Interacting C/EBPg and YBP regulate DNA methyltransferase 1 expression in Bombyx mori embryos and ovaries.

DNA methylation is an important epigenetic modification. DNA methyltransferases (Dnmts), which catalyze the formation of 5-methylcytosine, play a role in ovarian and embryonic development in some insects. However, the underlying mechanism of Dnmt in mediating ovarian and embryonic development remains unclear. In this study, the regulation and function of Bombyx mori Dnmt1 were investigated. By progressively deleting the sequence upstream of Dnmt1, a region located between -580 and -560 region from the transcription initiation site was found to have the most transcriptional activity. Electrophoretic mobility shift assay and chromatin immunoprecipitation demonstrated that transcription factor Y box binding protein (YBP), a homolog of human Y box binding protein 1 (YBX1), bound to the -580 to -560 region. YBP knockdown and overexpression in a Bombyx cell line indicated that YBP activates Dnmt1 expression. Furthermore, GST-pulldown and co-immunoprecipitation demonstrated that YBP and ovarian CCAAT/enhancer binding protein (C/EBPg) could bind each other. Simultaneous knockdown of C/EBPg and YBP was more effective than single-gene RNAi in inhibiting Dnmt1 expression and reducing the hatching rate. These results demonstrated that the interaction of C/EBPg and YBP activated Dnmt1 expression. Correlated with the expression profiles of the studies genes, our results suggest that high-level expression and interaction of C/EBPg and YBP in ovaries and embryos enhance the expression of Dnmt1, thus ensuring high reproduction rate in B. mori.

Changes in expressions of ecdysteroidogenic enzyme and ecdysteroid signaling genes in relation to Bombyx embryonic development.

Although the role of ecdysteroids in regulating egg diapause process in Bombyx mori is well documented, temporal changes in expression levels of genes involved in ecdysteroid biosynthesis and its downstream signaling are less well understood. In the present study, we studied changes in expression levels of genes involved in ecdysteroid biosynthesis and its downstream signaling during embryonic development of B. mori. Results showed that in diapause eggs, the expression of ecdysteroid-phosphate phosphatase (EPPase) gene and Halloween genes (Spook [Spo] and Shade [Shd]) remained at very low levels. However, in eggs whose diapause initiation was prevented by HCl, significant increases in the messenger RNA (mRNA) levels of EPPase, Spo, and Shd were detected during embryonic development. Other Halloween genes (Neverland [Nvd] and Phantom [Phm]) also showed different changes between diapause and HCl-treated eggs. However, genes of Disembodied (Dib) and Shadow (Sad) showed similar changes in both diapause and HCl-treated eggs. We further investigated changes in expression levels of ecdysone receptor genes (EcRA, EcRB1, and USP) and downstream signaling genes (E75A, E75B, E74A, E74B, Br-C, HR3, HR4, KR-H1, and FTZ-F1). Results showed that genes of EcRA and the other nuclear receptors (E75A, E75B, E74A, HR3, HR4, KR-H1, and FTZ-F1) exhibited significant differential patterns between diapause and HCl-treated eggs, with increased levels being detected during later stages of embryonic development in HCl-treated eggs. Differential temporal changes in expressions of genes involved ecdysteroid biosynthesis and its downstream signaling found between diapause and HCl-treated eggs were further confirmed using nondiapause eggs. Our results showed that nondiapause eggs exhibited the same changing patterns as those in HCl-treated eggs, thus clearly indicating potential correlations between expressions of these genes and embryonic development in B. mori. To our knowledge, this is the first comprehensive report to study the transcriptional regulation of ecdysteroidogenic and ecdysteroid signaling genes, thus providing useful information for a clearer understanding of insect egg diapause mechanisms.

Microsporidia infection upregulates host energy metabolism but maintains ATP homeostasis.

