Effects of photoperiod and acclimation temperature on heat and cold ...

Appl. Entomol. Zool. 43 (4): 547–551 (2008) http://odokon.org/

Effects of photoperiod and acclimation temperature on heat and cold tolerance in the terrestrial slug, Lehmannia valentiana (Pulmonata: Limacidae) Hiroko UDAKA, Shin G. GOTO and Hideharu NUMATA* Department of Biology and Geosciences, Graduate School of Science, Osaka City University; Sumiyoshi-ku, Osaka 558–8585, Japan (Received 5 March 2008; Accepted 9 June 2008)

Abstract Heat and cold tolerance was examined in the terrestrial slug, Lehmannia valentiana, in Osaka, Japan. In the field, both the heat and cold tolerance of slugs changes seasonally. Heat tolerance was maximal in summer and minimal in winter, whereas cold tolerance was maximal in winter and minimal in summer. To clarify the environmental factors by which temperature tolerance is affected, the effects of acclimation temperature and photoperiod were also examined in slugs hatched under laboratory conditions. Heat tolerance was enhanced by a higher acclimation temperature. Longday conditions also increased heat tolerance. Cold tolerance was enhanced by both short-day conditions and a low acclimation temperature. These results indicate that seasonal changes of heat and cold tolerance are promoted not only by acclimation to ambient temperature but also by the photoperiod. Key words: Heat tolerance; cold tolerance; terrestrial slug; temperature acclimation; photoperiod

interest because they lack physical protection, such as shells and epiphragms, in contrast to land snails. Because they lack protection, terrestrial slugs are directly exposed to hot air in summer and to freezing temperatures in winter, which can cause inoculative freezing of their moist skin when in contact with environmental ice crystals. Only a few studies have reported cold tolerance in terrestrial slugs (Mellanby, 1961; Cook, 2004; Storey et al., 2007). The terrestrial slug, Deroceras reticulatum, is partially freeze tolerant, and slugs collected in winter had a higher SCP and showed a higher survival rate in a frozen state than those collected in summer (Cook, 2004); however, the environmental factors influencing the seasonal changes in the SCP are still unclear. Few data are available regarding heat tolerance in terrestrial slugs (Carrick, 1941; Udaka et al., 2007). The terrestrial slug, Lehmania valentiana, is indigenous to Europe and was introduced into various countries in the temperate zone (Waldén, 1961). In the 1950s L. valentiana was introduced

INTRODUCTION The temperature tolerance of terrestrial mollusks and the environmental factors that influence it have been investigated mainly in land snails. Heat tolerance was investigated in the land snail Discus cronkhitei, and the effect of acclimation temperature on heat tolerance was confirmed (Riddle, 1990). Seasonal changes in cold tolerance have been investigated in some land snails, including Helix aspersa, Anguispira alternata, D. cronkhitei, and Gastrocopta armifera (Riddle, 1981; Riddle and Miller, 1988; Ansart et al., 2002). In these species, it was verified that acclimation temperature or photoperiod influences the supercooling point (SCP) (see Ansart and Vernon, 2003, for a review). Thus, much data is available regarding the temperature tolerance of land snails; however, data on the temperature tolerance of the other member of terrestrial mollusks, terrestrial slugs, are lacking. The temperature tolerance of terrestrial slugs is of

* To whom correspondence should be addressed at: E-mail: [email protected] DOI: 10.1303/aez.2008.547

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into Japan, and is now widely distributed and a common species (Kurozumi, 2002). Interestingly, this species develops and reproduces in autumn and winter in Osaka, Japan, in contrast to other terrestrial invertebrates in the temperate zone (Udaka et al., 2007). Generally, animals do not feed in autumn or winter to avoid the intake of ice-nucleating agents with food, because the intake of such agents results in a loss of cold tolerance. Thus, it seems that L. valentiana is much more susceptible to cold in winter and is maladaptive to survive winter. In the present study, we examined seasonal changes in heat and cold tolerance in L. valentiana. Slugs were collected from fields in Osaka, Japan over a period of one year. To clarify the environmental factors that affect seasonal changes in temperature tolerance, we also investigated the effects of photoperiod and acclimation temperature in L. valentiana hatched under laboratory conditions. MATERIALS AND METHODS Field-collected slugs. To examine seasonal changes in temperature tolerance, L. valentiana was collected at four-week intervals from the campus of Osaka City University, Osaka, Japan (34.6°N, 135.5°E), from August 2003 to July 2004, except in May 2004, and from April to June 2005. Slugs were left at room temperature until the experiments were conducted, and temperature tolerance was examined within 10 h of collection. Juvenile slugs collected in March were small, with body weights as light as 0.01 g; however, mature slugs collected in January were as heavy as 1.5 g. Although two generations overlapped in the field from November to June (Udaka et al., 2007), they could be distinguished based on their body color; the ground color of young (immature) slugs is pale reddish-brown and that of old (mature) slugs is dirty yellowish-brown (Kanô et al., 2001). In March 2004, the temperature tolerance of both old and young slugs was examined. From April to June 2004 and 2005, only young slugs were used in the experiment, because only a small number of old slugs had survived in the field. Slugs reared under laboratory conditions. Eggs laid by slugs collected from the campus of Osaka City University or by their progeny reared under short-day conditions (12 h light and 12 h darkness, LD 12 : 12) at 15
1°C were used. Eggs

