The variable diapause features of bivoltine silkworm (Bombyx mori) strains regulated by environmental signals in the embryonic stage are closely related to epigenetics. Previously, we showed that the expression of YTHDF3 is significantly different in the pupae of the bivoltine silkworm Qiufeng developed from eggs incubated at a normal temperature (QFHT, diapause egg producer) compared to those from eggs incubated at a low temperature (QFLT, nondiapause egg producer), indicating that the expression of diapause-associated genes is regulated by the m⁶A modification level. However, how YTHDF3 regulates the expression of diapause-related genes remains unclear. In this study, we observed that the knockdown of B. mori YTHDF3 resulted in delayed embryo development, while the overexpression of YTHDF3 resulted in the transformation of nondiapause-destined eggs into a mixture of diapause and nondiapause eggs. Further studies showed that YTHDF3, as a reading protein, can recognize the m⁶A site of Cyp307a1 and Cyp18a1 genes in the ecdysone synthesis pathway (ESP), and the overexpression of YTHDF3 affects the diapause traits of the silkworm by decreasing the stabilities of mRNAs of Cyp307a1 and Cyp18a1 and inhibiting their translation. The above results demonstrate that m⁶A modification mediates YTHDF3 to affect the expression levels of its target genes, Cyp307a1 and Cyp18a1, in the ESP to regulate diapause in bivoltine B. mori. This is the first report of the m⁶A methylation regulation mechanism in diapause in B. mori and provides new experimental data for clarifying the diapause regulation network.

Genetic mutations leading to premature termination codons are known to have detrimental effects. Using the Lepidoptera model insect, the silkworm (Bombyx mori), we explored the genetic compensatory response triggered by mutations with premature termination codons. Additionally, we delved into the molecular mechanisms associated with the nonsense-mediated mRNA degradation pathway. CRISPR/Cas9 technology was utilized to generate a homozygous bivoltine silkworm line BmTrpA1^−/− with a premature termination. Transcript levels were assessed for the BmTrpA paralogs, BmPyrexia and BmPainless as well as for the essential factors Upf1, Upf2, and Upf3a involved in the nonsense-mediated mRNA degradation (NMD) pathway. Upf2 was specifically knocked down via RNA interference at the embryonic stage. The results comfirmed that the BmTrpA1 transcripts with a 2-base deletion generating a premature termination codon in the BmTrpA1^−/− line. From day 6 of embryonic development, the mRNA levels of BmPyrexia, BmPainless, Upf1, and Upf2 were significantly elevated in the gene-edited line. Embryonic knockdown of Upf2 resulted in the suppression of the genetic compensation response in the mutant. As a result, the offspring silkworm eggs were able to hatch normally after 10 days of incubation, displaying a non-diapause phenotype. It was observed that a genetic compensation response does exist in BmTrpA1^−/− B. mori. This study presents a novel discovery of the NMD-mediated genetic compensation response in B. mori. The findings offer new insights into understanding the genetic compensation response and exploring the gene functions in lepidopteran insects, such as silkworms.

[Objective] Our group previously generated a BmTRPA1 nonsense mutant and observed no significant difference in the diapause phenotype of its progeny compared to the wild type, yet the underlying molecular mechanism remains unclear. This study aimed to elucidate the effects of the BmTRPA1 nonsense mutation on the ovarian proteomic profile at the pupal stage of bivoltine silkworm and to explore the potential genetic compensation involved. [Methods] Ovarian tissues were collected on day 3 post-pupation from wild-type (WT) and BmTRPA1-/- mutants of the bivoltine strain "Qiufeng", with three biological replicates per group. Differentially expressed proteins (DEPs) were profiled using tandem mass tag (TMT)-based quantitative proteomics coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS), followed by Gene Ontology (GO), KEGG pathway, and protein-protein interaction (PPI) network analyses. [Results] A total of 6,318 proteins were identified, and 192 DEPs were obtained using thresholds of fold change (FC) > 1.5 and Q-value < 0.05, comprising 96 upregulated and 96 downregulated proteins. Functionally, at terms of immunity and stress level, antimicrobial peptides (Glv1, Glv2, Glv3 and Attacin1), lysozyme (Lzm), and pattern recognition receptors (CTL19 and PGRP) were significantly upregulated, whereas Spätzle-like proteins, the key ligands of the Toll pathway, were markedly downregulated. At the energy and metabolic level, fat body protein 3Rev-G1, acetoacetyl-CoA thiolase (Aact), and trehalase-2 (Treh-2) were significantly upregulated, while glycogen synthase (GlyS), phosphoglycerate mutase (Pglym), and oxidative phosphorylation-related proteins were downregulated. At the signaling and transcriptional level, adenylyl cyclase (AC), Cad96Ca-like protein, and Par-3 homolog were upregulated, notably, a histone lysine N-methyltransferase was significantly upregulated, whereas the homeobox protein Nkx-6.2 and transcription factor Atf2 were significantly downregulated. [Conclusion] Combined with our previous finding that progeny of the BmTRPA1 deletion mutant showed no significant difference in diapause phenotype, this study revealed that the pupal ovarian proteome nevertheless underwent substantial reprogramming, suggesting that BmTRPA1 loss may sustain diapause through metabolic bypasses, signaling network adjustment, and genetic compensation. Histone lysine N-methyltransferase, Nkx-6.2, and Atf2 represent promising candidate molecules for further elucidating genetic compensation mechanisms in silkworms. These results provide candidate proteins and clues for further dissection of BmTRPA1-/- mediated diapause regulation and its compensatory mechanisms.