Calcium signaling mediates cold sensing and triggers rapid cold-hardening in insect tissues

The ability to rapidly respond to changes in temperature is a critical adaptation for insects and other ectotherms living in thermally variable environments. In a process called rapid cold-hardening (RCH), insects significantly enhance cold tolerance following brief (i.e. minutes to hours) exposure to non-lethal chilling. While the ecological relevance of RCH is well-established, the underlying physiological mechanisms that trigger RCH are poorly understood. Because of the speed at which RCH occurs, it is thought to be primarily regulated by post-translational cell-signaling events. Also, RCH can be elicited in isolated tissues ex vivo, suggesting cold-sensing and downstream hardening pathways are governed by signaling mechanisms that are independent of the brain and hormones. In this study, we test the hypothesis that calcium signaling mediates cold-sensing and is required for RCH in insect tissues. In tracheal cells of the freeze-tolerant goldenrod gall fly, Eurosta solidaginis, chilling to 0°C evoked a 40% increase in intracellular calcium concentration as determined by live-cell confocal imaging. Downstream of calcium entry, RCH conditions significantly increased the activity of calcium/calmodulin-dependent protein kinase II (CaMKII) while reducing phosphorylation of the inhibitory Thr306 residue. Pharmacological inhibitors of calcium entry, calmodulin activation, and CaMKII activity all prevented ex vivo RCH in midgut and salivary gland tissues, indicating that calcium signaling is required for RCH to occur. Similar results were obtained for a freeze-intolerant species, adults of the flesh fly, Sarcophaga bullata, suggesting calcium-mediated cold-sensing is a general feature of insects. Our results imply that insect tissues use calcium signaling to instantly detect decreases in temperature and trigger downstream cold-hardening mechanisms. Future efforts in this project seek to identify the downstream signaling targets and phosphorylation events that regulate rapid responses to low temperature.