within 24 h of oviposition were maintained under LD 12 : 12 at 20
1°C until they hatched. Juvenile slugs within 48 h after hatching were reared in a group of 10–40 individuals in a plastic pot (diameter150 mm, depth66 mm) under short-day (LD 12 : 12) or long-day conditions (LD 16 : 8) at 20
1°C. The slugs were fed insect food (Oriental Yeast, Tokyo, Japan). Thirty days after hatching, the slugs were transferred into a different plastic pot (10 slugs per pot), and were then fed carrots and insect food. Forty-five days after hatching, slugs were maintained at a temperature of 15 or 25°C or were kept continuously at 20°C under the same photoperiodic conditions. Temperature tolerance was examined 60 days after hatching. Determination of lethal temperatures. Seven to ten slugs were placed in a 50-ml plastic tube. For heat tolerance examinations, moist filter paper was placed in the tube to maintain a high level of humidity. The tube was sealed with Parafilm® M (Pechiney Plastic Packaging, Chicago, IL, USA) and placed in a climatic chamber for exposure between 11 and 4°C, or in a water bath or a glycerol bath for exposure between 31 and 37°C. Slugs were exposed to each test temperature for 1 h. After exposure to heat or cold, slugs were placed in a plastic pot with wet paper and maintained at LD 12 : 12 at 20°C; mortality was recorded 24 h after treatment. A total of 19 or more slugs were used in 2–4 replicates for each temperature exposure. The data from replicates were combined, and the mortality rate at each temperature was calculated. In field-collected slugs, the mortality was plotted against the exposure temperature, and temperatures that killed 25, 50, and 75% of the population (Lt25,50,75) were obtained by directly reading intercept. RESULTS Seasonal changes in temperature tolerance In field-collected slugs, heat tolerance was highest in July and August, and their higher Lt50 was 36.5°C (Fig. 1, upper panel). Heat tolerance gradually decreased thereafter until February, increasing slightly in March. In March, young slugs were more tolerant to heat than old slugs. From March to July, lethal temperatures increased gradually for young slugs. Cold tolerance in field-collected slugs was stable

Temperature Tolerance in a Slug

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Fig. 1. Seasonal changes in heat and cold tolerance in Lehmannia valentiana from August 2003 to July 2004 (circles) and from April to June, 2005 (triangles) in Osaka, Japan. Closed circles represent an old generation; open circles represent a new generation. The symbols indicate the lethal temperature (Lt) that causes 50% mortality (Lt50) and the bars indicate lethal temperature that causes 25% mortality (Lt25) and 75% mortality (Lt75). n19–40 for each test temperature.

in August and September, and the lower Lt50 was 5.6°C (Fig. 1, lower panel). Slugs acquired cold tolerance thereafter, and the lower Lt50 was 8.0°C in January and 7.8°C in February. In March, the lower Lt50 in old slugs suddenly increased to 5.8°C. Young slugs collected in March were highly susceptible to cold, and their lower Lt50 was 3.2°C. Cold tolerance of the new generation gradually increased, and the lower Lt50 was 7.2°C in May and thereafter decreased toward late July. Effects of acclimation temperature and photoperiod on temperature tolerance Temperature acclimation greatly affected heat tolerance, irrespective of the photoperiod (Fig. 2, right column). At test temperatures of 34 and 35°C, there were significant differences in mortality between the acclimation temperatures under both photoperiodic conditions. Slugs that were acclimated to 25°C were the most heat tolerant, those that were acclimated to 20°C showed intermediate levels of heat tolerance, and those acclimated to 15°C showed least heat tolerance under both photoperiodic conditions. Photoperiod also affected heat tolerance. Slugs reared under long-day conditions were significantly more tolerant to heat than those reared under short-day conditions at 20°C; however, the photoperiodic effect was less obvious at 25°C, and no significant differences were ob-

Fig. 2. Effects of photoperiod and temperature on heat and cold tolerance in Lehmannia valentiana. Slugs within 48 h of hatching were reared under long-day (LD 16 : 8) or shortday (LD 12 : 12) conditions at 20°C. Forty-five days after hatching, the slugs were transferred to 15 or 25°C temperature condition or were kept continuously at 20°C, under the same photoperiodic conditions. Sixty days after hatching, temperature tolerance was examined. Temperatures in the right margin show acclimation temperatures during the last 15 days. Statistical differences by Fisher’s exact test or the extended version of the test for three samples (http://aoki2.si.gunma-u.ac.jp/ exact/fisher/getpar.html; Mehta and Patel, 1998) are shown as ** (p0.01) and * (p0.05) between the photoperiodic conditions, and (**) (p0.01) and (*) (p0.05) between the acclimation temperatures in the middle panels. n20–35 for each symbol.

served between the photoperiods at 15°C. Cold tolerance was also affected by acclimation temperature (Fig. 2, left column). Slugs reared at 15°C were more tolerant to cold than those reared at 20 and 25°C; there were significant differences in mortality between the acclimation temperatures at most of the test temperatures under both photoperiodic conditions. The effect of photoperiod on cold tolerance was prominent when the slugs were acclimated to 15 or 25°C. DISCUSSION This is the first report to show annual changes in both heat and cold tolerance in terrestrial mollusks.

