Insect Bombyx Mori Research Articles

Larval development of a parasitoid depends on host ecdysteroids

Parasitoids often exhibit high flexibility in their development depending on stages of their host at the parasitism, yet little is known about the mechanism underlying such flexibility. In the study, we evaluated the larval development time of the parasitoid Exorista sorbillans (Diptera: Tachinidae) on the lepidopteran model insect Bombyx mori (Lepidoptera: Bombycidae). We found that the development duration of E. sorbillans larvae parasitizing on the late-developmental silkworms was significantly shorter than that of the larvae parasitizing on the early-developmental hosts. Intriguingly, the 2nd-3rd instar molting of parasitoid always occurred when the ecdysteroid titers in the host were increased to higher levels. Furthermore, inhibiting the release of ecdysteroids to parasitic abdomen by thorax-abdomen ligation of the host only repressed the 2nd-instar growth and molting of E. sorbillans larvae, but had no effect on their pupation. Meanwhile, the ecdysone synthesis and 20-hydroxyecdysone (20E) signaling in larval parasitoids were impeded after ligation treatment. Moreover, exogenous 20E application could largely rescue the defect in 2nd instar growth and molting through stimulating ecdysone synthesis and signaling in E. sorbillans. Our results indicate that the parasitoid requires the host ecdysteroids to stimulate 20E signaling and the subsequent 2nd- instar growth and molting. These findings will improve our understanding of the host utilization strategies of parasitoids, and contribute to the development of in vitro rearing procedures of tachinid parasitoids for biological control.

Content difference of 1-deoxynojirimycin in fifty varieties and its correlation with viability of the edible insectBombyx mori (Lepidoptera: Bombycidae)

Silkworm (Bombyx mori) is a traditional edible insect. Whole silkworm powder contains 1-deoxynojirimycin (DNJ). DNJ is an alpha-glucosidase inhibitor with hypoglycaemic activity. The silkworm cannot synthesise DNJ by itself, most of the DNJ in silkworm is obtained by the consumption of mulberry leaves. In this study, the DNJ content of silkworm in different developmental stages and that of 50 representative resource varieties were measured, its correlation with viability of the variety was analysed. The results showed that silkworms at the peak feeding point of the 3rd instar had the highest DNJ content in seven development stages from the 2nd instar to the pupa stage. There was a large difference in the content of DNJ among varieties. The average DNJ content was 7.528 mg/g and the distribution of DNJ content among varieties conformed to the normal distribution (P>0.05). The varieties could be divided into three clusters according to DNJ content, and the DNJ content in cluster III, which contained 11 varieties was higher than other clusters. There were significant differences among polyvoltine species, Chinese species, and Japanese species, and significant differences between colour-cocoon varieties and white-cocoon ones. DNJ content in silkworm larvae was significantly correlated with larval survival rate and larva-pupa rate. In this study, the cluster III with the highest DNJ content may have the potential to be used as preferred raw material for hyperglycaemia regulation or treatment and candidate parent materials for enriched material. Furthermore, DNJ may have a positive effect on the physical fitness of silkworm.

The morphogen Hedgehog is essential for proper adult morphogenesis in Bombyx mori

The well-known morphogen Hedgehog (Hh) is indispensable for embryo patterning and organ development from invertebrates to vertebrates. The role of Hh signaling pathway has been extensively investigated in the model organism Drosophila melanogaster, whereas its biological functions are still poorly understood in non-drosophilid insects. In the current study, we describe comprehensive investigation of Hh biological roles in the model lepidopteran insect Bombyx mori by using both CRISPR/Cas9-mediated gene ablation and Gal4/UAS-mediated ectopic expression. Direct injection of Cas9 protein and Hh-specific sgRNAs into preblastoderm embryos induced complete lethality. In contrast, Hh mutants obtained by the binary transgenic CRISPR/Cas9 system showed no deleterious phenotypes during embryonic and larval stages. However, mutants showed abnormalities from the pupal stage and most of adult body appendages exhibited severe developmental defects. Molecular analysis focused on wing development reveal that Hh signaling, Imd signaling and Wnt signaling pathways were distorted in Hh mutant wings. Ectopic expression by using the binary Gal4/UAS system induce early larval lethality. On contrary, moderate overexpression of Hh by using a unitary transgenic system resulted in severe defects in adult leg and antenna development. Our data directly provide genetic evidence that Hh plays vital roles in imaginal discs development and proper adult morphogenesis in B. mori.

