DNA methylation is an important epigenetic modification in plants and animals [1
]. It has been shown to play key roles in a broad range of biological processes, including embryonic development [5
], histone modification [6
], X chromosome inactivation [7
] and brain development [8
]. In mammals, a primary function of DNA methylation is to suppress gene expression through increased promoter DNA methylation [9
]. The roles of methylation include regulation of transcriptional activities [1
], alternative exon splicing [10
] and developmental activities [11
]. DNA methylation in animals is accomplished by two types of DNA methyltransferases (DNMTs), de novo and maintenance DNMTs [12
]. De novo DNMTs are responsible for establishing new methylation patterns by the Dnmt3 family of proteins, and maintenance DNMTs maintain previously established methylation patterns and are represented by the Dnmt1 family of proteins [13
]. In contrast to these DNMTs, Dnmt2 shows a weak methyltransferase activity towards DNA and has been characterized as an active RNA methyltransferase [12
]. Although Drosophila melanogaster
possesses only a single DNMT (Dnmt2), the DNA methylation rate in the genome is at a low detectable level [15
In insects, DNA methylation is found at appreciable levels in the genomes of many, but not all, insects [16
] and is primarily targeted to genes that are broadly expressed in the genome [17
]. The presence of a functional DNA methylation system across the insect class with conserved patterns of methylation [18
], suggests an important role for this epigenetic marker in insect biology. The importance of DNA methylation in insect development has been strikingly demonstrated in Apis mellifera
, which is capable of influencing developmental plasticity [21
]. Chen et al. provided evidence for a functional DNA methylation system in Plutellaxylostella
and its possible role in adaptation during host transfer [24
]. However, fundamental questions remain on the patterning of DNA methylation during insect development; in particular, how DNA methylation affects the functional mechanism of early insect embryonic development and insect diapause.
Advances in whole-genome sequencing, coupled with bisulfite DNA treatment (WGBS) have led to single-nucleotide resolution methylation maps in a wide range of invertebrates [10
]. Most recently, Jones et al. used WGBS and behavioural assays in a detailed analysis of the Helicoverpaarmigera
methylome to investigate potential methylation differences in a subset of genes, demonstrating distinct flight performances in insects [27
]. Similarly, Guan et al. examined the effects of Cd exposure on global DNA methylation using WGBS in Drosophila melanogaster
]. In the silkworm, WGBS was used to investigate the silkworm mid-gut, identifying 0.11% methylcytosine levels across genomic cytosines [28
]. Recently, Wu et al. used WGBS to demonstrate that epigenetic regulation may play roles in host–virus interactions in the silkworm [29
]. As a robust and effective technique, WGBS is becoming more and more popular for insect DNA methylation research, including in the silkworm.
The silkworm is an economically important model insect of the lepidopteran order. Previous silkworm studies have shown that ~0.11% of their DNA is methylated [28
], with only Bombyx moriDnmt1
) and Bombyx moriDnmt2
) proteins being reported [28
], and BmDnmt1
retains the function as maintenance DNMT, but its sensitivity to metal ions is different from mammalian Dnmt1 [31
]. Silkworm, like Polistes canadensis
], has lost orthologs of Dnmt3 [28
], and BmDnmt1 preferentially methylates hemimethylated DNA [31
]. Kay et al. suggests that Dnmt1
functions primarily as a maintenance DNMT in B. mori
, despite a putative secondary role in de novo methylation [33
]. By contrast, Dnmt2 has been characterized as an active RNA methyltransferase [14
], and the function of BmDnmt2 is unclear. Silkworms can revert to a diapause state, which arrests development in early embryonic development stages. The state is used to survive unfavourable environments such as low temperatures, drought and/or food shortages [34
]. Diapause occurs at specific embryonic stages, i.e., after formation of cephalic lobes and telson and sequential mesoderm segmentation [36
]. Diapause-destined eggs completely enter diapause three days after oviposition when non-HCl treated. If diapause-destined eggs are treated with 1.075 g/L HCl 24 h after oviposition, the eggs will terminate diapause 48 h after HCl treatment [34
]. The development of diapause-destined embryos is arrested during the G2 cell cycle stage, immediately after mesoderm formation [38
]. During sericulture, hydrochloric acid (HCl) is often used to treat silkworm eggs to disrupt diapause [39
]. Once diapause terminates, the embryos resume development at 25 °C, they enter M phase, and cell division resumes [38
]. The silkworm is an ideal model for studying relationships between DNA methylation, embryonic development and insect diapause.
Currently, functional analyses of DNA methylation during insect embryogenesis, especially insect diapause, are limited. Our previous study provided functional insights into BmDnmt1
in the regulation of silkworm embryonic development; we showed that the expression of BmDnmt1
was elevated in diapause-terminated eggs that were HCl-treated when compared with diapause-destined eggs [40
In this study, we investigate DNA methylation patterns using WGBS and RNA sequencing (RNA-seq) technologies in diapause-terminated eggs compared with diapause-destined eggs in B. mori. We explore different methylation patterns and methylation modification genes and pathways associated with embryonic development. Based on our findings, DNA methylation appears to be essential during B. mori diapause and embryogenesis.
DNA methylation is one of the most widespread epigenetic markers in the genome and has been linked to insect development, specifically embryonic development [16
]. Our previous study showed that BmDNMT
s were highly expressed during embryonic development, especially at early embryonic stages; its expression was significantly upregulated in diapause-terminated eggs after HCl-treatment [40
]. However, DNA methylation regulation in silkworm embryonic development and diapause still remains unclear. This study provided insights into the potential cytosine methylation in diapause-destined eggs and diapause-terminated eggs using WGBS in relation to methylation and embryonic development.