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In L. valentiana, heat tolerance was maximal in summer, decreased with decreasing ambient temperatures in autumn, and was minimal in winter. Heat tolerance increased from spring to summer. Conversely, cold tolerance was minimal in spring and summer and increased from autumn to winter. These patterns, except for the changes in cold tolerance from March to May, corresponded with ambient temperature changes, suggesting that seasonal changes in heat and cold tolerance are, at least partially, achieved via temperature acclimation. This finding was verified in our laboratory experiments, i.e., heat tolerance decreased and cold tolerance increased at lower ambient temperatures, whereas cold tolerance decreased and heat tolerance increased at higher ambient temperatures; however, the effect of ambient temperature is not always relevant in terrestrial mollusks. Heat tolerance decreased at lower acclimation temperature in the land snail, D. cronkhitei, but the effect of acclimation temperature was less obvious in another land snail, G. armifera (Riddle, 1990). Acclimation to lower temperatures decreases SCPs in the land snails D. cronkhitei, A. alternata, and G. armifera; however, it does not affect cold tolerance in H. aspersa (Riddle and Miller, 1988; Ansart et al., 2001). Even in the terrestrial slug, D. reticulatum, low temperature acclimation does not affect cold tolerance (Mellanby, 1961). Thus, the effect of temperature acclimation may be species-specific. Alternatively, photoperiodic conditions during acclimation or the duration of acclimation or both may greatly mask the acquisition of temperature tolerance, as pointed out by Ansart et al. (2001). Indeed, the present study revealed that the photoperiodic conditions affected temperature tolerance in L. valentiana (see below). Newly emerged slugs exhibited least cold tolerance in March; however, they gradually acquired higher cold tolerance toward May. In this period, slugs grow quickly in the field (data not shown; Kanô et al., 2001). In the intertidal snail, Littorina littorea, cold tolerance increases with body weight (Murphy and Johnson, 1980); therefore, the increase in body weight might contribute to the acquisition of cold tolerance from March to May in L. valentiana. Conversely, in our study, heat tolerance was rather stable during the same period. Body weight seemed to have little or no effect on heat tolerance.

Photoperiod affects heat tolerance in L. valentiana, i.e., long-day conditions promote the acquisition of heat tolerance. This is the first report to show the effect of photoperiod on heat tolerance in terrestrial mollusks. Both photoperiod and temperature conditions also probably contribute to seasonal changes in heat tolerance in the field. The effect of photoperiod on cold tolerance was obvious in L. valentiana, i.e., long-day conditions decreased cold tolerance, especially by acclimation to higher temperatures, whereas short-day conditions increased cold tolerance. This finding indicates that an increase in cold tolerance in autumn and winter is promoted not only by lower ambient temperatures but also by short-day conditions. In the land snail, H. aspersa, in which acclimation temperature does not affect the change in the SCP (Ansart et al., 2001), a decrease in photoperiod or short-day conditions lowers the SCP (Biannic and Daguzan, 1993; Ansart et al., 2001). The present results refuted the assumption that L. valentiana is susceptible to cold in winter because of the intake of ice-nucleating agents by active feeding. The species successfully acquired cold tolerance before winter, probably by temperature acclimation and the photoperiodic response. Although the physiological and biochemical mechanisms responsible for the acquisition of cold tolerance are still uncertain in this species, a cryoprotectant like glucose may contribute, as reported in the slug, Deroceras leave (Storey et al., 2007). REFERENCES Ansart, A. and P. Vernon (2003) Cold hardiness in molluscs. Acta Oecol. 24: 95–102. Ansart, A., P. Vernon and J. Daguzan (2001) Photoperiod is the main cue that triggers supercooling ability in the land snail, Helix aspersa (Gastropoda: Helicidae). Cryobiology 42: 266–273. Ansart, A., P. Vernon and J. Daguzan (2002) Elements of cold hardiness in a littoral population of the land snail Helix aspersa (Gastopoda: Pulmonata). J. Comp. Physiol. B 172: 619–625. Biannic, M. and J. Daguzan (1993) Cold-hardiness and freezing in the land snail Helix aspersa Müller (Gastropoda; Pulmonata). Comp. Biochem. Physiol. 104: 503–506. Carrick, R. (1941) The grey field slug Agriolimax agrestis L., and its environment. Ann. Appl. Biol. 29: 44–55. Cook, R. T. (2004) The tolerance of the field slug Deroceras reticulatum to freezing temperatures. CryoLetters 25: 187–194. Kanô, Y. et al. (2001) Distribution and seasonal maturation

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