A Comparative Analysis of Bombyx mori (Lepidoptera: Bombycidae) β-fructofuranosidase Homologs Reveals Different Post-Translational Regulations in Glyphodes pyloalis Walker (Lepidoptera: Pyralidae).

Simple SummaryThe β-fructofuranosidase (β-FFase) encoding gene BmSuc1 regulates the glycometabolism of silkworm larvae, and it participates in the resistance of mulberry alkaloids. However, there is no molecular or biochemical information available about the mulberry pest Glyphodes pyloalis Walker β-FFase homologs. In this paper, we have obtained five β-FFase homologous genes in G. pyloalis and characterized the expression and the localization of GpSUC1a in the midgut. The β-FFase activity in the midgut of G. pyloalis larvae and GpSUC1a were both confirmed, while recombinant GpSUC1a displayed little activity as compared with the higher activity of BmSUC1. Some putative N-glycosylation sites were found in GpSUC1a but none in BmSUC1, while there was more methylation in BmSUC1 than in GpSUC1a. The results indicate that such post-translational modifications (PTMs) are differentially supporting that β-FFase are active in these two mulberry feeding caterpillars, and the activation of GpSUC1a may be controlled by a more complex post-translational regulatory system in G. pyloalis larvae. This is the first report on the characterization of β-FFase genes from G. pyloalis and the first comparison of expression regulation between two mulberry feeding insects B. mori and G. pyloalis. Moreover, this research may provide new ideas for the management of mulberry borers.The silk-spinning and Lepidopteran model insect Bombyx mori (Bombycidae) is a mulberry specialist. The BmSuc1 gene is the first β-fructofuranosidase (β-FFase) encoding gene identified in animals, and β-FFase acts as an essential sucrase for glycometabolism modulation in the silkworm larvae, involved in resistance to mulberry alkaloids. Glyphodes pyloalis Walker (Lepidoptera: Pyralidae) is an important mulberry pest leading to heavy economic loss of sericulture. However, no molecular or biochemical information is available about G. pyloalis β-FFase homologs. In this study, five β-FFase homologous genes in G. pyloalis were obtained. The genes GpSuc1a and GpSuc2c were expressed in the midgut; GpSuc2c encodes a truncated polypeptide. The expression and the localization of GpSUC1a in the midgut was characterized. Whereas recombinant GpSUC1a expressed in both Escherichia coli and BmN cells displayed little activity as compared with higher activity of BmSUC1, β-FFase activity in the larval midgut of G. pyloalis and GpSUC1a purified from the midgut were both confirmed. The data suggested that the activation of GpSUC1a is probably controlled by a more complicated post-translational regulation system in G. pyloalis larvae than that of BmSUC1 in B. mori. To study post-translational modifications (PTMs), GpSUC1a and BmSUC1 were purified from larval midguts using immunoprecipitation and subjected to LC-MS to perform PTMs analysis. Some putative N-glycosylated sites were found in GpSUC1a but none in BmSUC1, while there was more methylation in BmSUC1 than in GpSUC1a, indicating that such PTMs were supporting the differential β-FFases activities in these two mulberry feeding caterpillars.

CRISPR/Cas9 Mediated Disruption of Seminal Fluid Protein Sfp62 Induces Male Sterility in Bombyx mori.

