J. Eukaryor. Microbid.. 49(1), 2002 pp. 11-16 0 2002 by the Society of Protozoologists Serological Affinities of the Oyster Pathogen Perkinsus marinus (Apicomplexa) with Some Dinoflagellates (Dinophyceae)

DAVID BUSHEK; CHRISTOPHER F. DUNGANband ALAN J. LEWITUS8,c 'Univer,sity of South Curolina, Belle W. Baruch Institute for Marine Biology und Coastal Research, Baruch Murine Field Laboratory, Georgetown, South Carolina 29442, USA, and bMuryland Department of Nutural Resources, Cooperative Oxford Laboratory, Oxjford, Maryland 21654, USA, and cSouth Carolina Depurtment of Nuhiml Resources, Marine Resources Institute, Charleston, South Carolina 29442-2559, USA

ABSTRACT. The protozoan oyster pathogen Perkinsus marinus is classified in the phylum Apicomplexa, although molecular-genetic and ultrastructural evidence increasingly concur on its closer phylogenetic relationship with the dinoflagellates. To test for evidence of serological epitopes common to P. marinus and dinoflagellates, we probed I9 free-living and 8 parasitic dinoflagellate, or dinoflagellate- like, species for cross-reactivity with polyclonal antibodies to P. marinus. Three of 19 free-living dinoflagellates (16%), and 7 of 8 parasitic dinoflagellates (88%) were labeled by anti-P. marinus antibodies. In reciprocal immunoassays using polyclonal antibodies to the Hematodinium sp. dinoflagellate parasite of Norway lobsters, Nephrops norvegicus, P. marinus and the same 7 parasitic dinofla- gellates labeled by anti-P. murinus antibodies, were again labeled. The dinoflagellate-like parasite of prawns Pandalus platyceros was not labeled by either antibody reagent. These reciprocal results confirm the presence of shared antibody-binding epitopes on cells of P. marinus and several dinoflagellates. The apparent widespread serological affinity between P. marinus and the parasitic dinoflagellates suggests a closer phylogenetic link to the syndinean dinoflagellate lineage. The consistent failure of the dinoflagellate-like prawn parasite to bind either antibody reagent shows that this parasite is serologically distinct from both P. marinus and Henzatodiiziinn-species parasitic dinoflagellates. Key Words. Antibody cross-reaction, dermo, epitope sharing, Hematodinium, immunoassay, protozoan phylogeny, oyster disease, parasitic dinoflagellates, spot prawn, Syndinideae.

ESTRUCTIVE impacts on aquatic habitats and living gellates suggested by nucleotide sequence homology data, and D aquatic resources by toxic algae, parasitic dinoflagellates, to localize possible shared antibody-binding epitopes, we and pathogenic protozoans in estuarine environments have been probed a variety of free-living and parasitic dinoflagellate cells increasingly recognized, yet the taxonomic status of some iden- with polyclonal rabbit anti-P. marinus antibodies (Dungan and tified agents remains controversial, and objective methods for Roberson 1993). Upon the unexpected detection of such cross- their rapid identification unavailable. Development of polyclon- reactivity, especially among the parasitic dinoflagellates, we al antibodies to the protozoan oyster pathogen Perkinsus mar- also tested the parasitic dinoflagellates and P. marinus for re- inus (Dungan and Roberson 1993) improved detection of this ciprocal cross-reactivity with polyclonal rabbit antibodies raised pathogen in host tissues and environmental samples, and pro- against a Hematodinium sp. dinoflagellate parasite of Norway vided a novel tool for testing long-standing hypotheses on the lobsters, Nephrops norvegicus (Field and Appleton 1996). We P. marinus life cycle, oyster disease transmission, and disease report here the results of those experiments. pathogenesis. Because Perkinsus karlssoni is no longer consid- ered a valid Perkinsus species (Goggin et al. 1996), these poly- MATERIALS AND METHODS clonal anti-P. marinus antibodies are now known to be broadly Viable and formaldehyde-fixed suspensions of free-living di- specific for all described, and most undescribed, Perkirzszis-spe- noflagellates were obtained from several sources (Table 1). Vi- cies mollusc parasites (Blackbourn, Bower, and Meyer 1998; able