139 Abstract.–On the eastern seaboard of Mortality and hematology of blue crabs, the United States, populations of the blue crab, Callinectes sapidus, experi- Callinectes sapidus, experimentally infected ence recurring outbreaks of a parasitic dinoflagellate, Hematodinium perezi. with the parasitic dinoflagellate Epizootics fulminate in summer and autumn causing mortalities in high- Hematodinium perezi* salinity embayments and estuaries. In laboratory studies, we experimentally investigated host mortality due to the Jeffrey D. Shields disease, assessed differential hemato- Christopher M. Squyars logical changes in infected crabs, and Department of Environmental Sciences examined proliferation of the parasite. Virginia Institute of Marine Science Mature, overwintering, nonovigerous The College of William and Mary female crabs were injected with 103 or P.O. Box 1346, Gloucester Point, VA 23602, USA 105 cells of H. perezi. Mortalities began E-mail address (for J. D. Shields): [email protected] 14 d after infection, with a median time to death of 30.3 ±1.5 d (SE). Sub- sequent mortality rates were greater than 86% in infected crabs. A relative risk model indicated that infected crabs were seven to eight times more likely to Hematodinium perezi is a parasitic larger, riverine (“bayside”) fishery; die than controls and that decreases in total hemocyte densities covaried signif- dinoflagellate that proliferates in it appears most detrimental to the icantly with mortality. Hemocyte densi- the hemolymph of several crab spe- coastal (“seaside”) crab fisheries. ties declined precipitously (mean=48%) cies. In the blue crab, Callinectes Outbreaks of infestation by Hema- within 3 d of infection and exhibited sapidus, H. perezi is highly patho- todinium spp. have caused concerns differential changes in subpopulations genic and usually kills the host. to several major crustacean fisher- of granulocytes and hyalinocytes that lasted throughout the course of the The main symptom of the infection ies. Significant population declines infection. Crabs that did not present is lethargy. Heavy infections are and economic losses have been infections after injection (i.e. “immune” characterized by discolored (brown, reported for the Tanner (Chion- hosts) did not show hemocytopenia and yellow, milky or chalky) hemolymph oecetes bairdi) and snow (C. opilio) exhibited significant long-term (21–27 d) that does not clot. The disease oc- crab fisheries of Alaska and New- granulocytemia. Detection of the para- site in the hemolymph of infected crabs curs in blue crabs in high-salinity foundland (Meyers et al., 1987, increased from approximately 30% after (>11‰) waters from Delaware to 1990; Taylor and Khan, 1995),3 the 14 d to 60% after 21 d to 100% after Florida, and in the Gulf of Mexico Norway lobster (Nephrops norveg- 35 d. Plasmodial stages were, however, (Newman and Johnson, 1975; Mes- icus) fishery of western Scotland detectable in histological preparations sick and Sinderman, 1992). In 1975, (Field et al., 1992), and the velvet of the heart within 3 days of infection and increased in number over 5 and Newman and Johnson (1975) re- crab (Necora puber) fishery of west- 7 days. Sporulation of the parasite ported a prevalence of 30% in blue ern France (Wilhelm and Miahle, occurred over a short time (at least 4 crabs from Florida; the effect of this 1996). The parasite causes a condi- d, after 43 d of infection) and did not disease on the blue crab population culminate in the immediate death of was thought to be high. the host. Hematodinium perezi repre- sents a significant threat to the blue In 1991 and 1992, prevalences of * Contribution 2241 from the Virginia Insti- crab fisheries in high-salinity estuar- infection up to 100% were found in tute of Marine Science, The College of ies. Although the parasite infects male blue crabs (mean prevalence=43%, William and Mary, Gloucester Point, VA and female crabs, it may have a greater several locations from 70% to 100%, 23602. 1 Rux, S. 1993. Red Bank Seafood Co., impact on mature females as they move n=971) from coastal bays in Mary- to higher salinities to breed. Box 37 Marionville, VA 23408. Personal land and Virginia (Messick, 1994). commun.; Oesterling, M. 1993. VASG, Commercial watermen reported re- Virginia Inst. Marine Science, Gloucester Point, VA 23062. Personal commun. duced catches, lethargic and mor- 2 Shields, J. D. 1997. An investigation into ibund crabs in pots and shedding the epizootiology of Hematodinium perezi, facilities, and crabs that died soon a parasitic dinoflagellate in the blue crab, after capture (Rux, Oesterling1). In Callinectes sapidus. Saltonstall-Kennedy Program, National Marine Fisheries Ser- 1996 and 1997, 10% to 40% of adult vice, NOAA. Final Report. crabs from the eastern portions of 3 Prevalences in Newfoundland are now at Chesapeake Bay in Virginia were 1–15% in the northern bays. Taylor, D. 2 1998. DFO, CP 5567, White Hills, St. Manuscript accepted 23 August 1999. infected. The disease has a low Johns, Newfoundland, Canada, A1C 5X1. Fish. Bull. 98:139–152 (2000). prevalence or does not occur in the Personal commun. 