SPSL1 is essential for spermatophore formation and sperm activation in Spodoptera frugiperda.

The reproductive process in various species has undergone evolutionary adaptations at both the physiological and molecular levels, playing a significant role in maintaining their populations. In lepidopteran insects, the spermatophore is a unique structure formed in the female reproductive system, in which sperm storage and activation take place. It is known that the formation of the spermatophore is regulated by seminal fluid proteins derived from males. However, studies investigating the genetic mechanisms behind spermatophore formation in lepidopterans have been limited. In this study, our focus was on SPSL1, a gene that encodes a trypsin-type seminal fluid protein in Spodoptera frugiperda, a pest species with global invasive tendencies. Our findings revealed that SPSL1 expression was predominantly observed in the male reproductive tracts, and the disruption of this gene resulted in male sterility. Surprisingly, fluorescence analysis indicated that the absence of SPSL1 did not affect spermatogenesis or sperm migration within the male reproductive system. However, when females mated with SPSL1-mutant males, several defects were observed. These included disruptions in spermatophore formation, sperm activation in the copulatory bursae, and sperm migration into the spermathecae. Additionally, mass spectrometry analysis highlighted reduced levels of energy-related metabolites, suggesting that SPSL1 plays an essential role in promoting hydrolysis reactions during copulation. Consequently, our study demonstrates that SPSL1 is crucial for male fertility due to its functions in spermatophore formation and sperm activation. This research provides valuable insights into the genetic factors underlying reproductive processes in lepidopteran insects and sheds light on potential strategies for controlling invasive pest populations.

Transgenic Clustered Regularly Interspaced Short Palindromic Repeat/Cas9-Mediated Viral Gene Targeting for Antiviral Therapy of Bombyx mori Nucleopolyhedrovirus.

We developed a novel antiviral strategy by combining transposon-based transgenesis and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system for the direct cleavage of Bombyx mori nucleopolyhedrovirus (BmNPV) genome DNA to promote virus clearance in silkworms. We demonstrate that transgenic silkworms constitutively expressing Cas9 and guide RNAs targeting the BmNPV immediate early-1 (ie-1) and me53 genes effectively induce target-specific cleavage and subsequent mutagenesis, especially large (∼7-kbp) segment deletions in BmNPV genomes, and thus exhibit robust suppression of BmNPV proliferation. Transgenic animals exhibited higher and inheritable resistance to BmNPV infection than wild-type animals. Our approach will not only contribute to modern sericulture but also shed light on future antiviral therapy.IMPORTANCE Pathogen genome targeting has shown its potential in antiviral research. However, transgenic CRISPR/Cas9 system-mediated viral genome targeting has not been reported as an antiviral strategy in a natural animal host of a virus. Our data provide an effective approach against BmNPV infection in a real-world biological system and demonstrate the potential of transgenic CRISPR/Cas9 systems in antiviral research in other species.

Masculinizer gene controls sexual differentiation in Hyphantria cunea.

The Masculinizer gene, Masc, encodes a lepidopteran-specific novel CCCH-type zinc finger protein, which controls sex determination and dosage compensation in Bombyx mori. Considering the potential application of it in pest control, it is necessary to investigate the function of Masc gene in Hyphantria cunea, a globally invasive forest pest. In the present study, we identified and functionally characterized the Masc gene, HcMasc, in H. cunea. Sequence analysis revealed that HcMASC contained the conserved CCCH-type zinc finger domain, nuclear localization signal, and male determining domain, in which the last was confirmed to be required for its masculinization in BmN cell line. However, expression data showed that unlike male-biased expression in B. mori, HcMasc gene expresses in main all developmental stages or tissues in both sexes. Clustered regularly interspaced palindromic repeats (CRISPR) / CRISPR-associated protein 9-based disruption of the common exons 1 and 3 of the HcMasc gene resulted in imbalanced sex ratio and abnormal external genitalia of both sexes. Our results suggest that the HcMasc gene is required for both male and female sexual differentiation and dosage compensation in H. cunea and provide a foundation for developing better strategies to control this pest.

