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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 | [email protected]
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 104 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.
Results
Experimental Design
A) Incidence of bacteremia and level of infection in Dungeness crabs during summer 2010
A) Culturing methods to isolate and enumerate bacteria from samples
Sample:
•Hemolymph
•Carapace
•Water
Count colonies
Streak plates from
(double layer plates) individual colonies
B) Molecular methods to identify isolates
Figure 1. Distribution of
infection levels in CFU/mL
of hemolymph.
25 crabs were sampled
each trip. Colonies were
counted from double layer
plates.
Grow isolates in
broth cultures
Extract DNA for
molecular studies
Table 1. Incidence of bacteremia and infection level.
Crabs were collected 3 times during summer 2010. Hemolymph of 25
crabs were sampled each time.
B) Diversity of isolates
Amplify 16S ribosomal
Sequence 16S ribosomal
Ligate PCR product Grow single
gene by PCR
white colony in gene (OHSU), and do
into plasmid and
(using 27 fw. and 1492 rev. transform E.coli
LB broth
BLAST search (NCBI)
universal primers)
C) Challenge experiments
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.
C) Effect of bacteremia on the crab’s immune response
•Inject with buffered saline (control) or buffered saline
with isolated Psychrobacter or Bacillus YT0027
•(2.5 X 104 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).
68 to 84% of crabs sampled had bacteria in their hemolymph, indicating that bacteremia is
common among Dungeness crabs.
8 different species of bacteria were isolated and identified from the hemolymph and the
presence of an additional uncultured bacterium was shown. Molecular studies indicated that
the carapace was not the source of hemolymph bacteria.
Infection levels ranged from 0-28760 CFU/ml.
Injections of a non-lethal dose of Psychrobacter sp. and Bacillus YT0027 bacteria decreased
hemolymph cell activity in the crabs, indicating that bacteremia presents a chronic challenge
for the crabs’ immune system.
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.
Acknowledgements:
MJ Murdock Charitable Trust
Pacific University College of Arts and Sciences