MFR PAPER 1340 Victor Sprague is with the Center for Environmental and Estuarine Studies, Chesapeake Biological Laboratory, University of Maryland, Solomons, MD 20688. This paper is Contribution No. 797, Center for En­ Comments on Trends in Research vironmental and Estuarine Studies of the University of Maryland, Sol­ on Parasitic Diseases omons, MD 20688. of Shellfish and Fish

In Fish Only very recently, amoebae have VICTOR SPRAGUE become generally recognized as impor­ tant mortality factors in fish. Some amoebae pathogenic to man also occur in fish. Disease conditions of epizootic This subject is arbitrarily limited in of enormous numbers of the parasites. proportions in rainbow trout on com­ scope to the and in time to the As the number of parasites increases, mercial trout farms of Italy and other past decade. It deals very briefly with the hemocytes decrease in number. European countries have been attrib­ recent developments in the biology and Heavy infections may be accompanied uted by Ghittino et al. (1977) to an systematics of some protozoan groups by lysis of the skeletal muscles. . Epizootics associated with the that are significant as pathogens. More Paramoeha perniciosa has been lim­ same amoeba were reported by Fergu­ detailed reviews have recently been ited to the higher salinity waters. It has son and McC Adair (1977) in Scotland published by Couch (1978), Hazard been found most frequently in and Ireland. It seems clear from these and Oldacre (1976), Sindermann "peeler" suffering heavy mor­ reports that an amoeba (apparently not (1970), Sprague (1970a, b, 1971, talities in holding tanks, but has been yet identified) is a very important factor (977), and Sprague and Couch (197 I). found also in "hard" crabs collected in in widespread mortalities of trout in The symposium edited by Snieszko enzootic areas. Lunz (1968) found it European fish farms. ( (970) contains numerous articles per­ associated with mortalities along Taylor ( 1977) isolated amoebae from tinent to "fish health," the two by the coast of South Carolina and Geor­ II species of fish in the southeastern Sprague being specifically cited above gia. Sawyer (1968) gave preliminary states. Among those isolated was as being particularly relevant to this data on the epizootiology and host­ Acanthamoeba polyphaga which presentation. parasite relations. Newman and Ward Taylor regarded as the cause of a large ( 1973) reported a modest epizootic in kill, involving mainly blue tilapia, in RECENT ADVANCES Chincoteague Bay. Sawyer (1968) Florida. This study has special interest found the parasite in the blood only because A. polyphaga is pathogenic to Amoebae during mortalities, which occurred man. In Crabs mainly in the spring. He postulated a Sawyer et at. (1974) described cryptic tissue phase in the parasite's life Thecamoeba hoffmani from the gills of Only one amoeba is believed to be a cycle during other parts of the year. fingerling rainbow trout, coho salmon, serious mortality factor in shellfish. Very recently, Johnson (1977) discov­ and chinook salmon. They concluded This parasite, Paramoeba perniciosa ered the' 'cryptic phase" in the connec­ that the species is essentially a free­ Sprague, Beckett, and Sawyer, 1969, tive tissues. She found that the parasite living form, although it seems to be a was first reported and briefly described is present in these tissues at all seasons contributory factor in mortalities. in 1966 by Sprague and Beckett who of the year and invades the circulating found it in the hemolymph of dead and blood in the vessels only when the dis­ moribund crabs, , ease is terminal. Johnson also trans­ In Bivalve Mollusks in Chincoteague Bay, Maryland and mitted the infection by inoculation, Virginia. These authors did not recog­ with fatal results to the inoculated Microsporidium sp. has been found, nize the parasite as an amoeba at that crabs. but not yet reported, by Brian Jones' in time, but in a later note (Sprague and The evidence that this amoeba is a the visceral mass of Ostrea lutaria in Beckett, 1968) assigned it to genus very significant mortality factor in Cal­ New Zealand. This is the third species' Paramoeba Schaudinn, 1896. linectes sapidus is overwhelming, if known in bivalve mollusks. These The parasite occurs in the largely circumstantial. The parasite has species are not significant pathogens. hemolymph of heavily infected crabs. not been grown in pure culture, and its Sometimes the body fluid of the host normal mode of transmission is un­ 'Jones, B., Zoology Department. Victoria Uni­ became mil ky white due to the presence known. versity, Wellington, N.Z.