Microsporidia are a group of obligate intracellular parasites which lack mitochondria and have highly reduced genomes. Therefore, they are unable to produce ATP via the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. Instead, they have evolved strategies to obtain and manipulate host metabolism to acquire nutrients. However, little is known about how microsporidia modulate host energy metabolisms. Here, we present the first targeted metabolomics study to investigate changes in host energy metabolism as a result of infection by a microsporidian. Metabolites of silkworm embryo cell (BmE) were measured 48 h post infection by Nosema bombycis. Thirty metabolites were detected, nine of which were upregulated and mainly involved in glycolysis (glucose 6-phosphate, fructose 1,6-bisphosphate) and the TCA cycle (succinate, α-ketoglutarate, cis-aconitate, isocitrate, citrate, fumarate). Pathway enrichment analysis suggested that the upregulated metabolites could promote the synthesization of nucleotides, fatty acids, and amino acids by the host. ATP concentration in host cells, however, was not significantly changed by the infection. This ATP homeostasis was also found in Encephalitozoon hellem infected mouse macrophage RAW264.7, human monocytic leukemia THP-1, human embryonic kidney 293, and human foreskin fibroblast cells. These findings suggest that microsporidia have evolved strategies to maintain levels of ATP in the host while stimulating metabolic pathways to provide additional nutrients for the parasite.

Expression of protein tyrosine phosphatases and Bombyx embryonic development.

Protein phosphorylation is an integral component of signal transduction pathways within eukaryotic cells, and it is regulated by coordinated interactions between protein kinases and protein phosphatases. Our previous study demonstrated differential expressions of serine/threonine protein phosphatases (PP2A and calcineurin) between diapause and developing eggs in Bombyx mori. In the present study, we further investigated expression of protein tyrosine phosphatases (PTPs) in relation to the Bombyx embryonic development. An immunoblot analysis showed that eggs contained the proteins of the 51-kDa PTP 1B (PTP1B), the 55-kDa phosphatase and tensin homologue (PTEN), and the 70-kDa Src homology 2 (SH2) domain-containing phosphatase 2 (SHP2), which undergo differential changes between diapause and developing eggs. Protein level of PTP1B and PTEN in eggs whose diapause initiation was prevented by HCl gradually increased toward embryonic development. The protein level of SHP2 also showed a dramatic increase on days 7 and 8 after HCl treatment. However, protein levels of PTP1B, PTEN, and SHP2 in diapause eggs remained at low levels during the first 9 days after oviposition. These differential changing patterns in protein levels were further confirmed using both non-diapause eggs and eggs in which diapause had been terminated by chilling of diapausing eggs at 5 °C for 70 days and then were transferred to 25 °C. Direct determination of PTP enzymatic activities showed higher activities in developing eggs (HCl-treated eggs, non-diapause eggs, and chilled eggs) compared to those in diapause eggs. Examination of temporal changes in mRNA expression levels of PTP1B, PTEN, and SHP2 did not show significant differences between diapause eggs and HCl-treated eggs except high expression in SHP2 variant B during the later embryonic development in HCl-treated eggs. These results demonstrate that higher protein levels of PTP1B, PTEN, and SHP2 and increased tyrosine phosphatase enzymatic activities in developing eggs are likely related to embryonic development of B. mori.

Maternal GABAergic and GnRH/corazonin pathway modulates egg diapause phenotype of the silkworm Bombyx mori.

Diapause represents a major developmental switch in insects and is a seasonal adaptation that evolved as a specific subtype of dormancy in most insect species to ensure survival under unfavorable environmental conditions and synchronize populations. However, the hierarchical relationship of the molecular mechanisms involved in the perception of environmental signals to integration in morphological, physiological, behavioral, and reproductive responses remains unclear. In the bivoltine strain of the silkworm Bombyx mori, embryonic diapause is induced transgenerationally as a maternal effect. Progeny diapause is determined by the environmental temperature during embryonic development of the mother. Here, we show that the hierarchical pathway consists of a γ-aminobutyric acid (GABA)ergic and corazonin signaling system modulating progeny diapause induction via diapause hormone release, which may be finely tuned by the temperature-dependent expression of plasma membrane GABA transporter. Furthermore, this signaling pathway possesses similar features to the gonadotropin-releasing hormone (GnRH) signaling system for seasonal reproductive plasticity in vertebrates.

Investigation of the molecular mechanism underlying the inhibitory activities of ethanol extract of Bombyx mori pupa-incubated Cordyceps militaris fruiting bodies toward allergic rhinitis.

