Meeting Abstract

P3.47  Sunday, Jan. 6  Response of the Giant Keyhole Limpet to Major Blood Loss MARTIN, G.G.*; KELLY, T.J.; SAFRAN, R.; Occidental College, Los Angeles; Occidental College, Los Angeles; Occidental College, Los Angeles gmartin@oxy.edu

Stellar Biotechnologies Inc. routinely bleeds limpets Megathura crenulata in order to extract and purify keyhole limpet hemocyanin (KLH), the respiratory pigment of the limpet. KLH is a molecule used in studies in vertebrate immunology, specifically in the treatment of cancers and allergies. One half of the limpet’s body weight is blood and approximately 50% of that may be withdrawn as a non-lethal bleed. This sets up an unusual situation for an animal that uses blood as its hydrostatic skeleton. Before and after a bleed the osmolarity of the blood and the concentration of hemocytes and KLH remain constant the total volume of blood is decreased. This prompts the question: when will body weight and blood volume return to pre-bleed levels? In our closed aquaria, this is a slow process. Weight increases slowly over several weeks presumably through an influx of seawater. This dilution of the blood should trigger production of hemocytes and KLH. We determined that there was an increase in hemocyte cell division 48 hours post bleeding followed by a return to pre-bleed level by the end of the first week. KLH levels showed no change. However, without a significant increase in body weight should there be a trigger to stimulate KLH synthesis? With the loss of so much oxygen carrying capacity, does the limpet need to resort to anaerobic metabolism? We are testing for indicators of this shift, such as succinate. We have also developed a procedure to bleed a limpet and then replace the lost volume with culture medium. This drops the THC and KLH levels to half their initial concentrations, yet maintains the pre-bleed hemolymph osmolarity. In preliminary trials, limpets have survived and were able to maintain their pre-bleed body weights. We will report on the response of hemocyte division and KLH synthesis to this novel transfusion.