26 Marine Fisheries Review Sprague (in press) as a new species (Fig. 2). Pleistophora penaei Con­ stransitch. 1970 was found in commer­ cial shrimp. Pleistophora crangoni Breed and Olson, 1977 was found in four species of Crangon. Some unde­ scribed species have been listed by Sprague ( 1977). In Fish Microsporidia are very common in fish; over 100 species of fish are now known to be parasitized. Some micro­ sporidia are also known to be serious mortality factors in fish. Delisle, 1972 found massive mortalities of smelt, as­ sociated with this parasite, in certain Canadian lakes. G/ugea stephani (l-Iagenmuller, 1899) has been found Figure 1.-Amesol1 pulvis (Perez, 1905), spores, in muscle of green crab. by different investigators to be an im­ portant mortality factor in young flatfish of different species. Sprague and Vernick (1968) found that Ichthyosporidium giganteul11 (Thelo­ In Crabs the latter, now Ameson pu/vis (Perez, han, 1895), long treated as haplospo­ Microsporidia are common patho­ 1905) Sprague, 1977 (Fig. I). ridium, is a microsporidium. This (or gens of crabs and shrimp, mainly in the a very similar species) is not uncom­ In Shrimp skeletal muscle. About a dozen species mon in ..spot," Leiostol11us xanthurus are now known in crabs, four of them in Nosema ne/soni recently became Lacepede, in tributaries of the Chesa­ the muscles of the blue crab, Cal­ Ameson ne/soni (Sprague, 1950) peake Bay. It produces a very large /inectes sapidus Rathbun. Three of the Sprague, 1977. This species, originally abdominal swelling, usually in young latter are rare, but one. Nosema found in the muscles of Penaeus az­ fish, that probably kills the host. michaelis Sprague, 1970a, is common, tents Ives, has now been identified in Haplosporidia widely distributed, and lethal to its six species of penaeid shrimp. host; the impact on the host population Thelohania macrocystis, in muscles of There has been much interest in hap­ has not been assessed. Nosema Palaemonetes varians, recently be­ losporidia during the past decade, michaelis, being readily obtainable in came Chapmanium macrocystis (Gur­ mai nly because they are very destruc­ great quantity, has become a useful ob­ ley, 1893) Hazard and Oldacre, 1976. ti ve pathogens of oysters. This has led ject for studies on cytology of the para­ Thelohania penaei, in ovary Penaeus to the discovery of a few new species, site (Sprague et aJ. 1968; Weidner, setiferus (L.), recently became Agma­ much better understanding of the 1970, 1976; Weidner and Trager, soma penaei (Sprague, 1950) Hazard group, and new ideas on classification. 1973) and on intracellular and Oldacre, 1976. In Biva/ve Mollusks (Trager, 1974). This species was re­ Quite recently, a relatively large cently made the type of a new genus, number of new species have been dis­ Perkins (1968, 1969) and Rosenfield now beingAmeson michaelis (Sprague, covered in shrimp. A new genus and et aJ. (1969) have contributed much to 1970) Sprague, 1977. One of the four new species, Inodosporus spraguei our understanding of haplosporidia by species known in Callinectes, Overstreet and Weidner, 1974, was making the first electron microscope The/ohania sp. Johnson, /972, is unde­ found in Pa/ael11onetes pugio Holthius studies on some typical forms-those scribed. The first two species of micro­ and P. kadiakensis. P/eistophora lin­ familiar species in Crassostrea vir­ sporidia reported in crabs, The/ohania toni Street and Sprague, 1974 was de­ ginica (Gmel in). maenadis Perez, 1904, and Nosema pu/­ scribed in Pa/aemonetes pugio. Several Minchinia arl110ricana van Banning, vis Perez, 1905, both in the green crab undescribed species were found in pan­ 1977, was found in the European flat Carcinus maenas (L.), were recently daJid shrimp sent to me from British oyster, Ostrea edulis. This is the only rediscovered, the first by Sprague and Columbia by T. H. Butler. One of known haplosporidian that has tails on Vivares independently and the second these, The/ohania sp. Vernick, its spores like those seen in the type by Vivares. Vivares (1975) has already Sprague. and Krause, 1977, in Pan­ species M. chitonis (Lankester, 1885). published on the former, while Vivares da/us jore/ani Rathbun, has been de­ Sprague (1970c) suggested that pres­ and Sprague (in press) have redescribed scribed by Johnston, Vernick, and ence or absence of these tails be used as

