Sex determination is an important and traditional biological process. In Lepidoptera, Masculinizer (Masc) and doublesex (dsx) are the essential genes for sex determination and play critical roles in sexual differentiation and development. The functions of Masc and dsx have been characterized in several model insect species. However, the molecular mechanism and sex determination functions of Masc and dsx in Ostrinia furnacalis, an agricultural pest, are still unknown. Here, we successfully used the CRISPR/Cas9 genome editing system to knock out OfMasc and Ofdsx. Mutation of OfMasc induced male external genital defects and sterility. Disruptions of the Ofdsx common region caused sex-specific defects in the external genitals and adult sterility. In addition, we found that OfMasc and Ofdsx can regulate the pigmentation genes that control wing pigmentation patterns. These results demonstrate that OfMasc and Ofdsx play key roles in the sex determination of O. furnacalis, and suggest novel genetic control approaches for the management of pests, including O. furnacalis.

Seminal fluid proteins provide factors necessary for development, storage, and activation of sperm. Altered expression of seminal fluid proteins can lead to defect in male infertility. We investigated the function of seminal fluid protein Sfp62 in the model lepidopteran insect Bombyx mori using CRISPR/Cas9 mediated mutagenesis. The knockout of BmSfp62 gene led to male sterility but has no effect on female fertility. The mutation did not affect growth and development of the silkworm of both sexes. Motility of sperm in male mutants was decreased and the mRNA expression levels of other genes encoding seminal fluid proteins were altered in BmSfp62 mutants compared to the wild-type controls. The male sterility caused by mutation of BmSfp62 was stably inherited. As the proteins encoded by Sfp62 genes are conserved among lepidopteran species, Sfp62 is a potential target for the biological management of lepidopteran pests.

Silkworm (Bombyx mori) is an economically significant insect that produces silk and makes important contributions to the development of silk-producing countries. The genetic diversity and unique adaptive traits of silkworm germplasm resources form the foundation for breeding efforts. In various geographical regions, silkworm have developed distinct traits through long-term adaptive selection. In this study, we focused on identifying and evaluating representative silkworm germplasm resources from both China and Uzbekistan. We discovered notable differences in the morphology and production traits of local silkworm strains, likely influenced by differing geographical environments. It is also possible that China has a long history of silkworm rearing and after a long time of breeding selection, the diversity of silkworm morphology is less than that of Uzbekistan, but the production characteristics are more suitable for silk utilization. Phylogenetic analysis based on genomic comparisons revealed that Uzbekistan’s local silkworm strains are positioned between China’s local and improved strains. This suggests that Uzbekistan silkworms primarily originated from China. The further analysis of genetic diversity and strain differentiation highlights the unique differences of each country’s silkworms, especially in terms of genetic diversity on chromosome 1 (sex chromosome). By identifying and evaluating these germplasm resources and linking them to unique advantageous traits, it provides a scientific basis for improving cocoon silk quality and optimizing sericulture productivity.

Artificial parthenogenetic induction via thermal stimuli in silkworm is an important technique that has been used in sericultural production. However, the molecular mechanism underlying it remains largely unknown. We have created a fully parthenogenetic line (PL) with more than 85% occurrence and 80% hatching rate via hot water treatment and genetic selection, while the parent amphigenetic line (AL) has less than 30% pigmentation rate and less than 1% hatching rate when undergoing the same treatment. Here, isobaric tags for relative and absolute quantitation (iTRAQ)-based analysis were used to investigate the key proteins and pathways associated with silkworm parthenogenesis. We uncovered the unique proteomic features of unfertilized eggs in PL. In total, 274 increased abundance proteins and 211 decreased abundance proteins were identified relative to AL before thermal induction. Function analysis displayed an increased level of translation and metabolism in PL. After thermal induction, 97 increased abundance proteins and 187 decreased abundance proteins were identified. An increase in stress response-related proteins and decrease in energy metabolism suggested that PL has a more effective response to buffer the thermal stress than AL. Cell cycle-related proteins, including histones, and spindle-related proteins were decreased in PL, indicating an important role of this decrease in the process of ameiotic parthenogenesis.

In most species, oocytes are arrested at the prophase or metaphase of meiosis I and require sperm-derived or external stimuli to resume meiosis. Maturation-promoting factor (MPF) is an oocyte maturation factor composing the catalytic subunit Cdc2 and the regulatory subunit CycB that can restart stalled meiosis. In this study, we demonstrated that MPF activity affected parthenogenesis induction in the model lepidopteran insect Bombyx mori using activator and inhibitor interference. We found that the upregulation of MPF activity significantly increased the parthenogenesis induction rate, whereas downregulation significantly reduced it. Furthermore, the inhibition of MPF activity also led to a delay in embryonic development. Given its evolutionary conservation, MPF emerges as a potential universal target for manipulating reproductive outcomes, offering broad applications in genetics and selective breeding.

Protein kinase A (PKA), commonly referred to as cAMP-dependent protein kinase, exists as a heterotetramer composed of two catalytic (C) and regulatory subunits (R). This versatile kinase exhibits regulatory functions in various biological processes including growth, division, and differentiation. Although PKA is well established as a master regulator of oocyte maturation across species, its functional role in insect parthenogenesis has remained enigmatic. Here, we systematically investigated the regulatory effect of PKA in the induction of parthenogenesis in model lepidopteran Bombyx mori. Our findings demonstrated an inverse correlation between PKA activity and parthenogenetic induction efficiency in silkworms. Notably, PKA activation resulted in delayed embryonic development, whereas PKA-C1 knockdown disrupted normal cell cycle progression. These results indicated that maintaining appropriate PKA activity is essential for ensuring proper cell division process, especially in the successful induction of silkworm parthenogenesis. The evolutionary conservation of PKA across species, coupled with its critical regulatory role in parthenogenesis, positions this kinase as a promising molecular target for breeding design. Our findings establish a foundation for developing silkworm strains with enhanced parthenogenetic capacity through PKA modulation, thereby facilitating the preservation of elite production traits. These results provide novel mechanistic insights into parthenogenesis while demonstrating the potential application of PKA regulation in both genetic studies and breeding programs.