Simple SummaryIn gamogenetic animals, seminal fluid proteins are essential for male fertility. In this study, we investigated the function of the seminal fluid protein Sfp62 by using the CRISPR/Cas9 system in lepidopteran model insect Bombyx mori. Sfp62 mutation led to male sterility and can be inherited stably. The mutation did not affect growth and developmental nor female fertility. These data indicate that Sfp62 is an ideal target for sterile insect technology (SIT), in which genetically modified insects are released on a large scale to mate with wild-type insects in order to reduce or even eradicate the target pests. The determining factors for the effective implementation of SIT include the strong competitiveness of the modified individuals and multi-generational effects resulting from the mutation. Sfp62 meets these criteria and is therefore a promising target for biological pest control.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.

BmPMFBP1 regulates the development of eupyrene sperm in the silkworm, Bombyx mori.

Sperm deliver the male complement of DNA to the ovum, and thus play a key role in sexual reproduction. Accordingly, spermatogenesis has outstanding significance in fields as disparate as infertility treatments and pest-control, making it a broadly interesting and important focus for molecular genetics research in a wide range of species. Here we investigate spermatogenesis in the model lepidopteran insect Bombyx mori (silkworm moth), with particular focus on the gene PMFBP1 (polyamine modulated factor 1 binding protein 1). In humans and mouse, PMFBP1 is essential for spermatogenesis, and mutations of this gene are associated with acephalic spermatozoa, which cause infertility. We identified a B. mori gene labeled as “PMFBP1” in GenBank’s RefSeq database and sought to assess its role in spermatogenesis. Like in mammals, the silkworm version of this gene (BmPMFBP1) is specifically expressed in testes. We subsequently generated BmPMFBP1 mutants using a transgenic CRISPR/Cas9 system. Mutant males were sterile while the fertility of mutant females was comparable to wildtype females. In B. mori, spermatogenesis yields two types of sperm, the nucleated fertile eupyrene sperm, and anucleated unfertile apyrene sperm. Mutant males produced abnormal eupyrene sperm bundles but normal apyrene sperm bundles. For eupyrene sperm, nuclei were mislocated and disordered inside the bundles. We also found the BmPMFBP1 deficiency blocked the release of eupyrene sperm bundles from testes to ejaculatory seminalis. We found no obvious abnormalities in the production of apyrene sperm in mutant males, and double-matings with apyrene-deficient sex-lethal mutants rescued the ΔBmPMFBP1 infertility phenotype. These results indicate BmPMFBP1 functions only in eupyrene spermatogenesis, and highlight that distinct genes underlie the development of the two sperm morphs commonly found in Lepidoptera. Bioinformatic analyses suggest PMFBP1 may have evolved independently in lepidoptera and mammals, and that despite the shared name, are likely not homologous genes.

Mutation of Serine protease 1 Induces Male Sterility in Bombyx mori.

Serine proteases are important in reproduction, embryonic development, cell differentiation, apoptosis, and immunity. The genes encoding some serine proteases are essential for male fertility in both humans and rodents and are functionally conserved among metazoan. For example, the Serine protease 1 (Ser1) gene determines male reproductive success in the model lepidopteran insect Bombyx mori. In this study, we explored the function of BmSer1 through transgenic CRISPR/Cas9 technology-mediated mutations in silkworm. We found that the mutation of BmSer1 gene resulted in male sterility but had no effect on female fertility. Male mutants produce normal eupyrene sperm bundles, but the sperm bundles do not dissociate into single sperm. Male sterility caused by the BmSer1 gene mutation was inherited stably through female individuals. Therefore, the serine protease encoded by BmSer1 is essential for male reproductive success in lepidopterans and is a potential target gene for biological reproductive regulation.

U1 small nuclear ribonucleoprotein is essential for early larval development in silkworm, Bombyx mori.

U1 small nuclear ribonucleoproteins (U1 snRNP) associates with 5' splice sites in the form of ribonucleoprotein particles and is highly conserved among species. The physiological functions of U1 snRNP in a lepidopteran model insect Bombyx mori is mostly unknown. Here, we showed that U1 snRNP plays an important role in the development of silkworm. Knockout of U1 snRNP in silkworm showed either delayed or stationary 1st instar larva development compared with the wild-type group. U1 snRNP deletion mutants exhibited abnormal cellular phenotypes with enlarged cell nucleus, scant cytoplasm and enlarged nuclei. RNA-seq analysis revealed that genes involved in metabolic pathway, biosynthesis of secondary metabolites and steroid hormone biosynthesis were significantly affected by U1 snRNP depletion. Taken together, our study suggests that U1 snRNP homeostasis plays an important role in silkworm development.

