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Fishery Bulletin 105(4) 



be attractive to mud snails (Ilyanassa obsoleta), and 

 because horseshoe crab eggs have been proposed as 

 a source of compounds for inclusion in manufactured 

 bait (Ferrari and Targett, 2003). Horseshoe crab he- 

 molymph was tested as a potential source of attractant 

 for inclusion in replacement bait because it is a readily 

 available natural product. It is a waste product pro- 

 duced during the preparation of Limulus amoebocyte 

 lysate (LAD, a clotting agent used worldwide to test 

 for bacterial contamination of pharmaceuticals and 

 implantable medical devices. More importantly, when 

 handled properly, horseshoe crabs used for hemolymph 

 collection show low rates of postrelease mortality (Walls 

 and Berkson, 2000). A patent has been issued for hemo- 

 lymph-based bait for whelks (U.S. Patent 6391295), but 

 we are unaware of any published studies documenting 

 its efficacy in the whelk pot fishery. Odorant solutions 

 prepared from hard clam tissue were tested because 

 bivalve mollusks are a common prey item for whelks 

 (Walker, 1988). 



Electrophysiological techniques were used to test 

 each proposed bait, rather than behavioral methods, 

 because the former allows precise control of stimulus 

 parameters and the rapid assay of various compounds 

 and concentrations on individual animals. Electrophysi- 

 ological techniques, moreover, have been used for many 

 years to investigate successfully chemosensory ques- 

 tions in both aquatic invertebrates (e.g., Borroni et al., 

 1986; Kamio et al., 2005) and vertebrates (e.g., Hara, 

 1975; Wilson, 2004). Behavioral methods, such as flume 

 choice (Y-maze) experiments, can also be difficult with 

 whelks. A large cross-sectional area within a flume is 

 necessary to accommodate adult whelks. This large 

 cross-sectional area requires a high-volume flow rate 

 and concomitantly large volumes of odorant solution to 

 achieve a detectable concentration over the long time 

 periods necessary for whelk to respond behaviorally 

 (Ferner and Weissburg, 2005); large volumes of odorant 

 solutions can be difficult and costly to produce (Ferrari 

 and Target, 2003; Ferner and Weissburg, 2005). Elec- 

 trophysiological techniques, however, cannot differen- 

 tiate between attractive and repulsive odors and must 

 subsequently be paired with behavioral studies. 



Activity in the pallial nerve was recorded while odor- 

 ant solutions were applied directly to the osphradium. 

 The pallial nerve connects the osphradium to the su- 

 praesophageal ganglia, which is part of the central 

 nervous system (Alexander, 1970). The osphradium is 

 considered to be the primary chemosensory organ of 

 prosobranch mollusks because its leaf-like structure 

 bears a strong resemblance to the nasal rosettes of 

 aquatic vertebrates (Hansen and Reutter, 2004), and 

 because its location at the base of the incurrent siphon 

 maximizes exposure to odorants (Bailey and Laverack, 

 1966; Emory, 1992). It should be noted, however, that 

 the osphradium is not the only chemosensory organ in 

 gastropod mollusks. The rhinophores may also provide 

 sensory information allowing gastropod mollusks to 

 track odor plumes (Levy et al., 1997, Rahman et al., 

 2000; Ferner and Weissburg, 2005). Indeed, the rhino- 



phores are considered a primary chemosensory organ in 

 nudibranch mollusks (Alexander, 1970; Wedemeyer and 

 Schild, 1995). A record of activity in the pallial nerve 

 during exposure of the osphradium to odorant solu- 

 tions derived from various sources therefore provides a 

 robust method to determine whether a specific odorant 

 solution is detectable by whelks and is a candidate for 

 subsequent behavioral testing. 



Materials and methods 



Knobbed and channeled whelks were obtained from local 

 processing plants or collected from estuaries behind the 

 barrier islands on the eastern shore of Virginia. They 

 were maintained at the Virginia Institute of Marine 

 Science (VIMS) (Gloucester Point, VA) and also at the 

 VIMS Eastern Shore Laboratory (Wachapreague, VA) in 

 tanks supplied with running water drawn directly from 

 the mouth of the York River or the Virginia eastern shore 

 estuaries, respectively. 



Horseshoe crab eggs were obtained from beaches bor- 

 dering the Chesapeake and Delaware Bays, sealed in 

 sterile 50-mL plastic tubes, and stored frozen. Hard 

 clams were obtained from stocks maintained by the 

 VIMS Eastern Shore Laboratory and only fresh tissue 

 was used. Stock odorant solutions were prepared ac- 

 cording to methods described by Ferrari and Targett 

 (2003). In brief, horseshoe crab eggs were mixed (1:2 

 egg to liquid volume) with aerated, filtered, and ul- 

 traviolet sterilized water (FSW) drawn from the same 

 sources supplying the holding tanks. The tissue was 

 then crushed with a clean mortar and pestle and the 

 mixture was allowed to sit overnight at 4°C. It was then 

 centrifuged to remove cellular debris and sand. Clam 

 tissue was treated likewise. In addition, horseshoe crab 

 extract solution was prepared as described above, with 

 the exception that 50 mM Tris buffer solution (pH 7.5, a 

 mixture of Trizma HCl and Trizma Base, Sigma Chemi- 

 cal Co, St. Louis, MO) was used instead of FSW. Two 

 molecular weight fractions (>3 kDa and <3 kDa) were 

 then generated by using stirred cell ultra-filtration with 

 YM-3 membranes (Millipore Inc., Billerica, MA). The 

 molecular weight fractions were tested individually on 

 channeled whelk, and after they were recombined. The 

 stock odorant solutions were diluted with FSW 1:10^, 

 1:10^, 1:10^, and 1:10^ (volume to volume) immediately 

 before experiments. Horseshoe crab hemolymph (free of 

 any anticoagulants or preservatives) was obtained from 

 Wako Chemicals (Cape Charles, VA) and stored at 4°C. 

 It was likewise diluted (1:100, 1:133, 1:200, and 1:400) 

 with FSW immediately before use. 



During experiments, an individual whelk was pre- 

 sented with at least four concentrations of an individual 

 odorant solution, plus controls consisting of FSW or a 50 

 mM Tris buffer solution in random order. The limited 

 funding available for this project precluded the testing 

 of all odorants on both species of whelks. The specific 

 odorants presented to each whelk species are summa- 

 rized in Table 1. 



