Heat shock proteins and resistance to desiccation in congeneric land snails

Abstract

Land snails are subject to daily and seasonal variations in temperature and in water availability and depend on a range of behavioral and physiological adaptations for coping with problems of maintaining water, ionic, and thermal balance. Heat shock proteins (HSPs) are a multigene family of proteins whose expression is induced by a variety of stress agents. We used experimental desiccation to test whether adaptation to different habitats affects HSP expression in two closely related Sphincterochila snail species, a desiccation-resistant, desert species Sphincterochila zonata, and a Mediterranean-type, desiccation-sensitive species Sphincterochila cariosa. We examined the HSP response in the foot, hepatopancreas, and kidney tissues of snails exposed to normothermic desiccation. Our findings show variations in the HSP response in both timing and magnitude between the two species. The levels of endogenous Hsp72 in S. cariosa were higher in all the examined tissues, and the induction of Hsp72, Hsp74, and Hsp90 developed earlier than in S. zonata. In contrary, the induction of sHSPs (Hsp25 and Hsp30) was more pronounced in S. zonata compared to S. cariosa. Our results suggest that land snails use HSPs as part of their survival strategy during desiccation and as important components of the aestivation mechanism in the transition from activity to dormancy. Our study underscores the distinct strategy of HSP expression in response to desiccation, namely the delayed induction of Hsp70 and Hsp90 together with enhanced induction of sHSPs in the desert-dwelling species, and suggests that evolution in harsh environments will result in selection for reduced Hsp70 expression.

Fig. 1

Changes in mass and in extrapallial fluid osmolality during 21 days of normothermic desiccation in S. cariosa and S. zonata. a Changes in mass. The results are summation of two to three independent experiments (n = 21 snails for S. zonata and 32 snails for S. cariosa). All values are expressed as percentage of initial fully hydrated mass (mean ± SE). Asterisks denote significant differences between the two species (*p < 0.05; **p < 0.01; ***p < 0.001). b Changes in extrapallial fluid osmolality. All values are expressed as mean ± SE (n = 6 snails, except for aestivation group where n = 9–14 snails and rehydration group n = 4–6 snails). Asterisks denote significant differences from hydration group (*p < 0.05; **p < 0.01; ***p < 0.001)

Fig. 2

Expression of Hsp70 isoforms (Hsp72 and Hsp74) in the foot tissue of S. cariosa and S. zonata. Total protein was extracted from fully hydrated control and desiccated snails during 10 days of desiccation and subjected to Western blotting. a A representative blot for tissue lysate of three independent experiments is shown. Arrows denote the point of connection, where two consecutive gels were combined. b and c are graphical presentations of the relative levels of Hsp74 (b) and Hsp72 (c) in tissue lysates. n = 6–15 snails. Different letters above the bars denote significant differences within each species (lower case—S. cariosa; upper case—S. zonata). Asterisks denote significant differences between the two species (*p < 0.05; **p < 0.01; ***p < 0.001). d Relative levels of Hsp72 in the foot cytoskeletal–nuclear fraction. n = 7–8 snails. Asterisks denote significant differences within each species and also between the two species (*p < 0.05; **p < 0.01)

Fig. 3

Expression of Hsp70 and sHSPs in the foot (F), kidney (K), and hepatopancreas (H) tissues of S. cariosa and S. zonata. Total protein was extracted from snail tissues and subjected to Western blotting using an anti-Hsp70 (a) and anti-Hsp25 (b) antibodies. Lanes contain equivalent amounts of protein from tissue extracts prepared from individual snails

Fig. 4

Relative endogenous levels of Hsp72 and Hsp90 in the foot, kidney, and hepatopancreas tissues of S. cariosa and S. zonata. Total protein was extracted from fully hydrated, control snails and subjected to Western blotting. Levels of Hsp72 (a) and Hsp90 (b) are expressed relative to an internal Hsp70 protein (250 ng) or Hsp90 protein (200 ng) standards. n = 8–14 snails. Different letters above the bars denote significant differences within each species (lower case—S. cariosa; upper case—S. zonata). Asterisks denote significant differences between the two species (**p < 0.01)

Fig. 5

Expression of Hsp90 in the foot, kidney, and hepatopancreas tissues of S. cariosa and S. zonata. Total protein was extracted from fully hydrated control and desiccated snails during 10 days of desiccation and subjected to Western blotting. A representative blot of three independent experiments in the foot (a) and kidney (b) is shown. Arrows denote the point of connection where two consecutive gels were combined. Relative levels of Hsp90 in the foot (c), kidney (d), and hepatopancreas (e) are shown. n = 6–15 snails. Different letters above the bars denote significant differences within each species (lower case—S. cariosa; upper case—S. zonata). Asterisks denote significant differences between the two species (*p < 0.05; **p < 0.01; ***p < 0.001)

Fig. 6

Expression of Hsp25 in the foot tissue of S. cariosa and S. zonata. Total protein was extracted from fully hydrated control and desiccated snails during 10 days of desiccation and subjected to Western blotting. a A representative blot for tissue lysate of three independent experiments is shown. Arrows denote the point of connection where two consecutive gels were combined. b Relative levels of Hsp25 protein in tissue lysate. n = 6–15 snails. Different letters above the bars denote significant differences within each species (lower case—S. cariosa; upper case—S. zonata). Asterisks denote significant differences between the two species (*p < 0.05; **p < 0.01; ***p < 0.001). c Relative levels of Hsp25 protein in the cytoskeletal–nuclear fraction. n = 3–8 snails. Asterisks denote significant differences within each species and also between the two species (*p < 0.05; **p < 0.01)

Fig. 7

Expression of sHSPs in the kidney tissue of S. cariosa and S. zonata. Total protein was extracted from fully hydrated control and desiccated snails during 10 days of desiccation and subjected to Western blotting. a Relative levels of Hsp25. b Relative levels of Hsp30. n = 6–15 snails. Different letters above the bars denote significant differences within each species (lower case—S. cariosa; upper case—S. zonata). Asterisks denote significant differences between the two species (*p < 0.05; **p < 0.01)

Fig. 8

Expression of sHSPs in the hepatopancreas tissue of S. cariosa and S. zonata. Total protein was extracted from fully hydrated control and desiccated snails during 10 days of desiccation and subjected to Western blotting. a, c A representative blot of three independent experiments is shown. Arrows denote the point of connection where two consecutive gels were combined. b Levels of Hsp30 protein. d Levels of Hsp17 protein. n = 6–15 snails of both species. Different letters above the bars denote significant differences within each species (lower case—S. cariosa; upper case—S. zonata). Asterisks denote significant differences between the two species (**p < 0.01)