Ultrasensitive detection by maxillary palp neurons allows non-host recognition without consumption of harmful allelochemicals

Most lepidopteran insect larvae exhibit stepwise feeding behaviors, such as palpation using the maxillary palps (MPs) followed by test biting and persistent biting. However, the purpose of palpation has been unclear. In particular, nothing is known about the neurons in the MP and their mode of recognition of undesired plants, although such neurons have been suggested to exist. In this study, we used larvae of the stenophagous insect Bombyx mori and compared the roles of palpation and test biting in the selection of feeding behavior. When the larvae were given non-host plant leaves, they did not initiate test biting, indicating that non-host plant leaves were recognized via palpation without biting, and that this behavior resulted in a lack of persistent biting, as the leaves were judged non-suitable for consumption. Surface extracts of inedible leaves significantly suppressed test biting of mulberry leaves, a host plant of B. mori, suggesting that secondary metabolites on the leaf surface of inedible leaves function as test biting suppressors, even when another conditions are suitable for test biting. The allelochemical coumarin, which is found in the inedible leaves of cherry, Cerasus speciosa, significantly suppressed test biting of mulberry leaves, suggesting that coumarin is a possible deterrent to the eating of cherry leaves. Using the electrophysiological method of tip recording and a leaf-surface extract as the test material, leaf-surface compound-responsive neurons were identified in the MP. In addition, several neurons that respond to coumarin in the attomolar range were identified, suggesting that the larvae use ultrasensitive neurons in the MP to recognize inedible leaves. In the HEK293T cell heterologous expression system, the B. mori gustatory receptors BmGr53 and BmGr19, which were previously found to be expressed in the MP and to respond to coumarin in the attomolar range, responded to a leaf-surface extract of C. speciosa, suggesting that these receptors may be present on the inedible-leaf-recognizing neurons of the MP. These findings suggest that ultrasensitive plant secondary metabolite-recognizing neurons in the MP allow for the recognition of non-host plants via palpation without risking damage caused by ingesting harmful allelochemicals.

The therapeutic potential and the health benefits of Morus indica Linn.: a mini review

Morus indica (L.) commonly known as Indian mulberry belonging to the family Moraceae is a fast growing perennial plant and is the primary food plant for the monophagous insect Bombyx mori has a special significance in the sericulture industry. Medicinal properties of this plant have been depicted in the ancient literature of Ayurveda. Some of the ayurvedic preparations use fruits, leaves, roots, bark or latex of the mulberry plant as potential treatment aid against various diseases. Because of the presence of several bioactive compounds like steroids, phenolics, alkaloids, reducing sugars, tannins, flavonoids, anthocyanins, terpenoids, cardiac glycosides, saponins, proteins, amino acids, carbohydrates etc., it is traditional being used as natural medicine for- anti-bacterial, anti-fugal, anti-hyperglycaemic, anti-hypoglycaemic, anti-hypolipidemic, hepatoprotective, anti-diabetic, anti-cancer, anti-pyretic, high antioxidant, and anti-inflammatory properties. The present review reveals the wide range of important uses of Indian mulberry including its phytochemical composition, antioxidants and medicinal properties and management towards human diseases.

