Turkish Journal of Zoology Turk J Zool (2017) 41: 370-374 http://journals.tubitak.gov.tr/zoology/ © TÜBİTAK Short Communication doi:10.3906/zoo-1601-77

The influence of temperature on the hibernation patterns and activity of moulinsiana (Dupuy, 1849) (: : )

Zofia KSIĄŻKIEWICZ-PARULSKA*, Katarzyna PAWLAK Department of General Zoology, Faculty of Biology, Adam Mickiewicz University, Poznań, Poland

Received: 31.01.2016 Accepted/Published Online: 22.06.2016 Final Version: 04.04.2017

Abstract: The aim of this study was to investigate the impact of different temperatures, i.e. 11 °C and 21 °C, in conditions of medium relative humidity on the rate of awakening of Vertigo moulinsiana after hibernation and its activity during the following 7 days. One- way ANOVA did not show statistically significant differences in timings of awakening and activity between individuals kept at 11 °C and 21 °C during the first day of the study. The 1-week observations of the activity of V. moulinsiana showed significant differences in relation to temperature. Snails were more active at the lower temperature, which may be explained by a lower loss of water from the snails and a higher availability of water as dew formed condensation on the inner walls of the test tubes. At both temperatures, not all of the specimens awoke from hibernation, which could be related to differences in hydration between particular individuals or a diversified bet-hedging strategy, implemented by the species in the face of unpredictable environmental conditions. Results of this study indicate that increasing temperatures due to climatic changes may reduce the activity of this species and therefore also its reproduction rates.

Key words: , wintering, biology, wetland, local extinctions

Vertigo moulinsiana (Dupuy, 1849) (Figure 1) is a flooded sites where water levels are at the ground surface pulmonate micromollusk (the height of its shell does not level or remain close to it (Killeen, 2003; Tattersfield and exceed 2.73 mm, while the breadth of the shell reaches McInnes, 2003; Książkiewicz-Parulska and Ablett, 2016). a maximum of 1.65 mm) considered to be an Atlantic- This micromollusk requires high ambient humidity Mediterranean species with a range extending from (Killeen, 2003; Książkiewicz et al., 2013). To satisfy its Ireland to Russia and south to North Africa (Pokryszko, requirements, an individual snail changes its position on 1990; Killeen, 1996). V. moulinsiana is listed in Annex II of a plant to cope with changing environmental variables (i.e. the EU Habitat Directive and is considered as Vulnerable Jankowski, 1939; Killeen, 2003; Myzyk, 2011). With the (VU) according to the IUCN Red List of Threatened onset of winter V. moulinsiana descends to the ground or Species. Currently this classification is based on the loss overwinters on tussock sedges (Pokryszko, 1990; Killeen, of individuals in sites and subpopulations from sites, 2003; Książkiewicz and Pawlak, 2016). and also due to the shrinking of its range (Killeen et al., The most critical challenge for terrestrial snails in terms 2012), which is caused by climatic change and/or human of temperature is the timing of winter hibernation (Szybiak activities (Pokryszko, 2003). At the same time, knowledge et al., 2009). Therefore, in this study we investigated the on the biology and ecology of this species is scarce, which impact of different temperatures on the awakening rate further hampers effective species protection. of V. moulinsiana after hibernation. We also compared V. moulinsiana is associated with moraine plateaus the activity of this species at 2 different temperatures, 11 dominated by sediments of impermeable clay in Poland °C and 21 °C, in conditions of medium relative humidity (Książkiewicz and Gołdyn, 2015) and it occurs in wetlands: (43%–49%) over 7 days after awakening. alder carr, marshes, and fens covered with monocots Snails were collected on 14 January 2014, after 2 such as sedges, Glyceria, or beds of reed (Pokryszko, weeks of frost, low temperatures, and snowfall (Table 1). 1990, 2003; Cameron, 1992; Książkiewicz et al., 2015). All of them were inactive and their bodies were deeply Previous studies have shown that the survival and density withdrawn into their shells. Thus, we assumed that the of V. moulinsiana highly depend on ground water levels: collected specimens were fully hibernating. Specimens highly numerous populations occur in periodically were collected in a wetland situated on the shore of a pond

