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Fishery Bulletin 104(2) 



ment of the higher-stressed group, mortality rates were 

 not significantly different between unbled crabs in the 

 two bleeding experiments (?!=79; P=0.4937). Of the bled 

 crabs, there was a mortality rate of 0% in the lower- 

 stressed group and a rate of 8.3% in the higher-stressed 

 group, indicating a significant difference in mortality 

 rates between the two groups (n = 316; P<0.0001). 



Discussion 



According to our study of 395 horseshoe crabs, no impact 

 on mortality was observed with blood extraction up to 

 407f of the horseshoe crab's blood volume under condi- 

 tions of lower stressors. Any mortality associated with 

 this maximum level of bleeding (40% of total blood 

 volume) was so low that it was not captured by our 

 sample size of 40 individuals per bleeding treatment. 

 This result also may indicate that despite hypovole- 

 mia, or low blood volume, and altered blood chemistry 

 (Hurton, 2003), horseshoe crabs may be relatively tol- 

 erant of the removal of a large amount of blood in the 

 absence of other stressors. 



Horseshoe crab mortality, however, was significant 

 in the presence of higher levels of stress, including 

 external stressors (i.e.. lengthy air exposure, elevated 

 temperatures, etc.) as applied in our study. Novitsky's 

 (1991) aquarium studies reported that up to 30% of 

 blood volume can be safely extracted, indicating that 

 the animals in that study may have been similar to 

 our lower-stress group; however, details regarding har- 

 vest method, transport, and handling procedures were 

 not specified (Novitsky, 1991). It is likely that, under 

 different conditions, horseshoe crab response to blood 

 extraction may be altered. Findings from our study 

 indicated that a combination of external effects and 

 sublethal hemorrhaging may result in significantly in- 

 creased mortalities, possibly from a synergistic inter- 

 action between the two types of stressors. Studies in 

 other species exposed to various stressors have also 



indicated that mortality may be affected by a syner- 

 gistic combination of effects from multiple stressors 

 (Schisler et al., 2000; Schulz and Dabrowski, 2001; 

 Hatch and Blaustein, 2003). Such results emphasize 

 the importance of considering the cumulative impact 

 of multiple stressors. 



Although the data in our study indicated a signifi- 

 cantly increased mortality rate in the higher-stressed 

 group as opposed to the lower-stressed group, it is im- 

 portant to note the variability in mortality through- 

 out the treatments. It was observed that the male 

 and female horseshoe crabs had different and vari- 

 able responses in the different bleeding treatments. 

 For example, males had two and three mortalities in 

 the 20% and 30% bleeding treatments, respectively; 

 yet, only one mortality in the 40% treatment (Table 1). 

 Females appeared to have a spike in mortality in the 

 40% treatment (Table 1). When viewing the data this 

 way, the variability in mortality could be in part due to 

 the health and precollection stress level of a horseshoe 

 crab, which can differ from its counterparts. Different 

 conditions may lead to different responses and not every 

 individual will react exactly alike. More than likely, 

 much of this variability is an artifact of a small sample 

 size. Once the male and female data were pooled, total 

 mortality appeared to proceed in an increasing trend 

 as bleeding amount increased (Table 1). If sample size 

 were increased enough, perhaps a strong trend would 

 emerge in mortality between males and females, or 

 perhaps not. Further investigation with larger sample 

 sizes and a different experimental design could shed 

 light on a possible trend in male and female mortality 

 rates and whether one sex may be more susceptible to 

 mortality than the other. 



The combination of multiple stressors could also affect 

 not only mortality, but possibly the amount of blood able 

 to be extracted by the biomedical industry. It is possible 

 that horseshoe crabs exposed to higher levels of external 

 stress, such as greater lengths of time to air and higher 

 temperatures, would lose moisture from their exposed 

 gills and possibly become dehydrated and thus have a 

 decreased blood volume such that a smaller amount 

 of blood is available for extraction. It is unknown how 

 this would affect the harvest of horseshoe crab blood 

 for the production of LAL. Dehydration rates in horse- 

 shoe crabs are unknown, but have been examined in a 

 freshwater crayfish {Austropotamobius pallipes) which 

 is a facultative air breather that can survive about 

 three days out of water (Taylor et al., 1987). Taylor et 

 al. reported that when exposed to air (70-80% relative 

 humidity) for 27 hours, crayfish became dehydrated and 

 had a 25% decreased blood volume. When exposed to 

 water-saturated air (100% relative humidity), crayfish 

 did not have a decrease in blood volume. Similarly, 

 horseshoe crabs exposed to water-saturated air may 

 be less likely to become dehydrated, thereby possibly 

 decreasing the effects of one type of stress. 



The results presented in the present study provide in- 

 sight into the possible combined effects of blood extrac- 

 tion and external stressors associated with biomedical 



