MFR PAPER 1145 Gilbert B. Pauley was with the Pathobiology Investigations sec­ tion of the Middle Atlantic Coastal Fisheries Center, National Marine Fisheries Service, NOAA, Oxford, Seru m Changes in the Blue Crab, MD 21654. He is now with the Washington Cooperative Fishery Callinectes sapidus, Associated With Unit, College of Fisheries, Univer­ Paramoeba perniciosa, the Causative sity of Washington, Seattle, WA 98195. Martin W. Newman is with Agent of Gray Crab Disease the Pathobiology Investigations section of the Middle Atlantic Coastal Fisheries Center, National Marine Fisheries Service, NOAA, GILBERT B. PAULEY, MARTIN W. NEWMAN, and EDITH GOULD Oxford, MD 21654. Edith Gould is with the Experimental Biology In­ ABSTRACT-Hemolymph from healthyblue crabs (Callinectes sapidus) was vestigations section of the Middle Atlantic Coastal Fisheries Center, compared with that ofanimals infected with Paramoeba perniciosa, the causa­ National Marine Fisheries Service, tive agent of gray crab disease. There was a significant difference (P = 0.01) NOAA, Milford, CT 06460. between normal and gray crab sera in both the protein and the glucose values. Analysis of infected blue crab serum by both immunoelectrophoresis and Newman and Ward (1973). Hemolymph acrylamide gel electrophoresis showed altered patterns of total protein and from healthy crabs, placed in a test hemocyanin that could be accurately correlated with the severity of the dis­ tube, was allowed to clot around an ap­ ease. plicator stick after which the clot was easily removed. Because the hemo­ A previously unknown disease of the MATERIALS AND METHODS lymph of most crabs infected with P. commercially important blue crab perniciosa does not clot, it was centri­ The crabs, C. sapidlls, used in this (Callinectes sapidlls) which killed 20-30 fuged at 5,000 rpm for 10 min at 4°C, percent of the animals held in commer­ study were obtained from Chincoteague to remove cellular debris. The resultant Bay, near Greenbackville, Va. Crab cial shedding tanks was described by sera were passed through Swinnex-25 sampling began on 26 May 1972, with Sprague and Beckett (1966). When millipore filters (0.45 fJ pore size) into six subsequent samplings during June viewed ventrally, moribund and dead sterile tubes, divided into 1.0 ml lots, 1972. The sample size varied from 4 to specimens had a translucent gray ap­ dated, and frozen at -25°C. Sera from 26 animals, with a total of 84 animals pearance, hence the name "gray crab individual crabs were always kept sepa­ being examined. Control sera for im­ disease." Sprague and Beckett (1968) rate. munoelectrophoresis were obtained ascertained that the etiological agent of Two rabbits were used for each anti­ from uninfected Chesapeake Bay crabs, the disease was an amoeba, subse­ serum produced. Freund's complete ad­ taken near Oxford, Md. quently named Paramoeba perniciosa. juvant (Difco) was mixed with an equal The posterior portion of the carapace The amoebae cause lysis of muscle amount ofcrab serum and administered was swabbed with 70 percent ethanol and blood cells and may completely re­ weekly for 5 weeks as 1.0 ml subcutane­ before withdrawing hemolymph from place the normal blood cells in the ous and 2.0 ml intramuscular injections the pericardial cavity with a sterile 5.0 hemolymph prior to death of the crabs in the rabbits. Two weeks after the final or 10.0 ml syringe and 20-gauge J1I2-inch (Sprague et aI., 1969; Sawyer et aI., injection, antiserum was collected by needle. A drop of hemolymph from 1970). Epizootiological studies have cardiac puncture, filter-sterilized, and each animal was placed on a microscope shown the disease is ephemeral, occur­ frozen at -25°C until used. Antisera slide and spread evenly in a thin film. ring each summer in the latter part of were made against the sera from 13 in­ This was allowed to air dry for 1-5 min June, and has a restricted range from fected crabs from Chincoteague Bay and then placed in 10 percent neutral­ Maryland to North Carolina (Sawyer, and from two normal male and two buffered formalin seawater. Smears 1969; Newman and Ward, 1973). normal female crabs from Chesapeake prepared from gray-appearing crabs In another commercially important Bay. Immunoelectrophoresis and stain­ were fixed in separate containers to crustacean, Homarus americanus, it ing were carried out as described by avoid the possibility ofamoebae floating has been found that bacteria, Gaffkya Pauley (1974). offthe slide and contaminating adjacent homari, cause serious alterations in The total protein concentration in the hemolymph chemistry (Stewart et aI., slides. Hemolymph smears were serum of 66 crabs was analyzed by the stained for 10 min with Giemsa l stain 1969; Stewart and Cornick, 1972). F olin-Ciocalteau method (Lowry et aI., diluted 1:5 with phosphate buffer, pH Therefore, a study was undertaken to 1951). Serum glucose was determined 6.8, and examined for the presence ofP. determine whether the hemolymph for 60 crabs by the method of Hultman perniciosa. The slides were then scored from blue crabs infected with P. (1959). Because ofthe small sample size as normal, light, moderate, or heavy in­ perniciosa differed from that of normal of some groups and because they were fection, based on the relative number of crabs. This paper reports our findings not homogeneous by analysis of the amoebae present as described by on the changes observed in the serum variance using the F distribution (Sne­ glucose, serum protein, and serum elec­ decor, 1956), no statistical tests for I Reference 10 lrade names does nol imply en­ trophoretic patterns in infected C. dorsemenl by the National Marine Fisheries Ser­ significance were attempted for the var­ sapidus. vice, NOAA. ious groups other than between pooled 34 normal and pooled heavily-infected Table 1.-Concentratlons of total protein and glucose In normal and heavily parasitized blue crab serum. gray crab samples. The I-test for com­ Total protein (mg/mll Glucose (mg/l00 ml) paring the means of two randomized Sid. Std. Sid. Sid. groups of unequal size was used for No. Range Mean dey. error No. Range Mean dey. error statistical analysis ofthis data according Normal to the procedure outlined by Snedecor Males 11 25.0.67.0 45,4 15.1 4.5 11 10.5-66,4 34.7 18,4 5.5 Females 11 5.3·22,4 14.6 5.6 1.7 15 n~-74,4 24.4 18.6 4.8 (1956). Sponge 10 3.9-55,4 31.0 15.7 5.0 6 31.8·130.3 74,4 37.2 15.2 Aliquots (ca. 3 /.II) of serum from All 32 3.9-67.0 30.3 18.0 3.2 32 7.9-130.3 37.3 28.9 5.1 individual blue crabs were subjected to Gray Males 23 2.8·18.5 9,4 4.6 1.0 18 0.61.1 14.1 15.8 3.7 electrophoresis at 4°C on 7 percent ac­ Females 11 2.8·13.7 7,4 3.9 1.2 9 0-29.2 9,4 10,4 3.5 rylamide gel columns, pH 9. I, with All 34 2.8·18.5 8.8 4,4 0.8 28 0-61.1 11.1 14.1 2.7 sample and stacker gels of 3 percent acrylamide, pH 5.2. Electrode buffer 70- 1.30- was 0.005M Tris (hydroxymethyl 1.40- 60­ aminomethane) - 0.038 M Glycine, pH 1.10- 8.3. Running time was 60 min at I ~ 50- ~I.OO- , milliampere/column followed by 70-75 E'" E ;; 40- -;;. .90- min at 3 milliamperes/column with con­ ~ stant current. Both gel formulas and ~ .80- o 30- l)l 70- electrophoretic procedure are based on 8: O' g 60- the work of Davis (1964) and have been ...I 20- ...I' ;~' ~ ~ < ?;;f fully described by Gould and Medler ~ .50- 10- ~ (1970). o·40- Subsequent to electrophoresis, gels F- .:30- 0- .i were stained for total protein or for cop­ .20- ~ per. The stain used for total protein was j ::l , ! .. .10- $ L.... _ NORMAL GRAY Amido Schwartz 10 B (Buffalo Black), I 0- ... ... ...J I~ percent in 7.5 percent acetic acid. Gels ... ~ .. '"z ...J .. Figure 1.-Graphlc presentation 01 the range, .. ...::l! f ::l .. ...::l! ::l were destained by passive diffusion in standard deviation, standard errOf, and mean of ::l! IL II> .. ::l! IL .. several changes of methanol-glacial total serum protein concentration In normal and NORMAL GRAY heavily parasitized blue crabs. acetic acid-distilled water (5: I:5) for a total ofabout 20 h. Hemocyanin sites on Figure 2.-Graphlc presentation 01 the range, normal and the gray crabs. Serum con­ standard deviation, standard error, and mean of . the gels were marked by a stain for cop­ centrations ofprotein and glucose cou Id total serum glucose concentration In normal and heavily parasitized blue crabs. per using an aqueous tetrazolium­ often be correlated with the intensity of cyanide solution (Gould and Karolus, infection (Table 2). In press), that proved to be faster and guishable (Fig. 5). Although no heavily more consistently reliable than the IMMUNOELECTROPHORESIS infected female crabs were analyzed by classic rubeanic acid stain (Horn and The normal immunoelectrophoretic this method, because of a shortage of Kerr, 1969). patterns of male and female crabs were hemolymph, there was never any doubt about a correct diagnosis with this BIOCHEMICAL ANALYSIS different (Figs. 3, 4). Infection of C. sapidus by P. perniciosa caused a pro­ method when hemolymph was tested Concentrations of total protein and gressive loss of serum protein as ob­ from moderately or heavily infected glucose in the serum of heavily infected served by immunoelectrophoresis, male crabs.