CRISPR/Cas9-Mediated Mutagenesis of Abdominal-A and Ultrabithorax in the Asian Corn Borer, Ostrinia furnacalis.

(1) Background: Abdominal-A (Abd-A) and Ultrabithorax (Ubx) are homeotic genes that determine the identity and morphology of the thorax and abdomen in insects. The Asian corn borer, Ostrinia furnacalis (Guenée) (Lepidoptera: Pyralidae), is a devastating maize pest throughout Asia, the Western Pacific, and Australia. Building on previous knowledge, we hypothesized that the knockout of Abd-A and Ubx would disrupt the abdominal body planning in O. furnacalis. (2) Methods: CRISPR/Cas9-targeted mutagenesis was employed to decipher the functions of these homeotic genes. (3) Results: Knockout insects demonstrated classical homeotic transformations. Specifically, the mutagenesis of OfAbd-A resulted in: (1) Fused segments and segmental twist during the larval stage; (2) Embryonic lethality; and (3) The pleiotropic upregulation of other homeotic genes, including Lab, Pd, Dfd, Antp, and Abd-B. The mutagenesis of OfUbx led to: (1) Severe defects in the wing pads, which limited the ability of the adults to fly and mate; (2) Female sterility; and (3) The pleiotropic upregulation of other homeotic genes, including Dfd, Abd-B, and Wnt1. (4) Conclusions: These combined results not only support our hypothesis, but they also strengthen the potential of using homeotic genes as molecular targets for the genetic control of this global insect pest.

piggyBac-based transgenic RNAi of serine protease 2 results in male sterility in Hyphantria cunea.

Fall webworm, Hyphantria cunea, is a global invasive forest pest that causes serious damage to the economy and ecosystem of agriculture and forestry. Due to the extent of the problem and the difficulty of conventional chemical control, new technologies must be pursued, such as genetic-based inheritable insect sterile technology (gSIT), which exhibits promise for pest control. In the present study, we established a piggyBac-based transgenic system in fall webworm and generated a dominant male-sterile strain by targeting the seminal fluid protein serine protease 2 (Hcser2), displaying an outstanding trait of gSIT. First, an RNA polymerase type III (Pol III) promoter, the HcU62 small nuclear RNA (snRNA) gene promoter, was identified and characterized through direct injection of RNAi plasmids in vivo. Quantitative real-time PCR revealed that HcU62 had the greatest knockdown efficiency of the Hcyellow gene among five short hairpin RNA (shRNA) plasmids tested, designated HcU61-HcU65. Second, subsequent application of piggyBac-based transgenic RNAi (HcU62: shHcyellow, Ysh2) significantly reduced the expression level of the Hcyellow gene, resulting in a stable yellow observable phenotype from the larval to pupal stages in Ysh2 transgenic mutants. Finally, an HcU62-driven transgenic RNAi strain targeting the Hcser2 gene was obtained, resulting in a dominant male-sterile phenotype. Significantly, this process did not affect the growth, development, mating behavior or egg laying of the mutants, and the dominant sterile trait could be inherited in the next generation through female Hcser2 mutants. Furthermore, CRISPR/Cas9-mediated disruption of the Hcser2 gene further confirmed the dominant sterile phenotype, supporting it as a generalized target for genetic control of H. cunea. This study reports the first piggyBac-mediated transgenic system in H. cunea, providing a promising genetic method for controlling this pest by targeting Hcser2 gene.

CRISPR/Cas9-mediated Serine protease 2 disruption induces male sterility in Spodoptera litura.