Cordyceps militaris has been widely studied for its various pharmacological activities such as antitumor, anti-inflammation, and immune regulation. The binding of an allergen to IgE-sensitized mast cells in nasal mucosa triggers allergic rhinitis. We found that oral administration of 300 mg/kg of the ethanol extract prepared from silkworm pupa-cultivated Cordyceps militaris fruiting bodies significantly alleviated the symptoms of ovalbumin-induced allergic rhinitis in mice, including sneeze/scratch, mast cell activation, eosinophil infiltration, and Syk activation. The treatment of ethanol extract significantly suppressed the release of ß-hexosaminidase (a degranulation marker) and mRNA expression levels of various cytokines, including IL-3, IL-10, and IL-13 in activated RBL2H3 cells. The ethanol extract and ß-sitostenone, which was purified from the extract, could respectively reduce the Ca2+ ion mobilization in activated RBL-2H3 cells. Furthermore, results collected from western immunoblotting demonstrated that ethanol extract significantly retarded Ca2+ ion mobilization-initiated signaling cascade, which provoked the expression of various allergic cytokines. Also, the extract incubation interfered with P38 as well as NF-kB activation and Nrf-2 translocation. Our study suggested that ethanol extract possessed some natural constituents which could inhibit immediate degranulation and de novo synthesis of allergic cytokines via inhibition of Ca2+ ion mobilization in mast cells in the nasal mucosa of allergic rhinitis mice.

Molecular basis of the silkworm mutant rel causing red egg color and embryonic death.

The coloration and hatchability of insect eggs can affect individual and population survival. However, few genetic loci have been documented to affect both traits, and the genes involved in regulating these two traits are unclear. The silkworm recessive mutant rel shows both red egg color and embryo mortality. We studied the molecular basis of the rel phenotype formation. Through genetic analysis, gene screening and sequencing, we found that two closely linked genes, BGIBMGA003497 (Bm-re) and BGIBMGA003697 (BmSema1a), control egg color and embryo mortality, respectively. Six base pairs of the Bm-re gene are deleted in its open reading frame, and BmSema1a is expressed at abnormally low levels in mutant rel . BmSema1a gene function verification was performed using RNA interference and clustered randomly interspersed palindromic repeats (CRISPR)/CRISPR-associate protein 9. Deficiency of the BmSema1a gene can cause the death of silkworm embryos. This study revealed the molecular basis of silkworm rel mutant formation and indicated that the Sema1a gene is essential for insect embryo development.

The receptor tyrosine kinase torso regulates ecdysone homeostasis to control developmental timing in Bombyx mori.

Insect growth and development are precisely controlled by hormone homeostasis. The prothoracicotropic hormone (PTTH) receptor, Torso, is a member of the receptor tyrosine kinase family in insects. Activation of Torso by PTTH triggers biosynthesis and release of the steroid hormone in the prothoracic gland (PG). Although numbers of genes functioning in steroid hormone synthesis and metabolism have been identified in insects, the PTTH transduction pathway via its receptor Torso is poorly understood. In the current study, we describe a loss-of-function analysis of Torso in the silkworm, Bombyx mori, by targeted gene disruption using the transgenic CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/RNA-guided Cas9 nucleases) system. Depletion of B. mori Torso (BmTorso) did not eventually affect larval ecdysis and metamorphosis processes. Instead, BmTorso deficiency resulted in significant extension of developing time during larval and pupal stages with increased pupa and cocoon sizes. The ecdysteriod titers in the hemolymph of BmTorso mutants sharpy declined. Transcriptional levels of genes involved in ecdysone biosynthesis and ecdysteroid signaling pathways were significantly reduced in BmTorso-deficient animals. Additionally, RNA-Seq analysis revealed that genes involved in the longevity pathway and protein processing in the endoplasmic reticulum pathway were affected after BmTorso deletion. These results indicate that Torso is critical for maintaining steroid hormone homeostasis in insects.

Semaphorin-1a-like gene plays an important role in the embryonic development of silkworm, Bombyx mori.

