Adaptability of antioxidant defence system in Helix pomatia snails: effect of forced aestivation during early spring

Brown garden snails (Helix pomatia L.), spend cold winter seasons as well as dry and hot summer seasons in torpor, which is characterized by a strong reduction of metabolic rate (Guppy &Withers, 1999). At the end of winter torpor the snails enhance the capacity of their antioxidant defence system to prevent oxidative stress, which accompanies return from hypometabolism to activity (Nowakowska et al., 2009a). The augmented antioxidant defence, including synthesis of glutathione and glutathione-related enzymes, has also been shown in Cornu aspersum (Ramos-Vasconcelos & Hermes-Lima, 2003; RamosVasconcelos, Cardoso & Hermes-Lima, 2005) during dormancy/ activity cycles. On the other hand, active H. pomatia exhibit a high level of antioxidant defence during summer (Nowakowska et al., 2009b), which can be regarded as a preadaptation to unpredictable, irregular episodes of aestivation. Because the capacity of antioxidant defence in active snails during spring was lower than that recorded at the same time in their counterparts continuing in torpor (Nowakowska et al., 2009a), we wondered how they would respond to aestivation, induced experimentally, in this period. It must be stressed that early spring weather in the north temperate zone of central Poland can occasionally be hot and dry enough to induce aestivation. Therefore, the ability of snails to survive under such stressful conditions should be determined by the adjustment to sudden environmental challenges. We examined the ability of H. pomatia to modulate their antioxidant defence in response to dehydration-induced torpor during early spring and we compared their response with that previously recorded in torpid snails during summer (Nowakowska, Caputa & Rogalska, 2011). The capacity of antioxidant defence was estimated as activities of antioxidant enzymes (catalase, CAT; total glutathione peroxidase, GPX; selenium-dependent glutathione peroxidase, Se-GPX and glutathione transferase, GST), as concentration of reduced glutathione (GSH) and as end products of lipid (thiobarbituric acid reactive substances, TBARS) and protein (carbonyl protein groups, CP) peroxidation during the aestivation/ arousal cycle in the kidney, hepatopancreas and foot of 30 adult specimens of H. pomatia. The snails were collected from their natural habitat in central Poland (538020N, 188350E) as soon as they emerged from winter torpor during spring (24 April) and they were immediately deprived of food and water to induce aestivation (n 1⁄4 18). The aestivating snails were used in one of three experimental treatments: (1) after 3 weeks of aestivation (n 1⁄4 6), (2) just after arousal from aestivation (n 1⁄4 6) and (3) 24 h postarousal (n 1⁄4 6). Snails in groups 2 and 3 were sprayed with water to interrupt aestivation, which happened within a few minutes. The control group (n 1⁄4 6) consisted of active snails, examined just after being collected from the field. In addition, snails in additional control group (n 1⁄4 6) collected in the field together with the experimental snails, were put into a cage, sprayed with water every day and fed ad libitum. These were used to examine the effect of laboratory conditions on the antioxidant defence system. The methods used to examine activities of antioxidant enzymes and concentrations of GSH, TBARS and CP have been described in detail elsewhere (Nowakowska et al., 2009a,b; Nowakowska, Caputa & Rogalska, 2010, 2011; Nowakowska et al., 2014). The results were analysed by two-way ANOVA (experimental conditions x organs as factors), followed by Tukey-Kramer post hoc test. The investigation showed that snails forced to aestivate in early spring did not show signs of preparatory changes for arousal-induced oxidative stress, such as increased level of CAT, which was observed in summer-torpid snails (Nowakowska et al., 2009b; Nowakowska et al., 2010, 2011), but forced aestivation led to a strong decrease in the kidney’s enzyme activity (Fig. 1). In both spring (Fig. 1) and summer aestivation (Nowakowska et al., 2011) the lowest activities of CAT were recorded in the foot and were significantly different (P , 0.001) from those in the hepatopancreas and kidney, but in spring there was no compensatory increase in activity of total GPX in the foot, which has been recorded in summer throughout both torpor and activity cycles and under control conditions (Nowakowska et al., 2011). In the present paper, activity of total GPX was found to be organ-dependent, but it was unaffected by experimental conditions; during the spring, the aestivation/arousal cycle activity of the enzyme remained relatively stable (except for transient changes in postarousal activity of the enzyme in the kidney indicated by the post hoc test, see Fig. 1). Our previous studies showed some discrepancies in activity of total GPX in summeraestivating snails; in one case (Nowakowska et al., 2009b) there was a transient, highly significant increase in the activity in the kidney of snails freshly aroused from aestivation, but in two other cases (Nowakowska et al., 2010, 2011) aestivation/arousal cycles did not influence activity of the enzyme in the kidney or that in two other organs. In the present study, activity of Se-GPX was influenced by experimental conditions and organ type, and the highest activity was recorded in the foot (Fig. 1). Moreover, in all experimental groups, activities of Se-GPX in