Come rain or shine: the combined effects of physical stresses on physiological and protein-level responses of an intertidal limpet in the monsoonal tropics

P>1. Traditional approaches to understanding species responses to environmental conditions have focused on the isolated effects of single stressors, despite the fact that in nature organisms experience a variety of conditions. 2. In tropical monsoonal areas, intertidal animals can face hot desiccating conditions during emersion preceded, or followed by, intense rainfall. The combined effects of these stresses on physiological responses and protein profiles were investigated in a limpet, Cellana grata. 3. With short exposure (60 min) to single stressors, heat stressed limpets had elevated heart rates and more concentrated haemolymph and mantle water osmolalities than under normal temperatures or awash. Animals under rain had reduced haemolymph and mantle water osmolalities, but similar heart rates to unstressed animals. 4. After 120 min, unstressed animals did not differ in their physiological responses. Heat stressed limpets, however, had faster heart rates and more concentrated haemolymph and mantle water osmolalities, whilst those under rain had the lowest osmolalities, but similar heart rates to unstressed animals. 5. Limpets under rain followed by heat stress had faster heart rates, but lower haemolymph and mantle water osmolalities compared to animals under normal temperatures or heat stress. Limpets that were heat stressed, followed by rain, had similar heart rates to animals awash, under rain or normal temperatures but lower haemolymph osmolalities than other treatments, with the exception of limpets under rain. 6. There was a positive relationship between haemolymph and mantle water osmolalities, except for animals under rain, where mantle water osmolality was lower than the haemolymph, suggesting some isolation of body fluids from the external medium. 7. Haemolymph protein/peptide mass spectra of heat stressed animals (either before or after rain) were similar, while all other treatments differed, suggesting differential expression and regulation of proteins. 8. Heat stress invokes a more active physiological and protein level response than rain, but their combination had an interactive effect on limpets' metabolism. 9. Identifying the effects of multiple stresses at a variety of biological levels highlights the interactive effects which impact species, and provides a more complete understanding of how species may respond to environmental changes in their natural habitats.