The transcriptomic and physiological basis of desiccation stress response in natural European D. melanogaster populations
Climate change is one of the main factors shaping the distributions and biodiversity of organisms, among others by greatly altering water availability, thus exposing species and ecosystems to harsh desiccation conditions. Insects are specially threatened by these challenging dry environments, thanks to their small size and thus large surface area to volume ratio. Drosophila melanogaster is a great model to study the response of populations to rapidly changing conditions, because of its southern African origin and recent and fast worldwide spreading. Desiccation stress response is a complex and extensively studied trait, however the natural variation and underlying molecular and physiological mechanisms responsible for the tolerance in natural European D. melanogaster populations has never been studied. Here we subjected to desiccation stress 74 natural D. melanogaster European strains, belonging to five different climate zones. We found that the strains belonging to the cold semi-arid climates are more tolerant compared to the others, and that this tolerance can be explained by the combination of altitude and evaporation. The tolerant strains had a lower level of initial water content and lost less water during desiccation stress. We found that the reduction in the water loss happens through a decrease in the respiration rate in desiccation stress conditions, and by having a less permeable cuticle prior the stress exposure. The decreased rate of respiration in the tolerant strains is a consequence of the down-regulation of genes related to diverse metabolic processes. Moreover, we found that the genes related to response to stimulus and environmental sensing are up-regulated only in the tolerant strains. We also identified four transposable element insertions, which might be responsible for changes in gene expression. Overall, our study for the first time described the physiological and transcriptomic changes underlying the desiccation tolerance of natural European D. melanogaster strains, and generated a list of putative mutations shaping desiccation stress response. Overall design: Six Drosophila melanogaster strains (three  tolerant to desiccation stress: ES_GIM_15_12 (GIM-012), ES_GIM_15_24 (GIM-024),ES_COR_15_23 (COR-023) and three sensitive to desiccation stress ES_TOM_15_8 (TOM-08), LUN_15_7 (LUN-07) and MUN_15_13 (MUN-013)) were chosen for RNA sequencing. For each of the six strains, three replicates were subjected to desiccation stress (treated samples) and other three acted as control. In treated conditions,  flies were placed in empty vials and between the cotton and the parafilm, three grams of silica gel (Merck) were placed, so they were starved and desiccated. Control vials were prepared similarly, except that it contained 1mL of 1% agar on the bottom of the vial to prevent desiccation. Agar in the tubes provides hydration but not food source for the control flies (Sinclair et al 2007).