In insects, sex determination pathways involve three levels of master regulators: primary signals, which determine the sex; executors, which control sex-specific differentiation of tissues and organs; and transducers, which link the primary signals to the executors. The primary signals differ widely among insect species. In Diptera alone, several unrelated primary sex determiners have been identified. However, the doublesex (dsx) gene is highly conserved as the executor component across multiple insect orders. The transducer level shows an intermediate level of conservation. In many, but not all examined insects, a key transducer role is performed by transformer (tra), which controls sex-specific splicing of dsx. In Lepidoptera, studies of sex determination have focused on the lepidopteran model species Bombyx mori (the silkworm). In B. mori, the primary signal of sex determination cascade starts from Fem, a female-specific PIWI-interacting RNA, and its targeting gene Masc, which is apparently specific to and conserved among Lepidoptera. Tra has not been found in Lepidoptera. Instead, the B. mori PSI protein binds directly to dsx pre-mRNA and regulates its alternative splicing to produce male- and female-specific transcripts. Despite this basic understanding of the molecular mechanisms underlying sex determination, the links among the primary signals, transducers and executors remain largely unknown in Lepidoptera. In this review, we focus on the latest findings regarding the functions and working mechanisms of genes involved in feminization and masculinization in Lepidoptera and discuss directions for future research of sex determination in the silkworm.

The silkworm Bombyx mori is an economically important insect. The sericulture industry is seriously affected by pathogen infections. Of these pathogens, Bombyx mori nucleopolyhedrovirus (BmNPV) causes approximately 80% of the total economic losses due to pathogen infections. We previously constructed a BmNPV-specific CRISPR/Cas9 silkworm line with significantly enhanced resistance to BmNPV. In order to optimize the resistance properties and minimize its impact on economic traits, we constructed an inducible CRISPR/Cas9 system for use in transgenic silkworms. We used the 39k promoter, which is induced by viral infection, to express Cas9 and the U6 promoter to express four small guide RNA targeting the genes encoding BmNPV late expression factors 1 and 3 (lef-1 and lef-3, respectively), which are essential for viral DNA replication. The system was rapidly activated when the silkworm was infected and showed considerably higher resistance to BmNPV infection than the wild-type silkworm. The inducible system significantly reduced the development effects due to the constitutive expression of Cas9. No obvious differences in developmental processes or economically important characteristics were observed between the resulting transgenic silkworms and wild-type silkworms. Adoption of this accurate and highly efficient inducible CRISPR/Cas9 system targeting BmNPV DNA replication will result in enhanced antivirus measures during sericulture, and our work also provides insights into the broader application of the CRISPR/Cas9 system in the control of infectious diseases and insect pests.

Uric acid (UA) is the end-product in the human purine metabolism pathway. The UA that accumulates in silkworm tissues is excreted as a nitrogen waste product. Here, we first validated that Bombyx mori has a homolog of the human gene that encodes the 5′-nucleotidase (5′N) involved in purine metabolism. The B. mori gene, Bm5′N, is located upstream of other genes involved in UA metabolism in the silkworm. Disruption of Bm5′N via the CRISPR/Cas9 system resulted in decreased UA levels in the silkworm epidermis and caused a translucent skin phenotype. When Bm5′N mutant silkworms were fed with the uric acid precursor inosine, the UA levels in the epidermis increased significantly. Furthermore, the metabolomic and transcriptomic analyses of Bm5′N mutants indicated that loss of the Bm5′N affected purine metabolism and the ABC transport pathway. Taken together, these results suggest that the UA pathway is conserved between the silkworm and humans and that the Bm5′N gene plays a crucial role in the uric acid metabolism of the silkworm. Thus, the silkworm may be a suitable model for the study of UA metabolism pathways relevant to human disease.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a pathogen that causes severe disease in silkworms. In a previous study, we demonstrated that by using the CRISPR/Cas9 system to disrupt the BmNPV ie-1 and me53 genes, transgenic silkworms showed resistance to BmNPV infection. Here, we used the same strategy to simultaneously target lef8 and lef9, which are essential for BmNPV replication. A PCR assay confirmed that double-stranded breaks were induced in viral DNA at targeted sequences in BmNPV-infected transgenic silkworms that expressed small guide RNAs (sgRNAs) and Cas9. Bioassays and qPCR showed that replication of BmNPV and mortality were significantly reduced in the transgenic silkworms in comparison with the control groups. Microscopy showed degradation of midgut cells in the BmNPV-infected wild type silkworms, but not in the transgenic silkworms. These results demonstrated that transgenic silkworms using the CRISPR/Cas9 system to disrupt BmNPV lef8 and lef9 genes could successfully prevent BmNPV infection. Our research not only provides more alternative targets for the CRISPR antiviral system, but also aims to provide new ideas for the application of virus infection research and the control of insect pests.

