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Insect juvenile hormones (JHs) prevent precocious metamorphosis and allow larvae to undergo multiple rounds of status quo molts. However, the roles of JHs during the embryonic and very early larval stages have not been fully understood. We generated and characterized knockout silkworms (Bombyx mori) with null mutations in JH biosynthesis or JH receptor genes using genome-editing tools. We found that embryonic growth and morphogenesis are largely independent of JHs inBombyxand that, even in the absence of JHs or JH signaling, pupal characters are not formed in first- or second-instar larvae, and precocious metamorphosis is induced after the second instar at the earliest. We also show by mosaic analysis that a pupal specifier genebroad, which is dramatically up-regulated in the late stage of the last larval instar, is essential for pupal commitment in the epidermis. Importantly, the mRNA expression level ofbroad, which is thought to be repressed by JHs, remained at very low basal levels during the early larval instars of JH-deficient or JH signaling-deficient knockouts. Therefore, our study suggests that the long-accepted paradigm that JHs maintain the juvenile status throughout larval life should be revised because the larval status can be maintained by a JH-independent mechanism in very early larval instars. We propose that the lack of competence for metamorphosis during the early larval stages may result from the absence of an unidentifiedbroad-inducing factor, i.e., a competence factor.

It is known that in the silkworm ( Bombyx mori ), males have two Z sex chromosomes whereas females have Z and W and the W chromosome has a dominant role in female determination; here a single female-specific W-chromosome-derived PIWI-interacting RNA is shown to be the feminizing factor in B. mori . The silkworm feminizing factor Susumu Katsuma and colleagues have answered a question that has perplexed insect geneticists for more than eight decades — how does the W chromosome determine femaleness in the silkworm Bombyx mori and many other Lepidoptera? In this system, males have two Z sex chromosomes and females have one Z and one W. The authors show that the feminizing factor is a single W chromosome-derived PIWI-interacting RNA (piRNA). The piRNA silences the product of a gene located on the Z chromosome (named Masc ) that encodes a CCCH-type zinc finger protein. This silencing is in turn important for the production of female-specific isoforms of the doublesex gene that acts at the downstream end of the sex differentiation cascade in the embryo. In male embryos Masc protein controls both dosage compensation and masculinization. The silkworm Bombyx mori uses a WZ sex determination system that is analogous to the one found in birds and some reptiles. In this system, males have two Z sex chromosomes, whereas females have Z and W sex chromosomes. The silkworm W chromosome has a dominant role in female determination 1 , 2 , suggesting the existence of a dominant feminizing gene in this chromosome. However, the W chromosome is almost fully occupied by transposable element sequences 3 , 4 , 5 , and no functional protein-coding gene has been identified so far. Female-enriched PIWI-interacting RNAs (piRNAs) are the only known transcripts that are produced from the sex-determining region of the W chromosome 6 , but the function(s) of these piRNAs are unknown. Here we show that a W-chromosome-derived, female-specific piRNA is the feminizing factor of B. mori . This piRNA is produced from a piRNA precursor which we named Fem . Fem sequences were arranged in tandem in the sex-determining region of the W chromosome. Inhibition of Fem -derived piRNA-mediated signalling in female embryos led to the production of the male-specific splice variants of B. mori doublesex ( Bmdsx ), a gene which acts at the downstream end of the sex differentiation cascade 7 , 8 . A target gene of Fem -derived piRNA was identified on the Z chromosome of B. mori . This gene, which we named Masc , encoded a CCCH-type zinc finger protein. We show that the silencing of Masc messenger RNA by Fem piRNA is required for the production of female-specific isoforms of Bmdsx in female embryos, and that Masc protein controls both dosage compensation and masculinization in male embryos. Our study characterizes a single small RNA that is responsible for primary sex determination in the WZ sex determination system.

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.

Insects have evolved diapause to cope with harsh environmental conditions, during which their metabolism undergoes significant remodeling. The silkworm, Bombyx mori , enters diapause in the early embryonic stage, a process critically regulated by the sorbitol metabolism pathway and its key enzyme, sorbitol dehydrogenase (SDH). Nevertheless, the precise involvement of SDH in the diapause regulation of the silkworm remains to be fully understood. In this study, we identified that BmSdh2 is highly expressed in diapause-destined silkworm embryos through RNA-seq analysis. Genetic manipulation of BmSdh2 expression significantly influenced diapause progression: complete homozygous knockout of BmSdh2 led to diapause termination, whereas partial loss-of-function mutations maintained the wild-type diapause phenotype. Furthermore, integrative LC-MS/MS, metabolomic, and lipidomic analyses demonstrated that BmSdh2 dosage critically modulates diapause maintenance. These findings highlight BmSdh2 as a novel and potentially central molecular regulator in the silkworm diapause pathway.

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; however, its molecular mechanisms are largely unknown. Here, we show that the Bombyx TRPA1 ortholog (BmTrpA1) acts as a thermosensitive transient receptor potential (TRP) channel that is activated at temperatures above ∼21 °C and affects the induction of diapause in progeny. In addition, we show that embryonic RNAi of BmTrpA1 affects diapause hormone release during pupal-adult development. This study identifying a thermosensitive TRP channel that acts as a molecular switch for a relatively long-term predictive adaptive response by inducing an alternative phenotype to seasonal polyphenism is unique.