October /978 27 the basis for distinguishing Minchinia Labbe, 1896 from Haplosporidium Caullery and Mesnil, 1899. Two species of a previously unfamil­ iar kind have been found in association with oyster mortalities. They are Marteilia refringens Grizel et al., 1974 (type species), in the European flat oys­ ter Ostrea edulis L., and M. sydneyi Perkins and Wolf, 1976, in the Austra­ lian oyster, Crassostrea commercialis. Perkins (1976a) suggested that they are related to the haplosporidia, mainly be­ cause they have "haplosporosomes." /n Decapods "Minchinia sp." Marchand, 1974, was found in the mud crab, Rhithro­ panopeus harrisii, in France. Plas­ modia of a haplosporidian were found Figure 2.-The/ohania sp. Vernick et al., 1976, various sporulation stages, by Newman et al. (1976) in Callinectes in muscle of the shrimp Panda/us jordani. Bouin, section, Heidenhain; sapidus from Virginia and North 1,300 x. Carolina. Myxosporidia John Finlay 2 for identification. I noted a Drastic changes are taking place in New species are frequently added to resemblance to intracellular stages of the classification of the higher the long list of myxosporidian species Hexamita but, being unable to make categories of protozoa, since recent ad­ in fish. Most are not noted for positive identification, sent the sl ides to vances in knowledge indicate that the pathogenicity, but one highly patho­ Glenn Hoffman. At first, Hoffman3 old Protozoa is an artificial as­ genic form, Myxosoma eerebralis suspected the parasite might be identi­ semblage of several phyla. A . 'Com­ (Hofer, 1903), has been the object of cal with the amoebae reported by Ghit­ mittee on Classification and Nomencla­ many recent studies. An interesting tino et al. (1977), but after further con­ ture" of the Society of Protozoologists, species recently described is Unieap­ sideration, he doubted that it is an headed by N. D. Levine, is making a sula pflug/elderi Schubert, Sprague, amoeba. The identity of the parasite revision. Microsporidia, haplo­ and Reinboth, 1975. It has spores with remains undetermined. sporidia, myxosporidia, and some two rudimentary polar capsules, as well other groups will probably become as the fully formed one that is charac­ CLASSIFICAnON distinct phyla, as ciliates have already teristic of the genus. Another species, done. Classification of microsporidia interesting because it infects an impor­ Some minor changes in classifica­ probably will be based on that of tant sport fish, is Henneguya sp. tion, particularly the transfer of some Sprague (1977). Classification of hap­ Meyers, Sawyer, and MacLean, 1977, microsporidian species to new genera, losporidia will probably include a in the heart of the bluefish, Pomatomus have already been mentioned. One of major new taxon to include Marteilia. saltatrix (L.). the most spectacular of the minor de­ Myxosporidia are now considered by velopments in recent years was the many protozoologists to be metazoa Protista Incertae Sedis finding by Perkins (1976b) that the with coelenterate affinities, but they An unidentified parasite with vague flagellated zoospore of Dermocys­ will probably remain for awhile in the resemblance to the Coccidia was re­ tidium marinum Mackin, Owen, and system of the protista. cently found by Peter Wolf (1977) in Collier, 1950 has an apical complex. This is a convenient occasion to take ova of the blacklipped oyster, Cras­ This led Perkins to conclude that the specific action upon my previous sostrea echinata, from Australia. parasite, long regarded as a , is (Sprague, 1970c) suggestion and trans­ Another unidentified protist was found related to the Coccidia. fer from genus Minchinia Labbe, 1896 in the digestive epithelium of the pearl to Hap/osporidium Caullery and Mes­ oyster, Pinctada maxima, in Australia 'Finlay, J., Fish Diseases Laboratory, Ministry nil, 1899 species which, like the type (Wolf and Sprague, 1978). of Agriculture, Fisheries and Food, The Nothe, species H. sea/opli Caullery and Mes­ A parasite in spleen and liver of rain­ Weymouth, Dorset. England DT4 8UB. nil, 1899, have spores without tails. 3 Hoffman, G. A., Fish Farming Experimental Sta· bow trout, causing epizootic disease in tion. U.S. Fish and Wildlife Service, SIUllgart, Accord ingly, Minchinia louisiana England, was recently sent to me by AR 72160. Pers. commun. Sprague, 1963b becomes Haplo-

