Thermal profiling of novel and known miRNAs during cold acclimation in Aedes aegypti

The disease vector, Aedes aegypti, can better survive low temperature exposure following cold acclimation. This plasticity may be contributing to this species' recent range expansion. However, the molecular mechanisms behind this ability remain unexplored. MicroRNAs (miRNAs) can drive rapid changed in gene expression and are known to regulate thermal plasticity in other animals. We used a bioinformatic approach to identify three novel miRNAs in Ae. aegypti. After acclimating adult female Ae. aegypti to cold (15 °C) and warm (25 °C) conditions, we used RT-qPCR to perform differential expression analysis on these novel miRNAs and four other miRNAs downregulated during diapause in another mosquito species. Two novel miRNAs, aae-miR-novel2 and aae-miR-novel3, were upregulated in cold-acclimated individuals. Two described miRNAs associated with lipid metabolism and reproduction, aae-miR-8-3p and aae-miR-13b-3p, were downregulated in the cold-acclimated group, suggesting these processes are affected during cold acclimation. Temperature alone can alter miRNA target genes, and the thermodynamic parameters used to predict miRNA targets are typically established assuming body temperatures relevant to endotherms, rather than those applying to poikilothermic ectotherms. Here, we instead tested for cold-influenced targets of miRNAs of interest, predicted to only become thermodynamically stable at 15 °C. This approach provides insight into the putative roles that these novel miRNAs could play in cold acclimation. Many predicted targets were involved in processes associated with insect low temperature survival, like ion homeostasis and membrane stability, while others suggest roles for understudied processes. Our results suggest that miRNAs play a regulatory role in mosquito thermal plasticity that deserves further attention.