Microsporidia are a group of intracellular and unicellular eukaryotic parasites, which can nearly infect all animals, including human beings. As the first identified microsporidia, Nosema bombycis is a world-wide threat for silkworm eggs production, it can cause the congenital infection via transovarial transmission. It is important for pathogenesis elucidation to unravel the molecular characteristics of N. bombycis proliferation and host immune responses to the congenital infection in embryo and larva stage. Here, we adopted dual RNA-seq approach to investigate and compare the dynamic molecular pattern of pathogen proliferation and host immune responses between diapause and non-diapause silkworm eggs. Our results showed the N. bombycis proliferation in non-diapause silkworm eggs is a continuous process, many parasites enter the sporogony stage at 2 days post-oviposition (dpo). For newly hatched larva (1 dph), the abundance of pathogen mRNA sequences is up to 2.32% in non-diapause strain, far higher than 0.34% of diapause strain, the main reason is the hot HCl bath treatment at 24 h post-oviposition for diapause silkworm eggs with the aim to free the egg diapause. As to immune responses, whatever for diapause strain or non-diapause strain, there is stronger immune responses to congenital infection in larva stage than that of embryo stage, however, the host immune responses to congenital infection are fairly different between non-diapause and diapause strains of silkworms, especially in embryo stage. We found the surprising “First day Chaos” that there are 6,071 differential expressed genes (DEGs) at 1 dpo for non-diapause strain between infection group and uninfected group, but decreases dramatically to 109 DEGs at 2 dpo. In non-diapause strain, the earliest DEGs of antimicrobial peptides were up-regulated at 1 dpo, then is 5 dpo with up-regulated lebocin, 7 dpo with morLP-B1, morLP-B4. For non-diapause strain, the well-established immune responses were observed in newly hatched larvae. On the contrast, for diapause strain, the earliest DEGs of AMPs appear at 5 dph, the mature immune responses are well established at 5 dph too. In non-diapause silkworms, we observed obvious pathogen’s regulation in the main immune pathways including Toll, IMD, JAK-STAT and melanization at the different steps such as immune recognition, signal modulation and transduction, effectors. Taken together, our results for the first time provide a global molecular view of microsporidia proliferation and innate immunity responses in a congenital infection system and provide some new insights into immune development and establishment in the embryo and early larva stage of Bombyx mori.

The bivoltine strain of the domestic silkworm, Bombyx mori, has two generations per year. It shows a facultative diapause phenotype determined by environmental conditions, including photoperiod and temperature, and nutrient conditions during embryonic and larval development of the mother. However, it remains unclear how the environmental signals received during development are selectively utilized as cues to determine alternative diapause phenotypes. We performed a comparative analysis between the Kosetsu strain of B. mori and a Japanese population of the wild mulberry silkworm B. mandarina concerning the hierarchical molecular mechanisms in diapause induction. Our results showed that for the Kosetsu, temperature signals during the mother’s embryonic development predominantly affected diapause determination through the thermosensitive transient receptor potential ankyrin 1 (TRPA1) and diapause hormone (DH) signaling pathways. However, embryonic diapause in B. mandarina was photoperiod-dependent, although the DH signaling pathway and thermal sensitivity of TRPA1 were conserved within both species. Based on these findings, we hypothesize that TRPA1-activated signals are strongly linked to the signaling pathway participating in diapause induction in Kosetsu to selectively utilize the temperature information as the cue because temperature-dependent induction was replaced by photoperiodic induction in the TRPA1 knockout mutant.

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.

Cyclin-dependent protein kinase 2 (Cdk2), an important member of the serine/threonine-specific protein kinase family, plays a critical regulatory role in biological processes. Previous studies have demonstrated that Cdk2 is involved in the arrest and resumption of meiosis in mammalian oocytes. In this study, we explored the function of Cdk2 through parthenogenetic lines (PLs) and corresponding amphigonic lines (ALs) in a model lepidopteran insect silkworm, Bombyx mori L. Our findings revealed a positive correlation between Cdk2 activity and the parthenogenesis induction rate. The pharmacological inhibition of Cdk2 using the specific inhibitor AUZ454 not only significantly reduced the parthenogenesis induction rate but also caused developmental delays in embryos. These results demonstrate that Cdk2 is essential for parthenogenesis success and is a potential target gene for biological reproductive regulation.

Background Transcription factor lark has been demonstrated to play multiple functions in Drosophila , but the function of this gene in embryonic development remains to be elucidated. Results In this study, the CRISPR/Cas9 gene-editing method was used to construct a Bmlark mutant strain of Bombyx mori to investigate the roles of this gene. The results showed that the homozygous mutant Bmlark −/− was lethal. The Bmlark −/− embryos showed obvious developmental defects, such as defective sclerotization and melanization of the exoskeleton. A transcriptomic comparison of Bmlark −/− and wild-type embryos showed that the differentially expressed genes were mainly enriched in the structure and metabolic processes of chitin and cuticles. While the expression levels of chitin metabolism-related enzyme genes did not significantly change, in the mutant embryos compared to the wild-type embryos, the expression levels of 63 putative cuticle protein genes showed significant differences. Among which, 35 genes were downregulated and 28 genes were upregulated. The expression levels of the transcription factor BmPOUM2 and eight wing disc cuticle protein genes ( WCP ) also changed. BmPOUM2 , WCP5 , WCP9 , WCP10 , WCP11 were downregulated and WCP1 , WCP2 , WCP3 , WCP6 were upregulated in Bmlark −/− embryos. While the expression level of TH in the tyrosine-mediated pigmentation pathway was upregulated in the mutant embryos, the expression levels of the four key pigment synthesis genes DDC , aaNAT , Laccase2A , and yellow-f2 were significantly downregulated. Conclusions The expression levels of 63 putative cuticle protein genes, eight WCP genes, and five pigment synthesis genes significantly changed in Bmlark mutant B. mori compared to those of the wildtype. These results suggest that Bmlark is essential for normal development of cuticle and tyrosine-mediated melanization in silkworm embryos.