1. Bacterial Infection in the Dungeness Crab, Cancer magister Sarah Dunn, Hannah Pramuk, David Scholnick and Györgyi Nyerges Pacific University, Department of Biology | 2043 College Way | Forest Grove, OR 97116 | nyerges@pacificu.edunyerges@pacificu.edu Acknowledgements:  MJ Murdock Charitable Trust  Pacific University College of Arts and Sciences Abstract The health of the marine environment is deteriorating, and coastal regions are showing increasing signs of pollution and bacteria. Our previous work showed that the Dungeness crab maintains low levels of a Psychrobacter strain in the hemolymph. In the current study we determined the presence of bacteria in the hemolymph from 75 crabs sampled during the summer months of 2010. The level of infection and the strain of bacteria present were identified. Bacteria were isolated from the majority of crabs sampled (68 to 84%). The infection level was assessed by plate counting method and varied between: 0-28760 CFU/ml. The identity of the isolates was determined from 16S rDNA sequence. Psychrobacter was the most prevalent strain, although we obtained 7 other isolates by traditional culturing methods (tryptic soy agar with 2.5% NaCl and marine agar), and the presence of an unculturable strain was shown by molecular methods. In order to test effects of bacteria in the hemolymph, crabs were injected with buffered saline (control) or buffered saline with isolated Psychrobacter or Bacillus YT0027 (2.5 X 10 4 g-1 body weight), hemolymph samples were taken before injection and 80 min afterward. Hemolymph cell activity was estimated by mitochondrial enzyme activity using Cell Proliferation Reagent WST-1. Total hemolymph cell activity decreased after injection of bacteria. These data demonstrate that the chronic presence of a variety of bacteria in the hemolymph is common in the Dungeness crab and may present a constant challenge for their immune system. Research Objectives  Sample Dungeness crabs throughout summer for bacteria in hemolymph and determine infection incidence and level.  Isolate and identify bacteria from hemolymph.  Study the crabs’ immune response when exposed to bacteria isolated from the hemolymph. Experimental Design Sample: Hemolymph Carapace Water Count colonies (double layer plates) Streak plates from individual colonies Grow isolates in broth cultures Extract DNA for molecular studies Amplify 16S ribosomal gene by PCR (using 27 fw. and 1492 rev. universal primers) Ligate PCR product into plasmid and transform E.coli Grow single white colony in LB broth Sequence 16S ribosomal gene (OHSU), and do BLAST search (NCBI) A) Culturing methods to isolate and enumerate bacteria from samples B) Molecular methods to identify isolates C) Challenge experiments Inject with buffered saline (control) or buffered saline with isolated Psychrobacter or Bacillus YT0027 (2.5 X 10 4 g-1 body weight) Sample hemolymph before and 80 minutes after challenge Asses total hemocyte activity by measuring mitochondrial enzyme activity, using Cell Proliferation Reagent WST-1. Change in mitochondrial activity results in change of absorbance (450 nm). Results Figure 3. Hemolymph cell activity before and after bacterial challenge, as a function of absorbance. Data are means of each treatment group (n=6) and asterisks indicate statistically significant difference. Figure 1. Distribution of infection levels in CFU/mL of hemolymph. 25 crabs were sampled each trip. Colonies were counted from double layer plates. Table 1. Incidence of bacteremia and infection level. Crabs were collected 3 times during summer 2010. Hemolymph of 25 crabs were sampled each time. A) Incidence of bacteremia and level of infection in Dungeness crabs during summer 2010 B) Diversity of isolates C) Effect of bacteremia on the crab’s immune response Figure 2. Phylogenetic tree of bacterial isolates from the hemolymph of Dungeness crabs. Tree was constructed with PhyML and TreeDyn programs based on partial 16S rDNA sequences (~800 bp) aligned using MUSCLE program.