Fuyin-lethal red egg (Fuyin-lre) is a red egg mutant discovered from the germplasm resource Fuyin of Bombyx mori. The embryo of Fuyin-lre stops developing at the late stage of gastrulation due to chromosome structural variation. In this work, precise mutation sites at both ends of the mutated region were determined, and two inserted sequences with lengths of 1232 bp and 1845 bp were obtained at both ends of the mutation region. Interestingly, a bmmar1 transposon was detected in the inserted 1845 bp sequence. Bmmar1 possesses features of the Tcl/mariner superfamily of transposable elements (TEs), which belongs to class II TEs that use a DNA-mediated "cut and paste" mechanism to transpose. This finding suggests that Fuyin-lre mutation might be related to the "cut and paste" action of bmmar1. The mutation resulted in the deletion of 9 genes in the mutation region, of which the red egg gene re (BMSK0002766) did not affect embryonic development of B. mori, and the BMSK0002765 gene was unexpressed during the early stage of embryonic development. The RNA interference results of the remaining 7 genes suggest that the semaphorin-1a-like gene (BMSK0002764) had a major contribution to the embryonic lethality of Fuyin-lre.

Twenty-hydroxyecdysone produced by dephosphorylation and ecdysteroidogenesis regulates early embryonic development in the silkmoth, Bombyx mori.

Ecdysteroids are key regulators of embryonic development as well as molting and metamorphosis in insects. Although an active form of ecdysteroids, 20-hydroxyecdysone (20E) is known to be produced through ecdysteroidogenesis from cholesterol and dephosphorylation of 20E-phosphate during embryogenesis in Lepidoptera, the importance of these production mechanisms in embryonic development has been unclear. Here, we investigated the activation timing of ecdysteroidogenesis from cholesterol and 20E-phosphate dephosphorylation during early embryogenesis in non-diapause eggs of the silkmoth Bombyx mori by observing morphological development, quantifying 20E and 20E-phosphate, measuring transcripts of enzymes involved in 20E production, and detecting activity of these enzymes using egg extracts. Stage-dependent 20E fluctuation and changes in mRNA amounts of enzymes suggest that the two 20E-producing mechanisms are activated at different stages during embryogenesis. Furthermore, knockdown of a dephosphorylation enzyme delayed development at early embryogenesis, whereas knockdown of an ecdysteroidogenic enzyme delayed development at early-middle embryogenesis. These results suggest that 20E is primarily produced initially by dephosphorylation of 20E-phosphate, and then by ecdysteroidogenesis from cholesterol to induce progression of embryonic development in B. mori.

A mitochondrial phosphatase PTPMT1 is essential for the early development of silkworm, Bombyx mori.

Juvenile hormone (JH) plays important roles in the control of many biological processes in insects, such as development, reproduction, and polyphenism. JH is primarily produced in the corpora allata (CA) by specific JH biosynthetic enzymes under strict temporal regulation. In a previous study, we identified a novel putative JH biosynthetic gene, protein tyrosine phosphatase, mitochondrial 1 (PTPMT1), from silkworm, Bombyx mori, whose expression is nearly exclusive in the CA and is correlated with JH synthetic activities during late larval development. In this study, to reveal the function of PTPMT1 in vivo, we generated PTPMT1 knockout silkworms using TALEN. In the knockout mutants, no signs indicating defects in JH activity were observed. Instead, PTPMT1 knockout silkworms showed embryonic lethality, developmental arrest, and 3rd-instar lethality not only in mutants lacking total enzymatic activity but also in mutants lacking mitochondrial translocation signals. Moreover, in PTPMT1 knockout embryos, the expression of two genes encoded by the mitochondrial genome, CYTB and ND3, was decreased, indicating a mitochondrial disorder. These results suggested that PTPMT1 plays conserved vital role(s) reported in vertebrates in insect mitochondria.

Pupae protein extracts exert anticancer effects by downregulating the expression of IL-6, IL-1ß and TNF-α through biomolecular changes in human breast cancer cells.