Insect courtship and mating depend on integration of olfactory, visual, and tactile cues. Compared to other insects,Bombyx mori, the domesticated silkworm, has relatively simple sexual behaviors as it cannot fly. Here by using CRISPR/Cas and electrophysiological techniques we found that courtship and mating behaviors are regulated in male silk moths by mutating genes in the sex determination cascade belonging to two conserved pathways. Loss ofBmdsxgene expression significantly reduced the peripheral perception of the major pheromone component bombykol by reducing expression of the product of theBmOR1gene which completely blocked courtship in adult males. Interestingly, we found that mating behavior was regulated independently by another sexual differentiation gene,Bmfru.Loss ofBmfrucompletely blocked mating, but males displayed normal courtship behavior. Lack ofBmfruexpression significantly reduced the perception of the minor pheromone component bombykal due to the down regulation ofBmOR3expression; further, functional analysis revealed that loss of the product ofBmOR3played a key role in terminating male mating behavior. Our results suggest thatBmdsxandBmfruare at the base of the two primary pathways that regulate olfactory-based sexual behavior.Author Summary: The fundamental insect sexual behaviors, courtship and mating, result from successful integration of olfactory, vision, tactile and other complex innate behaviors. In the widely used insect model,Drosophila melanogaster, the sex determination cascade genesfruitlessanddoublesexare involved in the regulation of courtship and mating behaviors; however, little is known about the function of these sexual differentiation genes in regulating sex behaviors of Lepidoptera. Here we combine genetics and electrophysiology to investigate regulation pathway of sexual behaviors in the model lepidopteran insect, the domesticated silk moth,Bombyx mori. Our results support the presence of two genetic pathways inB. mori, namedBmdsx-BmOR1-bombykol andBmfru-BmOR3-bombykal, which control distinct aspects of male sexual behavior that are modulated by olfaction. This is the first comprehensive report about the role of sex differentiation genes in the male sexual behavior in the silk moth.

Spermatogenesis is a key process for the sexual reproduction species. In lepidopteran insects, dichotomous spermatogenesis is a notable feature, which produces eupyrene (nucleate) and apyrene (anucleate) spermatozoa. Both sperm morphs are essential for fertilization, as eupyrene sperm fertilizes the egg, while apyrene sperm is necessary for eupyrene sperm migration. In Drosophila , Prmt5 acts as a type II arginine methyltransferase to catalyze the symmetrical dimethylation of arginine residues (sDMA) in Vasa. However, Prmt5 is involved in regulating spermatogenesis, but Vasa is not. To date, the functional genetic study on dichotomous spermatogenesis in the lepidopteran model Bombyx mori has been limited, thus the underlying mechanism remains largely unknown. In this study, we report that both BmPrmt5 and BmVasa act as essential components in the regulation of dichotomous spermatogenesis in Bombyx mori . The loss-of-function mutants of BmPrmt5 ( ΔBmPrmt5 ) and BmVasa ( ΔBmVasa ) derived from CRISPR/Cas9-based gene editing show similar male and female-sterile phenotypes. Through immunofluorescence staining analysis, we found that the morphs of both ΔBmPrmt5 and ΔBmVasa sperm show severe defects, indicating an essential role for both BmPrmt5 and BmVasa in the regulation of dichotomous spermatogenesis. RNA-seq analyses indicate that the defects in dichotomous spermatogenesis observed for ΔBmPrmt5 and ΔBmVasa mutants could be attributed to the reduced expression of the spermatogenesis-related genes including Sex-lethal ( BmSxl ), implying that BmSxl may act downstream of Prmt5 and Vasa in regulating apyrene sperm development. These findings suggest that BmPrmt5 and BmVasa constitute an integral regulatory module essential for dichotomous spermatogenesis in Bombyx mori , in which BmPrmt5 may promote BmVasa activity through sDMA modification. Author Summary: Dichotomous spermatogenesis is a notable feature in Lepidoptera insects, in which a single male produces nucleated and anucleated dimorphic sperm. Both sperm morphs are essential for fertilization, as the eupyrene sperm fertilizes the egg, while the apyrene sperm assists in transporting the eupyrene sperm to the female sperm storage organs. To date, the mechanism of dichotomous spermatogenesis is still unclear, as very limited components participating in this process have been reported. Here we defined the function of BmPrmt5 (Protein arginine methyltransferase 5) and its substrate BmVasa in dichotomous spermatogenesis in the lepidopteran insect Bombyx mori . Our genetic analyses revealed that both BmPrmt5 and BmVasa are essential for dichotomous spermatogenesis. Moreover, RNA-seq analyses, in conjunction with the genetic and immunofluorescence staining analyses, suggest a potential regulatory pathway consisting of the BmPrmt5–BmVasa module in dichotomous spermatogenesis in Bombyx mori . This study expands the knowledge of reproduction biology in insects, and may provide potential gene targets for pest control.