populations were propagated in vitro and fixed for 1 h with Dungan and Roberson 1993; Maeno, Yoshinaga, and Nakajima 4% (w/v) buffered formaldehyde or (w/v) paraformalde- 1999; Dungan et al. 2002), but not for other protozoan patho- 2% hyde, diluted in culture-medium seawater. Fixed cells were im- gens of fish and shellfish. mobilized on track-etched, I-km pore-size, black polycarbonate Perkinsus murinrrs is currently classified as an apicomplexan membranes clamped in 13-mm swinnex filter housings, and based on zoospore apical complex ultrastructures, including an by sequential, unidirectional pas- atypical conoid apparatus (Levine 1978). However, recent anal- fluorescence-immunostained sage of immunoassay reagents through the sample membranes, yses of P. marinus rRNA and actin gene nucleotide sequences (Fong et al. 1994; Goggin and Barker 1993; Reece et al. 1997; as described (Dungan and Roberson 1993). Membrane-immo- Siddall et al. 1997) and morphological attributes (Perkins 1996; bilized cells were washed with 0.15 M phosphate-buffered sa- Siddall et al. 1997; Sunila, Hamilton, and Dungan 2001; Vivier line (PBS) containing 0.05% (v/v) Tween-20 (PBST), and in- 1982) have challenged inclusion of Perkinsus species in the cubated in blocking buffer for 30 min. Blocking buffer was Apicomplexa. Several independent analyses that have increas- PBST containing 2% (w/v) bovine serum albumin (BSA) and ingly indicated that the strongest phylogenetic affinities of Per- 0.2% (v/v) normal goat serum. Sample cells were then sequen- kinsus species lie with dinoflagellate taxa (Siddall et al. 1997; tially exposed to a dilution (- 10 pg IgG ml-I) of the Wolters 1991), have inspired Cavalier-Smith (1998) to propose rabbit anti-P. marinus antiserum of Dungan and Roberson erection of the phylum Dinozoa to house proposed subphyla (1993) for 30 min, goat anti-rabbit IgG-FITC conjugate (1 pg Dinoflagellata and Protalveolata (Perkinsus et al.) together. To- TgG rn-I) for 30 min, and 0.5% (w/v) Evan's blue counterstain. ward a similar taxonomic objective, NorBn, Moestrup, and Antibody reagents were diluted in serum diluent (PBST + 1% Rehnstam-Holm (1 999) proposed separate phylum status for BSA). Samples were washed with PBST between immunoassay both the Perkinsozoa and the Dinoflagellata, within their com- reagent incubations, and immunostained membranes were mon alveolate clade. mounted in glycerol mounting medium pH 9, for examination To test for phenotypic evidence supporting or refuting phy- by epifluorescence microscopy under 480-nm blue light exci- logenetic affinities between Perkinsus species and the dinofla- tation. Histological samples of parasitic dinoflagellates infecting a variety of hosts were obtained from cooperating colleagues Corresponding author: C. Dungaii-Telephone number: 410-226- worldwide (Table 2). Histological samples were sectioned and 5 193; FAX number: 410-226-5925; E-mail: [email protected] immunostained as described (Dungan and Roberson 1993), with 11 12 J. EUKARYOT. MICROBIOL., VOL. 49, NO. I, JANUARY-FEBRUARY 2002 the following differences. Dewaxed and rehydrated sections were absent from autofluorescence and non-specific antibody were incubated for 1 h in blocking buffer. Primary antibodies binding control replicates. were protein A-affinity purified IgG from either normal rabbit Similar histological immunoassay methods were used to stain serum (negative control normal IgG) or from the anti-P. mari- P. marinus-infected oyster tissues, and fish and crustacean tis- nus antiserum of Dungan and Roberson (1 993), both incubated sues infected by dinoflagellate, or dinoflagellate-like, parasites, at 10 pg IgG rn-' for 1 h. Secondary antibodies were affinity- using the anti-Hematodinium sp. antibodies of Field and Ap- purified, FITC-conjugated goat anti-rabbit IgG, which were in- pleton (1996). This rabbit antiserum, raised against a lysate cubated with sections for I h at 1 pg IgG rn-'. Other reagents from in vitro propagated Hematodinium sp. vegetative cells, were identical to those employed for membrane-immobilized was diluted 1:500 in serum diluent, to an estimated concentra- free-living cells. tion of 10 pg IgG d-l. Positive control sections were from Formalin-fixed P. marinus hypnospores from infected oyster Hematodinium sp.