* Correspondence: [email protected] 370 KSIĄŻKIEWICZ-PARULSKA and PAWLAK / Turk J Zool

a heated room. The humidity in the rooms was relatively constant and varied between 44% and 47%. Both humidity and temperature in the rooms were controlled with a TFA Digital Thermo-Hygrometer (±1 °C temperature accuracy; ±5% relative humidity accuracy). Since no literature on the impact of temperature on V. moulinsiana is available, we decided to choose experimental conditions that we were able to maintain at a constant level. Tubes were numbered and the position of snails on the leaves was noted. On the day of collection, individuals were observed for 320 min at intervals of 20 min. Over the next 7 days the activity of snails from both groups was recorded twice a day, at around 0700 and 1900 hours, and the number of individuals that had awoken was noted (i.e. they were active, they were inactive but had changed position, or new mucus trails had appeared). A snail was Figure 1. Shell of V. moulinsiana collected at the studied site. only regarded as active if it was crawling or if, although immobile, its body was extended and the tentacles fully everted (Cameron, 1970). Since no data on the activity rhythms of V. moulinsiana (geographical coordinates: 52°28′07.05″N, 16°56′02.18″E) were available in the literature, we decided to exclude the 2 in Wielkopolska (W Poland). The site covers ca. 500 m impact of light and dark from the observations and thus and is partially shaded by alders (Alnus glutinosa) and both groups were stored in dark conditions. Snails were, densely overgrown with sedges (Carex sp.) and reeds however, exposed to the light of a flashlight for a short time (Phragmites australis) (Figure 2A). Snails were searched twice a day when the hibernation patterns and activity of for by eye on the leaves of plants within a relatively the snails were recorded. The tubes were oxygenated twice homogeneous fragment of the site (the plot was densely a day while the activity of the snails was monitored. On covered with sedges; it was not inundated but litter and soil the first day of the study, tubes were oxygenated once, 2 were waterlogged). Individuals were harvested by cutting h after the individuals were collected. Relative humidity fragments of decaying leaves to which they were attached. in the tubes was checked twice during the 8 days: on the Each specimen was placed into separate test tubes of 12 first day of the study, 2 h after snails were collected (mean mL (Figure 2B). relative humidity in the test tubes at 11 °C: 44.9%; σ = 0.8; Individuals were transported to the lab at the outdoor 21 °C: 44.3%, σ = 0.8), as well as on the 4th day of the study temperature shortly after they were collected. In the lab, (mean relative humidity in the test tubes at 11 °C: 45.6%, tubes with snails were split into two equal groups of 33 σ = 1.7; 21 °C: 45.1%; σ = 1.5). The relative humidity in individuals: one group was placed at a temperature the tubes was checked with a digital thermos-hygrometer of 11 °C and the second at a temperature of 21 °C. The WSD 12A (±5% accuracy). temperature of 11 °C was maintained in a refrigerated After the last observation on the 8th day, we provided room while the temperature of 21 °C was maintained in a couple of drops of water to the tubes where snails had

Table 1. Weather conditions in the fortnight (1–14 January 2016) of the study before specimen collection and 2 months before (November and December 2015): average minimum temperature and minimum temperate (min temp), average maximum temperature and maximum temperature (max temp), and sum of precipitations (sum prec).

Average Max temp Min temp Sum prec Min temp Max temp Month [°C] [°C] [mm] [°C] [°C] November 2015 4 8.8 15 –3 45.1 December 2015 4 7.6 10.5 –4.7 22.5 1–14 January 2016 –5.6 –1.7 5 –14.5 26

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Table 2. Percent of snails active and awoken at 11 °C and 21 °C observed in intervals of 20 min during 320 min in the first day.

Snails active [%] Snails awoken [%] Min 11 °C 21 °C 11 °C 21 °C 20 0.0 0.0 0.0 0.0 40 6.1 20.0 6.1 20.0 60 36.4 36.7 30.3 13.3 80 42.4 33.3 9.1 6.7 100 51.5 53.3 18.2 20.0 120 48.5 43.3 12.1 6.7 140 51.5 43.3 0.0 6.7 160 48.5 53.3 3.0 3.3 180 27.3 50.0 6.1 3.3 200 30.3 26.7 0.0 0.0 220 36.4 26.7 3.0 0.0 240 45.5 26.7 0.0 0.0 260 42.4 26.7 0.0 3.3 280 30.3 33.3 0.0 0.0 300 27.3 40.0 0.0 3.3 320 27.3 6.7 0.0 0.0

