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Home , Argopecten, Argopecten irradians, Eastern oyster

DISEASES OF AQUATIC ORGANISMS Vol. 24: 77-80, 1996 Published January 4 Dis aquat Org

NOTE

An assessment of lesions in bay irradians attributed to karlssoni (, Apicomplexa)

C. L. Gogginl*', S. E. McGladdery 2, S. K. whyte3, R. J. cawthorn3

'Department of Parasitology, The University of Queensland. Brisbane, Queensland 4072, Australia 'Department of Fisheries and , Gulf Fisheries Centre, PO Box 5030. Moncton, New Brunswick, Canada ElC 9B6 3Department of PaUlology and Microbiology, Atlantic Veterinary College. University of Prince Edward Island, 550 University Avenue. Charlottetown, Prince Edward Island, Canada CIA 4P3

ABSTRACT- Leslons found in bay scallops Argopecten ~rradi- more than 1 organism associated with this name which ans imported into Atlantic Canada for culture were attributed may later be described outside the Perkinsus, to a parasite Perklnsus karlssonj It appears, however, that Bay scallops imported into stages froin several organisms were combined to create the

life cvcle of P karlsson~.The ultrastructure of the aaentd In Canada for in and suffered heavy lesions of bay scallops IS not documented adequately to dlag- POSt-Spawnlng nlortalltles McGladdery et al. (1991) nose affinities to the genus Perklnsus Furthermore, there was described 'swlrl' leslons from the bay scallops whlch discrepancy as to whether P karlsson~enlarged in flu~dthio- they attributed to the parasite perkinsus karlssoni, glycollate medium, and the shape and structure of the pre- zoosporangia as well as the movement and life span of the 'S"ir1' lesions most numerous in P~~~-~P~~~~~~ zoospores were not consistent with the genus Perkinsus P scallops. Bay scallo~s,however, usually die after karlssonj cannot be considered as a of Perk~nsus. spawning and P. karlssoniwas not associated with the pathogenesis. The 'swirl' lesion found in A. irradiansis KEY WORDS: Perkinsus karlssoni . Apicomplexa - Parasite a generalised response to a agent and is not Bay . Argopecten irradlans diagnostic of an infection by parasites in the genus Perkinsus.P. karlssoniwhich were healthy when pre- served have not been seen under the electron micro- Perk~nsusis a genus of protistan parasite which scope. The organisms identified as P. karlssoniin elec- infects and kills marine molluscs. It has been found in tron micrographs published by Whyte et al. (1994) more than 30 species of molluscs around the world (Fig. 18a, b) are dead organisms encapsulated by (Goggin & Lester 1987, Perkins 1988). Four species ceroid and do not resemble Perkinsus. have been described: P. marinusfrom eastern In histological sections, the meront of species of virginica in America (Mackin et al. 1950, Perkinsusis distinguished by a large eccentric vacuole Levine 1978); P. atlanticus from Rudltapes which sometimes contains a vacuoplast. A vacuole was decussatusin Portugal (Azevedo 1989); P. olseni from rarely observed in P. karlssoni,but a dense basophilic in Australia (Lester & Davis body was found next to the nucleus which could be 1981); and P. karlssoni from bay scallops Argopecten equivalent to the vacuoplast of P. marinus (McGlad- irradians in eastern Canada and adjacent United dery et al. 1991). The basophilic body of P. karlssoni, States (McGladdery et al. 1991, McGladdery et al. however, was not contained in a large eccentric vac- 1993). There are significant differences, however, uole, which makes this association unlikely. Further- between P. karlssoniand the other 3 described species more, the presence of a basophilic body does not diag- of Perkinsus.P. karlssoniis used hereafter as a matter nose a Perkinsus spp. because the vacuoplast can be of convenience with the recognition that there may be either basophilic or eosinophilic in P. marinus(Mackin et al. 1950) or slightly eosinophilic (when present) in P. olseni(Lester & Davis 1981). 'Present address CSIRO Divls~on of Fishenes, GPO Box Perkinsuskarlssoni differs from described species of 1538, Hobart, Tasmania 7001, Australia Perkinsus in other ways. P. karlssoni was not recog- E-mall louise goggin@ml csiro au nised by an antibody raised against the meront of P.