The Pupae of Bombyx mori and Samia ricini are a source of high-quality proteins and essential nutrient elements for human. Recent studies revealed that protein extracted from pupae possessed therapeutic benefits for the treatment of many diseases. However, the anticancer activity of protein extracts from the pupae of B. mori and S. ricini has been rarely reported. Our objective was to study the effect of protein extracts from the pupae of B. mori and S. ricini on cytotoxicity and expression of pro-inflammatory cytokines; IL-6, IL-1ß and TNF-α, in breast cancer cells (MCF-7). Additionally, anticancer action of protein extracted from the pupae was further investigated through biomolecular changes in MCF-7 cells using Fourier transform infrared (FTIR) spectroscopy. Pupae protein extracts of B. mori exhibited cytotoxic effects with an IC50 value of 15.23 + 0.4 µg/mL with higher selectivity than doxorubicin on MCF-7 cells. Fourier transform infrared (FTIR) spectroscopy revealed that lipid contents in MCF-7 cells treated with pupae protein extracts of B. mori were higher than untreated cells. Treatment with protein extracts from pupae of B. mori or S. ricini caused significantly reduced protein and nucleic acid contents of MCF-7 cells. The expression of IL-6, IL-1ß and TNF-α in MCF-7 treated cells was investigated using RT-qPCR and ELISA. Our results revealed that protein extracts from the pupae of B. mori or S. ricini significantly decreased IL-6, IL-1ß and TNF-α in MCF-7 cells both at mRNA and protein levels. Expression of IL-6 and IL-1ß in MCF-7 treated cells, especially IL-6, was strongly reduced compared to untreated cells, while TNF-α expression was slightly decreased. These findings suggest that pupae protein extracted from B. mori or S. ricini may play a role in breast cancer through a down-regulatory action on the expression of IL-6, IL-1ß and TNF-α, and may also exert anticancer effects by causing biochemical changes of lipids, proteins and nucleic acids. These findings indicate that pupae protein extracted from B. mori or S. ricini may provide a potential novel therapeutic target for breast cancer.

Toxicology study of fraxinellone as ovicidal agents against Mythimna separata Walker and Bombyx mori Linaeus.

Fraxinellone is a naturally occurring degraded limonoid isolated from many species of plants in Meliaceae and Rutaceae. Besides structural modification of the lead compounds, the toxicology study of the lead compounds is also a very important procedure to develop insecticidal agents. Herein the toxicology study of fraxinellone was carried out as the ovicidal agent against the eggs of two lepidopteran insects Mythimna separata Walker and Bombyx mori Linaeus. Fraxinellone selectively exhibited an ovicidal activity against the eggs of M. separata. After treatment with fraxinellone, the eggshells of M. separata were shrinked, whereas those of B. mori had no obvious change. The dynamic process of M. separata embryo development demonstrated that the distinct difference between the treated eggs and the control ones was obvious at the second day after treatment, especially, the control embryo finished blastokinesis, whereas the treated ones were still laid at pre-reversion status and a lot of yolk can be seen around the embryo. It ultimately resulted in the eggshell withered and the egg hatching inhibited.

miR-34 regulates larval growth and wing morphogenesis by directly modulating ecdysone signalling and cuticle protein in Bombyx mori.

microRNAs (miRNA) are small non-coding RNAs that modulate the myriad biological activities by targeting genes, and many studies showed that miRNAs played a pivotal role in insect development. Here, we find that Bm-miRNA (miR-34) controls larval growth and wing morphology by targeting BmE74 and BmCPG4. Overexpression of miR-34 in the whole body caused a smaller body size, partially displays deformed wings and venation defects in adults. Ablation of miR-34 by transgenic CRISPR/Cas9 technology resulted in a severe developmental delay during the larval stage. Moreover, we confirmed that miR-34 directly targeted BmE74 and BmCPG4 by using a dual luciferase reporter assay in HEK293T cells. Remarkably, loss-of-function of BmCPG4 caused wing defects, which was similar to the phenotype of miR-34 overexpression in animals. In addition, our analysis revealed that ecdysone strongly inhibited miR-34 expression in vivo. Taken together, our study identifies miR-34 as a modulator that regulates larval growth and wing morphogenesis by directly modulating ecdysone signalling and cuticle protein in Bombyx mori.