Male fertility is essential for reproduction and population growth in animals. Many factors affect male fertility, such as courtship behavior, sperm quantity, and sperm motility, among others. Seminal Fluid Proteins (SFPs) are vital components of seminal fluid in the male ejaculate, which affect male fertility, sperm activation, and female ovulation. However, the knowledge of SFPs is insufficient; the function of many SFPs remains unknown, and most described functions were mainly characterized in Drosophila or other laboratory models. Here, we focus on the Serine protease 2 (Ser2) gene in the lepidopteran pest Spodoptera litura. The Ser2 gene was specifically expressed in male adults. Disruption of the Ser2 gene mediated by CRISPR/Cas9 induced male sterility but females remained fertile. PCR-based detection of the next-generation mutants showed that male sterility was stably inherited. The qRT-PCR analysis of SlSer2 mutants showed that motor protein family genes and structural protein family genes were down-regulated, while protein modification family genes were up-regulated, suggesting that SlSer2 may be involved in sperm movement and activity. These results demonstrate that Ser2 is an important component of SFPs in seminal fluid and was identified for a useful sterile gene for pest control that may lead to new control strategies for lepidopteran insect pests such as S. litura.

Masculinizer and Doublesex as Key Factors Regulate Sexual Dimorphism in Ostrinia furnacalis.

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.

CRISPR/Cas9-Mediated Mutagenesis of Sex-Specific Doublesex Splicing Variants Leads to Sterility in Spodoptera frugiperda, a Global Invasive Pest.

Spodoptera frugiperda (J. E. Smith), an emerging invasive pest worldwide, has posed a serious agricultural threat to the newly invaded areas. Although somatic sex differentiation is fundamentally conserved among insects, the sex determination cascade in S. frugiperda is largely unknown. In this study, we cloned and functionally characterized Doublesex (dsx), a "molecular switch" modulating sexual dimorphism in S. frugiperda using male- and female-specific isoforms. Given that Lepidoptera is recalcitrant to RNAi, CRISPR/Cas9-mediated mutagenesis was employed to construct S. frugiperda mutants. Specifically, we designed target sites on exons 2, 4, and 5 to eliminate the common, female-specific, and male-specific regions of S. frugiperda dsx (Sfdsx), respectively. As expected, abnormal development of both the external and internal genitalia was observed during the pupal and adult stages. Interestingly, knocking out sex-specific dsx variants in S. frugiperda led to significantly reduced fecundity and fertility in adults of corresponding sex. Our combined results not only confirm the conserved function of dsx in S. frugiperda sex differentiation but also provide empirical evidence for dsx as a potential target for the Sterile Insect Technique (SIT) to combat this globally invasive pest in a sustainable and environmentally friendly way.

Mutation of P-element somatic inhibitor induces male sterility in the diamondback moth, Plutella xylostella.

BACKGROUND: Genetic manipulation of sex determination pathways in insects provides the basis for a broad range of strategies to benefit agricultural security and human health. The P-element somatic inhibitor (PSI) protein, an exon splicing silencer that promotes male-specific splicing of dsx, plays a critical role in male sexual differentiation and development. The functions of PSI have been characterized in the lepidopteran model species Bombyx mori. However, the molecular mechanism and functions of PSI in Plutella xylostella, a worldwide agricultural pest and taxonomically basal species, are still unknown. RESULTS: Here we identified PxPSI transcripts and analyzed their spatiotemporal expression pattern in P. xylostella. Multiple sequence alignment revealed that PxPSI contains four KH domains and is highly conserved in lepidopterans. We used the CRISPR-Cas9 system to generate mutations of the PxPSI genomic locus. Disruptions of PxPSI caused male-specific defects in internal and external genitals. In addition, we detected female-specific Pxdsx transcripts in PxPSI male mutants. Mutations also caused changes in expression of several sex-biased genes and induced male sterility. CONCLUSION: Our study demonstrates that PxPSI plays a key role in male sex determination in P. xylostella and suggests a potential molecular target for genetic-based pest management in lepidopteran pests. © 2021 Society of Chemical Industry.