O Inter-Research 1996 Resale of full art~clenot perm~tted 78 Dis aquat Org 24: 77-80, 1996

marinus that binds to the 3 other described species of media (La Peyre et al. 1993).Whether this hyphal form Perkinsus in histological sections (Dungan & Roberson is a phase of the life cycle in P. marinus or a contami- 1993). The 'swirl' lesion which surrounds P. karlssoni nant is not clear. Cells with hyphal-like outgrowths, does not account for the failure of the immunological however, are neither the usual nor the dominant form test because the antibody recognised Perkinsus sp. in of the parasite and failure to observe the typical pre- similar lesions in clams Mya arenana (C. Dungan pers. zoosporangia of Perkinsus casts doubt on the diagnosis comm.). These results suggest that P. karlssoni does of McGladdery et al. (1991). not belong to the genus Perkinsus. Perkinsus spp. release zoospores from the prezoo- Perkinsus infections are diagnosed by incubating sporangia (developed in FTM) after several days in host tissues in fluid thioglycollate medium (FTM) for seawater. The zoospores are biflagellate with filamen- several days. The parasites enlarge and can be stained tous mastigonemes on one side of the anterior flagel- with Lugol's iodine after incubation (Ray 1966). Blue- lum. Whyte et al. (1993a) found that the most efficient black spheres were found in tissues of Argopecten method to collect 'zoosporangia' and zoospores in- irradians from Canada using this procedure (McGlad- cluded the maceration of the scallop tissue followed by dery et al. 1991, Whyte et al. 1994). McGladdery et incubation in seawater at 26°C for 3 d. We attempted a!. (1991) reported that mcronts increased from to isolate zoospores of P. karlssoni using the same 4.3-23.11 pm to reach 80.0-138.3 pm after incubation procedure. We placed tissues directly into sterile in FTM. Whyte et al. (1993a, p. 203) found, in contrast, artificial seawater and biflagellates appeared in our that P. karlssoni did not swell in FTM. The difference cultures after 4 d. DNA was extracted from these in the results of the FTM test suggest that Whyte et al. organisms and the small subunit ribosomal DNA (SSU (1993a, b) isolated a different organism from that rDNA) amplified by polymerase chain reaction (PCR) described by McGladdery et al. (1991). Indeed, the (Goggin & Barker 1993). The product was sequenced 'prezoosporangium' isolated from bay scallop tissues using automatic sequencing (Applied Biosystems, Inc.) that had not been incubated in FTM (Whyte et al. and the data compared to those held in computer data- 1993a, Fig. lb) does not belong to the genus Perkinsus. bases. The sequence (1680 bp) was most similar to the The organism has membranous plates or scales around SSU rDNA data from Cafeteria roenbergensis (EMBL the cell, multivesicular bodies in the cytoplasm and accession number L27633; Leipe et al. 1994) with dif- mitochondrial structure more closely resembling that ferences in 5 positions (2 additional, 2 different, 1 miss- of the thraustochytrids than that of Perkinsus spp. (see ing). Fourteen ambiguous positions in the SSU rDNA Moss 1985). data from C. roenbergensis were resolved by our work. Meronts of Perkinsus spp. enlarge in FTM to form These molecular analyses indicate that the biflagel- zoosporangia when released into seawater. Cells lates found in our seawater cultures were members of divide internally to produce thousands of motile zoo- the genus Cafeteria and were most likely C. roenber- spores which later escape through a discharge tube or gensis. plug on the surface of the zoosporangia. Prezoo- Cafeteria roenbergensis is a bicosoecid flagellate sporangia of Perkinsus spp. do not multiply when reported from waters near Denmark, Australia and released in seawater. In contrast, bay scallop tissues Hawaii (Fenchel & Patterson 1988). The cell body of incubated in FTM at 22OC develop aggregations of C. roenbergensis measures 4-6 X 4-4.5 pm, which is several hundred large P. karlssoni when placed in similar in size to that of (4-6 X 2-3 seawater (McGladdery et al. 1991). Some thraus- pm) and P. karlssoni (5.8 X 3.2 pm). C. roenbergensis tochytrids appear viable after incubation in FTM is a heterokont and has a bilateral array of tubular (Quick 1972). they could proliferate in seawater and mastigonemes on the antenor flagellum (5 to 8 pm) account for this observation. and a trailing posterior flagellum (5 to 8 pm) which The prezoosporangia described by McGladdery et lacks mastigonemes. The zoospores of P, marinus al. (1991) differ from those of Perkinsusspecies in other have an anterior flagellum with filamentous mastigo- ways. Successive bipartitioning of the protoplast of the nemes (10 to 18 pm) and a posterior, naked flagellum cell as described for Perkinsus spp. (Perkins & Menzel (6 to 10 pm) (Perkins & Menzel 1966). The zoospores 1966) was not observed in P. karlssoni. Furthermore, of P. karlssoni are also biflagellate (McGladdery et al. the discharge tube of the 'zoosporangium' of P. karls- 1991), with a posterior flagellum (2.7 to 5.4 pm) of soni was filled with cytoplasm which is unlike other similar length and an anterior flagellum (11.9 to Perkinsus spp. (Perkins & Menzel 1966, Lester & Davis 17.3 pm) that is longer than that of C. roenbergensis. 1981, Azevedo et al. 1990). The 'zoosporangia' de- It is possible that Whyte et al. (1993a) confused C. scribed by McGladdery et al. (1991) may be equivalent roenbergensis with P. karlssoni zoospores under the to the hyphal form of P. marinus found occasionally in light microscope, particularly as they had not incu- FTM (Ray & Chandler 1955, Quick 1972) and culture bated tissues in FTM. Goggin et al.: Perkinsus kadssoni lesions in bay scallops

