Does Inhibition of Hemolymph Clotting Affect Circulating Immune Cell Response to Acute Bacterial Exposure in the American Lobster?

Author: Cici Montalvo
Major: Biology
Approved: Spring 2021
Status: In progress

The American lobster (Homarus americanus) has an open circulatory system compared to a closed one. They are then able to mount a rapid and effective immune response to an acute bacterial challenge, which can be seen with Vibrio campbellii, a marine bacterium that H. americanus is exposed to in natural conditions. Lobster vascular fluid, hemolymph, is capable of clotting rapidly at a wound site. Previous work has also shown that hemocytes (phagocytic cells) extend projections called filopodia when they encounter a foreign material in the hemolymph, and they migrate towards each other to form nodules as they engulf this material and remove it. Therefore, I hypothesize that this is the part of the regular clotting process and that it is essential to the clearance of foreign material in the hemolymph. The main goal behind my project is to investigate whether inhibition of the clotting process will affect the response of hemocytes to the presence of foreign material by using an anti-coagulant, glycine methyl ester (GME), with hemolymph exposed to V. campbellii that have been bioengineered to fluoresce so they can be visualized with a fluorescence microscope. This will be done by setting up four different conditions in which hemocyte filopodial extension is measured over time in vitro. These conditions include hemolymph, hemolymph infected with V. campbellii, hemolymph with GME, and hemolymph with bacteria and GME. Hemolymph will be drawn from a live, unaffected animal and placed on a microscope slide to then be challenged with bacteria. Hemocyte levels will be both monitored and recorded. The filopodial extensions will be observed using fluorescence microscopy, and possibly confocal microscopy. Since GME is an anti-coagulant and H. americanus gets rid of bacteria through clotting, I am interested in seeing how this affects hemocyte structure/function and the rate at which this process occurs.