-infected N. norvegicus tissues. Otherwise, tissues, which were enlarged in Ray's fluid thioglycollate me- immunoassays were identical to those performed with anti-P. dium (RFTM) and isolated after host tissue hydrolysis (Bushek, marinus antibodies. Ford, and Allen 1994), were immobilized on membranes as positive controls for cytological immunoassays of suspended RESULTS cells. Sections of P. marinus-infected Crassostrea virginica oyster tissues were used as positive controls for histological The rabbit polyclonal antibodies to P. marinus used in this immunoassays performed with anti-P. marinus antibodies. Rep- study label all described, and several undescribed, Perkinsus- licate preparations of each tested sample omitted the primary species mollusc parasites (Blackbourn, Bower, and Meyer antibody, or substituted normal rabbit IgG, to control for pos- 1998; Dungan and Roberson 1993; Maeno, Yoshinaga, and Na- sible non-specific binding of either rabbit primary antibodies or kajima 1999; Dungan et al. 2002). Using the methods described FITC-congugated secondary antibodies. Autofluorescence con- here for histological immunoassays, these antibodies were pre- trol replicates of each tested sample were not exposed to any viously shown not to label the protistan pathogens of finfish antibodies, but were blocked, counterstained, and analyzed for and shellfish, Dermocystidium salmonis, Haplosporidium nel- autofluorescence under blue light excitation. Positive immuno- soni, and a Dermocystidium-like systemic salmon pathogen assay results, including sites of antibody labeling, were record- (Dungan and Roberson 1993). We have subsequently deter- ed only for immunofluorescence signals detected in samples mined that these antibodies are also unreactive with the protists stained with both primary and secondary antibodies, but which Labyrinthuloides hnliotidis (Bower 1987) and the undescribed

Table 1. Antibody binding by free-living dinoflagellates assayed with anti-Perkinsirs niarznus antibodies.

Dinoflagellate species Isolate code Isolate source Antibody binding phototroph, toxic Alexandriuin af%ne Balech AABBO 1/2 a Alexandriuin catenella Balech GCRUX a Alexandriuin catenella OF87842 a Alexandrium fundyense Balech AFNS85 a A lexnndrium jiindyense GTCA29 d Alexandrium minutum Balech GTPORT a Alexnndriuni niiniihtni AMDOl a Alexaizdriuin tamarense Balech OF84423-03 a Alexandrium taninreiise AT-9 d Alexandrium tamnrense PGT183 a Alexandrium taniarense ATGHOPEI a Amphidinium cnrterne Hulburt CCMP13 14 b Gymnodinirim breve Davis GB, Willon C Gymnodinium breve d Gymnodinium catenntum Graham CCMP414 b Gyrodinium mikimotoi Miyake & Kominari CCMP430 b Gyrodinium galatlieanum Baarud CCMP4 16 b Lingulodinium polyedru Dodge CCMP407 b Prorocentruin minimum Schiller CCMP698 b Prorocentrum minimum PROR6 17-2 e Procentrum minimum I? min. 2A e phototroph, nontoxic Ceratium fusus Dujardin CCMP 1758 Gyrodinium uncateimm Hulburt CCMP1310 Gyrodinium sp. Kofoid & Swezey G. est. Heterocapsa pygnzaea Loeblich, Schmidt & Sherley 242 1 Peridinium foliaceum Lemmerman 1688 Symbiodinium sp. Freudenthal CCMP832 heterotroph, nontoxic Oxyrrhis murina Dujardin 1974 f Isolate Sources: a. G. Doucette, NOAA/NOS, Charleston, SC, USA; b. Provasoli-Guillard Center for Culture of Marine Phytoplankton, West Boothbay Harbor, ME, USA; c. E Van Dolah, NOAANOS, Charleston, SC, USA; d. I? Tester, NOAA/NMFS, Beaufort, NC, USA; e. A. Lewitus. Belle W. Baruch IMBCR, University of South Carolina, Georgetown, SC, USA; f. The Culture Collection of Algae at the University of Texas, Austin, TX, USA. BUSHEK, DUNGAN & LEWITUS-P. MARZNUSDINOPHYCEAE SHARED EPITOPES 13