28 Marine Fisheries Review sporidium louisiana (Sprague. 1963b) tion of the Committee on Merchant Marine and Sprague. V. 196.1a. Rcvision of genus Huplo­ comb. n. and Haskin. Fisheries-House of Represenlatives. Com­ sporidium and rc:storation of genus Millchi"ia M. nelsoni mer. Fish. Res. Legisl. H. R. 18008. Ser. No. (Haplosporidia. Haplosporidiidae). 1. Proto­ Stauber. and Mackin 1966 becomes H. 90-29, p. 99-104. U.S. Gov. Print. Ofr.. zool. 10:263-266 nelsoni (Haskin et al.. 1966) comb. n. Wash .. D.C. ____. 196.1b. Millchilli" louisialla n. sp. Other species which Sprague (1963a: Marchand. J. 1974. Presence de Minchiniu sp. (Haplosporidia. Haplosporidiidac). a parasite (Haplosporida - Haplosporidiidae) chez Ie of Pallopeus herbslii. J. Protozool. 10:267­ 265-266) transferred (for reasons later xanthide Rhilhrop(//lOpeu.\· harrisii (Gould) 274. found to be invalid) from Haplo­ fridenlalUS (Maitland) dan Ie canal de Caen a ____. /970a. Some protozoan parasites sporidium to Minchinia are now re­ la mer. Rev. Trav. Insl. Peches Marit. and hyperparasites in marine decapod Crus­ turned to 38:209-213. tacea. In S. F. Snkszko (editOr). A symposium Haplosporidium. Meyers. T. R.• T. K. Sawyer. and S. A. Mac­ on diseases of fishes and shellfishes. p. 416­ Lean. 1977. Hennegu)'u sp. (Cnidospora: 4.10. Am. Fish. Soc. Spec. Publ. 5. LITERATURE CITED Myxosporida) parasitic in the heart of the 1970b. Some protozoan parasitcs bluefish. PomalOmus salTatrix. J. Parasitol. and hyperparasites in marine bivalve molluscs. 63:890-896. In S. F. Snieszko (editor). A symp