Mutation of the seminal protease gene, serine protease 2, results in male sterility in diverse lepidopterans.

Sterile insect technology (SIT) is an environmentally friendly method for pest control. As part of our efforts to develop a strategy that results in engineered male-sterile strains with minimum effects on viability and mating competition, we used CRISPR/Cas9 technology to disrupt Ser2, which encodes a seminal fluid protein, in the model lepidopteran insect, Bombyx mori, and an important agricultural pest, Plutella xylostella. Disruption of Ser2 resulted in dominant heritable male sterility. Wild-type females mated with Ser2-deficient males laid eggs normally, but the eggs did not hatch. We detected no differences in other reproductive behaviors in the mutant males. These results support the conclusion that Ser2 gene is necessary for male reproductive success in diverse lepidopterans. Targeting Ser2 gene has the potential to form the basis for a new strategy for pest control.

Disruption of the ovarian serine protease (Osp) gene causes female sterility in Bombyx mori and Spodoptera litura.

BACKGROUND: Precise regulation of oogenesis is crucial to female reproduction. Seventy percent of pests belong to lepidopteran species, so it would be interesting to explore the highly conserved genes involved in oogenesis that do not affect growth and development in the lepidopteran model, Bombyx mori. This can provide potential target genes for pest control and promote the development of insect sterility technology. RESULTS: In lepidopteran species, ovarian serine protease (Osp), which encodes a member of the serine protease family, is essential for oogenesis. In this study, we used transgenic CRISPR/Cas9 technology to obtain Osp mutants in the model lepidopteran insect Bombyx mori and in the lepidopteran agricultural pest Spodoptera litura. Sequence analysis of mutants revealed an array of deletions in Osp loci in both species. We found that the deletion of Osp resulted in female sterility, whereas male fertility was not affected. Although B. mori and S. litura mutant females mated normally, they laid fewer eggs than wild-type females and eggs did not hatch. CONCLUSION: Osp is crucial for female reproductive success in two species of Lepidoptera. As the Osp gene is highly conserved in insect species, this gene is a potential molecular target for genetic-based pest management. © 2019 Society of Chemical Industry.

Mutation of doublesex in Hyphantria cunea results in sex-specific sterility.

BACKGROUND: The gene doublesex (dsx) plays pivotal roles in sex determination and controls sexually dimorphic development in certain insects. Importantly, it also displays a potential candidate target for pest management due to its sex-specific splicing. Therefore, we used CRISPR/Cas9-mediated gene disruption to investigate the function of dsx in Hyphantria cunea, an invasive forest pest. RESULT: In the present study, we identified the dsx gene from H. cunea which showed a sex-biased expression pattern that was different from other lepidopteran insects. Referring to sex-specific functional analyses in Bombyx mori, we performed a site-specific knockout of the Hcdsx gene by using a CRISPR/Cas9 system, which induced severe abnormalities in external genitalia and some incomplete sex reversal phenotypes, which in turn led to reduced sex-specific fecundity. An alternative splicing pattern of Hcdsx was altered by CRISPR/Cas9-induced mutation, and alterations in splicing affected expression of downstream genes encoding pheromone binding protein 1, vg1 and vg2 (encoding vitellogenin), which contributed to the sex-specific sterility phenotypes in the Hcdsx mutants. CONCLUSION: The Hcdsx gene plays important roles in sexual differentiation in H. cunea. Disruption of Hcdsx induced sex-specific sterility, demonstrating a potential application in control of this pest. © 2019 Society of Chemical Industry.

CRISPR Disruption of BmOvo Resulted in the Failure of Emergence and Affected the Wing and Gonad Development in the Silkworm Bombyx mori.