Figure 2. (A) The site of V. moulinsiana; (B) awoken individuals Table 3. Percent of snails active and awoken at 11 °C and 21 °C of V. moulinsiana in test tubes. observed in intervals of 12 h in 2–8 days (18–174 h) after snail collection. Snails awoken were inactive at the time of control. remained inactive during the whole study period to check Snails active [%] Snails awoken [%] Day Hour if they were alive. To check if the temperature had an 11 °C 21 °C 11 °C 21 °C influence on the rate of V. moulinsiana awakening after 18 0.0 0.0 3.0 0.0 hibernation and its activity on the first day of the study, 2 and if the temperature had an impact on the activity 7 30 0.0 0.0 0.0 3.3 days after the day of awakening, we performed a one-way 42 0.0 0.0 0.0 0.0 3 ANOVA test (randomized version). For calculations we 54 9.1 3.3 0.0 0.0 used RundomPro 3.14 software and we considered P < 66 6.1 0.0 0.0 0.0 0.05 as the minimum level determining significance. 4 During the 8 days of observations, 29 individuals 78 0.0 0.0 0.0 0.0 awoke at 11 °C and 4 remained alive but inactive until 90 0.0 0.0 0.0 0.0 5 the end of the study. At 21 °C, 27 individuals awoke, 3 102 6.1 0.0 0.0 0.0 remained inactive, and 3 were found to be dead. One-way 114 0.0 0.0 0.0 0.0 ANOVA did not show statistically significant differences 6 in the rate of awakening (P = 0.89; F = 0.04) and activity (P 126 0.0 0.0 0.0 0.0 = 0.37; F = 0.92) between individuals kept at 11 °C and 21 138 0.0 0.0 0.0 0.0 7 °C on the first day of the study (Table 2). During the 320 150 12.1 0.0 0.0 0.0 min of observations a total of 26 snails (86.7%) awoke at 162 3.0 0.0 0.0 0.0 21 °C and 28 (84.8%) at 11 °C. On the following day, one 8 more individual awoke at both 11 °C and 21 °C (Table 3). 174 6.1 0.0 0.0 0.0

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Observation of activity on days 2–8 (18 to 174 h) showed particular species of land snail at a given temperature may statistically significant differences in the species’ activity be different (Cameron, 1970), the temperature has a strong (P = 0.027; F = 6.29); the species was more active at 11 °C effect on the rate of water loss at constant saturation deficit (Table 3). (Warburg, 1965). Thus, we suspect that V. moulinsiana In conclusion, the results of our studies show that was more active at 11 °C due to a lower loss of water in after an initial rise in temperature to 11 °C or 21 °C, the cooler conditions and additionally the higher availability majority of V. moulinsiana awoke during the first 2 h of of water, as dew formed condensation on the inner walls observations (i.e. 75% of snails at 11 °C and 66% at 21 °C). of test tubes. In the case of both experimental temperatures, not all of the The results of this study also show how the warming specimens awoke, which could be related to the hydration of the climate that we are now facing (IPCC, 2014) may of particular individuals. On the other hand, it could be impact this species. House et al. (2016) showed that climate also a sign of a form of diversified bet-hedging strategy, change, causing changes in hydrological conditions, implemented by the species in the face of unpredictable contributes to transformations of plant communities in environmental conditions (e.g., Haccou and Iwasa, 1995; wetlands, which also causes changes in temperature and Cáceres and Tessier, 2003). In temperate climate zones, humidity within microhabitats (Ruckli et al., 2013). For winter temperatures can sometimes change rapidly, with Vertigo moulinsiana, which is strictly related to particular heavy frosts coming after a period of mild thawing. In such microhabitat conditions (i.e. Jankowiak and Bernard, 2013; cases, awoken/active individuals may not have enough time to fall back into hibernation and may die due to rapid Książkiewicz et al., 2013), it means the lowering of activity freezing. Snails that stay hibernating during the preceding and reproduction rates, which may lead to a decrease in period of temperature increase would probably provide the number of individuals within populations and finally a safeguard, preventing the population from becoming local extinctions of V. moulinsiana populations. locally extinct. This is especially important since only a few individuals are needed to recreate an entire population Acknowledgments (Pokryszko, 1987). The authors would like to thank to Bartłomiej Gołdyn On the other hand, observations of V. moulinsiana (Adam Mickiewicz University, Poznań) for valuable during the 7 days after the day of awakening from suggestions during the manuscript preparation and hibernation showed that the activity of V. moulinsiana Jon Ablett (Natural History Museum, London) for his is highly dependent on the temperature in conditions corrections of the English as well as for useful comments of average relative humidity. Although the activities of a on the draft of the present paper.

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