Uridine diphosphate glucosyltransferase is vital for fenpropathrin resistance in Bombyx mori (Lepidoptera).

The silkworm (Bombyx mori) is an important model lepidopteran insect and can be used to identify pesticide resistance-related genes of great significance for biological control of pests. Uridine diphosphate glucosyltransferases (UGTs), found in all organisms, are the main secondary enzymes involved in the metabolism of heterologous substances. However, it remains uncertain if silkworm resistance to fenpropathrin involves UGT. This study observes significant variations in BmUGT expression among B. mori strains with variable fenpropathrin resistance post-feeding, indicating BmUGT's role in fenpropathrin detoxification. Knockdown of BmUGT with RNA interference and overexpression of BmUGT significantly decreased and increased BmN cell activity, respectively, indicating that BmUGT plays an important role in the resistance of silkworms to fenpropathrin. In addition, fenpropathrin residues were significantly reduced after incubation for 12 h with different concentrations of a recombinant BmUGT fusion protein. Finally, we verified the conservation of UGT to detoxify fenpropathrin in Spodoptera exigua: Its resistance to fenpropathrin decreased significantly after knocking down SeUGT. In a word, UGT plays an important role in silkworm resistance to fenpropathrin by directly degrading the compound, a function seen across other insects. The results of this study are of great significance for breeding silkworm varieties with high resistance and for biological control of pests.

Bombyx mori voltage-dependent anion-selective channel induces programmed cell death to defend against Bombyx mori nucleopolyhedrovirus infection.

BACKGROUND: Voltage-dependent anion-selective channels (VDACs) serve as pore proteins within the mitochondrial membrane, aiding in the regulation of cell life and cell death. Although the occurrence of cell death is crucial for defense against virus infection, the function played by VDAC in Bombyx mori, in response to the influence of Bombyx mori nucleopolyhedrovirus (BmNPV), remains unclear. RESULTS: BmVDAC was found to be relatively highly expressed both during embryonic development, and in the Malpighian tubule and midgut. Additionally, the expression levels of BmVDAC were found to be different among silkworm strains with varying levels of resistance to BmNPV, strongly suggesting a connection between BmVDAC and virus infection. To gain further insight into the function of BmVDAC in BmNPV, we employed RNA interference (RNAi) to silence and overexpress it by pIZT/V5-His-mCherry. The results revealed that BmVDAC is instrumental in developing the resistance of host cells to BmNPV infection in BmN cell-line cells, which was further validated as likely to be associated with initiating programmed cell death (PCD). Furthermore, we evaluated the function of BmVDAC in another insect, Spodoptera exigua. Knockdown of the BmVDAC homolog in S. exigua, SeVDAC, made the larvae more sensitive to BmNPV. CONCLUSION: We have substantiated the pivotal role of BmVDAC in conferring resistance against BmNPV infection, primarily associated with the initiation of PCD. The findings of this study shine new light on the molecular mechanisms governing the silkworm's response to BmNPV infection, thereby supporting innovative approaches for pest biocontrol. © 2024 Society of Chemical Industry.

Studying the role of Bombyx mori molybdenum cofactor sulfurase in Bombyx mori nucleopolyhedrovirus infection.

Molybdenum cofactor sulfurase (MoCoS) is a key gene involved in the uric acid metabolic pathway that activates xanthine dehydrogenase to synthesise uric acid. Uric acid is harmful to mammals but plays crucial roles in insects, one of which is the immune responses. However, the function of Bombyx mori MoCoS in response to BmNPV remains unclear. In this study, BmMoCoS was found to be relatively highly expressed in embryonic development, gonads and the Malpighian tubules. In addition, the expression levels of BmMoCoS were significantly upregulated in three silkworm strains with different levels of resistance after virus infection, suggesting a close link between them. Furthermore, RNAi and overexpression studies showed that BmMoCoS was involved in resistance to BmNPV infection, and its antivirus effects were found to be related to the regulation of uric acid metabolism, which was uncovered by inosine- and febuxostat-coupled RNAi and overexpression. Finally, the BmMoCoS-mediated uric acid pathway was preliminarily confirmed to be a potential target to protect silkworms from BmNPV infection. Overall, this study provides new evidence for elucidating the molecular mechanism of silkworms in response to BmNPV infection and new strategies for the prevention of viral infections in sericulture.