DNA Methylation Is Correlated with Gene Expression during Diapause Termination of Early Embryonic Development in the Silkworm (Bombyx mori).

DNA modification is a naturally occurring DNA modification in prokaryotic and eukaryotic organisms and is involved in several biological processes. Although genome-wide methylation has been studied in many insects, the understanding of global and genomic DNA methylation during insect early embryonic development, is lacking especially for insect diapause. In this study, we analyzed the relationship between DNA methylomes and transcriptomes in diapause-destined eggs compared to diapause-terminated eggs in the silkworm, Bombyx mori (B. mori). The results revealed that methylation was sparse in this species, as previously reported. Moreover, methylation levels in diapause-terminated eggs (HCl-treated) were 0.05% higher than in non-treated eggs, mainly due to the contribution of CG methylation sites. Methylation tends to occur in the coding sequences and promoter regions, especially at transcription initiation sites and short interspersed elements. Additionally, 364 methylome- and transcriptome-associated genes were identified, which showed significant differences in methylation and expression levels in diapause-destined eggs when compared with diapause-terminated eggs, and 74% of methylome and transcriptome associated genes showed both hypermethylation and elevated expression. Most importantly, Kyoto Encyclopaedia of Genes and Genomes (KEGG) analyses showed that methylation may be positively associated with Bombyx mori embryonic development, by regulating cell differentiation, metabolism, apoptosis pathways and phosphorylation. Through analyzing the G2/M phase-specific E3 ubiquitin-protein ligase (G2E3), we speculate that methylation may affect embryo diapause by regulating the cell cycle in Bombyx mori. These findings will help unravel potential linkages between DNA methylation and gene expression during early insect embryonic development and insect diapause.

Proliferation characteristics of the intracellular microsporidian pathogen Nosema bombycis in congenitally infected embryos.

Nosema bombycis is an obligate intracellular pathogen that can be transmitted vertically from infected females to eggs, resulting in congenital infections in embryos. Here we investigated the proliferation characteristics of N. bombycis in silkworm embryos using a histopathological approach and deep RNA sequencing. We found that N. bombycis proliferated mainly around yolk granules at the early stage of the embryonic development, 1-2 days post oviposition (dpo). At 4-6 dpo, a portion of N. bombycis in different stages adjacent to the embryo were packaged into the newly formed intestinal lumen, while the remaining parasites continued to proliferate around yolk granules. In the newly hatched larvae (9 dpo), the newly formed spores accumulated in the gut lumen and immediately were released into the environment via the faeces. Transcriptional profiling of N. bombycis further confirmed multiplication of N. bombycis throughout every stage of embryonic development. Additionally, the increased transcriptional level of spore wall proteins and polar tube proteins from 4 dpo indicated an active formation of mature spores. Taken together, our results have provided a characterization of the proliferation of this intracellular microsporidian pathogen in congenitally infected embryos leading to vertical transmission.

Estrogen-related receptor participates in regulating glycolysis and influences embryonic development in silkworm Bombyx mori.

Estrogen-related receptors (ERRs) play indispensable roles in development, energy metabolism, and cancers and are metabolic switches in Drosophila. However, the mechanism underlying their metabolic role is unknown in insects. This study analysed the expression profiles of Bombyx mori ERR (BmERR), hexokinase (BmHK), pyruvate kinase (BmPK) and phosphofructokinase (BmPFK) during embryonic development. The expression of BmERR tended to be similar to that of the other genes. We observed a regulatory association between BmERR and glycolytic rate-limiting enzymes by BmERR overexpression, RNA interference (RNAi), and ERR inhibitors in B. mori embryo cells. Subsequently, ERR cis-regulation elements (ERREs) were predicted and identified in the BmPFK promoter. Transfection assays, electrophoretic mobility shift assays and chromatin immunoprecipitation showed that BmERR can bind to one of these elements to regulate the expression of BmPFK. ERREs were also predicted in the BmHK and BmPK promoters. In the eggs, the expression of glycolytic rate-limiting enzyme genes was suppressed when the expression of BmERR was interference by double-stranded BmERR, the glucose levels also was increased. Meanwhile, the development of silkworm embryos was delayed by about 1 day. These results indicate that BmERR can bind to the ERREs of glycolytic gene promoters and regulate the expression of glycolytic genes, ultimately affecting embryonic development in silkworms.