Fig. 1. Phototrophic dinoflagellate Gyi-odinium uncareiicm immunostained on black polycarbonate membrane with normal rabbit serum. Only cytoplasmic chlorophyll (orange) autofluorescence is seen in this non-specific antibody-binding negative control treatment. Digital gray scde conversion from original color print. N, nucleus; arrowhead, sulcus. Bar = 20 pm. Fig. 2. Phototrophic dinoflagellate Gyodiiziunt uncatenunz immunostained on black polycarbonate membrane with anti-Perkinsus mcrrinus antiserum. Besides cytoplasmic chlorophyll (orange) autofluorescence, the feeding veil (arrow) and cell wall were fluorescence-labeled (green) by antibodies. Digital gray-scale conversion from original color print. N, nucleus; arrowhead, sulcus. Bar = 20 pm. labyrinthomorphid quahog clam parasite QPX (Whyte, Caw- sick 1994; Newman and Johnson 1975) showed strong labeling thorn, and McGladdery 1994). of both cytoplasmic and nuclear epitopes. The plasmalemma, Twenty-eight strains of free-living dinoflagellates, represent- nuclear membrane, and nucleoplasm were labeled in Hemato- ing 19 phototrophic, heterotrophic, toxic, or non-toxic species, dinium australis infecting Australian sand crabs Portunus pe- were tested for labeling by anti-P. marinus antibodies (Table lagicus (Hudson and Shields 1994). Nuclear and cytoplasmic 1). Three planktonic phototrophs (16% of tested species) epitopes were labeled in a Hematodinirinz sp. parasite of velvet showed immunofluorescence signal intensities similar to posi- swimming crabs Necora puber from France (Wilhelm and tive control P. marinus hypnospores. Both cell walldmem- Mialhe 1996). Nuclear, cell wall, and cytoplasmic epitopes branes and feeding veils of Gymnodinium catenatum and Gy- were labeled in a Hematodinium sp. infecting an unidentified rodiniunz uncatenum bound anti-P. marinus antibodies (Fig. 1, gammaridean amphipod from Maryland coastal bays (Johnson 2). Cell walls or membranes of Gyrodiniurn galatheanum were 1986). In Amyloodinium occelatum ectoparasites infesting gills stained, as were cytoplasmic epitopes of G. uncatenuni. Nuclear in Gulf of Mexico red drum Sciaenops ocellatus (Brown and immunostaining in free-living suspended dinoflagellate cells Hovasse 1946; Nigrelli 1936), cell walls, reticular cytoplasm, was never observed. Sixteen species (84%) of free-living di- nuclear membranes, and non-chromosomal nucleoplasm were noflagellates failed to bind anti-P. marinus antibodies, indicat- labeled. Repeated efforts to immunostain a dinoflagellate-like ing that P. marinus-like epitopes labeled in immunopositive parasite in histological samples of infected spot prawns P. pla- species are neither broadly conserved nor generally expressed tyceros from both southeast Alaska (Meyers, Lightner, and by free-living dinoflagellates. Redman 1994) and British Colombia (Bower, McGladdery, and Seven of eight (88%) tested dinoflagellate, or dinoflagellate- Price 1994), consistently failed. like parasites of marine crustacea and fish were labeled by anti- Tests for reciprocal immuno-staining by anti-Hematodinium P. marinus antibodies (Table 2), and intracellular locations of sp. rabbit antiserum were performed only on histological sec- labeled epitopes varied between parasites. Like anti-P. marinus tions from P. marinus-infected oysters and from dinoflagellate- antibody labeling in cognate P. marinus cells (Fig. 3), a He- infected hosts, using the antiserum described by Field and Ap- matodinium sp. infecting Alaska tanner crabs Chionoecetes pleton (1996). These antibodies label Hematodinium sp. para- bairdi (Meyers et al. 1987) showed strong labeling of cell wall, site cells in infected Norway lobster tissues, but not host lobster cytoplasmic and nuclear epitopes. Parasite cell wall and cyto- tissues, or a co-infecting Paranophrys-like ciliate also present plasmic epitopes, but not nuclear epitopes, were labeled in a in some affected lobsters. Hematodinium sp. infecting Scottish Norway lobsters N. now- Perkinsus marinus (Fig. 4) and the same seven parasitic di- egicus (Field et al. 1992) (Fig. 5). Another Henzatodiniunz sp. noflagellates that were labeled by anti-P. marinus antibodies, infecting Chesapeake Bay blue crabs Callinectes sapidus (Mes- were also reciprocally labeled by antibodies to the Hematodi- 14 J. EUKARYOT. MICROBIOL., VOL. 49, NO. 1, JANUARY-FEBRUARY 2002

