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2 Department of Biological Sciences Plymouth State University 17 High Street Plymouth, New Hampshire 03264 Abstract—When the blood of American horseshoe crabs (Limulus polyphemus) is extracted to create Limulus amebocyte lysate, the crabs are often subjected to warm temperatures and time out of water, in addition to blood loss. The goal of this study was to determine the effects of these 3 stressors on the locomotor activity and hemocyanin (HCY) levels of American horseshoe crabs. We found that a full bleeding treatment involving all 3 stressors had the biggest impact on mortality and HCY levels, followed by bleeding along with at least 1 other stressor. All of the treatments resulted in similar small changes in their overall activity and in the types of biological rhythms expressed. There was greater overall activity in animals that had higher HCY levels (P<0.0001). We also found that HCY levels were lowest in the spring and early summer and highest in the late summer and fall. In each month, males had higher HCY levels than females. These results indicate that low HCY concentrations can negatively affect the health of horseshoe crabs. If biomedical facilities take sex differences and seasonal fluctuations in HCY levels into account, they might be able to reduce some of the deleterious effects of the bleeding process.

Effects of the biomedical bleeding process on the behavior and hemocyanin levels of the American horseshoe crab (Limulus polyphemus)