Possible mechanisms by which silkworm faeces extract ameliorates adenine-induced renal anaemia in rats

Ethnopharmacological relevanceSilkworm faeces are the dry faeces of the insect Bombyx mori (Linnaeus) and have historically been used in traditional Chinese medicine to treat blood deficiency and rheumatic pain. Silkworm faeces extract (SFE) is derived from silkworm faeces.Aim of the study: Clinical observations of patients in the Department of Nephrology have shown that SFE effectively improves renal anaemia. However, the molecular mechanism remains unclear. This article mainly explores the regulatory effects of SFE on erythropoietin (EPO) and hepcidin to identify the molecular mechanism of SFE. Materials and methodsA rat model of renal anaemia was established by feeding rats food containing 0.75% adenine. SFE was orally administered to the rats, while recombinant human erythropoietin (rhEPO) was used as a positive control drug. Haematological parameters and inflammation levels were compared between rats from each group, and pathological kidney sections from each rat were observed. The serum EPO and hepcidin levels were detected using enzyme-linked immunosorbent assay (ELISA) kits, while Western blot analyses were performed to detect the levels of proteins involved in the EPO-related hypoxia-inducible factor 2α (HIF-2α)/prolyl hydroxylase 2 (PHD2) signalling pathway and hepcidin-related BMP6/SMAD4 and interleukin-6 (IL-6)/STAT3 signalling pathways. ResultsSFE significantly ameliorated haematological parameters, renal function, and inflammation levels in the rats. A mechanistic study showed that SFE promoted EPO expression by upregulating HIF-2α expression and inhibiting the expression of NF-κB and GATA2 both in vivo and in vitro. In particular, SFE inhibited PHD2 expression, resulting in a decrease in the enzymatic reaction of HIF-2α to increase EPO expression. Furthermore, SFE inhibited hepcidin expression by blocking the BMP6/SMAD4 and IL-6/STAT3 pathways. ConclusionsSFE regulated iron metabolism by inhibiting hepcidin and simultaneously promoted EPO synthesis to improve renal anaemia in rats.

Insulin-like peptides in model insects

Recent years, invertebrate animals with clear genetic background and completed genome sequence have become very popular in biological research. Insulin and insulin-like peptides (ILPs) are a class of peptides having important physiological functions, including promotion of cell proliferation and differentiation, and body growth and development. To date, many invertebrates have been identified with multiple ILP families, whose structures and functions have become increasingly clear to scientists. This review summarizes the ILPs identified in the model Lepidopteran insect Bombyx mori, in the aspects of structures and classification, functions and pathways. A brief discussion on the ILPs from several other model insects, including Drosophila melanogaster, Aedes aegypti, and Apis mellifera, was also included.

Biological activities of melanin pigment extracted from Bombyx mori gut-associated yeast Cryptococcus rajasthanensis KY627764.

Melanin pigment has been produced and extracted from a wide variety of living forms ranging from microorganisms to higher organisms. Owing to the therapeutic nature of the pigment, various microbial populations have been explored for its production. Hence, we isolated a melanin producing yeast from the insect Bombyx mori gut microflora and identified it as Cryptococcus rajasthanensis based on the molecular characterization. The isolated yeast produced enhanced melanin pigment when cultured in the minimal L-tyrosine broth as compared to the Saboraud medium. The pigment was extracted and characterized as melanin based on UV-Visible spectroscopy, FTIR (Fourier-transform infrared) spectroscopy and 1H NMR (Nuclear magnetic resonance). The melanin pigment was evaluated as a potent bioactive molecule with bioactivity like antimicrobial, antioxidant, anti-inflammatory, and anticancer activity that describes the therapeutic nature of the extracted melanin pigment. Distinct from the biologically active role the melanin pigment isolated from the yeast, the Cryptococcus extract also exhibited killer toxin activity against the pathogenic yeast Candida albicans.

Transcriptomic analysis at the first instar larval stage of nonmolting Bombyx mori mutant (a42).

The life cycle of the holometabolous insect Bombyx mori (Linnaeus) consists of the embryo, larva, pupa, and adult stages with six larval molts. Ecdysone and juvenile hormones play important roles in the growth and development of the silkworms. The a42 silkworm mutant is recessive and homozygous lethal by exhibiting a dark-colored and small body size and fails to molt to second instar. We compared the gene expression of a42 mutants with normal individuals at the first larval molting stage to elucidate the physiological influence of the a42 mutation on the growth and development of silkworms. The transcriptomic sequencing results revealed that 1,411 genes are differentially expressed in a42 mutants, compared with wild-type control silkworms, in which 791 genes are upregulated and 620 genes are downregulated. Gene Ontology/Kyoto Encyclopedia of Genes and Genomes analyses identified differentially expressed genes (DEGs) assigned to biological pathways, such as pentose and glucoronate interconversions, glycerolipid metabolism, folate biosynthesis, amino sugar, and nucleotide sugar metabolism. Two hydroxylases of phenylalanine hydroxylase (BmPAH) and tyrosine hydroxylase (BmTh) are upregulated in a42 mutants. The influence of a42 mutation on these DEGs reveals that melanin metabolism plays an important role during the molting process in silkworms.