Advances in the extracellular signal-regulated kinase signaling pathway in silkworms, Bombyx mori (Lepidoptera).

Signaling pathways regulate the transmission of signals during organism growth and development, promoting the smooth and accurate completion of numerous physiological and biochemical reactions. The extracellular signal-regulated kinase (ERK) signaling pathway is an essential pathway involved in regulating various physiological processes, such as cell proliferation, differentiation, adhesion, migration, and more. This pathway also contributes to several important physiological processes in silkworms, including protein synthesis, reproduction, and immune defense against pathogens. Organizing related studies on the ERK signaling pathway in silkworms can provide a better understanding of its mechanism in Lepidopterans and develop a theoretical foundation for improving cocoon production and new strategies for pest biological control.

Transcriptomic analysis of the fat body of resistant and susceptible silkworm strains, Bombyx mori (Lepidoptera), after oral treatment with fenpropathrin.

The widespread use of pyrethroid pesticides has brought serious economic losses in sericulture, but there is still no viable solution. The key to solving the problem is to improve silkworm resistance to pesticides, which depends on understanding the resistance mechanism of silkworms to pesticides. This study aimed to use transcriptomes to understand the underlying mechanism of silkworm resistance to fenpropathrin, which will provide a theoretical molecular reference for breeding pesticide-resistant silkworm varieties. In this study, the fat bodies of two strains with differential resistance after 12 h of fenpropathrin feeding were analyzed using RNA-Seq. After feeding fenpropathrin, 760 differentially expressed genes (DEGs) were obtained in the p50(r) strain and 671 DEGs in the 8y strain. The DEGs involved in resistance to fenpropathrin were further identified by comparing the two strains, including 207 upregulated DEGs in p50(r) and 175 downregulated DEGs in 8y. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that these fenpropathrin-related DEGs are mainly enriched in the metabolism and transporter pathways. Moreover, 28 DEGs involved in the metabolic pathway and 18 in the transporter pathway were identified. Furthermore, organic cation transporter protein 6 (BmOCT6), a transporter pathway member, was crucial in enhancing the tolerance of BmN cells to fenpropathrin. Finally, the knockdown of the expression of the homologs of BmOCT6 in Glyphodes pyloalis (G. pyloalis) significantly decreased the resistant level of larvae to fenpropathrin. The findings showed that the metabolism and transporter pathways are associated with resistance to fenpropathrin in silkworm, and OCT6 is an effective and potential target not only for silkworm breeding but also for pest biocontrol.

Transcriptomic dissection of termite gut microbiota following entomopathogenic fungal infection.

Termites are social insects that live in the soil or in decaying wood, where exposure to pathogens should be common. However, these pathogens rarely cause mortality in established colonies. In addition to social immunity, the gut symbionts of termites are expected to assist in protecting their hosts, though the specific contributions are unclear. In this study, we examined this hypothesis in Odontotermes formosanus, a fungus-growing termite in the family Termitidae, by 1) disrupting its gut microbiota with the antibiotic kanamycin, 2) challenging O. formosanus with the entomopathogenic fungus Metarhizium robertsii, and finally 3) sequencing the resultant gut transcriptomes. As a result, 142531 transcripts and 73608 unigenes were obtained, and unigenes were annotated following NR, NT, KO, Swiss-Prot, PFAM, GO, and KOG databases. Among them, a total of 3,814 differentially expressed genes (DEGs) were identified between M. robertsii infected termites with or without antibiotics treatment. Given the lack of annotated genes in O. formosanus transcriptomes, we examined the expression profiles of the top 20 most significantly differentially expressed genes using qRT-PCR. Several of these genes, including APOA2, Calpain-5, and Hsp70, were downregulated in termites exposed to both antibiotics and pathogen but upregulated in those exposed only to the pathogen, suggesting that gut microbiota might buffer/facilitate their hosts against infection by finetuning physiological and biochemical processes, including innate immunity, protein folding, and ATP synthesis. Overall, our combined results imply that stabilization of gut microbiota can assist termites in maintaining physiological and biochemical homeostasis when foreign pathogenic fungi invade.

