Magel et al.: Activity in the pallial nerve of Busycon conca and Busycotypus canoliculatum 



491 



5- 1.; 



1.6 



e 1.2 



trafiltration procedures destroy the stimula- 

 tory components present within the horse- 

 shoe crab egg extract. These results also 

 imply that channeled whelk have broadly 

 tuned chemoreceptors requiring a range 

 of low and high molecular weight constitu- 

 ents to elicit a response. Channeled whelk 

 thus appear to differ from other aquatic 

 animals that generally respond to a narrow 

 spectrum of chemical stimulants (e.g., fish- 

 es, Carr et al., 1996; Zielinski and Hara, 

 2007, and crustaceans, Borroni et al., 1986; 

 Kozlowksi et al., 2001). Our results from 

 whelk also differ from behavioral experi- 

 ments on mud snails, in that mud snails 

 are apparently capable of detecting the low 

 molecular weight fractions (<10 kDa) of 

 horseshoe crab egg extracts prepared by 

 ultrafihration (Ferrari and Targett, 2003). 



Knobbed whelk responded to an odorant 

 solution prepared from hard clam tissue 

 (Fig. 3). Because hard clams are a com- 

 mon prey item of whelks (Magalhaes, 1948; 

 Walker, 1988), we surmise that channeled 

 whelk would respond similarly. Bait based 

 on hard clam tissue, or some extract there- 

 of, could potentially serve as replacement 

 for horseshoe crabs in the whelk pot fishery. 

 The cost of raw material may be prohibi- 

 tive, however, because hard clams are also 

 harvested for human consumption. Like- 

 wise, knobbed whelk responded to horse- 

 shoe crab hemolymph even when diluted 

 133:1 (Fig. 4). Surprisingly, hemolymph did 

 not induce a response in channeled whelk 

 at even the highest concentration tested. There is no 

 apparent explanation for this difference in response by 

 the two species. Horseshoe crab hemolymph may offer 

 some promise as the basis for manufactured bait for the 

 whelk pot fishery primarily because it can be obtained 

 in large quantities from an ecofriendly source (i.e., from 

 the pharmaceutical companies producing LAD, and 

 probably at a reasonable cost. It should be noted that 

 horseshoe crab blood is generally treated with propri- 

 etary chemical mixtures during LAL production to 

 prevent clotting and to prepare the amoebocyte cells 

 for further processing. If the chemicals are toxic, this 

 could prevent the use of waste hemolymph directly in 

 manufactured baits and preclude their eventual release 

 into the environment. 



In summary, electrophysiological techniques record- 

 ing afferent activity in the pallial nerves of whelks can 

 be used to identify effective odorant solutions, as well 

 as approximate detectable odor concentration limits. 

 Knobbed and channeled whelks responded to extracts 

 prepared from horseshoe crab eggs, and the former 

 responded to horseshoe crab hemolymph diluted up to 

 133 times, as well as to extracts prepared from hard 

 clam tissue. Channeled whelk do not respond to in- 

 dividual molecular weight fractions of horseshoe crab 



3.6 

 2,7 



1.0 



10 







Horseshoe crab eggs 

 ,5 IZZZ3 > 3 kDa fraction 

 J VTZ/^^ < 3 kDa fraction 

 K X XI Fractions recombined 



]/^mA 



50nl\/l 

 Tris buffer 



lilO" 



1:10^ 1:10'' 



Stimulus dilution 



lilO-" 



Figure 5 



Mean responses (±95'>J confidence interval) of channeled whelk (Busy- 

 cotypus canoliculatum) expressed as the ratio of integrated activity 

 in the pallial nerve before the introduction odorant solutions or 50 

 mM Tris buffer solution. The odorant solutions were prepared from 

 horseshoe crab (Limulus polyphemus) eggs extracted with 50 mM Tris 

 buffer solution, two molecular fractions (>3 kDa and <3 kDa) of this 

 odorant solution prepared by ultrafiltration, and the two molecular 

 weight fractions recombined. The numbers of individuals exposed to 

 various odorant solutions are shown above each bar (n). A significant 

 response (indicated by "*") was determined to have occurred when 

 the mean ratio of integrated nerve activity was significantly differ- 

 ent from 1.0 (based on the 95% CI). The solid line indicates a ratio 

 of integrated activity value = 1.0. 



egg extracts, and this result implies that whelks have 

 broadly tuned chemoreceptors and that manufactured 

 baits may need to mimic the complex mixture of odors 

 derived from natural sources. 



Acknowledgments 



This project was supported by grants from the Na- 

 tional Oceanic and Atmospheric Administration Chesa- 

 peake Bay Office (PO no. DG133F04SE1208), the Na- 

 tional Marine Fisheries Service (Northeast Fisheries 

 Science Center), Delaware Sea Grant (RF-9 05/07), 

 Delaware Department of Natural Resources and Envi- 

 ronmental Control, and the National Fish and Wildlife 

 Foundation. We acknowledge R. Fisher (Virginia In- 

 stitute of Marine Science Sea Grant Program) for his 

 support throughout the project; R. Robins Jr. and M. 

 Pegg for donations of live whelk and horseshoe crab he- 

 molymph; J. Harding, R. Mann, and J. Shields (VIMS) 

 for their help in getting us started; the VIMS Eastern 

 Shore Laboratory for providing live whelk, hard clams, 

 animal holding space, and laboratory facilities. This 

 is contribution no. 2828 from the Virginia Institute of 

 Marine Science. 