nium sp. parasite of N. norvegicus (Table 2). Immunostaining munopositive samples, and broadly prevalent among the para- of Hematodinium sp. cells by their cognate antibodies included sitic dinoflagellates. The relatively high apparent frequency of cell wall and cytoplasmic epitopes (Fig.6), strikingly similar to shared epitopes among parasitic dinoflagellates further suggests the result obtained in Hematodinium sp. cells immunostained that Perkinsus species protozoan parasites may reflect a stron- with anti?. marinus antibodies (Fig. 5). The dinoflagellate-like ger phylogenetic affinity with an evolutionary lineage common parasite infecting spot prawns P. platyceros, also failed to label to the parasitic dinoflagellates. Alternatively, expression and de- with anti-Hematodinium sp. antibodies. Although the resolution volution of these shared epitopes may reflect phenotypic con- of light microscopy for epitope localization and differentiation vergence associated merely with common parasitism of marine is low, the subcellular locations of binding epitopes for both hosts. This interpretation seems unlikely, in light of the lack of antibody reagents appeared similar, and both antibodies gave reactivity documented for the anti-P. marinus antibodies used similar reciprocal staining reactions when reacted with each during this investigation with a broad range of protistan pcara- others' cognate antigens (Fig. 3-6). These similar reciprocal sites of marine invertebrates and finfish. staining results confirm that P. marinus and several parasitic Despite assertions that described P. marinus ultrastructural dinoflagellates share common antibody binding epitopes. characteristics merely limit its taxonomic assignment to a di- verse alveolate protozoan clade, and the suggestion that P. mar- DISCUSSION inus is a dinoflagellate, per se (Siddall et al. 1997), unique di- Binding of anti-P. marinus antibodies to the majority of test- nokaryon nuclear morphology common in all parasitic and free- ed dinoflagellate parasites of crustacea and fish collected world- living dinoflagellates tested during this study, are absent in P. wide, indicates a widespread presence of shared epitopes con- marinus. Unlike the dinoflagellates, and despite numerous ob- sistent with phylogenetic affinities suggested by DNA nucleo- servations of proliferating cell populations, condensed chro- tide sequence homologies among members of these different mosomes are never reported in ultrastructural or histological parasitic taxa. Nearly identical reciprocal labeling of epitopes studies of Perkinsus species nuclei (Sunila, Hamilton, and Dun- on P. marinus and the same seven parasitic dinoflagellates, by gan 2001), indicating that chromosome condensation during antibodies to the Norway lobster Hematodinium sp. parasite, Perkinsus species cell division is either absent or highly tran- confirms epitope sharing between these groups as the basis for sient. If demonstration of common antibody-binding epitopes serological cross-reactions. Several phototrophic dinoflagellates supports the hypothesis that P. marinus evolved from a com- were also labeled by anti-P. marinus antibodies, indicating that mon basal lineage shared by dinoflagellates, and particularly by P. marinus-like epitopes also occur among some free-living di- parasitic dinoflagellates, then its complete lack of dinokaryon noflagellates, as well. Together, these results provide strong nuclear structures clearly differentiates P. marinus from most phenotypic evidence supporting a proposed phylogenetic link dinoflagellates. between these taxonomically diverse protists (Siddall et al. By its consistent failure to label with antibodies to either P. 1997). marinus or the Norway lobster Hematodinium sp. parasite, the Although the chemical identities of shared epitopes were not dinoflagellate-like parasite of northeast Pacific pandalid shrimp investigated during this study, it is possible that one or more Pandalus borealis and P. platyceros was clearly differentiated represent conserved phenotypic elements common to all im- serologically from both P. marinus and the universally immu-

Table 2. Labeling of dinoflagellate, and dinoflagellate-like parasites of fish and crustaceans by anti-Perkinsus marinus (+, -) and anti- Hematodiriiaim sp. (+, -) antibodies.