The domesticated silkworm is an economically important insect that is widely used as a lepidopteran insect model. Although somatic sex determination in the silkworm is well characterized, germline sex determination is not. Here, we used the transgenic-based CRISPR/Cas9 genome editing system to study the function of the Ovo gene in Bombyx mori. BmOvo is the homolog of a factor important in germline sex determination in Drosophila melanogaster. BmOvo mutants had abnormally shaped eggs that were disordered in the ovarioles, and gonad development was abnormal. Interestingly, wing discs and wings did not develop properly, and most of the mutants failed to eclose. Gene expression analyses by qRT-PCR showed that BmOvo gene was highly expressed in the wing disc and epidermis. Genes involved in the WNT signaling pathway and wing development genes BmWCP10 and BmE74 were downregulated in the BmOvo mutants when compared with wild-type animals. These results demonstrate that the BmOvo gene product plays an important role in wing metamorphosis. Thus, this study provides new insights into the multiple functions of BmOvo beyond germline sex determination.

CRISPR/Cas9-mediated ebony knockout results in puparium melanism in Spodoptera litura.

Insect body pigmentation and coloration are critical to adaption to the environment. To explore the mechanisms that drive pigmentation, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing system to target the ebony gene in the non-model insect Spodoptera litura. Ebony is crucial to melanin synthesis in insects. By directly injecting Cas9 messenger RNA and ebony-specific guide RNAs into S. litura embryos, we successfully induced a typical ebony-deficient phenotype of deep coloration of the puparium and induction of melanin formation during the pupal stage. Polymerase chain reaction-based genotype analysis demonstrated that various mutations had occurred at the sites targeted in ebony. Our study clearly demonstrates the function of ebony in the puparium coloration and also provides a potentially useful marker gene for functional studies in S. litura as well as other lepidopteran pests.

Identification and functional characterization of doublesex gene in the testis of Spodoptera litura.

Fusion of the testis occurs in most Lepidoptera insects, including Spodoptera litura, an important polyphagous pest. Testicular fusion in S. litura is advantageous for male reproduction, and the molecular mechanism of fusion remains unknown. Doublesex influences the formation of genitalia, the behavior of courtship, and sexually dimorphic traits in fruit-fly and silkworm, and is essential for sexual differentiation. However, its purpose in the testis of S. litura remains unknown. The doublesex gene of S. litura (Sldsx) has male-specific SldsxM and female-specific SldsxF isoforms, and exhibits a higher expression level in the male testis. At the testicular fusion stage (L6D6), Sldsx attained the highest expression compared to the pre-fusion and post-fusion periods. Moreover, Sldsx had a higher expression in the peritoneal sheaths of testis than that of germ cells in the follicle. CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Cas9) was applied to S. litura to determine the role of Sldsx. A mixture of single guide RNA messenger RNA and Cas9 protein (300 ng/µL each) was injected into eggs within 2 h following oviposition. CRISPR/Cas9 successfully induced genomic mutagenesis of Sldsx at G0 generation. The mutant males had smaller testis surrounded by less tracheae. Moreover, the mutant males had abnormal external genitalia and could not finish mating with wild-type females. Additionally, testes were fused for almost all mutant males. The results showed that Sldsx was not related to testicular fusion, and is required for both testis development and the formation and function of external genitalia in S. litura. The main roles of doublesex on the male are similar to other insects.

Mutation of doublesex induces sex-specific sterility of the diamondback moth Plutella xylostella.

DOUBLESEX (DSX): the downstream gene in the insect sex determination pathway, plays a critical role in sexual differentiation and development. The functions of dsx have been characterized in several model insect species. However, the molecular mechanism and functions of sex determination of dsx in Plutella xylostella, an agricultural pest, are still unknown. In present study, we identified a male-specific and three female-specific Pxdsx transcripts in P. xylostella. Phylogenetic analyses and multiple sequence alignment revealed that Pxdsx is highly conserved in lepidopterans. The CRISPR/Cas9 technology was used to induce mutations in the male-specific isoform, the female-specific isoform, and common regions of Pxdsx. Disruptions of Pxdsx sex-specific isoforms caused sex-specific defects in external genitals and partial sexual reversal. In addition, we found that female specific transcripts were detected in PxdsxM male mutants and male-specific transcripts were detected in PxdsxF female mutants. Mutations also caused changes in expression of several sex-biased genes and induced sex-specific sterility. This study demonstrates that Pxdsx plays a key role in sex determination of P. xylostella and suggests novel genetic control approaches for the management of P. xylostella.

