BULLETINOFMARINESCIENCE,64(3): 543-548, 1999 BACTERIA ASSOCIATED WITH HEMOLYMPH IN THE CRAB CALLINECTES BOCOURTI IN PUERTO RICO A. Rivera, K. Santiago, J. Torres, M. P. Sastre and F. Fuentes Rivera Crabs of the genus Callinectes are commonly found in coastal, estuarine and lagoon waters of the Caribbean, the Gulf of Mexico, and the Atlantic and Pacific coasts of Central America (Williams, 1974; Norse, 1978). One of the most abun- dant and commercially important species is the blue crab C. sapidus. At least six species of Callinectes are naturally found in Puerto Rico (Buchanan and Stoner, 1988; Stoner and Buchanan, 1990). Bacteria associated with Callinectes spp. he- molymph is of public health significance due to the presence of potential human pathogens (Sizemore et aI., 1975). Hemolymph from sea hares and sipunculid worms has been shown to be sterile (Pauley et aI., 1971; Bang and Krassner, 1958). However, bacteria in hemolymph of invertebrate blood systems have been isolated from lobsters (Stewart et aI., 1966), dungeness crabs (Faghri et al., 1984) and blue crabs (Callinectes sapidus) (Sizemore et aI., 1975; Davis and Sizemore, 1982). Naturally occurring bacterial flora isolated from blue crab hemolymph include several potential human patho- gens such as Aeromonas sp., Pseudomonas sp., Flavobacterium sp., Bacillus sp., and Vibrio sp.; the latter being the predominant bacterial genus (Sizemore et al., 1975; Colwell et aI., 1975). Bacterial species V. cholerae, V. vulnificus and V. parahaemolyticus have been isolated from blue crab hemolymph (Davis and Siz- emore, 1982). Densities in excess of 105 bacteria ml-J have been reported in the hemolymph of blue crabs in Galveston Bay, Texas. Higher water temperatures apparently enhance bacterial proliferation in the hemolymph of live crabs (Davis and Sizemore, 1982). Literature describing the microflora of blue crabs is limited and, to our knowl- edge, no microbiological study has been performed in the hemolymph of Calli- nectes spp. living in the tropics. This study attempts to quantify total bacterial densities and identify species of bacteria in the hemolymph of C. bocourti in a high nutrient eutrophic system in Puerto Rico. MATERIALS AND METHODS STUDY AREA, Crabs and water quality data were gathered from a site in Mandry Channel, in the Department of Natural and Environmental Resources Reserve, Humacao, Puerto Rico. This channel is approximately 10 m wide, 3 km long and receives runoff mainly from rural and agricultural areas. Depth in the sampling site ranges from 1.5 to 1.7 m. Previous data from the same sampling site in Mandry Channel indicates lower average dissolved oxygen concentrations (2.5 mg L-I) and much higher nitrates (0.16 mg L~I),ammonia (7.50 mg L-I) and total organic carbon (14.21 mg L-I) levels as compared to coastal lagoons and estuaries in Puerto Rico. Acridine orange direct counts (AODC) averaged 4.70 x 109 bacteria ml-I (Fuentes Rivera, 1987). COLLECTIONOF CRABS. Crabs (c. bocourti) were collected using a dip net and by taking special care not to damage the carapaces or break the appendages. After each catch, crabs were rapidly placed in a clean stainless steel pail (one crab per pail) with water from the study site and taken alive into the laboratory within 30 min, The temperature of the water in the pail remained within :!: 5°C. BACTERIAEXTRACTIONANDPLATE COUNTS.A sample of six individuals were used for hemolymph extraction, Hemolymph was extracted from crabs within I h of collection by means of sterile syringes using a 37-mm 23-gauge needle (Thbiash et aI., 1975). Prior to extraction, 0.5 ml of sterile citrate! EDTA buffer (0.14 M NaCl; 0.1 M glucose; 30 mM trisodium citrate; 26 mM citric acid and 10 mM EDTA) was inserted into the syringe as an anti-coagulant (Sizemore et aI., 1975). The needle insertion point and the surrounding area of the crab's carapace was thoroughly cleansed with 70% ethanol. The 543 544 BULLETINOFMARINESCIENCE,VOL.64, NO.3,1999 needle was inserted either in the membrane between the first leg merus and carpus; or in the membrane between the abdomen and the intestinal region of the carapace, thus allowing the extraction of he- molymph directly from or near the heart. Special care was taken to extract pure hemolymph and not other fluids from the crab. Two milliliters of hemolymph were collected from each crab and rapidly placed (without storage) in a sterile container. A 0.1 ml hemolymph sub-sample was used for making serial dilutions (10-2 to 10-8) using a sterile 1% NaCI solution as the diluent. A specific volume of each dilution was filtered through a sterile, gridded 47-mm membrane filter with pore diameter of 0.45 /Lm.Each filter was then placed on Tryptic