Title page for ETD etd-12172003-122534

Type of Document Master's Thesis
Author Hurton, Lenka
Author's Email Address lhurton@vt.edu
URN etd-12172003-122534
Title Reducing post-bleeding mortality of horseshoe crabs (Limulus polyphemus) used in the biomedical industry
Degree Master of Science
Department Fisheries and Wildlife Sciences
Advisory Committee
Advisor Name Title
Berkson, James M. Committee Chair
Hallerman, Eric M. Committee Member
Smith, Stephen A. Committee Member
  • Limulus polyphemus
  • amebocyte
  • hemolymph
  • blood volume
  • mortality
  • horseshoe crab
Date of Defense 2003-12-03
Availability unrestricted

HORSESHOE CRABS (Limulus polyphemus)


Lenka Hurton


This study examined the effects of blood extraction on the survival of horseshoe crabs and performed a preliminary investigation into amebocyte maintenance in vitro. Hemolymph volume of L. polyphemus was estimated over a representative size range of adults. Hemolymph volume expressed as a percentage of wet body weight was 25 ± 2.2% (mean ± S.D.) for males and 25 ± 5.1% for females.

Mortality associated with blood extraction was evaluated for horseshoe crabs bled 0, 10, 20, 30, and 40% of their estimated hemolymph volume (unstressed group, N = 200). Mortality associated with the same bleeding levels was evaluated in horseshoe crabs that underwent simulated transport and handling procedures of the biomedical industry's bleeding process (stressed group, N = 195). Mortality rates of the unbled crabs were not significantly different between the stressed group and unstressed group. Of the bled animals, there was a higher (8.3%) mortality rate in the stressed group, than that (0%) in the unstressed group (P < 0.0001). Within the stressed group, mortality was significantly associated with bleeding (P = 0.0088).

Horseshoe crab serum and a variety of standard insect cell culture media were evaluated for their effects on amebocyte morphology and viability after 7 days of maintenance in vitro. Horseshoe crab serum-supplemented cultures had significantly higher cell viability than serum-free cultures (N = 6; P = 0.0147). Significant differences in amebocyte viability were identified among the six insect cell culture media tested (N = 36; P < 0.0001), with the highest amebocyte viability of 77.2 ± 5.1% (mean ± S.D.) in Grace's Insect Medium without serum.

Information gained from this study provides guidance on altering biomedical bleeding protocols to decrease horseshoe crab stress and mortality, and advances information on amebocyte culture medium selection, both of which contribute to decreasing the biomedical industry's impact on the horseshoe crab population.

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