CRISPR/Cas9-based mutation reveals Argonaute 1 is essential for pigmentation in Ostrinia furnacalis.

Ostrinia furnacalis (Lepidoptera: Pyralidae) is one of the most destructive agricultural pests in Asia. Traditional pest-management methods include sex pheromone capture, transgenic crops that produce Bacillus thuringiensis toxin, and pesticides. Although these strategies control pest populations effectively, they also cause negative side effects, including dramatically increased pesticide resistance, severe pollution, and hazards for human health. Recently developed genome editing tools provide new prospects for pest management and have been successfully used in several species. However, few examples have been reported in the agricultural pest O. furnacalis due to a lack in genomic information. In this report, we identified only one transcript of O. furnacalis Argonaute 1 (OfAgo1) gene from the genome and cloned the open reading frame. OfAgo1 presented the maximum expression at the embryo stage or in the fat body during the larval stages. To understand its function, an OfAgo1 mutant was constructed using the Clustered Regularly Interspaced Short Palindromic Repeat/RNA-guided Cas9 nuclease (CRISPR/Cas9). Mutagenesis of OfAgo1 disrupted cuticle pigmentation by down-regulating micro RNAs and pigmentation-related genes. This is the first report for the cloning and functional analysis of OfAgo1, revealing a role of OfAgo1 in cuticle pigmentation. The current report also established a CRISPR/Cas9 system in O. furnacalis, providing a new insight for pest management.

BmHpo mutation induces smaller body size and late stage larval lethality in the silkworm, Bombyx mori.

As a core member of the Hippo signaling pathway, Hpo plays a critical role in regulating growth and development. Previous studies reported that loss of function of Hpo results in increased proliferation, reduced apoptosis and induction of tissue overgrowth in Drosophila. In this study, we used CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Cas9) to study Hpo gene (BmHpo) function in the lepidopteran insect Bombyx mori, known commonly as the silkworm. Sequence analysis of BmHpo revealed an array of deletions in mutants. We found that BmHpo knockout resulted in defects in body size regulation, in developmental defects and pigment accumulation and early death. Our data show that BmHpo is essential for regulation of insect growth and development and that CRISPR/Cas9 technology can serve as a basis for functional analysis of target genes in lepidopteran insects.

CRISPR/Cas9-mediated targeted gene mutagenesis in Spodoptera litura.

Custom-designed nuclease technologies such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) system provide attractive genome editing tools for insect functional genetics. The targeted gene mutagenesis mediated by the CRISPR/Cas9 system has been achieved in several insect orders including Diptera, Lepidoptera and Coleoptera. However, little success has been reported in agricultural pests due to the lack of genomic information and embryonic microinjection techniques in these insect species. Here we report that the CRISPR/Cas9 system induced efficient gene mutagenesis in an important Lepidopteran pest Spodoptera litura. We targeted the S. litura Abdominal-A (Slabd-A) gene which is an important embryonic development gene and plays a significant role in determining the identities of the abdominal segments of insects. Direct injection of Cas9 messenger RNA and Slabd-A-specific single guide RNA (sgRNA) into S. litura embryos successfully induced the typical abd-A deficient phenotype, which shows anomalous segmentation and ectopic pigmentation during the larval stage. A polymerase chain reaction-based analysis revealed that the Cas9/sgRNA complex effectively induced a targeted mutagenesis in S. litura. These results demonstrate that the CRISPR/Cas9 system is a powerful tool for genome manipulation in Lepidopteran pests such as S. litura.