The short-term effects of foot clipping as a nonlethal method of obtaining tissue samples from terrestrial gastropods

The most widely used method to obtain DNA for molecular analyses of terrestrial gastropods involves the death of the individual animal under study (Campbell, 2006). While the loss of a few individuals may not be important for the viability of populations of abundant species, this is not the case for rare or threatened species. Therefore, several recent studies have obtained tissue samples from terrestrial gastropods using an apparently nonlethal foot-clipping method. Most of the previous studies using foot clipping have examined endangered or localized gastropod species. Thacker & Hadfield (2000) and Holland & Hadfield (2002, 2004, 2007) studied the highly endangered Achatinella genus of Hawaiian tree snails, while Anderson (2007) used the same method to obtain tissue samples from the very localized polygyrid Euchemotrema hubrichti in southwestern Illinois. Wade & Mordan (2000) and Wade, Mordan & Naggs (2006) conducted phylogenetic analyses of pulmonate snails using pieces of tissue clipped from the foot of both common and rare species. It is likely that continuing advances in conservation genetics, combined with continuing degradation of the conservation status of many gastropods, will increase the need for molecular information from endangered species. Quantitative information about the effects of foot clipping would therefore be helpful to assess whether the method is appropriate for use and what its limitations might be. None of the previous studies reported experimental data on the behaviour or survival of clipped snails, although Thacker & Hadfield (2000) and Holland & Hadfield (2007) stated that no deaths followed the procedure in laboratory populations. In order to investigate the effects of clipping on the behaviour and survival of land snails, we conducted a randomized experiment on the discid Anguispira cumberlandiana (Lea, 1840), a nonthreatened species that is a close relative of the endangered Anguispira picta (Clapp, 1920). We gathered live A. cumberlandiana snails from the field by searching a limestone outcrop in Shakerag Hollow, Sewanee, Franklin County, TN, USA (location in decimal degrees: 35.226944, 285.909722, 365 m elevation). We assigned 23 snails to two groups (treatment and control), using a random numbers table to randomize assignments. We then placed all snails on individual pieces of parafilm. Snails at this point were withdrawn into their shells. We waited for the snails to emerge, which took from 2 min to 2 h. When they emerged, snails assigned to the treatment group (n 1⁄4 11 snails) had a piece of tissue about 1 mm sliced from the posterior tip of their foot with a sterile scalpel blade. The tissue sample was then immediately stored at 2208C until DNA was extracted. Snails that had been assigned to the control group (n 1⁄4 12 snails) were not clipped, but received the same handling as the treatment snails. After clipping and/or handling, all snails were replaced in cleaned, individual glass containers with a moist tissue paper and slices of carrot and cucumber. The jars were covered with perforated parafilm and stored on a laboratory bench that received indirect natural light and sporadic artificial light from laboratory overhead lights. We made behavioural observations of the snails after 6 h and 18 h, then at approximately daily intervals for 2 weeks. At each observation time we noted whether each snail was alive and, if so, whether it was active (i.e. the head and/or foot emerged from the shell) or inactive (snail withdrawn into shell). No snails died over the 2-week period of the experiment. The behaviour of clipped snails differed from unclipped snails at 6 h after the procedure, but thereafter we did not detect differences in behaviour (Fig. 1). Specifically, the portion of snails that were active was significantly lower for clipped than for unclipped snails after 6 h (Fisher’s exact test, P 1⁄4 0.012), but this difference was no longer significant at 18 h after clipping (Fisher’s exact test, P 1⁄4 0.68) or thereafter (Fisher’s exact test, P . 0.2 for all times). Our results accord with the lack of mortality reported by Thacker & Hadfield (2000) in clipped Achatinella. However, it appears that the foot-clipping procedure caused a short-term change in behaviour of the snails, with clipped snails remaining withdrawn in their shells for the first half-day after the procedure. Thereafter, there did not appear to be any differences in the behaviours that we observed. The observed short-term change in behaviour could result in loss of feeding opportunity, or even increase risk of predation if withdrawn snails are left in exposed areas. However, these indirect negative fitness effects are speculative. Our findings are also consistent with the results of clipping experiments in bivalves. Berg et al. (1995) examined the effects of mantle clipping in freshwater mussels,