Uric acid metabolism promotes apoptosis against Bombyx mori nucleopolyhedrovirus in silkworm, Bombyx mori.

The white epidermis of silkworms is due to the accumulation of uric acid crystals. Abnormal silkworm uric acid metabolism decreases uric acid production, leading to a transparent or translucent phenotype. The oily silkworm op50 is a mutant strain with a highly transparent epidermis derived from the p50 strain. It shows more susceptibility to Bombyx mori nucleopolyhedrovirus (BmNPV) infection than the wild type; however, the underlying mechanism is unknown. This study analysed the changes in 34 metabolites in p50 and op50 at different times following BmNPV infection based on comparative metabolomics. The differential metabolites were mainly clustered in six metabolic pathways. Of these, the uric acid pathway was identified as critical for resistance in silkworms, as feeding with inosine significantly enhanced larval resistance compared to other metabolites and modulated other metabolic pathways. Additionally, the increased level of resistance to BmNPV in inosine-fed silkworms was associated with the regulation of apoptosis, which is mediated by the reactive oxygen species produced during uric acid synthesis. Furthermore, feeding the industrial strain Jingsong (JS) with inosine significantly increased the level of larval resistance to BmNPV, indicating its potential application in controlling the virus in sericulture. These results lay the foundation for clarifying the resistance mechanism of silkworms to BmNPV and provide new strategies and methods for the biological control of pests.

Fumarate mitigates disruption induced by fenpropathrin in the silkworm Bombyx mori (Lepidoptera): A metabolomics study.

The silkworm Bombyx mori L. is a model organism of the order Lepidoptera. Understanding the mechanism of pesticide resistance in silkworms is valuable for Lepidopteran pest control. In this study, comparative metabolomics was used to analyze the metabolites of 2 silkworm strains with different pesticide resistance levels at 6, 12, and 24 h after feeding with fenpropathrin. Twenty-six of 27 metabolites showed significant differences after fenpropathrin treatment and were classified into 6 metabolic pathways: glycerophospholipid metabolism, sulfur metabolism, glycolysis, amino acid metabolism, the urea cycle, and the tricarboxylic acid (TCA) cycle. After analyzing the percentage changes in the metabolic pathways at the 3 time points, sulfur metabolism, glycolysis, and the TCA cycle showed significant responses to fenpropathrin. Confirmatory experiments were performed by feeding silkworms with key metabolites of the 3 pathways. The combination of iron(II) fumarate + folic acid (IF-FA) enhanced fenpropathrin resistance in silkworms 6.38 fold, indicating that the TCA cycle is the core pathway associated with resistance. Furthermore, the disruption of several energy-related metabolic pathways caused by fenpropathrin was shown to be recovered by IF-FA in vitro. Therefore, IF-FA may have a role in boosting silkworm pesticide resistance by modulating the equilibrium between the TCA cycle and its related metabolic pathways.

Bombyx mori transcription factor, E74A, beneficially affects BmNPV infection through direct interaction.

