans. As such this is considered the first record from Iraqi fishes 339 Photograph of freshwater fish, Barbus esocinus. Rahemo and Taha (2013) El- Bouhy and Mahboub (2014) colleected 100 Nile tilapia (Oreochromis niloticus) from Edco private fish farms in Behiera governorate during the period between July and September 2013 and screened them for branchiomyces infection. The infected fish were suffering from respiratory distress (resulted from gill tissue damage); gasping air from the water surface, rapid movement of operculum and massive mortality, which resulted in the loss of 90% of the collected fish. Squash preparations from the infected gill tissue revealed brown, broad, branched and non-septated hyphae. On Sabouraud's dextrose agar (SDA) medium with 10% duck decoction bright white colonies appeared after 2 days which reached their maximum growth 8 days post inoculation. Microscopical examination of stained preparations with Lactophenol cotton blue revealed branched hyphae at their tips which were characteristic for Branchiomyces sp. The causal pathogen was identified as Branchiomyces demigrans, in which the diameter of spores and non-septated hyphae were 4-10 μm and 16-24 μm respectively. The fungus was confirmed using polymerase chain reaction (PCR). Experimental infection and reisolation of fungus revealed the same findings of natural infection. Clotrimazole was more effective than clove oil, while using both of them revealed higher lysozyme activity and phagocytic activity. Histopathological examination from naturally and experimentally infected fish gills revealed non-septated hyphae and spores were embedded inbetween affected gill tissues, which confirmed that the isolated organism was Branchiomyces demigrans. Khalil et al. (2015) identified several cases of branchial mycosis caused by Branchiomyces spp. in fingerlings specimens of Nile tilapia (Oreochromis niloticus) and Common carp (Cyprinus carpio) obtained from different 4 private farms at Damietta, Port-said, El-Behera and Kafr-El Sheikh governorates, Egypt during the summer of 2014. Mortalities ranged from a few hundred to several thousand fish per pond. Significant gross and histopathological findings from these cases were limited to the gills. The infected fishes were suffering from respiratory distress; gasping air from the water surface and rapid movement of opercula. Squash preparations from the infected gill tissue revealed brown, broad, branched and non-septated hyphae. On Sabouraud's dextrose agar (SDA) medium with 10% duck decoction showed bright white colonies after 2 days which reached its maximum growth 8 days post inoculation. Microscopical examination of stained growth with lactophenol cotton blue, branched hyphae at their tips were identified which were characteristic for Branchiomyces sp. The causal pathogen was identified as Branchiomyces demigrans, in which the diameter of spores and non-septated hyphae were 4-10μm and 16-24μm respectively. Histopathological examination from infected 340 fish gills revealed that all examined fishes had fungal mycelia that were mainly but not entirely confined to the base of the primary lamellae and the gill arches. These fungal hyphae were intravascular and occluded vessels in the gill tissues. It was found that ammonia, nitrite and organic matter were elevated over the permissible levels in the surveyed localities. The present paper describes the fungal characteristics and pathology of branchial mycosis caused by Branchiomyces spp. in these freshwaterfishes and the relation of Branchiomyces infection with water quality parameters. Naturally examined C. carpio showing marbled appearance of the gills with the pale and necrotizing areas (Photos 1 and 2), and congestion