NOTE Berkson and Hurton Potential causes of mortality for Limulus polyphemus 



297 



transport and holding methods on horseshoe crab mor- 

 tality. Of the various protocols followed in the bleeding 

 process, we simulated an example of transport, holding, 

 and bleeding methods that provided poor conditions for 

 the horseshoe crabs and that resulted in significant 

 mortality rates. In the typical biomedical bleeding pro- 

 cess, horseshoe crabs undergo time on a boat deck or in 

 collection bins, transport, storage in a coldroom, bleed- 

 ing, holding time, and transport back to the ocean. At 

 each of these stages of processing, the animals are ex- 

 posed to a number of stressors of varying magnitudes, 

 including exposure to air for extended time periods, 

 elevated temperatures, dehydration, hypovolemia, and 

 likely other unknown stressors. However, these factors 

 can be controlled to a certain extent by providing condi- 

 tions for the horseshoe crabs that could alleviate much 

 of the stress. For example, holding horseshoe crabs in 

 an air-conditioned environment (i.e., during time in the 

 truck and in the cold room), covering the animals with 

 wet burlap while outside to keep them moist and shaded 

 from the sun, and increasing the relative humidity of 

 the air in the holding room could help to decrease the 

 physiological stress the animals experience throughout 

 the bleeding process. Because blood volume estimates 

 have recently been determined (Hurton et al., 2005), 

 the blood volume extracted by biomedical companies po- 

 tentially could be optimized according to average stress 

 conditions and attempts could be made to decrease 

 stress as much as feasibly possible, including keeping 

 horseshoe crabs hydrated and bleeding a prescribed 

 amount from individuals, as opposed to withdrawing 

 a wide range of blood volumes. Another important con- 

 sideration is stress arising from harvest methods. Our 

 study did not test the effects of this factor, but it may 

 have an effect on the condition of horseshoe crabs col- 

 lected for bleeding. Biomedical companies should in- 

 clude the potential effects of harvest methods when 

 determining what may be typical stressful conditions 

 for the horseshoe crabs, or they could consider using a 

 harvesting method that would be the least stressful on 

 the animals. All LAL producers can potentially imple- 

 ment some form of these recommendations, thereby al- 

 tering bleeding process protocols to decrease the stress 

 and mortality levels that these horseshoe crabs experi- 

 ence. Implementation of these recommendations would 

 decrease the impact of the biomedical industry on the 

 potentially declining horseshoe crab population and aid 

 in conservation of this species. 



Acknowledgments 



This work is a result of research sponsored in part by 

 the NOAA Office of Sea Grant, U.S. Department of Com- 

 merce, under Grant no. NA96RG0025 to the Virginia 

 Graduate Marine Science Consortium and Virginia Sea 

 Grant College Program. Cambrex Bio Science Walkers- 

 ville. Inc. generously supplied us with horseshoe crabs 

 for the study. We wish to thank Stephen Smith of the 

 Virginia-Maryland College of Veterinary Medicine at 



Virginia Tech for his help in the planning of this study. 

 We also thank Eric Hallerman of the Department of 

 Fisheries and Wildlife Sciences of Virginia Tech for his 

 helpful comments throughout this project. In addition, 

 Xin Zhong and Li Wang of the Department of Statistics 

 at Virginia Polytechnic Institute and State University 

 were the statistical consultants for this project. We offer 

 a special thanks to numerous colleagues for their assis- 

 tance in transporting, tagging, and bleeding horseshoe 

 crabs for this study. We greatly appreciate the time and 

 effort of all involved. 



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