Parallel ionoregulatory adjustments underlie phenotypic plasticity and evolution of Drosophila cold tolerance

Low temperature tolerance is the main predictor of variation in the global distribution and performance of insects, yet the molecular mechanisms underlying cold tolerance variation are poorly known, and it is unclear whether the mechanisms that improve cold tolerance within the lifetime of an individual insect are similar to those that underlie evolved differences among species. The accumulation of cold-induced injuries by hemimetabolous insects is associated with loss of Na(+) and K(+) homeostasis. Here we show that this model holds true for Drosophila; cold exposure increases haemolymph [K(+)] in D. melanogaster, and cold-acclimated flies maintain low haemolymph [Na(+)] and [K(+)], both at rest and during a cold exposure. This pattern holds across 24 species of the Drosophila phylogeny, where improvements in cold tolerance have been consistently paired with reductions in haemolymph [Na(+)] and [K(+)]. Cold-acclimated D. melanogaster have low activity of Na(+)/K(+)-ATPase, which may contribute to the maintenance of low haemolymph [Na(+)] and underlie improvements in cold tolerance. Modifications to ion balance are associated with both phenotypic plasticity within D. melanogaster and evolutionary differences in cold tolerance across the Drosophila phylogeny, which suggests that adaptation and acclimation of cold tolerance in insects may occur through similar mechanisms. Cold-tolerant flies maintain haemolymph osmolality despite low haemolymph [Na(+)] and [K(+)], possibly through modest accumulations of organic osmolytes. We propose that this could have served as an evolutionary route by which chill-susceptible insects developed more extreme cold tolerance strategies.