and mottled gills of O. niloticus (Photo 3).Khalil et al., 2015 341 Spores and hyphae of Branchiomyces demigrans(Photos 4 and 5) stained with Lactophenol cotton blue stain fromcultivation Gills of O. niloticus showing dilation and congestion of blood vessels of primary lamellar epithelium, aneurism, swelling of secondary lamellae as well as Telangiectasis (arrows) (60X, H& E) (Photo 6) and hematomas (arrows) and curling of secondary lamellae (40X, H&E) (Photo 7). Khalil et al., 2015 Gills of C. carpio showing severe hyperplasia in the epithelial lining the secondary lamellae, degenerative changes, necrosis and edema as well as the blood capillaries became large cysts (Arrows), (H&E stain)(X 250) (Photo 8), and complete destruction and proliferation in the epithelium of gill filament (Arrows), dilation and congestion of blood vessels in primary lamellae, hyperplasia and swelling of secondary lamella, thickening of primary lamellae, proliferation in the epithelium of gill filaments and shortening of secondary lamellae , necrotic changes in secondary lamella, curling & edema (Arrows) (60 X, H&E) (Photo 9). Khalil et al., 2015 References: 1. Andrew E. Branchiomycosis, Goodwin University of Arkansas at Pine Bluff Aquaculture/Fisheries Center 1200 N University Dr., Pine Bluff, AR 71601 Phone: 870-489-5997 agoodwin@uaex.edu 2. Chau-Heng CHIEN, Teruo MIYAZAKI and Saburoh S. KUBOTA. Branchiomycosis of the Reared Japanese Eel in Taiwan. Fish Pathology 13(4)179-182,1979. 3. Chien, C - H, T. Miyazaki, and S. S. Kubota. 1978. The histopathology of branchiomycosis of eel in Taiwan. JCRR Fish Series (Taiwan) 34: 97-98 [in Chinese, English abstract]. 4. El- Bouhy, Heba and H Mahboub. Branchiomyosis In Nile tilapia (Oreochromis niloticus) in Behiera Governorate With Trials For Treatment. November 2014 5. Easa, M. EI-S (1984):Branchiomycosis (mycolic disease of Egyptian fish farms) (Mugil cephalus and Mugil capitto) grey mullet.Assuit Vet. Med. J., 12, 109-114. 6. Ibrahim, Kh. S. Isolation and pathological study of branchiomycosis from the commercial pond of common carp (Cyprinus carpio) fish, in Governorate of Duhok / Iraq. The Iraqi Journal of Veterinary Medicine; 35 ( 1 ): 1 – 9: 2011 7. Khoo , A T Leard , P R Waterstrat , S W Jack and K L Camp. Branchiomyces infection in farm-reared channel catfish, Ictalurus punctatus (Rafinesque) .J. Fish Dis. Volume 21, 6 November 1998, 423–432 8. Klein, U. . Branchiomyces sanguinis als Ursache einer sekundären Mykose. Österr. Fischereiverband u. Bundesamt f. Wasserwirtschaft, download unter www.zobodat.at 9. Meyer, F. P. and J. A. Robinson. 1973. Branchiomycosis: A new fungal disease of North American fish . Progressive Fish Culturist 35:74-77. 10. Paperna, I and M. Smirnova. 1997. Branchiomyces-like infection in a cultured tilapia (Oreochromis hybrid, Cichlidae). Diseases of Aquatic Organisms 31:233-238. 11. Paperna, I. and D. di Cave. 2001. Branchiomycosis in an amazonian fish, Baryancistrus sp. (Loricariidae). Journal of Fish Diseases 24: 417- 420. 12. Riad H. Khalil, Talaat T. Saad, Talal A. M. Abo Selema, Hany M. R. Abdel-Latif. Branchiomyces Demigrans Infection in Farm‐Reared Common Carp (Cyprinus Carpio L.) and Nile Tilapia (Oreochromis Niloticus) at Different Localities in Egypt, 342 With Special Emphasis to the Role of Environmental Stress Factors . International Journal of Innovative Studies in Aquatic Biology and Fisheries (IJISABF) 1, 1, June 2015, PP 15-23 www.arcjournals.org 13. Zohair I.F.Rahemo and Khalid H.Taha. FIRST RECORD OF BRANCHIOMYCES DEMIGRANS FROM BIZZ FISHES, BARBUS ESOCINUS CAUGHT FROM MOSUL DAME LAKE. Trend i n Fishery Research. Vol. 2 Issue 1 (2013) ISSN: 2319–474X (Print); 2319–4758 (Online) © 2013 DAMA International 343