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.

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.

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.

Sperm deliver male genomic DNA to the ovum, playing a pivotal role in sexual reproduction across the animal kingdom. The molecular regulation of sperm morphogenesis has consequently become a focal point of genetic research, with dual implications for both reproductive medicine and sustainable agriculture. Here, we characterize the functional role of the testis-specific serine/threonine protein kinase-like ( TSSKL ) gene in the model lepidopteran insect Bombyx mori and the globally destructive crop pest Plutella xylostella . RNA-seq and qPCR analyses revealed TSSKL ’s testis-specific expression pattern. Using CRISPR/Cas9-mediated mutagenesis, we demonstrate that TSSKL knockout induces complete male sterility, while female fertility remains unaffected compared to wild-type. Fluorescence microscopy and ultrastructural analyses revealed that TSSKL deletion leads to severe morphological defects in both eupyrene and apyrene sperm, accompanied by impaired mitochondrial dynamics and aberrant autophagy. Comparative transcriptome and functional analyses linked these phenotypes to dysregulated energy metabolism pathways. Crucially, this sterility phenotype is conserved in P. xylostella , recapitulating the findings in B. mori . Our study demonstrates that TSSKL is crucial for male fertility, coordinating both structural and metabolic aspects of sperm development. Beyond advancing fundamental knowledge of insect reproductive biology, this work also identifies TSSKL as an ideal target for lepidopteran pest control through precision sterility induction.

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.