Antibody binding site Parasite (host) Sample code Antibody binding Wall Cytoplasm Nucleus Perkinsus maririus SMCC- 18- 14 + + + + + + + + (Crassostrea virginicn) Hematodinirim sp. 990427 Nnor-1" + + + + + + (Nephrops norvegicus) Hematodinium sp. 98-5 13-lb + + + + + + + + (Chionoecetes bnirdi) Hematodiniuni sp. CS 2809' + + ~ + + + + (Callinectes sapidus) Hematodinium australis 205d + + + + + + (Portunu.r pelagicus) Hematodinium sp. 475e + + + + (Necorn puber) Hemntudinium sp. OC819T019L + + + - + + (garnnaaridean amphipod) Amyloodinium occelatum 990409 Rdrum-2b' + + + + + + (Sciaenops occelatus) dinoflagellate-like sp. 6668-38 (Pandalus platvcerus) 90-5685 LMIh

~ ~~ ~ ~ a G. Stentiford, DEEB, University of Glasgow, Glasgow, Scotland, UK. T. Meyers, ADF&G, Juneau, AK, USA. . 'G. Messick, NOAA/NOS, Oxford, MD, USA. J. Shields, VTMS, Gloucester Point, VA, USA. G. Wilhelrn, IFREZMER, Lorient, Brittany, France. C. Harris, Texas A&M University, College Station, TX, USA. p S. Bower, DFO Pacific Biological Station, Nanaimo, BC, Canada. BUSHEK, DUNCAN & LEWITUS-P. MARINUSlDINOPHYCEAE SHARED EPITOPES 15

Fig. 3. Perkinsus marinus trophozoite. labeled by anti-P. marinus antibodies in histological section from infected Crassosrrea virginica. Fluorescent parasite cell phagocytized by host hemocyte, inside oyster mantle vein. Parasite nucleus (arrowhead) and cytoplasm are labeled. H. hemocyte nucleus; v, parasite vacuole. Bar = 5 pm. Fig. 4. Perkinsus marinus trophozoite labeled by anti-Hemntodinium sp. antibodies in histological section from infected Crnssostrea virginica. Fluorescent parasite cell in oyster visceral connective tissue. Non-nucleolar parasite nucleoplasm (arrowhead) and cytoplasm are labeled. H, hemocyte nucleus; v, parasite vacuole; Bar = 5 pm. Fig. 5. Hematodinium sp. labeled by anti-Perkinsus marinus antibodies in histological section from infected Nephrops non:egicus. Isolated parasite cell in lobster gill. Parasite nucleus (N) is not labeled, but cytoplasm and cell wall are. Bar = 5 pm. Fig. 6. Hematodinium sp. labeled by anti-Heinutodiiiiurn sp. antibodies in histological section from infected Neyhi-ops norvegiczrs. Isolated parasite cell in lobster muscle. Parasite nucleus (N) is not labeled, but cytoplasm and cell wall are. Bar = 5 pm. 16 J. EUJSARYOT. MICROBIOL., VOL. 49, NO. 1, JANUARY-FEBRUARY 2002 nopositive parasitic syndinean dinoflagellates infecting other Field, R. H., Chapman, C. J., Taylor, A. C., Neil, D. M. & Vickerman, crustacean hosts. These results challenge previous descriptions K. 1992. Infection of the Norway lobster Nephrops norvegicus by a of this prawn parasite as dinoflagellate-like (Meyers, Lightner, Henzatudinium-like species of dinoflagellate on the west coast of Scotland. Dis. Aquat. Org., 13:l-15. and Redman 1994), and Hematodiniurn-like (Bower, Mc- Fong, D., Rodriguez, R., Koo, K., Sun, J., Sogin, M. L., Bushek, D., Gladdery, and Price 1994), but provide no guidance on its true Littlewood, D. T. J. & Ford. S. E. 1993. Small subunit ribosomal taxonomic affinities. RNA gene sequence of the oyster parasite Perkinsus marinus. Mul. These findings confirm the presence of shared antibody-bind- Mar. Biol. Biotechnol., 2:346-350. ing epitopes on cells of the apicomplexan oyster pathogen, P. Goggin, C. L. & Barker, S. C. 1993. Phylogenetic position of the genus marinus, and several dinoflagellates. The phenomenon was ob- Perkinsus (Protista, Apicomplexa) based on