Field testing two individual marking methods for land snails, including description of a new PIT-tagging method

Recognition of individual animals is required for a variety of research and management purposes where detailed information on demography, life history, habitat use, or movements is needed. For gastropods, it usually involves marking the animals in some way so that they can be recognized at future detections. A wide range of marking methods have been deployed, each with their pros and cons (reviewed in Gerlacks, 2017). For snails, these include various paint marks and external attachments glued onto the shell (e.g. Henry & Jarne, 2007; Servens, 2009). Here, we describe field application of two new methods used as part of conservation projects for Poecilozonites bermudensis (IUCN critically endangered) in Bermuda and for Allogona townsendiana (endangered in Canada) in British Columbia: fluorescent, plastic “alpha tags” glued onto the shell and PIT tags (passive integrated transponders) inserted into the umbilical cavity of the shell.

Alpha tags: We glued a numbered, fluorescent microtag (1.2 mm × 2.7 mm ‘alpha tag’; Northwest Marine Technology Inc. 2016) onto the shell with Gorilla™ super glue (Fig.1). We carefully clipped the tag from the manufacturer's sheet with scissors, dipped it in glue and manipulated it with fine, blunt-tipped forceps onto a dried and cleaned shell, blotting off extra glue with a cotton swab. We kept the snail on a sheet of paper towel or in an individual plastic container until the glue was dry. Following our initial success with this method on P. bermudensis in the field, Flewitt et al., (2023) examined tag retention in a captive colony of this species at Chester Zoo. The field application described here augments this study and expands the results to field conditions.

PIT tags: We used the smallest available tags (full-duplex; 8.0 mm long, 1.4 mm wide, weight 0.027 g; Trovan Electronic Identification Systems in 2017, Oregon RFID™, thereafter). First, we measured the height of the spire of the shell to ensure that the tag would fit properly. We then inserted a twist of tissue paper through the open umbilicus and twirled it around to clean and dry the area. We dipped the end of the tag into Gorilla™ super glue and gently pushed it into the umbilical cavity. We kept the snail upside down on paper towel for a few minutes until the glue was deemed dry. In both of our study species with an open umbilicus, the tag fitted snuggly inside the shell without touching or damaging any soft parts (Fig. 2). The PIT tag constituted <2% of the mass of adult P. bermudensis and <1% of that of adult A. townsendiana.

We initially developed both marking methods for conservation studies of reintroduced P. bermudensis on Nonsuch Island, Bermuda, and tracked marked snails on two study plots during annual survey periods (the plots were: ‘Wall’, established in 2016, a self-sustaining population; ‘Grid’, established in 2017, maintained only by periodic releases, no reproduction confirmed; Ovaska, 2017; Outerbridge, Ovaska & Garcia, 2019, 2021). We marked snails with one or both of these methods mostly from 2017 to 2019 but continued to check the plots for marked snails annually in 2020, 2022, and 2023; there were no surveys in 2021 due to COVID-19 restrictions. We captured snails when they were active after dark and brought them to the laboratory for tagging either the same night or following day, and returned them to their original locations. We detected snails for recapture at night with illumination from visible spectrum and UVa flashlights; UV lights facilitated redetections due to the glow from the fluorescent alpha tags that was visible within a distance of about 2 m (Fig. 1B). We read PIT tags with a handheld scanner after a snail was detected. Detection distance of this reader ranged from 10–15 cm.

Poecilozonites bermudensis: In total, we marked 221 snails at the Grid and 234 at the Wall site (Table 1; all years combined). Most larger snails (16.5–23.5 mm in shell width at the widest point, SW) received both an alpha tag and a PIT tag, but some were not PIT tagged due to lack of tag availability; also, a low spire or deformed umbilical cavity prevented the insertion of a PIT tag on some larger snails. The smallest snail with an alpha tag was 11.0 mm SW and the largest 23.7 mm SW. The percentage of snails recaptured at least once within the same month when tagged was similar for snails that were marked with alpha tags and with both alpha and PIT tags (Table 1). Overall, the percentage of recaptured snails declined markedly after 1 year and again after 2 years of tagging; only one snail was detected after 3 years of tagging (Table 1). After 1 year from tagging, snails with both an alpha tag and a PIT tag were recaptured at a significantly lower rate than those with only an alpha tag (χ² = 4.68, df = 1, n = 455, P < 0.05, both sites combined). Only adult snails were PIT tagged, and it is likely that their low recapture success after 1 year reflects age-related attrition, as the life span of the snails in captivity is about 2–2.5 years (K.T. Papp et al., unpublished). All snails marked with a combination of both types of tags retained them at recaptures during the tagging month, indicating no tag loss. One double-tagged snail had lost its alpha tag after 1 year of tagging. The Grid site provided additional information on loss of alpha tags. All 41 snails (30 juveniles and 11 adults) released at this site in February 2017 were alpha tagged, but only 6 of 16 snails caught in February 2018 had a tag. All except one of the unmarked snails (14.5 mm SW) were large (16.4–22.5 mm SW) and were probably from the original batch of released snails, and one large snail had a tag-shaped scar on the shell, suggesting tag loss. Assuming that the smallest unmarked snail was born at the site, tag loss could have been as high as 60% over a year. At the two study sites, we found seven empty shells with alpha tags still attached, three after 1 year, one after 2 years and three after 3 years of tagging; three shells showed signs of rodent predation, while the remaining four were intact.

