Abstract

In order to develop tools for monitoring heath of American Lobsters, we developed assays to evaluate lobster immune functions and validated them in field studies (an EPA study on contamination of lobsters around dredge material dumpsites and an UConn study monitoring LIS lobster health). Hemolymph of lobsters was aspirated and mixed with acid citrate dextrose as Hemocytes were counted with a hemocytometer. Phagocytosis of hemocytes was determined by flow cytometry. A cell culture system maintaining lobster hemocytes in vitro was established. Our field study results showed that although hemolymph of lobsters varied from gray to greenish or orange, there was no statistical difference between the different locations. Phagocytosis o lobsters from one location was statistically higher than that from any other locations, and interestingly, hemocyte counts of lobsters from this location were significantly lower than 4 out of the 7 other groups. Even though phagocytic index using two different parameters correlated very well (r=0.82), there was no correlation between phagocytosis, hemocyte counts, and hemolymph appearance. Our lab work demonstrated that sea water with 10% FCS at 12°C represented the best in vitro cell culture conditions for lobster hemocytes. Cells adhered to the surface of the 24 well plate and adapted amoeboid shape. Some cells were elongated or fusiform and had prominent filamentous or round pseudopodium. According to cell size and cytoplasm granule, the cells might be differentiated into at least 2 types: hyaline hemocytes and granulocytes. Live cells were found for as long as 48 hours of incubation. Results of analytical toxicology and histophathology will allow the final validation of our field results on the significance of immune function assays. The correlation between immunology and toxicology will provide useful information for the evaluation of lobster health. Our future work will focus on developing other immune function assays such as respiratory burst, NK cell-like activity, hemocyte proliferation, and apoptosis, in addition to experimental immunotoxicology. We expect that those studies will be helpful in understanding the health status of lobsters and identifying the cause(s) and contributing factors involved in the recent LIS lobster die off.

Acknowledgements

This work was supported in part by grants from the Connecticut Department of Environment Protection and US Environmental Protection Agency. The authors wish to thank the Environmental Protection Agency for providing samples through ENSR.