Effects of the biomedical bleeding process on the behavior and physiology of the American horseshoe crab, Limulus polyphemus

Horseshoe crabs are harvested by the biomedical industry in order to create Limulus Amebocyte Lysate (LAL) to test medical devices, vaccines and pharmaceutical drugs for pathogenic gram-negative bacteria. Previous studies of the impacts of the biomedical bleeding process on horseshoe crabs have primarily focused on mortality rates and sublethal impacts, using animals held in the laboratory. Therefore, the first goal of this project was to determine the effects of the bleeding process on horseshoe crab behavior once they are released back into their natural environment. In addition, previous studies have typically only investigated the impacts of the full bleeding procedure, or just hemolymph extraction, on horseshoe crab mortality and behavior. Therefore, my second objective was to determine the relative impacts of the three main stressors (aerial exposure, increased temperatures, and blood loss) on the locomotor activity and hemocyanin levels of horseshoe crabs. Finally, previous studies have demonstrated that horseshoe crabs held in captivity, even if they are not bled, experience a decline in hemocyanin levels. Therefore, the third objective of this study was to test a food supplement that might reduce these sustained decreases in hemocyanin levels, along with the associated behavioral impacts. We found that once horseshoe crabs were released back into their natural habitat there were some immediate differences between bled animals and controls. Bled animals appeared to mate significantly less than control animals within the first week post-release, with the largest differences between bled and control females. However, the only other significant difference we observed between 14 bled and 14 control animals was a tendency for bled animals to remain significantly deeper during the two-year study. Our laboratory studies revealed that the full bleeding procedure typically used commercially (i.e., all three stressors) had the largest impact on mortality, hemocyanin levels, overall activity, and expression of biological rhythms, followed by bleeding along with at least one of the other stressors. We also saw a seasonal trend in hemocyanin levels and a strong, significant relationship between hemocyanin levels and overall activity. Our data also revealed that animals with starting hemocyanin levels of 0.13 mg/mL or less were more likely to die or be impaired by the bleeding process. Therefore, it appears that an awareness of the overall health and hemocyanin levels in animals captured for bleeding might help reduce mortalities and other impacts if companies would avoid using these borderline animals. Another approach might be to use a food supplement to prevent sustained reductions in hemocyanin levels. Our last study showed that animals that were fed had an increase in their hemocyanin levels and overall activity, and maintained the same biological rhythms they had prior to being bled. Therefore, providing dietary supplements to horseshoe crabs either before or after bleeding them might be a logistically realistic way to improve physiological status and maintain a healthy population of this important species. In summary, we saw negative impacts from the biomedical bleeding procedure on the behavior and mortality of horseshoe crabs both in the laboratory in the field. Our data should provide insight into ways that biomedical facilities can modify the process in order to alleviate these issues. Moreover, these data indicate that one approach would be to develop a food supplement that would help animals maintain healthy hemocyanin levels. Given the importance of horseshoe crabs to coastal and estuarine ecosystems, and human health, it is crucial to determine a sustainable practice for bleeding these animals to create LAL