THE TOLERANCE of the FIELD SLUG Deroceras Reticulatum to FREEZING TEMPERATURES
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CryoLetters 25, 187-194 (2004) Ó CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK THE TOLERANCE OF THE FIELD SLUG Deroceras reticulatum TO FREEZING TEMPERATURES R T Cook Kingston University, Kingston upon Thames, Surrey KT1 2EE, United Kingdom Abstract Cold hardiness of ectotherms has been widely studied in arthropods, but there is a more limited literature on the survival of molluscs at low temperatures. A number of intertidal species have been examined in detail, but terrestrial molluscs have largely been overlooked until recently. This paper reports results of laboratory experiments to evaluate the cold hardiness of the terrestrial slug, Deroceras reticulatum. The mean supercooling point (SCP) rose from -4.2°C in summer to -3.6°C in winter. The SCP that caused 50% mortality (LSCP50) remained constant at -4.7 to -4.8°C in both seasons, but slugs were able to survive the frozen state for longer in winter (LD50 of 31.8 minutes compared with 17.0 minutes in summer). Slug survival at freezing temperatures was prolonged to at least five hours when placed on a moist, absorbent substrate. D. reticulatum exhibits partial freeze tolerance, with an increased survival in winter. The results are discussed in relation to the slugs' natural environment. Keywords: cold hardiness, Deroceras, freezing, molluscs, slugs, supercooling INTRODUCTION In order to survive exposure to freezing temperatures, ectothermal organisms require physiological mechanisms to prevent or tolerate the formation of ice in their tissues. Such 'cold hardiness' has been reported in a number of invertebrates, and has been most extensively studied in arthropods (8, 17). Organisms that survive freezing temperatures have classically been categorised as either 'freeze susceptible' (also known as a 'freeze avoidance' strategy), in which body fluids undergo extensive supercooling without ice formation, or 'freeze tolerant' if the organism can tolerate the formation of extracellular ice in its tissues. However, other classifications have also been proposed to account for intermediate responses to low temperatures seen in a number of organisms (6, 16). Among gastropod molluscs, cold hardiness has been studied in intertidal species that are exposed to freezing temperatures for periods of up to a few hours at low tide (10), but relatively little is known about cold hardiness of terrestrial molluscs (4). The land snails Helix aspersa, H. pomatia and Arianta arbustorum have been classified as partially freeze tolerant, exhibiting a limited ability to supercool and survival of ice crystal formation in its tissues for between 40 minutes to a few hours (1, 4, 20). The few other species studied, Discus cronkhitei, Anguispira alternata, Gastrocopta armifera and Vallonia perspectiva are all small snails that have adopted a freeze avoidance strategy and can supercool to temperatures as low as -25°C (13, 15). Although closely related to pulmonate snails, terrestrial slugs possess two differences that are relevant in the context of survival at low temperatures. The first is that, despite some reports to the contrary (eg 9), they are not thought to hibernate in winter (18, 19), although 187 activity does decline with temperature and usually ceases as it approaches 0°C (12). The second is that, since they lack a shell into which they can retreat, they are susceptible to inoculative freezing. A freeze avoidance strategy would therefore seem an unlikely mechanism to survive freezing temperatures. The field slug, Deroceras reticulatum, is a common, geographically widespread pest of agricultural and horticultural crops in moist, temperate environments (19), but little is known about its over-wintering behaviour, survival and ecology. It has often been assumed that adults are killed during very cold winters, with subsequent generations being supplemented from over-wintering eggs (18). This slug species appears to be well adapted to cold temperatures, and is active at temperatures of around 0°C (12, 21). Furthermore, it has also been reported that D. reticulatum adults have a limited ability to survive freezing (5). However, personal observations (unpublished) revealed that field slugs survived freezing overnight in boxes of soil within an arable field, suggesting a greater tolerance of freezing. In order to provide further insights in to the cold hardiness of adult D. reticulatum at low temperatures, work was undertaken to investigate the supercooling points and survival of these slugs in winter and summer, as well as some observations of their behaviour in laboratory conditions. MATERIALS AND METHODS Adult slugs, weighing between 30 mg and 75 mg, were collected from gardens and a cemetery in Surrey, UK, on the day that they were to be used in