Maintenance of hindgut reabsorption during cold exposure is a key adaptation for Drosophila cold tolerance.

Maintaining extracellular osmotic and ionic homeostasis is crucial for organismal function. In insects, hemolymph volume and ion content is regulated by the secretory Malpighian tubules and reabsorptive hindgut. When exposed to stressful cold, homeostasis is gradually disrupted, characterized by a debilitating increase in extracellular K+ concentration (hyperkalemia). Accordingly, studies have found a strong link between species-specific cold tolerance and the ability to maintain ion and water homeostasis at low temperature. This is also true for drosophilids where inter- and intra-specific differences in cold tolerance are linked to the secretory capacity of Malpighian tubules. There is, however, little information on the reabsorptive capacity of the hindgut in Drosophila To address this, we developed a novel method that permits continuous measurements of hindgut ion and fluid reabsorption in Drosophila We demonstrate that this assay is temporally stable (∼2 h) and responsive to cAMP stimulation and pharmacological intervention in accordance with the current insect hindgut reabsorption model. We then investigated how cold acclimation or cold adaptation affected hindgut reabsorption at benign (24°C) and low temperature (3°C). Cold-tolerant Drosophila species and cold-acclimated D. melanogaster maintain superior fluid and Na+ reabsorption at low temperature. Furthermore, cold adaptation and acclimation caused a relative reduction in K+ reabsorption at low temperature. These characteristic responses of cold adaptation/acclimation will promote maintenance of ion and water homeostasis at low temperature. Our study of hindgut function therefore provides evidence that adaptations in the osmoregulatory capacity of insects are critical for their ability to tolerate cold.