Expression Analysis of mRNA Decay of Maternal Genes during Bombyx mori Maternal-to-Zygotic Transition.

Maternal genes play an important role in the early embryonic development of the silkworm. Early embryonic development without new transcription depends on maternal components stored in the egg during oocyte maturation. The maternal-to-zygotic transition (MZT) is a tightly regulated process that includes maternal mRNAs elimination and zygotic transcription initiation. This process has been extensively studied within model species. Each model organism has a unique pattern of maternal transcriptional clearance classes in MZT. In this study, we identified 66 maternal genes through bioinformatics analysis and expression analysis in the eggs of silkworm virgin moths (Bombyx mori). All 66 maternal genes were expressed in vitellogenesis in day eight female pupae. During MZT, the degradation of maternal gene mRNAs could be divided into three clusters. We found that eight maternal genes of cluster 1 remained stable from 0 to 3.0 h, 17 maternal genes of cluster 2 were significantly decayed from 0.5 to 1.0 h and 41 maternal genes of cluster 3 were significantly decayed after 1.5 h. Therefore, the initial time-point of degradation of cluster 2 was earlier than that of cluster 3. The maternal gene mRNAs decay of clusters 2 and 3 is first initiated by maternal degradation activity. Our study expands upon the identification of silkworm maternal genes and provides a perspective for further research of the embryo development in Bombyx mori.

The role of N6-methyladenosine modification on diapause in silkworm (Bombyx mori) strains that exhibit different voltinism.

N6-methyladenosine (m6 A) plays a key role in regulating gene expression in myriad organisms. Diapause is an important plastic phenotype that allows insects to survive under specific environmental conditions. However, the diapause molecular mechanism remains unknown. In this study, we analyzed the phylogenetics of genes related to the m6 A modification complex in the silkworm (Bombyx mori) based on identified sequences from other organisms. We detected the expression of these genes during different developmental phases from four strains with different voltinism. We also determined total m6 A content in cells treated with different diapause hormone concentrations or eggs exposed to hydrochloric acid. Our data revealed that m6 A-modification-related gene expression and m6 A content were greater in diapause-destinated compared to nondiapause-destined strains. Our findings suggest that m6 A modification may provide significant epigenetic regulation of diapause-related genes in the silkworm.

Involvement of Ecdysone Signaling in the Expression of the doublesex Gene during Embryonic Development in the Silkworm, Bombyx mori.

Steroid hormones, represented by estrogen and testosterone, act as sex hormones that play an essential role in the sexual differentiation of vertebrates. However, it remains unclear whether ecdysteroids, typical steroid hormones in insects, function as sex hormones. In this study, we investigated whether ecdysteroids or ecdysone signals are involved in the sexual differentiation of the silkworm (Bombyx mori) embryo. Quantitative analysis using LC-MS/MS demonstrated that there was no significant difference in the 20-hydroxyecdysone (20E) titer between sexes during embryonic development. Consistent with this result, expression levels of 2 genes encoding ecdysteroid-phosphate phosphatase (EPPase) and ecdysone 20-hydroxylase (E20OHase), which are essential for the biosynthesis of ecdysone and 20E in eggs, did not show a significant difference between male and female embryos. Expression levels of ecdysone receptor (EcR) and E75, which is one of a small set of genes induced directly by 20E, were also similar between the 2 sexes. However, knockdown of EPPase and one isoform of EcR (EcR-A) resulted in decreased expression of Bombyx doublesex (Bmdsx), a master regulatory gene for sexual differentiation of the silkworm in both male and female embryos. In vitro analysis with cultured testes revealed that expression levels of Bmdsx were increased in a dose-dependent manner of the ecdysone analog, ponasterone A. These results suggest that ecdysone signaling may play a role in indirectly regulating the expression of some genes involved in sexual differentiation through inducing expression of Bmdsx in the silkworm.