the experiments. They were housed in an incubator set at 10°C for a maximum duration of four hours before use to minimize any possibility of becoming acclimatised to the artificial environment. For the purpose of this work, winter was defined as between November and February, and summer was defined as from May to August. Three experiments were performed over a 16-month period from November 1999 to February 2001. Experiment 1: supercooling points and lethal freezing temperatures This experiment was designed to measure the temperature at which slugs froze in winter and summer, their survival, and to identify if the rate of cooling influences the supercooling point (SCP). Individual slugs were placed in a 1 ml microfuge tube from which the closed end had been cut open and sealed with a fine cotton mesh to allow air circulation. The slug was gently pushed in to the bottom of the tube which was plugged with cotton wool to hold the slug firmly in place. Two miniature catheter thermistor probes (Grant Instruments (Cambridge) Ltd, UK) were then gently inserted into the tube so that they pressed against the slug, and were held in place in the tube with insulation tape. Consequently, the slug was firmly confined in the tube, allowing very little movement and with the temperature probes constantly pressed against the body. The tube was placed in a 12 x 9 cm plastic bag which was suspended in a programmable cooling bath (Julabo Labortechnik GmbH, Seelbach, Germany, model HP-F32) containing Thermistor H5S oil (Julabo Labortechnik GmbH), ensuring that no oil entered the bag. A third thermistor in a second empty microfuge tube was also placed in the plastic bag so that the air temperature in the tube could be monitored. The thermistor probes were connected to a Squirrel SQ1002 data logger (Grant Instruments (Cambridge) Ltd, UK), and the SCP of the slug was recorded as the temperature at which the latent heat of crystallization was first detected. Once the slugs had frozen, the temperature of the cooling bath oil was maintained so that the air temperature in the microfuge tube was at, or just below, the slug’s SCP for five minutes. The cooling bath was programmed to produce a cooling and warming rate of the air in the microfuge tube at 1 deg C per minute or 1 deg C per five minutes. Once the body temperature of the slug had returned to 5°C, the tube containing the slug was taken out of the cooling bath, 188 and the cotton wool plug and thermistor probes were removed. The tube was placed in a clear plastic box lined with moist tissue and placed in a fridge at 5°C for one hour prior to being placed in an incubator at 15°C, when the condition of the slug was noted. The survival and physical condition of the slug was then monitored over the next five days. Although a five day duration does not conclusively demonstrate lack of a detrimental effect from freezing, it was sufficient to identify any acute damage. Indeed, all slugs that exhibited signs of damage, such as limited locomotory ability, died within the five day period. A control slug was placed in a sealed and plugged microfuge tube in an incubator with an air temperature of 15°C for the duration that the slugs were contained within the cooling bath. In addition, the data were submitted to probit analysis to estimate the SCP at which fifty percent of the slugs survived, without any apparent damage, for five days after being frozen for five minutes (lethal supercooling point, or LSCP50) in both summer and winter. Experiment 2: duration of survival The second experiment was designed to assess the duration that the slugs could withstand in the frozen state in winter and in summer. Slugs were cooled at a rate of 1 deg C per minute, using the same method as above, but maintaining the air temperature to which the slugs were exposed at between 0.5°C and 1°C below their SCP for 5, 15, 30, 45 or 60 minutes. By maintaining the temperature at just below the SCP for each slug, the proportion of body tissue ice formed was likely to be comparable for all slugs frozen for the same time. The duration at which fifty percent of the slugs survived in the frozen state (lethal duration, or LD50) was calculated from the results by probit analysis. As in experiment 1, only slugs that remained active and apparently unharmed for five days after freezing were considered to have survived the treatment. Experiment 3: behaviour The final experiment involved filming groups of between four and six adult slugs on fourteen separate occasions (sixty slugs used in total) when the air temperature was reduced from 100C to –90C. The slugs were placed in plastic, open-topped boxes (14 x 7.5 x 5 cm) which were filled to a depth of 3.5 cm with florists’ oasis material soaked with water. The upper edges of the box were greased with petroleum jelly to which salt was applied as a barrier to prevent slugs from escaping.