BACKGROUND: Nucleopolyhedrovirus (NPV), one of the baculoviruses, is a promising biopesticide for pest control. Lepidopteran account for 70% of pests, therefore investigation on highly conserved genes associated with viral infections in the lepidopteran model, the silkworm, will serve as a valuable reference for improving the effectiveness of pest management. BmE74A is a member of the erythroblast transformation-specific (ETS) family of transcription factors in Bombyx mori, which we previously found to be highly conserved and closely associated with BmNPV. This study aimed to elucidate the role of BmE74A in viral infection. RESULTS: A significantly high expression of BmE74A in eggs indicated its important role in embryonic development, as did relatively high expressions in the hemolymph and midgut. Significant differences in BmE74A expression in different resistant strains after BmNPV infection suggested its involvement as a response to viral infection. Moreover, RNA interference (RNAi) and overexpression experiments confirmed the important role of BmE74A in promoting viral infection. BmNPV infection was significantly suppressed and enhanced by BmE74A knockdown and overexpression, respectively. Besides, BmE74A was found to regulate the expression of BmMdm2 and Bmp53. Furthermore, the binding of ETS, the functional domain of BmE74A, to occlusion-derived virus proteins was confirmed by far-western blotting, and four viral proteins that may interact with ETS proteins were identified by mass spectrometry. Similarly, a homolog of BmE74A in Spodoptera litura was also found to be involved in larval susceptibility to BmNPV. CONCLUSION: BmE74A promotes BmNPV proliferation by directly interacting with the virus, which may be related to the suppression of the p53 pathway. © 2022 Society of Chemical Industry.

Functional characterization of a low-density lipoprotein receptor in the lepidopteran model, Bombyx mori.

The growth and development of metabolous insects are mainly regulated by ecdysone and juvenile hormone. As a member of the low-density lipoprotein receptor (LDLR) family, megalin (mgl) is involved in the lipoprotein transport of cholesterol which is an essential precursor for the synthesis of ecdysone. Despite extensive studies in mammals, the function of mgl is still largely unknown in insects. In this study, we characterize the function of mgl in the silkworm Bombyx mori, the model species of Lepidoptera. We find that mgl is broadly present in the genomes of lepidopteran species and evolved with divergence between lepidopterans and Drosophila. The expression pattern suggests a ubiquitous role of mgl in the growth and development in the silkworm. We further perform clustered regularly interspaced palindromic repeats (CRISPR) / CRISPR-associated protein 9-based mutagenesis of Bmmgl and find that both the development and the silk production of the silkworm are seriously affected by the disruption of Bmmgl. Our results not only explore the function of mgl in Lepidoptera but also add to our understanding of how cholesterol metabolism is involved in the development of insects.

Identification of optimal reference genes in Bombyx mori (Lepidoptera) for normalization of stress-responsive genes after challenge with pesticides.

Pesticides are frequently used to control pests in agriculture due to their ease of use and effectiveness, but their use causes serious economic losses to sericulture when their production overlaps with agriculture. However, no suitable internal reference genes (RGs) have been reported in the study of silkworms in response to pesticides. In this study, a standard curve was established to detect the expression levels of seven RGs in different tissues of different silkworm strains after feeding with pesticides using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), including BmGAPDH, BmActin3, BmTBP, BmRPL3, Bm28sRNA, Bmα-tubulin, and BmUBC, and the stability of them was evaluated by using NormFinder, geNorm, Delta CT, BestKeeper, and RefFinder. The results showed that BmGAPDH and Bmα-tubulin were relatively stable in the midgut after feeding with fenvalerate, BmGAPDH and Bmactin3 were relatively stable in the fat body, and Bmα-tubulin and Bmactin3 were relatively stable in the hemolymph, indicating that Bmactin3 was the most suitable RG when evaluating fenvalerate, followed by BmGAPDH and Bmα-tubulin. Besides, BmGAPDH and Bmactin3 were relatively stable in the midgut after treatment with DDVP, BmGAPDH and Bmα-tubulin were relatively stable in the fat body, and BmGAPDH and Bmα-tubulin were relatively stable in the hemolymph, indicating that Bmα-tubulin was the most stable RG when evaluating DDVP, followed by BmGAPDH and Bmactin3. Of note, BmGAPDH was shared by the two pesticides. The results will be valuable for RG selection in studying the pesticide response mechanism of silkworms and other lepidopteran insects.

