Identification and profiling of miRNAs in the freeze-avoiding gall moth Epiblema scudderiana via next-generation sequencing.

The rapid development of high-throughput next-generation sequencing approaches in recent years has facilitated large-scale discovery and expression analysis of non-coding RNAs, including miRNAs, in traditional and non-traditional animal models. Such an approach has been leveraged to amplify, identify, and quantify miRNAs in several models of cold adaptation. The present study is the first to investigate the status of these small RNAs in an insect species that uses the freeze avoidance strategy of cold hardiness, the gall moth Epiblema scudderiana. To characterize the overall miRNA expression profile and to identify cold-modulated miRNAs in control (5 °C) and cold-exposed (-15 °C) E. scudderiana larvae, a next-generation sequencing-based approach was undertaken. A total of 44 differentially expressed miRNAs were identified between the two conditions; 21 up-regulated miRNAs and 23 down-regulated miRNAs in -15 °C-exposed larvae as compared with controls. Among the most significant changes observed in miRNAs with potential relevance to cold adaptation were elevated miR-1-3p, miR-92b-3p, and miR-133-3p levels as well as reduced miR-13a-3p and miR-13b-3p levels in E. scudderiana larvae exposed to cold temperatures. Expression values obtained from next-generation sequencing were also validated by a quantitative PCR approach for five miRNAs; miR-34-5p, miR-274-5p, miR-275-3p, miR-307a-3p, and miR-316-5p. Overall, this work provides the first description of a miRNA signature for subzero survival by a freeze-avoiding insect using a high-throughput approach and positions a new group of miRNAs at the forefront of the molecular changes underlying cold adaptation.