1. Immune response to acute bacterial exposure in the American lobster: Do the tissues play a role in clearing bacteria from the hemoylmph? Alyssa McManaway Advisor : Dr. Jorgensen http://www.123rf.com/photo_7980498_american-lobster-homarus- americanus-in-front-of-white-background.html

2. Content Introduction Materials and Methods Results Conclusions http://www.seatreasures.ch/1000_624_csupload_42499575.jpg? u=3648795608

3. The American Lobster Homarus americanus Lives along the Atlantic Coast of North America Migratory animals Commercially important Open circulatory system http://img.geocaching.com/cache/168507fc-7396- 402b-b9df-aa2260dee374.jpg?rnd=0.01596797

4. Comparative Circulatory System Information Lobsters have an open circulatory system as compared to other organisms As compared to the closed system of vertebrates Vertebrates have a microcirculation Our vascular system is completely confined to a tube network Compared to Lobsters-

5. Lobster Circulatory system Hemolymph is not confined only in the vessel network The hemolymph leaves the arterial network and bathes the tissue cells directly before being collected in the venous network Material exchanged happens across the tissue cell membranes The space outside the tube network is called the hemocoel, essentially the extracellular space The ventricle is contractive and pumps hemolymph through the circulatory system. Gills are the site of gas exchange, see all of cardiac output Gills are immune organs http://betournay.wikispaces.com/Cir culation+and+Gas+ExchangeA http://www2.gsu.edu/~bioasx/closeopen.html

6. Lobster Immune System Two part system – Circulating hemocytes – analogs of our white blood cells Fixed phagocytes

7. Circulating Hemocytes Circulating hemolymph cells are able to differentiate self from foreign When a pathogen is sensed hemocytes quickly mobilize to the site of infection Hemocytes adhere to the cell surface of the pathogen (Caiwen and Shields,2007)

8. Nodule Formation (Martin, et al. 1998) In vitro experiment Hemocytes become sticky and clump together to form nodules These nodules get stuck in the gills and perhaps other tissues?

9. Fixed Phagocytes Phagocytotic cells that are stationary on tissue membranes As hemolymph baths the tissues these cells phagocytose pathogenic particles Have been found in the Hepatopancreas/digestive glad Hepatopancreas is a digestive gland– functions as a liver and in digestion http://encyclopedia.lubopitko-bg.com Body_Defenses.html

10. My Research Question Do the tissues of the lobster play a part in bacterial clearance from the hemolymph? If so, which tissues are important?

11. Materials and Methods

12. Prep Lobster by drilling 0.5mm holes into the carapace until the endomembrane was visible using sterile techniques The day prior to the experiment GM Vibrio campbellii was plated and allowed to grow for 24 hours Resistant to two antibiotics – for pure cultures On the day of the experiment the bacteria was suspended in a saline solution until the number of bacteria was equal to 2x10 8 Colony forming units Allows for bacterial infection, but not a lethal dose

13. GM Vibrio campbellii Naturally occurring ocean bacterium Bio-engineered to be resistant to kanamycin and chloramphenicol Expresses GFP This allows us to track the bacteria in whole animals Free from the masking effects of other bacteria

14. Hemolymph Sampling Taken from the pericardial sinus around the heart Used as an indicator of successful bacterial exposure Gills Hegner and Engemann 1968 (from Barth and Broshears,1982)

15. Tissue Sampling After 50 minutes the lobster was sacrificed At 60 minutes the heart was removed to stop circulation The tissues were removed, homogenized in HEPES (volume was mass dependent) and a subset of the homogenate was plated Bacterial colonies were counted after 24 hours

16. Tissues Sampled Ruppert and Barnes, 1994 Herrick, 1909 (From Factor, 1995) Gills Hegner and Engemann 1968 (from Barth and Broshears,1982)

17. Results

18. Antennal Gland, Heart, and Hepatopancreas have highest number of culturable bacteria Determined significance using an ANOVA statistical test and post 2-sample T-tests

19. n=6 * * *

20. Discussion and Conclusions

21. Discussion The results of the hepatopancreas were consistent with previous research In vitro studies, with inert particles, not bacteria The numbers of culturable bacteria in the heart and the antennal gland were surprising Tiny mass – lots of bacteria Possible explanations Bacteria may not have been phagocytosed Nodules may become trapped in the tissues

22. Discussion See very little culturable bacteria in the gills Possible explanations Nodules in the gills are not culturable Gills lack fixed phagocytes (Martin, et al. 1998)

23. Future Studies Interested in other tissues – tail muscle Leg muscle, cheliped muscle Using the GM vibrio to observe bacterial cells in the organs Future research with hypoxia

24. Acknowledgements Dr. Jorgensen Professor Thibodeaux Roanoke College Biology Department and the Thornhill Endowment at Roanoke College Grice Marine Laboratory at the College of Charleston, SC Current Jorgensen Lab members animal maintenance and care Jorgensen Lab Seniors: Ben Shapiro, Cullen Truett, and Shana Hensley

25. Questions? http://image1.masterfile.com/em_w/04/26/46/400-04264640w.jpg

26. References Burgents, J.E., Burnett, L.E., Stabb, E.V., and K.G. Burnett. (2005). Localization and bacteriostasis of Vibrio introduced into the Pacific white shrimp, Litopanaeus vannamei. Developmental & Comparative Immunology 29: 681-691. Holman, J.D., Burnett, K.G., and L.E. Burnett. (2004). Effects of Hypercapnic Hypoxia on the clearance of Vibrio campbellii in the Atlantic Blue Crab, Callinectes sapidus Rathbun. Biological Bulletin 206:188-196. McKay, D. and C.R. Jenkin. (1970). IMMUNITY IN THE INVERTEBRATES. CORRELATION OF THE PHAGOCYTIC ACTIVITY OF HAEMOCYTES WITH RESISTANCE TO INFECTION IN THE CRAYFISH (PARCHARAPS BICARINATUS). Aust. J. exp. Biol. Med. Sci. 48: 609-617 McKay, D. and C.R. Jenkin. (1970). IMMUNITY IN THE INVERTEBRATES. THE FATE AND DISTRIBUTION OF BACTERIA IN NORMAL AND IMMUNISED CRAYFISH (PARACHAERAPS BICARINATUS). Aust. J. exp. Biol. Med. Sci. 48: 599-607 McKay, D. and C.R. Jenkin. (1969). Immunity in the Invertebrates II. ADAPTIVE IMMUNITY IN THE CRAYFISH (PARACHAERAPS BICARINATUS). Immunology 17:127-137. McKay, D., Jenkins, C.R., and D. Rowley. (1969). Immunity in the invertebrates 1. Studies on the naturally occurring haemagglutinins in the fluids from invertebrates. Aust. J. exp. Biol. Med. Sci. 47: 125- 134. Martin, G.G., Kay, J., Poole, D., and C. Poole. (1998). In vitro nodule formation in the ridgeback prawn, Sicyonia ingentis, and the American lobster, Homarus americanus. Invertebrate Biology 117(2): 155-168. Reade, P.C. (1965). PHAGOCYTOSIS IN INVERTEBRATES. Aust. J. exp. Biol. med. Sci. 46: 219-229.