Map-based cloning and functional analysis revealed ABCC2 is responsible for Cry1Ac toxin resistance in Bombyx mori.

Bt toxins are parasporal crystals produced by Bacillus thuringiensis (Bt). They have specific killing activity against various insects and have been widely used to control agricultural pests. However, their widespread use has developed the resistance of many target insects. To maintain the sustainable use of Bt products, the resistance mechanism of insects to Bt toxins must be fully clarified. In this study, Bt-resistant and Bt-susceptible silkworm strains were used to construct genetic populations, and the genetic pattern of silkworm resistance to Cry1Ac toxin was determined. Sequence-tagged site molecular marker technology was used to finely map the resistance gene and to draw a molecular genetic linkage map, and the two closest markers were T1590 and T1581, indicating the resistance gene located in the 155 kb genetic region. After analyzing the sequence of the predicted gene in the genetic region, an ATP binding cassette transporter (ABCC2) was identified as the candidate gene. Molecular modeling and protein-protein docking result showed that a tyrosine insertion in the mutant ABCC2 might be responsible for the interaction between Cry1Ac and ABCC2. Moreover, CRISPR/Cas9-mediated genome editing technology was used to knockout ABCC2 gene. The homozygous mutant ABCC2 silkworm was resistant to Cry1Ac toxin, which indicated ABCC2 is the key gene that controls silkworm resistance to Cry1Ac toxin. The results have laid the foundation for elucidating the molecular resistance mechanism of silkworms to Cry1Ac toxin and could provide a theoretical basis for the biological control of lepidopteran pests.

Microbial Response to Fungal Infection in a Fungus-Growing Termite, Odontotermes formosanus (Shiraki).

The crosstalk between gut microbiota and host immunity has emerged as one of the research foci of microbiome studies in recent years. The purpose of this study was to determine how gut microbes respond to fungal infection in termites, given their reliance on gut symbionts for food intake as well as maintaining host health. Here, we used Metarhizium robertsii, an entomopathogenic fungus, to infect Odontotermes formosanus, a fungus-growing termite in the family Termitidae, and documented changes in host gut microbiota via a combination of bacterial 16S rDNA sequencing, metagenomic shotgun sequencing, and transmission electron microscopy. Our analyses found that when challenged with Metarhizium, the termite gut showed reduced microbial diversity within the first 12 h of fungal infection and then recovered and even surpassed pre-infection flora levels. These combined results shed light on the role of gut flora in maintaining homeostasis and immune homeostasis in the host, and the impact of gut flora dysbiosis on host susceptibility to infection.

Identification of the in vitro antiviral effect of BmNedd2-like caspase in response to Bombyx mori nucleopolyhedrovirus infection.

Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the most serious pathogens in sericulture, and the underlying antiviral mechanism in silkworm is still unclear. Bombyx mori Nedd2-like caspase (BmNc) has been identified as a candidate antiviral gene from previous transcriptome data, since it is differentially expressed in the midgut of differentially resistant silkworm strains following BmNPV infection. However, the molecular mechanism by which BmNc responds to BmNPV is unknown. In this study, the relationship between BmNc and BmNPV was confirmed by its significantly different expression in different tissues of differentially resistant strains after BmNPV infection. Moreover, the antiviral role of BmNc was confirmed by the significantly higher fluorescence signals of BV-eGFP after knockdown of BmNc in BmN cells, and a reduced signal after overexpression. This was further verified by the capsid gene vp39 expression, DNA copy number, and GP64 protein level in the RNAi and overexpression groups. Furthermore, the antiviral phenomenon of BmNc was found to be associated with apoptosis. In brief, BmNc showed a relatively high expression level in the metamorphosis stages, and the effect of BmNc on BmNPV infection following RNAi and overexpression was eliminated after treatment with the inducer, Silvestrol, and the inhibitor, Z-DEVD-FMK, respectively. Therefore, it is reasonable to conclude that BmNc is involved in anti-BmNPV infection via the mitochondrial apoptosis pathway. The results provide valuable information for elucidating the molecular mechanism of silkworm resistance to BmNPV infection.