Soy Agar (TSA) medium (Difco Laboratories, Detroit, Michigan) and incubated at 25°C for 48 h. Following incubation, colonies were counted with the aid of a magnifying glass. All density estimates were made in duplicate. DIRECTCOUNT.Serial dilutions of 10-2 to 10-8 were prepared in four test tubes, each containing 9.9 ml of 1% NaCI solution prepared with autoc1avedand filtered water (0.45 /LmMillipore Type HA filter). The total number of bacteria in the hemolymph was determined by using a modification of the method described by Zimmermann et al. (1978). One milliliter of each 10-4 to 10-8 dilution was placed in a test tube containing 5 ml of sterile water. Two milliliters of acridine orange solution (1: 10,000 in 6.6 mM phosphate buffer, pH 6.7) were added to each test tube and after 2 min the solution was stirred. The solution was filtered through a O.4-mm Millipore-isopore filter stained with Sudan Black to create contrast. The filter was placed under a fluorescent microscope to estimate total count. Ten optical fields were randomly selected per sample and all bacteria in each field were counted. One serum sample was performed for each of six crabs. CULTURE.Undiluted hemolymph-EDTA samples were taken using a sterile cotton swab and inoc- ulated in Petri dishes containing Tryptic Soy Agar (TSA), Marine Agar or Blood Agar (all from Difco Laboratories, Detroit, Michigan). All inoculations were performed in triplicate. Samples were incu- bated at 25°C for 48 h. After incubation morphologically similar colonies were isolated in TSA. The following selective media were used: thiosulfate-citrate-bile salts-sucrose agar (TCBS), Shigella-Sal- monella, Pseudomonas agar and Strepto agar (all from Difco Laboratories). API 20-E and API NFl' (Analytap Products, Plainview, New York) were used for bacterial identification. The API identification system has been used for the identification of bacteria living in hemolymph of crabs by Faghri et al. (1984). Colonies which could be isolated and/or identified were counted and the relative percent of isolated colonies was calculated for each identified taxa. The percent of infected crabs also was calculated for each identified taxa. RESULTS Water column temperature taken during sample collection ranged from 28.5 to 30.0° C and averaged 29.2°C (n = 5), Water quality data from the same sampling site (Fuentes Rivera, 1987) suggests Mandry Channel is a tropical high nutrient eutrophic system. Acridine orange direct counts (AODC) of bacteria in the water column during sampling procedures averaged 4.71 X 1011bacteria ml-I (n = 2). AODC of bacteria in hemolymph of C. bocourti ranged from 3.53 X 109 to I 4.64 X 1011bacteria ml- , and averaged 8.89 X 1010bacteria ml-I (n = 6) (stan- dard error = 8.53 X 1010).Thirteen genera of bacteria were identified from cul- tures of C. bocourti hemolymph (Table 1). Within these genera 15 species were identified. Incidence of Haemophilus sp., Pseudomonas sp. and Salmonella sp. were the highest in C. bocourti hemolymph. The relative percent of isolated col- onies also was the highest for these species (Table 1). A null association model (Schluter, 1984) indicated no significant associations of bacteria species among the individual hosts (variance ratio = 1.2937; W = 7.7622; 0.1 < P < 0.5). Eleven gram positive (41%) and 16 gram-negative (59%) morphotypes were de- tected from unidentified colonies. Gram positive colonies were more abundant only in one of six crabs. DISCUSSION The average density of bacteria in the water column (4.71 X 1011bacteria ml-I) is two orders of magnitude higher as compared to results from the same sampling site (4.70 X 109 bacteria ml-I) reported by Fuentes Rivera (1987), High numbers of total bacteria and total coliforms are not unusual in high nutrient tropical NOTES 545 Table I. Relative percent of isolated colonies, percent of C. bocourti infected and specific hosts infected for each bacterial taxa. Relative % of % of crabs Hosts isolated infected infected Taxa in hemolymph colonies (n = 6) (A to F) Aeromonas hydrophyla 4.5 16.7 E Aeromonas salmonicida 2.3 16.7 C Bacillus sp. 6.8 33.3 E,F Hemophilus paracuniculus 4.5 33.3 B,E Hemophilus sp. 11.4 50.0 A,D,E Legionella sp. 2.3 16.7 E Pasteurella multocida 4.5 33.3 A,C Pasteurella sp. 2.3 16.7 A Proteus sp. 2.3 16.7 F Pseudomonas cepacia 6.8 33.3 B,C Pseudomonas mallei 2.3 16.7 A Psuedomonas putrefasciens 9.1 50.0 A,D,F Pseudomonas sp. 6.8 50.0 A,B,F Salmonella sp. 9.1 50.0 A,D,F Serratia rubidaea 2.3 16.7 F Shigella flexeri 2.3 16.7 A Vibrio cholerae 2.3 16.7 B Vibrio fisheri 2.3 16.7 A Vibrio fluvialis 2.3 16.7 E Vibrio metschnicovic 2.3 16.7 D Vibrio proteoliticus 2.3 16.7 E Yersinia pseudotuberculosis 2.3 16.7 B Zoogloea sp.