Differential expression of miRNAs during diapause
We began by looking at global patterns of miRNA expression in order to gain an overview of temporal changes across diapause. General expression patterns observed via multidimensional scaling (MDS) showed that direct developing individuals have a linear development pattern largely moving along dimension 2 in both head and abdomen tissue. Diapause individuals have a divergent development that moves along dimension 1 reaching a maximal divergence at day 114-144, after which miRNA expression converges with the developmental trajectory of the direct developing individuals at diapause day 155 and direct day 3 (Figure 2).
Continuing with our global perspective, differential expression analysis found that there were 117 DE miRNAs between all comparisons in the head tissue, and 49 in abdomen tissue (Table S4). In order to identify clusters of similarly expressed miRNA during the course of diapause development, differentially expressed miRNAs among timepoints of diapause were used as input for a cluster analysis. The optimal number of clusters for each of the analyses was 7 for the head tissue (Figure 3) and 5 for the abdomen tissue (Figure 4). When DE miRNAs were filtered to fit a membership of > 0.8 to one of the clusters, the number of DE miRNAs were reduced to 83 in the head, and 25 in the abdomen. Of these filtered DEGs, 19 were in both head and abdomen samples.