Allogona townsendiana: We marked snails with alpha tags and/or PIT tags as part of a conservation project on five 10 m-radius study plots near Crofton, Vancouver Island, British Columbia. In total, we marked 120 snails with three different tag combinations (Table 2): (1) 24 snails with a PIT tag only (all were adults based on thickened apertural lip of the shell; 24.7–31.8 mm SW); (2) 24 snails with an alpha tag only (19 adults, 5 juveniles; 15.5–28.0 mm SW); (3) 73 snails with both an alpha and a PIT tag (65 adults, 7 juveniles; 23.0–32.2 mm SW). We surveyed the plots seven times from April to October 2019 and once in March 2020; we had no access to the site subsequently due to COVID-19 restrictions. We located snails when they were active during daylight hours by visually searching the ground and vegetation. In addition, we scanned the plots with portable readers (Biomark™ HPR Plus reader and BP Lite portable antenna), which allowed us to detect PIT-tagged snails that were hidden from view within the vegetation or the litter layer (Fig. 3). The detection distance of this reader was about 20–25 cm. We processed the snails, including tag attachment, in the field and released them to their original locations after completing a plot search. Of 120 snails, 60.8% were recaptured at least once during the study period (Table 2). The recapture frequency differed among the three tagging groups with only one recapture of a snail marked with only an alpha tag (χ² = 37.9, df = 2, P < 0.00001). The low recapture rate of this tagging group was almost certainly due to the relative ineffectiveness of visual searches in detecting juvenile snails. Recapture rates of snails with a PIT tag and those with both a PIT tag and an alpha tag that were detected at least once after tagging were similar, both at 75% (Table 2). Individual snails were detected up to eight times, and their recapture frequency was also similar (χ² = 4.77, df = 3, P > 0.05; snails detected > 3 times combined). The number of days between the first and last detection for individuals recaptured at least once during the study period ranged from 14 to 178 days (Table 3). The mean number of days was similar between the two tagging groups (2-tailed t-test: P = 0.359). We recorded no loss of PIT tags for snails marked with both methods, but the alpha tag of one snail was obscured after 14 days from tagging by excess glue, which had also caused a dent in the shell at the point of application. Several snails from this site had unusually thin shells, which may have contributed to this condition. Eight adult snails with both a PIT tag and an alpha tag died from unknown causes during the study; we found their shells intact with both tags still attached.

Alpha tags and PIT tags provide additional methods for tagging snails for studies where individual recognition is needed. Alpha tags have the advantage of extremely small size, allowing their use on snails as small as c. 11 mm SW, including juveniles of larger species. Being fluorescent, they also facilitate the detection of nocturnal species active in the environment, as noted for P. bermudensis. Snails retained their tags for up to 2 years (and in one case 3 years) in Bermuda, but information from the initial batch tagged with this method had a worrisome tag loss rate of up to 60%. In contrast, Flewitt et al. (2023) found no tag loss in captive P. bermudensis in a controlled experiment over a 6-month period. Tag loss in our study may have resulted from improper application of the initial batch of snails marked with this method or because of semi-fossorial behaviour of the snails coupled with harsh field conditions. In contrast, A. townsendiana on Vancouver Island experienced minimal tag loss over the 11-month duration of the study. Henry & Yarn (2007) recommended glued plastic tags for long-term studies of hard-shelled gastropods but stressed the importance of controlling for tag loss and examining potential impacts on life history. Where possible, we recommend using a combination of two tagging methods to document possible tag loss over time.

PIT tagging provided an excellent method both for individual recognition and for detecting marked snails through telemetry. To our knowledge, our innovative method of tag attachment in the umbilical cavity of snails is new; it often results in a vertical orientation of the tag for optimal detection distance with scanners. Among gastropods, PIT tags have been used previously with success to track moments of arionid slugs (Grimm, 1996; Ellis et al., 2020; Nyqvist et al., 2020). The tag was inserted internally into the foot of the slug, a procedure that required healing, and in some cases resulted in mortality. Baminger (2000) glued a PIT tag onto the dorsal surface of the shell of the land snail Arianta arbustorum and found adverse effects on mating behaviour of the snails. Parkyn, Brooks & Newell (2014) glued much larger radio-tags (including external antenna and battery) onto the shell of Thersites mitchellae. Harmonic direction finders with the tag glued onto the shell have also been used to track movements of land snails (Hall & Hadfield, 2009; Steensma, Lilley & Zandberg, 2009), but this method does not provide individual identification. The advantage of our method is that the tag does not touch any soft parts, and unlike an external attachment, it does not protrude from the shell and is invisible, hence, removing the possibility of attracting predators. However, the method applies only for larger snails (umbilical cavity ≥ 8 mm deep) and for species with an open umbilicus. With further miniaturization of tags, the method may become feasible for smaller snails, and future technological advances could increase the currently short detection distance. PIT-tag telemetry is an underused but powerful method to track movements of relatively sedentary small animals that cannot be easily radio-tagged, and it has been used for variety of small vertebrates with success (salamanders: Cucherousset et al., 2008; Brown, 2017; small snakes: Oldham et al., 2016; L. Sopuck et al., unpubl.). Use of stationary automated PIT tag detection systems also provides exciting possibilities for snails both in the context of conservation and pest management.

ACKNOWLEDGEMENTS

We very much appreciate assistance in the field by Heather Prince and Thomas Papp in Bermuda and by Laura Matthias and Sam Norris on Vancouver Island. Geraldo Garcia provided field support and captive-bred P. bermudensis from Chester Zoo for population augmentation at the Grid site on Nonsuch Island. We thank Eric Gross for loan of portable PIT-tag reader and assistance in securing funding for the Vancouver Island study.

FUNDING

Funding was obtained from the Bermuda Zoological Society grants to K.O. in 2018 and 2020 for studies in Bermuda and as part of Contract 300068623 from the Canadian Wildlife Service to Biolinx Environmental Research Ltd. for the study in British Columbia

CONFLICT OF INTEREST

The authors have no conflicts of interest to declare.

DATA AVAILABILITY

Data used for this study can be made available on request by contacting the first author.

REFERENCES