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.

The apoptosis pathway is highly conserved between invertebrates and mammals. Although genes encoding the classical apoptosis pathway can be found in the silkworm genome, the regulatory pathway and other apoptotic network genes have yet to be confirmed. Consequently, characterizing these genes and their underlying mechanisms could provide critical insights into the molecular basis of organ apoptosis and remodeling. A homolog of p53, a key apoptosis regulator in vertebrates, has been identified and cloned from Bombyx mori (Bmp53). This study confirmed via gene knockdown and overexpression that Bmp53 directly induces cell apoptosis and regulates the morphology and development of individuals during the metamorphosis stage. Furthermore, yeast two-hybrid sequencing (Y2H-Seq) identified several potential apoptotic regulatory interacting proteins, including the MDM2-like ubiquitination regulatory protein, which may represent an apoptosis factor unique to Bmp53 and which differs from that in other Lepidoptera. These results provide a theoretical basis for analyzing the various biological processes regulated by Bmp53 interaction groups and thus provide insight into the regulation of apoptosis in silkworms. The global interaction set identified in this study also provides a basic framework for future studies on apoptosis-dependent pupation in Lepidoptera.

In the silkworm, 20-hydroxyecdysone (20E) induces apoptosis and autophagy, driving larval organ degeneration and remodeling. This mechanism may be a potential target for eco-friendly strategy for insect pests. However, a major challenge in harnessing this approach lies in the insufficient understanding of 20E’s regulatory process in such a cell death mechanism. Our previous research has identified Bmp53 as a crucial gene in promoting the development of Bombyx mori during the pupal stage by inducing apoptosis, and has predicted the potential apoptotic regulatory network of Bmp53, wherein Mdm2-like ubiquitinating structural protein serves as a key component of this network. This study demonstrates that Mdm2-like acts as a ubiquitination regulatory protein, controlling its apoptosis-inducing activity via interaction with Bmp53. Moreover, co-expression of Mdm2-like and Bmp53 indirectly affects gene expression in the 20E-mediated apoptosis pathway. Further investigation revealed that Mdm2-like suppresses 20E-induced apoptosis by downregulating Bmp53 expression. This study reveals that the ubiquitination-mediated Mdm2-like/Bmp53 apoptosis pathway is a novel mechanism regulating silkworm apoptosis, with 20E playing a crucial role in this process. These findings enhance our understanding of the genetic mechanisms underlying tissue degradation during the metamorphic stage of the Bombyx mori. Additionally, these insights provide a theoretical reference for the development of environmentally friendly, hormone-based control strategies targeting protein modification as a means of managing lepidopteran pests.