DESICCATION TOLERANCE AND HEAT-SHOCK PROTEINS EXPRESSION IN ACTIVE AND DORMANT AMPHIBOLUS VOLUBILUS (EUTARDIGRADA)

Invertebrates living in extreme habitats at high altitudes or latitudes, as those living in ephemeral habitats, must be able to survive environmental stresses such as desiccation and freezing. In this contest several important scientific issues are emerging. Organisms can have four responses to overcome environmental stresses: regulative, acclimation, developmental, and evolutionary responses. Regulative and acclimation responses are carried out by the organisms in short or very short time to maintain their internal conditions for appropriate optimal functioning. Both responses are reversible in such a way as to follow the environmental fluctuations. They are also measurable according to a space-time scale depending on the size and the length of the life cycles of the organisms. Considering tardigrades, their persistence in unpredictable habitats is due to two widespread adaptive strategies, which are regulative responses: ability to enter cryptobiosis (both anhydrobiosis and cryobiosis) and/or to enter cyst. Anhydrobiosis and encystment are certainly characterized by several molecular events only partly identified. Other than the disaccharide trehalose, several stress proteins seem to be further keys to understand anhydrobiotic mechanisms. In particular, heat shock proteins (Hsps) and their molecular partners, which play diverse roles, including that of molecular chaperons, even in unstressed cells, in successful folding, assembly, intracellular localization, secretion and degradation of other proteins.A moss-dwelling eutardigrade collected in Northern Apennines (Modena, Italy; 1700 m a.s.l.), Amphibolus volubilis, has been utilized in our lab to evaluate the survival strategies in unpredictable habitats. It is known that this species is able to enter anhydrobiosis and cryobiosis and to form cysts. Lab experiments on desiccation tolerance have been carried out considering three different values of air relative humidity (RH). Survival resulted directly related to the RH values, and high survival (about 80%) was obtained only with the highest RH value tested (85%). Desiccated animals were utilized to evaluate the Hsp70 and Hsp90 expression by means of SDS-Page and Western blotting analysis. Hsp expression was also evaluated in active animals and (only Hsp70) in encysted (type 2 cyst) animals. Quantitative comparisons of protein expression have been made among these three conditions. The level of both Hsp70 and Hsp90 is higher in desiccated animals than in active ones. Encysted animals have a Hsp70 level lower than both active and desiccated animals. These results indicates that Hsp induction due to desiccation is necessary to preserve cells from desiccation damages. Instead, the lower Hsp level found in cysts with respect to active animals could be explained hypothesizing a little need of these proteins in animals already protected from environmental stresses by strong cuticular layers.