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).