There are other differences between the zoospores Dungan CF, Roberson BS (1993) Binding specificities of of Perkinsus karlssoni and those of other species of mono- and polyclonal antibodies to the protozoan Perkinsus. The behaviour of the zoospores of P. karls- pathogen Perkinsus marinus. Dis aquat Org 15: 9-22 soni, as described by McGladdery et al. (1991), is not Fenchel T, Patterson DJ (1988) Cafeteria roenbergensis nov. typical of Perkinsus spp. The zoospores of P. marinus gen., nov. sp., a heterotrophic microflagellate from marine swim with a jerky motion (Perkins & Menzel 1966), do . Mar microb Food Webs 3.9-19 not adhere usually to the substrate and live for several Goggin CL, Barker SC (1993) Phylogenetic position of the genus Perkinsus (Protista, Apicomplexa) based on the hours (F. 0. Perkins pers. comm.). Zoospores of P. small subunit ribosomal RNA. Mol Blochem Parasitol 60: karlssoni, in contrast, remained close and adhered 65-70 weakly to the bottom and were viable for up to 9 wk at Goggin CL, Lester RJG (1987) Occurrence of Perkinsus spe- 4°C (McGladdery et al. 1991). Interestingly, Cafeteria cies (Protozoa, Apicomplexa) in bivalves from the Great roenbergensis can swim but usually adhere to the sub- Barrier Reef. Dis aquat Org 3:113-117 La Peyre JF, Faisal M, Burreson EM (1993) In vitro propaga- strate by the posterior flagellum. The zoospores of P. tion of the protozoan Perkinsusmarinus, a pathogen of the karlssoni were also unlike those of other species of , Crassostrea virginica. J Euk Microbiol 40: Perkinsus (Perkins 1988, 1991) because they lacked an 304-310 apical complex, mastigonemes on the anterior flagel- Leipe DD, Wainwright PO, Gunderson JH, Porter D, Patterson DJ, Valois F, Himmerich S, Sogin ML (1994) The stra- lum and a large cylindrical inclusion in each kineto- menopiles from a molecular perspective: 16s-like rRNA some lumen (McGladdery et al. 1991). sequences from Labyrinthulo~des minuta and Cafeteria Apparently, the life cycle of Perkinsus karlssoni com- roenbergensis. Phycologla 33369-377 prises several unrelated organisms. It is not likely that Lester RJG, Davis GHG (1981) A new Perkinsus species (Api- the differences observed between P, karlssoni and complexa, Perkinsea) from the abalone, Haliotis ruber. J Invertebr Path0137 :l81-187 Perkinsus species result from 'ten years of transmission Levine ND (1978) Perkinsus gen. n. and other taxa in the pro- via hatchery manipulated spawning' as postulated by tozoan phylum Apicomplexa. J Parasitol 64:549 McGladdery et al. (1991). The 'prezoosporangia' and Mackin JG, Owen HM, Collier A (1950) Preliminary note on 'zoospores' attributed to P. karlssoni do not belong to the occurrence of a new protistan parasite, Dermocystid- ium marinum n. sp. in Crassostrea virginica (Gmelin). Sci- the genus Perkinsus. Perhaps the lesions in bay scal- ence 111:328-329 lops are caused by a Perkinsus sp. and most parasites McGladdery SE, Bradford BC, Scarratt DJ (1993) Investiga- die. The hepantotype of P. karlssoni deposited in the tions into the transmission of parasites of the bay scallop Canadian Museum of Nature (CMNP1990-0027) has Argopecten irradians (Lamarck, 1819), during quarantine 'swirl' lesions and is infected by a coccidian as de- introduction to Canadian waters. J Res 12: 49-58 scribed by Whyte et