140 Fishery Bulletin 98(1) tion known as bitter crab disease in snow and Tanner mm carapace width including epibranchial spines) crabs (Meyers et al., 1987). Low prevalences (1–4%) were used in the experiments. Females were used of another species, H. australis, have been reported to limit the number of treatment effects (e.g. poten- in sand (Portunus pelagicus) and mud (Scylla ser- tial differences between sexes) and to improve sample rata) crabs from Australia (Shields, 1992; Hudson sizes given the laborious nature of the experiments. and Shields, 1994). Hematodinium perezi was maintained in the lab- Infections of Hematodinium spp. or Hematodin- oratory by serial passage of infected hemolymph. ium-like species have been reported from a variety of Hemolymph from naturally infected crabs was in- different hosts (see Shields, 1994, for review). There jected directly into uninfected crabs. Naïve (unex- are, however, only two described species of Hemato- posed) crabs and crabs used for inoculation experi- dinium: H. perezi Chatton and Poisson, 1931, and H. ments were obtained from low-salinity non-enzootic australis Hudson and Shields, 1994. By convention locations. Infected and inoculated crabs were housed (Newman and Johnson, 1975; MacLean and Rud- separately and used as hemolymph donors to inject dell, 1978) and from its distinct morphological fea- naïve hosts (105–106 parasites per host). Injections tures, we concur that Hematodinium perezi is the were given in the arthrodial membrane of the fifth infectious species in the American blue crab. leg at the juncture of the basis with the cara- Blue crabs sustain one of the largest fisheries in pace. We have maintained H. perezi for over seven Chesapeake Bay. Current management plans and months using this method with no apparent loss from state regulations are based on population assess- pathogenicity. ments that include numbers of juvenile and adult Two mortality experiments and one early life his- crabs found during winter, spring, and summer sur- tory experiment were undertaken. The mortality-I veys (Lipcius and Van Engel, 1990; Abbe and Stagg, experiment used raw, infected hemolymph as the inoc- 1996; Rugolo et al., 1998). Although these projections ulant. Although appropriate for maintaining infec- include estimates of natural mortalities, they do tions in the laboratory, raw hemolymph cannot be not account for the potential epizootics and mortali- adjusted to manipulate parasite densities without ties caused by Hematodinium perezi. In this study, the use of physiological buffers, nor can it be guar- we examined host mortality in controlled laboratory anteed as sterile without appropriate assessment experiments and documented changes in the hemo- (see Welsh and Sizemore, 1985). Preliminary experi- grams (total cell counts, and differential counts) of ments with sterile sea water, physiological buffers, inoculated crabs versus uninfected crabs. We also and infected hemolymph indicated that buffer-washed examined proliferative growth of H. perezi at approx- parasites remained infectious, and could, therefore, imately weekly intervals and made observations on be adjusted to consistent densities appropriate to the biology and life history of the parasite. controlled experiments. The mortality-II experiment used buffer-washed parasites adjusted to a density similar to that used in the mortality-I experiment. Materials and methods Mortality-II experiment closely resembled mortality-I experiment except for 1) handling (buffer washes with Blue crabs were collected from Chesapeake Bay and centrifugation) and 2) the use of plasmodial versus several of its subestuaries during the annual VIMS uninucleate stages of the parasite. Uninfected crabs Winter Dredge Survey (part of the Chesapeake Bay served as controls in both experiments. Controls were Stock Assessment Program) with a 1.83-m-wide Vir- used to assess handling effects and to establish base- ginia crab dredge fitted with 0.5-inch (1.25-cm) Vexar line densities of hemocytes. The early infection exper- mesh dragged on the bottom for one minute at three iment was designed to examine the effects of early knots. Crabs were also taken with commercial crab infections on the hematology of the host and the pots from two reference locations on the Delmarva early life history of the parasite. Experimental den- Peninsula, Red Bank and Hungars Creeks, Virginia. sities in the early infection experiment were four Uninfected crabs were housed together for three to times higher than those in the previous experiments seven days prior to treatment to ensure acclimation (4.1 × 105 vs. approx. 1.0 × 105 parasites/crab, respec- and absence of overt bacterial or protozoal diseases tively) and were arbitrarily higher to insure obser- (as assayed below). During the experiments, crabs vation of parasites prior to their proliferation. were fed fish and squid semiweekly and held individ- In the mortality-I and mortality-II experiments ually in aquaria (5 gal., 19 liter) at 20° to 21°C, and 24 different proportions of trophonts and plasmodia ppt salinity.