Hypothesis testing of major transitions during diapause
In order to gain additional insights beyond our GSEA, we next identified differentially expressed miRNAs during the period when pupae transitioned from the deepest portion of diapause to post diapause quiescence (day 24-144). During this period butterflies were held at 2°C, allowing us to identify miRNAs associated with diapause progression independent of temperature change. There were eight DEGs identified in both the head and abdomen tissue, and of those, only two were differentially expressed in both tissues, miR-14-5p and miR-2a-3p. These two miRNAs have inverse normalized expression patterns, where miR-14-5p increases in expression early in diapause, and declines to a minimum expression after 114 days (Figure 6). miR-2a-3p has an expression pattern that starts off at the lowest expression, and increased through time until day 114, where it stays until 144, afterwards it starts to decline (Figure 6).