small subunit ribosomal served especially, but not exclusively, among the heterotrophic, RNA. Mol. Biochem. Parasitol., 60:65-70. parasitic Syndinidae. These results provide phenotypic evidence Goggin, C. L., McGladdery, S. E., Whyte, S. K. & Cawthorn, R. J. supporting recent molecular genetic nucleotide sequence data 1996. An assessment of lesions in bay scallops Argopecten irradians attributed to Perkinsus karlssoni (Protozoa, Apicomplexa). Dis. suggesting phylogenetic affinities between Perkinsus species Aquut. Org., 24:77-80. mollusc pathogens and dinoflagellates. Identification of this Hudson, D. A. & Shields, J. D. 1994. Hematodinium australis n. sp., a cross-reactivity by antibodies previously determined to react parasitic dinoflagellate of the sand crab Portunus pelagicus from only with members of the genus Perkinsus, cautions diagnos- Moreton Bay, Australia. Dis. Aquut. Org., 19:109-119. ticians to employ independent confirmatory or differential cri- Johnson, P. T. 1986. Parasites of benthic amphipods: dinoflagellates teria, such as nuclear morphology, photosynthetic pigment con- (Duboscquodinida: Syndinidae). Fisheiy Bull., 84:605614. tents, and cell Ornamentation, when interpreting results of im- Levine, N. D. 1978. Perkinsus gen. n. and other new taxa in the pro- tozoan phylum Apicomplexa. f. Purasitol., 64:549. munoassays using these or similar reagents for differentiation Mackin, J. G. H., Owen, M. & Collier, A. 1950. Preliminary note on of organisms in these groups. the occurrence of a new protozoan parasite, Dermocystidium marin- urn n. sp. in Crassostrea uirginicu (Grnelin), Science, 111:328-329. ACKNOWLEDGMENTS Maeno, Y., Yoshinaga, T. & Nakajima, K. 1999. Occurrence of Per- This research was cooperatively funded, in part, by the South kinsus species (Protozoa, Apicomplexa) from Manila clam Tapes phi- Carolina Sea Grant Consortium (PM-Zg) and the Maryland Sea lippinurunt in Japan. Fish Pnthol., 34: 127-13 1. Grant College Program (lUP-107-PD). We thank J. Cardinal Messick, G. A. 1994. Hematodinium pereri infections in adult and ju- and R. Hamilton for their fastidious execution and analysis of venile blue crabs Callinectes supidus from coastal bays of Maryland and Virginia, USA. Dis. Aquat. Org., 19:77-82. immunoassays. Parasitic and free-living dinoflagellate samples Meyers, T. R., Lightner, D. V. & Redman, R. M. 1994. A dinoflagellate- were kindly provided by P. Appleton, S. Bower, G. Doucette, like parasite in Alaskan spot shrimp Pandalus plaryceros and pink C. Harris, G. Messick, T. Meyers, J. Shields, P. Tester, F. Van shrimp P. borealis. Dis. Aquar. Org., 18:71-76. Dolah, and G. Wilhelm. G. Stentiford generously provided both Meyers, T. R., Koeneman, T. M., Botelho, C. & Short, S. 1987. Bitter tissues from Hernatodiniurn sp.-infected Norway lobsters, and crab disease: a fatal dinoflagellate infection and marketing problem Field & Appleton (I 996) rabbit antibodies to its Hemntodiniurn for Alaskan Tanner crabs Chionoecetes bairdi. Dis. Aquut. Org., 3: sp. parasite. Harry Jordan, Jordan Photography, generously dig- 1 95-2 16. itized and converted color photomicrograph prints of Fig. 1 and Newman, M. W. & Johnson, C. A. 1975. A disease of blue crabs (Cal- linectes .sapidus) caused by a parasitic dinoflagellate, Hematodiniurn 2 to gray scale images. This is Contribution No. 1296 of the sp. J. Parusitol., 61554-557. Belle W. 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