Replacement of loop2 and 3 of Cry1Ai in domain II affects specificity to the economically important insect Bombyx mori

Bacillus thuringiensis Cry1Ai belongs to three-domain Cry toxins and only shows growth inhibition effects against the agricultural pest Helicoverpa armigera, although it exhibits high toxicity against the non-target insect Bombyx mori. In previous studies, loop2 and loop3 on domain II from Cry1Ah were found to be related to binding and high toxicity against H. armigera. However, toxicity for B. mori of Cry1Ai-h-loop2, obtained by replacing loop 2 from Cry1Ah into Cry1Ai, was not modified. In this study, to further characterize the role of loop2 and loop3 in Cry1Ai, all of the amino acids in these two loops were substituted with the same amount of alanine residues. The Cry1Ai-loop3 mutant exhibited significantly lower toxicity against B. mori, but the toxicity of the loop2 mutant was not significantly changed. Furthermore, the double-exchange mutant Cry1Ai-h-loop2&3, replacing loop2 and loop3 from Cry1Ah into Cry1Ai, showed decreased toxicity against B. mori related to Cry1Ai. In addition, we found that the binding affinity of Cry1Ai-h-loop2&3 with brush border membrane vesicles (BBMVs) from the midgut of B. mori was lower than that of Cry1Ai, which correlates with the reduced toxicity.

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

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. 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. 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.

The CncC/keap1 pathway is activated in high temperature-induced metamorphosis and mediates the expression of Cyp450 genes in silkworm, Bombyx mori

Global warming is known to affect the growth, development and reproduction of insects. In this study, the larvae developmental process and endogenous hormone levels under high temperature (36 °C) stress were investigated in the lepidopteran model insect Bombyx mori (B. mori). After high temperature treatment, the duration of 5th instar larvae was shortened by 28 ± 2 h, the content of 20-hydroxyecdysone(20E) in hemolymph was significantly increased, and the transcription levels of the 20E response genes E93, Br–C, USP and E75 were up-regulated by 1.35-, 1.25-, 1.28-, and 1.27-fold, respectively. High temperature treatment also elevated the phosphorylation level of Akt and activated the downstream BmCncC/keap1 pathway, and the transcription levels of the 20E synthesis-related genes cyp302a1, cyp306a1, cyp314a1 and cyp315a1 were up-regulated by 1.12-, 1.51-, 2.17- and 1.23-fold, respectively. The transcription levels of cyp302a1 and cyp306a1 were significantly decreased in BmN cells after treatment with the double stranded RNA of BmCncC (dsBmCncC), whereas their transcription levels were significantly increased (2.15- and 1.31-fold, respectively) after treatment with the CncC agonist Curcumin. These results demonstrated that high temperature treatment promoted the metamorphosis and the BmCncC/keap1 pathway played a role in the metamorphosis of B. mori. Our results provided clues for understanding the CncC/keap1 pathway-mediated regulation of metamorphosis of Lepidopteran insects.

