Microscopic and metabolomic investigation of Hematodinium sp.\ninfections in Newfoundland snow crabs, Chionoecetes opilio

Bitter Crab Disease (BCD) is a fatal disease of crustaceans caused by parasitic syndinian\ndinoflagellates of the genus Hematodinium. Over forty species of crustaceans have been\nreported as hosts for Hematodinium or Hematodinium-like infections with a worldwide\ndistribution concentrated in North Pacific and Atlantic oceans. In eastern Canada BCD was first\nreported in snow crabs Chionoecetes opilio from Conception Bay, Newfoundland in 1990 with\nvery low disease prevalence (<0.11 – 3.7%). Subsequently, BCD has spread rapidly within the\neastern and northeastern bays of Newfoundland and Labrador and has resulted in at least three\nepizootic disease outbreaks with prevalence up to 25% in some subpopulations of snow crabs.\nHematodinium infections can be presumptively diagnosed macroscopically via visual\nexamination in many affected species, including snow crabs, due to characteristic discolorations\nof the carapace and/or arthrodial membranes. Specific detection of Hematodinium spp. via\nPolymerase Chain Reaction (PCR) assays is another diagnostic method used for the diagnosis of\ninfection in various crustacean hosts. In our study, 100% of mild infections (1/1), 40% of\ninfections with moderate infection intensity (2/5), 11-22% of advanced infections (1 or 2/9) had\nfalse negative test results while no very advanced infections (0/13) had false-negative test\nresults for diagnosis by visual exam or PCR assay. These findings suggest that diagnosis by visual\nexam or PCR assay is less accurate in the early stages of BCD and both diagnostic tests\nunderestimated the prevalence of Hematodinium infection in Atlantic Canadian snow crab by\n1.25 – 1.76%.\nLight microscopic examination of parasite life stages of Hematodinium sp. infections of varying\nintensity revealed changes in proportions of multinucleate plasmodia. In snow crabs with\ninfections of mild intensity (early BCD infections) the parasitic life stages had a high proportion\nof multinucleate plasmodia and had enhanced cytoplasmic basophilia, changes suggestive of\nrapid cellular proliferation. Moderately intense infections were predominated by uninucleate\ncells. In advanced and very advanced infection intensities (late BCD infections) there appeared\nto be a progressive increase in proportion of multinucleate life stages, suggestive of a\nresurgence of parasitic proliferation and/or impaired cytokinesis (possibly due to reduced host\nresources).\nThe uninucleate and multinucleate life stages were most often amoeboid in shape; vermiform\nplasmodial life stages were also observed, typically in heavily infected animals. The vermiform\nplasmodia were often intimately associated with cell surfaces, most commonly in the heart and\nalso rarely in the eyestalk, gill, and leg. Ultrastructural examination of these attached\nvermiform plasmodia revealed cytoplasmic extensions which interdigitated with host cell\nmembranes and which occasionally appeared to form connections between parasite cell\nbodies. Comparison of the in vivo and in vitro life stages of Hematodinium isolated from snow\ncrabs suggests that in vitro sheet-like and arachnoid plasmodia may represent exaggerations of\namoeboid plasmodia and attached vermiform plasmodial life stages observed in vivo. The rapid proliferation of the Hematodinium parasites in hemolymph and the parasites’ high\nmetabolic requirements during growth have been reported to decrease the hosts’ protein and\ncarbohydrate reserves and contribute to host morbidity and eventual mortality. Our research\nconcurs with these findings and indicates that BCD is associated with depletion of protein\n(reduction in hemolymph refractive index) and carbohydrate (reduced reserve inclusion scores)\nmetabolic reserves as well as potentially altering host lipid metabolism. Several BCD-associated\nphospholipids were identified in this study. These BCD-associated phospholipids could\ncontribute to the bitter taste associated with this disease. Additionally, BCD-associated\nphospholipids could contribute to the apparent lack of a cellular immune response against\nHematodinium.\nA histopathologic study of Newfoundland snow crabs confirmed that BCD was the most\nsignificant infectious cause of internal disease in the populations examined. Two patterns of\nnecrotizing and ulcerative enteritis were also observed in this study: focal ulcers at the level of\nthe midgut-hindgut junction and more extensive areas of enteritis in the non-chitinized midgut\nepithelium. The cause of these lesions is unclear; hypoxia/ischemia and/or infection with\npathogenic Vibrio bacteria are possible contributing factors in lesion development. There was\nno significant difference in the prevalence of hemocytic aggregates between Hematodinium\ninfected snow crabs and uninfected snow crabs suggesting that the hemocytic aggregates were\nbackground lesions unrelated to Hematodinium infection. No significant difference in the\nprevalence of intestinal inflammation (midgut-hindgut junctional ulcers or midgut necrotizing\nenteritis) or in the prevalence of hemocytic aggregates between BCD- and BCD+ snow crabs\nwas observed. When the data from both bays that contain cases of BCD were combined, higher\nprevalence of one pattern of inflammation (midgut-hindgut junctional ulcers) was seen in snow\ncrabs without BCD. There was also no significant difference in the prevalence of hemocytic\naggregates between Hematodinium-infected snow crabs and uninfected snow crabs, suggesting\nthat hemocytic aggregates observed in animals with BCD were background lesions unrelated to\nHematodinium infection.\nThis study confirms that BCD is associated with alterations in protein, glycogen, and lipid\nmetabolism consistent with parasite-associated negative energy balance (i.e., starvation) in\naffected snow crabs. Histologic examination of BCD infected snow crabs revealed vacuolar\ndegeneration in hepatopancreatic epithelium although there was no definitive evidence of\nparasite-associated inflammation or tissue necrosis. These findings indicate that the main\npathologic effects of BCD are metabolic in nature.

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