Magel et al.: Activity in the pallial nerve of Busycon canco and Busycotypus canaliculatum 



487 



Table 1 



Summary of odorant solutions derived from various sources tested on knobbed (Busycon carica) and channeled (Busycotypus 

 canaliculatum) whelks. The odorant solutions that were tested on a species are indicated by an "X". FSW = aerated, filtered, and 

 ultraviolet sterilized water. 



Knobbed whelk 



Channeled whelk 



Horseshoe crab (Limulus polyphemus) eggs extracted with FSW 



Hard clam (Mercenaria mercenaria ) tissue 



Horseshoe crab hemolymph 



Horseshoe crab eggs extracted with 50 mM Tris buffer solution, <30 kDa and 



>30 kDa molecular weight fractions of horseshoe crab eggs produced by ultrafiltration, 



and the molecular weight fractions recombined 



X 

 X 

 X 



X 

 X 



Quantifying activity in the pallial nerve in response 

 to odorant solutions required that the whelk be removed 

 from its shell, the pallial nerves exposed, and the os- 

 phradium isolated. To remove the animal from its shell, 

 the apex of the shell was cut away at the spire, the 

 columella muscle detached from the central column, and 

 the animal gently pulled through the opercular opening. 

 The individual was then immediately submerged in a 

 large dish of aerated FSW. The viscera and the majority 

 of the foot were removed and the mantle cavity opened 

 with a dorsal incision starting medially between the 

 cephalic eye stalks. The preparation was pinned to a 

 Sylgard®(Dow Corning Corp., Midland, MI) lined Petri 

 dish such that the body cavity was open and the buccal 

 mass held out of the way. The overlying connective tis- 

 sue was carefully dissected to expose the circumesopha- 

 geal ganglia, specifically the supraesophageal ganglion, 

 and the Petri dish holding the preparation was moved 

 to a second plastic chamber (filled with FSW) mounted 

 under a dissecting microscope. A modified 10-mL plastic 

 syringe barrel with a small amount of quick-setting 

 silicon elastomer (Kwik-Cast"', World Precision Instru- 

 ments, Sarasota, FL) around the base was placed over 

 the osphradium to isolate it from the fluid in the cham- 

 ber containing the animal (Fig. lA) . 



The odorant delivery system consisted of a glass res- 

 ervoir (filled with FSW) and polyethylene and stainless 

 steel tubing (Fig. lA). FSW was continuously delivered 

 to the osphradium chamber by gravity at approximately 

 6 ml/min. Odorant solutions were switched into the 

 delivery system without a change in fiow rate by using 

 a three-way stopcock. The 10-mL volume odorant solu- 

 tion was therefore delivered to the osphradium chamber 

 within approximately 90 seconds, and then flushed out 

 of the osphradium chamber by switching the flow back 

 to FSW. Because the volume of liquid in the osphradium 

 chamber was maintained as close to 2 mL as possible, 

 peak stimulus concentration in the osphradium chamber 

 reached approximately 60% of the original stimulus 

 concentration (Fig. IB) (Steffensen, 1989). 



To record activity in the pallial nerve associated with 

 the exposure of the osphradium to odorant solutions, 

 the nerve was cut and the afferent end was drawn into 



a suction electrode (Fig. IB). The resulting signal was 

 amplified (80 dB gain) and filtered (10 Hz high pass and 

 1 k Hz low pass) by using a DAM-50 amplifier (World 

 Precision Instruments. Sarasota, FL). The signal was 

 further conditioned to remove 60 Hz noise with the use 

 of a Humbug® active electronic filter (Quest Scientific, 

 North Vancouver, B.C., Canada), and then displayed on 

 a digital storage oscilloscope. The signal was digitized 

 (at 1 kHz sampling rate) with a USB analog to digital 

 I/O interface (model 1208LS, Measurement Comput- 

 ing, Middleboro, MA). The digital data were filtered (5 

 Hz high pass and 75 Hz low pass filters), processed, 

 recorded, and displayed on a computer screen by using 

 a custom designed computer program developed within 

 Dasylab (version 7.0, National Instruments Corp., Aus- 

 tin, TX). 



It was not practical to quantify responses by discrimi- 

 nation and by counting single nerve spikes because the 

 recordings contained a broad amplitude signal due to 

 activity in multiple nerve fibers, and because of the long 

 intervals of activity in response to odorant solutions 

 (Fig. 2) . Instead, the differentiation and integration 

 module within Dasylab was used to generate an output 

 value proportional to the integral of the filtered nerve 

 signal (Fig. 2B). The module was programmed to inte- 

 grate over 10.2-second intervals (i.e., 20 data blocks, 

 as defined within the Dasylab program) to reset the 

 output to zero, and then resume integrating. The digital 

 system was thus functionally equivalent to an analog 

 electronic "leaky RC integrator" circuit commonly used 

 to measure the magnitude of nerve activity (e.g., Hara, 

 1975; Kamio et al., 2005). 



For each trial, data were recorded for 12 minutes: 6 

 minutes before the introduction of an odorant solution 

 into the osphradium chamber, and 6 minutes after. 

 This recording interval was chosen on the basis of long 

 integration times reported for the chemosensory abili- 

 ties of whelks (Ferner and Weissburg, 2005) and other 

 mollusks (Murphy and Hadfield, 1997). An additional 

 3 minutes were allowed between odor trials to ensure 

 that the odorant solution was flushed from the osphra- 

 dium chamber and that the activity in the pallial nerve 

 had returned to prestimulus levels (Fig. 2). Only data 