October /978 29 Vivares, C. P. 1975. Elude comparative faile en 1976. Interaction of microsporidia Wolf, P. H. 1977. An unidentified protistan para­ microscopies photonique et electronique de trois wilh host cells. Proc. 1st Inl. Colloq. Inver­ site in the ova of th~ bla~klipp~d oyster. Cras­ especes de microspiridies appartenant au genre tebr. Pathol. and IXth Annu. Meel. Soc. Inver· wstr('u e('hil/uw. fmm n()rth~rn Australia. J. Thelohallia Henneguy. 1892, parasites de crus­ lebr. Palhol., p. 82-84. In\'ert~br. Pat hoI. 29:244-246. taces decapodes marins. Ann. Sci. Nal. Zool. ____, and W. Trager. 1973. Adenosine _____. and V. Sprague. 1978. An uniden· BioI. Anim. Ser. 12,17:141-178. triphosphate in the extracellular sun'ival of an tilicd protistan parasite of th~ p~arl oyster, ____, and V. Sprague. In press. The fine intracellular parasite (Nosema michaelis, Mi· Pillcwda maxima. in tropical Australia. J. In­ structure of Amesoll pu/vis (Perez. 1905) (Mi­ crosporidial. J. Cell BioI. 57:586-591. vertebr. Pat hoI. 31 :262-263. crospora, Microsporida) and its implication re­ garding classification and chromosome cycle. MFR Paper 1340 From Marine Fisheries Review. Vol. 40, No. 10. October J. Invenebr. PathoI. 1978. Copies of this paper, in limited numbers, are available from 0822, User Weidner, E. 1970. Ultrastructural study of mi­ Services Branch. Environmental Science Information Center, NOAA, Rockville. crosporidian developmenl. J. Nosema sp. MO 20852 Copies of Marine Fisheries Review are available from the Superin­ Sprague. 1965 in Callillectessl1pidus Rathbun. tendent of Documents, U.S. Government Printing Office, Washington, DC Z. lellforsch. Mikrosk. Anal. 104:33-54. 20402 for $1. 10 each.

MFR PAPER 1341

Susceptibility Studies of Various Salmonids to Whirling Disease: Histological Staining and Spore Concentration Procedures

JOSEPH O'GRODNICK

Since 1968, research on the trans­ the spinal skeleton if fish are exposed as mission, life history, and control of fry. "Blacktail," which is caused by whirling disease in trout has been con­ pressure on the nerves that control the ducted by the Pennsylvania Fish Com­ caudal pigment cells, may be seen in mission. Whirling disease is caused by some fish. a myxosporidian parasite, Myxosoma In this research, two methods were Figure I.-The plankton centrifuge. The cerebralis, which invades and destroys used to monitor the development of the centrifuge removes water through the cen­ trifugal force created by the revolving cartilage of susceptible salmonid whirling disease parasite in various drum. The excess water is carried away species. The parasite was introduced salmonid species: I) examination of through a rubber tube situated below the into the United States from Europe and histological sections, and 2) quantita­ drum. Solid particles are deposited on the wall of the revolving drum. has been established as a persistent tive estimates of spores determined by hatchery disease in several areas. the use of the plankton centrifuge pro­ Affected fish exhibit rapid, tail chas­ cedure (Fig. I). (This method involves ing behavior from the disintegration of homogenization of head skeletons, the cartilaginous support skeleton ofthe screening out tissue shreds, and fi sh at gi ven intervals, averaging 3 days organs of equilibrium. Also, severe concentrating with a continuous plank­ from initial exposure to the develop­ crippling may result from destruction of ton centrifuge. ment of spores at 120 days. The follow­ The life cycle of Myxosoma cere­ ing staining methods were used in the bralis was monitored in rainbow trout study: I) Hematoxylin and Eosin, 2) (highly susceptible species) and brown Mallory Heidenhain (Casson­ Joseph O'Grodnick is with the Ben­ ner Spring Fish Research Station, trout (highly resistant species) by the Modification), 3) Wright's stain, and 4) Pennsylvania Fish Commission, Bel­ examination of histological sections. Ziehl-Neelson (acid fast staining for lefonte, PA 16823. Tissues were prepared from infected spores) .

30 Marine Fisheries Review