Intersex regulates female external genital and imaginal disc development in the silkworm

As a component of the mediator complex, the intersex (ix) gene product is involved in the sex determination pathway of the Drosophila melanogaster. IX functions together with the female-specific product of doublesex (dsx) at the bottom of the hierarchy to implement female sexual differentiation. Here we analyzed the functions of the ix gene in the model lepidopteran insect Bombyx mori. We found that Bmix is expressed in many tissues and is highly expressed in early pupal stages. We used the transgene-based CRISPR/Cas9 system to generate mutants of the Bmix gene. The Bmix female mutants were sterile and had irregular external genitalia, whereas in the mutant males external genitalia were normal. Mutants of both sexes had normal gonad development and normal splicing of the Bmdsx pre-mRNA, suggesting that Bmix functions independently of Bmdsx. Interestingly, both male and female mutants had defective development of the imaginal disc including wing, antenna, and leg. RNA-seq and gene expression analyses indicated that genes involved in WNT, Hippo, and Hedgehog signaling pathways and wing development genes Bmawd and Bmfng were up-regulated or down-regulated in the Bmix mutants compared with wild-type animals. Our data provide insights into the multiple functions of Bmix in female external genital and imaginal disc development in the silkworm.

Complexities in Bombyx germ cell formation process revealed by Bm-nosO (a Bombyx homolog of nanos) knockout

Inheritance (sequestration of a localized determinant: germplasm) and zygotic induction are two modes of metazoan primordial germ cell (PGC) specification. vasa and nanos homologs are evolutionarily conserved germline marker genes that have been used to examine the ontogeny of germ cells in various animals. In the lepidopteran insect Bombyx mori, although the lack of vasa homolog (BmVLG) protein localization as well as microscopic observation suggested the lack of germplasm, classical embryo manipulation studies and the localization pattern of Bm-nosO (one of the four nanos genes in Bombyx) maternal mRNA in the egg raised the possibility that an inheritance mode is operating in Bombyx. Here, we generated Bm-nosO knockouts to examine whether the localized mRNA acts as a localized germ cell determinant. Contrary to our expectations, Bm-nosO knockout lines could be established. However, these lines frequently produced abnormal eggs, which failed to hatch, to various extent depending on the individuals. We also found that Bm-nosO positively regulated BmVLG expression at least during embryonic stage, directly or indirectly, indicating that these genes were on the same developmental pathway for germ cell formation in Bombyx. These results suggest that these conserved genes are concerned with stable germ cell production. On the other hand, from the aspect of BmVLG as a PGC marker, we showed that maternal Bm-nosO product(s) as well as early zygotic Bm-nosO activity were redundantly involved in PGC specification; elimination of both maternal and zygotic gene activities (as in knockout lines) resulted in the apparent lack of PGCs, indicating that an inheritance mechanism indeed operates in Bombyx. This, however, together with the fact that germ cells are produced at all in Bm-nosO knockout lines, also suggests the possibility that, in Bombyx, not only this inheritance mechanism but also an inductive mechanism acts in concert to form germ cells or that loss of early PGCs are compensated for by germline regeneration: mechanisms that could enable the evolution of preformation. Thus, Bombyx could serve as an important organism in understanding the evolution of germ cell formation mechanisms; transition between preformation and inductive modes.

Silkworm genetic sexing through W chromosome-linked, targeted gene integration.

Sex separation methods are critical for genetic sexing systems in commercial insect production and sterile insect techniques. Integration of selectable marker genes into a sex chromosome is particularly useful in insects with a heterogametic sex determination system. Here, we describe targeted gene integration of fluorescent marker expression cassettes into a randomly amplified polymorphic DNA (RAPD) marker region in the W chromosome of the lepidopteran model insect Bombyx mori using transcriptional activator-like effector nuclease (TALEN)-mediated genome editing. This silkworm strain shows ubiquitous female-specific red or green fluorescence from the embryonic to adult stages. Furthermore, we developed a binary, female-specific, embryonic lethality system combining the TALEN and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology. This system includes one strain with TALEN-mediated, W-specific Cas9 expression driven by the silkworm germ cell-specific nanos (nos) promoter and another strain with U6-derived single-guide RNA (sgRNA) expression targeting transformer 2 (tra2), an essential gene for silkworm embryonic development. Filial 1 (F1) hybrids exhibit complete female-specific lethality during embryonic stages. Our study provides a promising approach for B. mori genetic sexing and sheds light on developing sterile insect techniques in other insect species, especially in lepidopteran pests with WZ/ZZ sex chromosome systems.