3.3 ∣ Methylome
Recent studies have highlighted the importance of DNA methylation for understanding insect phenotypic plasticity and biological complexity (Lyko et al., 2011; Wang et al., 2014). To investigate the potential involvement of epigenetic regulation in metamorphosis of WWS, we first performed a comparative methylome analysis for male and female first instar nymphs by whole genome bisulfite sequencing (WGBS).
We assessed the DNA methylation patterns of the WWS genome and assessed that 4.42% of genomic cytosines are methylcytosines in males and 3.90% in females (Figure 3a). There are three types of methylation sites in the WWS genome (CG, CHH, CHG), and nearly all methylcytosines were found in CG dinucleotides, which were substantially enriched in gene bodies (Supplementary Figs. 12-13, Supplementary Table 16). CG and non-CG (CHH, CHG) methylation analysis indicated a mosaic pattern that fluctuated drastically across the linkage groups (Figure 3b, Supplementary Figs. 14-18, Supplementary Table 17), with high methylated domains interspersed with regions of low methylation. Interestingly, we found that repetitive elements were highly methylated and exons had higher methylation levels than introns (Figure 3a), which is different from what has been observed from other insect species in which introns have higher levels of methylation except for in Locusta migratoria (Wang et al., 2014). Although WWS appears to harbor only one copy of the methyltransferase gene (Dnmt1 ), we observed much higher methylation levels (~4.2%) in comparison with what (0.1-1.6%) have been reported from other insect species such as silkworm and honeybees (Phalke et al., 2009; Regev et al.,1998; Xiang et al., 2010) (Figure 3b, Supplementary Table 18).
Through WGBS, a total of 385,025,000 CpG sites were identified, covering 14,020 genes in the WWS genome (Supplementary Table 17). Among the methylated genes, 1,699 genes showed significant methylation differences between males and females. The differentially methylated genes were distributed throughout the genome, and were mainly classified into 14 categories, including metabolic process, developmental process and response to stimulus (Figure 3c, Supplementary Figs. 19, Supplementary Table 19). Many of these categories have been documented in various hexapoda species to have crucial roles in lipid and protein metabolism, tissue morphogenesis and organ development, which are all tied to metamorphosis in insects and other animals (Williams & Carrol, 2009). Term enrichment analysis using KEGG pathways yielded seven terms that were significantly different, including i) the renin–angiotensin system (RAS), ii) renin secretion, iii) glutathione metabolism, iv) fatty acid biosynthesis, v) starch and sucrose metabolism, vi) neuroactive ligand-receptor and vii) insulin secretion (Figure 3d). Intriguingly, the genes associated with the top seven KEGG terms all displayed higher methylation levels in males compared to females (Figure 3d, Supplementary Table 20). Our results suggest that males undergo more physiological changes in the first instar nymph, with concomitant high DNA methylation levels, while females appear to continue having the same methylation levels in different developmental stages. In addition, our data indicated that differential expression of genes with roles in hormone and energy metabolism were the basis for sexually dimorphic development. Methylation differences in muscle development genes were enriched in the first instar nymph, which may be linked with dimorphic development, particularly in wing-formation in males (which requires wing muscles) and in leg degeneration in females (which equates the loss of muscle mass) (Supplementary Figs. 2, Supplementary Table 21-22).