Overall, the levels of DNA methylation in most insects is <1% [28
]. This research has shown ~0.21%–0.26% mC methylation levels on the total sequenced C sites, and more than ~98.5% mC methylation in CG sequences. Based on previous methylome studies in the silkworm, the average methylation levels in the mid-gut and fat body at mC methylation in the genome were approximately 1% [29
], and ~0.11% for silk glands [28
]. When comparing eggs, the mid-gut, fat body and silk gland, it has been shown that DNA methylation levels have tissue specificity in the silkworm. Moreover, mC rates in sequenced C sites were significantly increased in diapause-terminated eggs (HCl-treated) when compared with diapause-destined eggs (non-treated), and change in methylation levels mainly comes from CG sites but not from CHG and CHH sites. It is suggested that when compared with other plants and animals which have higher methylation levels at CHG and CHH sites [50
], DNA methylation works primarily through CGs site in the silkworm.
DNA methylation generally functions as a repressive transcriptional signal, but it is also known to activate gene expression [52
]. Here, we calculated methylation levels in the context of gene regions and ~2 kb upstream and downstream regions. Consistently, CG sites showed more regularity than CHG and CHH sites. CG methylation, but not CHG or CHH methylation, exhibited a characteristic peak in the body of protein-coding genes and showed significant increases after TSS and TES, which maintain low methylation levels. These results are similar to those previously reported for other species [54
]. Moreover, CG methylation levels in the HCl-treated group were generally higher than those in the non-treated group. Thus, we speculate that CG methylation occurs in gene body regions, enhancing gene transcription for embryonic development.
To investigate the specific modification regions of DNA methyl groups in the silkworm genome, methylation profiles of genetic elements were analyzed, including CDS, downstream, upstream, exons, etc. Notably, methylation levels in all genetic elements were increased in HCl-treated samples when compared with non-treated samples, and methylation mainly occurred in coding sequences such as transcript, CDS and exons. Previous studies in silkworms have shown that CG methylation is substantially enriched in gene bodies and is positively correlated with gene expression levels, suggesting positive roles in gene transcription [26
]. Therefore, hypermethylation of coding sequences may be positively associated with the terminating of diapause in silkworm embryos.
DNA methylation patterns are prevalent in TEs and REs [17
]. Our results show that methylation in TEs, especially SINEs, DNA transposable, and RC are higher than in others. SINEs are an abundant class of TEs found in a wide variety of eukaryotes, mainly integrate into hypomethylated DNA regions and are targeted by methylases for de novo methylation [55
]. Previous studies have shown that SINEs promote the amplification of gene enrichment region fragments or stress-induced gene expression, which increase gene expression [58
]. Based on these analyses, hypermethylated SINEs may promote gene transcription and expression during embryo development.
Differentiation and organogenesis are two key phases of embryogenesis in the silkworm. Metabolic biosynthesis is also an essential activity during this period. Metabolic pathways were significantly activated after HCl-treatment, especially for glycogenolysis, protein synthesis and lipolysis. It is worth noting that HS6ST2
homologous gene (GeneID: LOC101737390) regulates energy metabolism and promoted embryonic development in Drosophila
]. Moreover, the Pi3k60
homologous gene (GeneID: LOC100158253), which was upregulated and hypermethylated, reportedly promotes the decomposition of lipids into energy for embryonic development [61
]. Thus, these results suggest that methylation modification may influence embryo development by regulating metabolic biosynthesis phases.
Enzyme inactivation via protein phosphorylation regulates embryo development during embryogenesis [44
]. Here, six MTGs were identified as being involved in phosphatidylinositol signalling. These genes showed increased methylation and expression level in diapause-terminated eggs when compared with diapause-destined eggs. Ponnuvel et al. reported that enzyme inactivation via protein phosphorylation during early silkworm embryogenesis was followed by dephosphorylation in later stages [44
]. On the other hand, INPP genes are important in regulating many cellular activities, including vesicle transport, cytoskeletal dynamics, protein synthesis, cell proliferation and survival and phosphatidylinositol signalling [45
]. Here, Inpp5j
, three of the six MTGs involved in the phosphatidylinositol dephosphorylation pathway, showed increased expression and methylation levels. It was also reported that Inpp5e promotes fundamental metabolism in fat body via phosphorylation [63
]. These results indicate that phosphorylation pathways are activated after gene methylation, thereby promoting embryonic signal transduction, material synthesis and other key activities during embryo development.
Our previous data showed that embryonic cells are arrested in G2 at diapause, and cell cycles become slower in proportion to increasing G1 length. Once diapause terminates, the embryos resume development at 25 °C, cells enter M phase, and cell division resumes in the embryos [38
]. GO analysis revealed that six MTGs were enriched in the G1/S transition of the mitotic cell cycle. It was interesting that gene 101739208, one of the six MTGs, was a homologue of the G2/M phase-specific E3 ubiquitin-protein ligase (G2E3
) and was upregulated and hypermethylated by HCl-treatment. BS-PCR results showed that non-treated and HCl-treated sample methylation levels were 33.3% and 48.1%, respectively for G2E3
), consistent with WGBS data. Previous research has shown that G2E3
is essential for early embryonic development in preventing apoptotic death, and it strengthens the synthesis and transport of genetic material and energy in the M phase [64
]. This indicates that hypermethylation is associated with embryonic diapause and may be regulated through the cell cycle and apoptosis inhibition.