al. (1994). Whyte et al. (1994), McGladdery SE, Cawthorn RJ, Bradford BC (1991) Perkinsus however, reported no apparent response of the bay karlssoni n. sp. (Apicomplexa) In bay scallops Argopecten scallop to the coccidian. Whether the coccidian causes irradians. Dis aquat Org 10.127-137 bay scallops to produce 'swirl' lesions under some con- Moss ST (1985) An ultrastructural study of taxonomically sig- nificant characters of the Thraustochytriales and the ditions is unknown. The agent that causes the bay scal- Labyrinthulales. Bot J Llnn Soc 91:329-357 lop to produce granulomas needs further investigation Perkins F0 (1988) Parasite morphology, strategy and evolu- to determine its taxonomic affinities. It is not certain tion. Structure of protistan parasites found in bivalve mol- that it is a species of Perkinsus and we do not recognise luscs. In: Fisher WS (ed) Disease processes in marine P. karlssoni as a species of Perkinsus. bivalve molluscs. Am Fish Soc Spec Pub1 18:93-111 Perkins F0 (1991) Sporozoa. In: Harrison FW, Corliss JO (eds) Microscopic anatomy of invertebrates, Vol 1. Wiley-Liss, New York, p 261-331 Perkins FO, Menzel RW (1966) Morphological and cultural Acknowledgements. We thank MS Janet Munholland and MS studies of a motile stage in the life cycle of Dermocystid- Colleen Murphy. IMB/NRC, Halifax, Canada, for sequencing ium marinum. Proc natl Shellfish Ass 56:23-30 Cafeteria roenbergensis and Drs S. M. Bower, R. J. G. Lester, Quick JA Jr (1972) Fluid th~oglycollate medium assay of F. Perkins and an anonymous reviewer for helpful com- 0. Labyrinthomyxa parasites in oysters. Florida Dept of Nat- ments on the manuscript. C.L.G, was supported by a Natural ural Resources Leaflet Series, Vol 6, Part 4, No 3. Florida Science and Research Council (NSERC) of Canada Interna- Dept of Natural Resources, St Petersburg tional Fellowship. Ray SM (1966) A review of the culture method for detecting Dermocystidium marinum, with suggested modifications LITERATURE CITED and precautions. Proc natl Shellfish Ass 54:55-69 Ray SM, Chandler AC (1955) Dermocystidium marinum, a Azevedo C (1989) Fine structure of Perkinsus atlanticus n. sp. parasite of oysters. Exp Parasitol 4:172-200 (Apicomplexa, Perkinsea) parasite of the Ruditapes Whyte SK, Cawthorn RJ, MacMillan RJ, Despres B (1993a) decussatus from Portugal. J Parasitol75527-635 Isolation and purification of developmental stages of Azevedo C, Corral L, Cachola R (1990) Fine structure of zoo- Perkinsus karlssoni (Apicomplexa: Perkinsea), a parasite sporulation in Perkinsus atlanticus (Apicomplexa: Perkin- affecting bay scallops Argopecten irradians. Dis aquat sea). Parasitology 100:351-358 Org 15:199-205 80 Dis aquat Org 24: 77-80, 1996

Whyte SK, Cawthorn RJ. McGladdery SE (1994) CO- Whyte SK, Cawthorn RJ, McGladdery SE, MacMillan RE, infection of bay scallops Argopecten jrradians with Montgomery DM (1993b) Cross-transmission studies of Perkinsus karlssoni (Apicomplexa, Perkinsea) and an Perkinsus karlsson~ (Apicomplexa) from bay scallops unidentified coccidian parasite. Dis aquat Org 18: Argopecten irradians to native Atlantic Canadian shellfish 53-62 species. Dis aquat Org 17:33-39

Responsible Subject Editor A. K Sparks, Seattle, Manuscript first received: ~Varch13, 1995 Wash~ngton,USA Rev~sed version accepted: September 18, 1995