Bmcas-1 plays an important role in response against BmNPV infection in vitro.

Apoptosis, as one kind of innate immune system, is involved in host response against pathogens innovation. Caspases play a vital role in the execution stage of host cell apoptosis. It has been reported that Bmcaspase-1 (Bmcas-1) has a close relationship with Bombyx mori nucleopolyhedrovirus (BmNPV) infection for its differentially expressed patterns after viral infection. However, its underlying response mechanism is still unclear. The significant differential expression of Bmcas-1 in different tissues of differentially resistant strains revealed its vital role in BmNPV infection. To further validate its role in BmNPV infection, budded virus (BV)-eGFP was analyzed after knockdown and overexpression of Bmcas-1 by small interfering RNA and the pIZT-mCherry vector, respectively. The reproduction of BV-eGFP obviously increased at 72 h after knockdown of Bmcas-1, and decreased after overexpression in BmN cells. Moreover, the conserved functional domain of Cas-1 among different species and the closed evolutionary relationship of Cas-1 in Lepidoptera hinted that Bmcas-1 might be associated with apoptosis, and this was also validated by the apoptosis inducer, Silvestrol, and the inhibitor, Z-DEVD-FMK. Therefore, Bmcas-1 plays an essential antiviral role by activating apoptosis, and this result lays a fundament for clarifying the molecular mechanism of silkworm in response against BmNPV infection and breeding of resistant strains.

The validation of the role of several genes related to Bombyx mori nucleopolyhedrovirus infection in vivo.

Bombyx mori nucleopolyhedrovirus (BmNPV) is one of primary silkworm pathogens and causes a serious damage of cocoon losses every year. Recent years, many works have been done to clarify the silkworm anti-BmNPV mechanism, and a significant progress has been made in screening and studying of genes and proteins related to BmNPV infection, but several of them lacked the proofs in vivo. In this study, to further validate the function of seven newly reported genes in vivo, including BmAtlatin-n, Bmferritin-heavy chain (BmFerHCH), Bmthymosin (BmTHY), Bmseroin1, Bmseroin2, Bmnuclear hormone receptors 96 (BmNHR96), and BmE3 ubiquitin-protein ligase SINA-like 10 (BmSINAL10), the response of them in the midgut, fat body, and hemolymph of differentially resistant strains (resistant strain YeA and susceptible strain YeB) at 48 h following BmNPV infection were analyzed. The results showed that the relative stable or upregulated expression level of BmAtlatin-n, BmTHY, Bmseroin1, and Bmseroin2 in YeA resistant strain following BmNPV infection further indicated their antiviral role in vivo, compared with susceptible YeB strain. Moreover, the significant downregulation of BmFerHCH, BmNHR96, and BmSINAL10 in both strains following BmNPV infection revealed their role in benefiting virus infection, as well as the upregulation of BmFerHCH in YeB midgut and BmSINAL10 in YeB hemolymph. These data could be used to complementary the proofs of the function of these genes in response to BmNPV infection.

CRISPR/Cas9-based knockout reveals that the clock gene timeless is indispensable for regulating circadian behavioral rhythms in Bombyx mori.

Circadian rhythms, which are ubiquitous and adaptive, occur across all species, from microbes to humans, in which they organize and modify behavior and physiology. timeless (tim) is a canonical clock gene. The core composition of the Drosophila melanogaster endogenous circadian clock has been extensively investigated; however, in lepidopteran insects, including Bombyx mori, the mechanism is complicated and little is known regarding the participation of tim in the negative feedback loop responsible for behavioral activities. To arrive at a comprehensive understanding of the role of tim in the B. mori endogenous circadian clock, we exploited the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 gene editing system. We attempted to elucidate the functions of tim in the circadian clock of B. mori using Bmtim mutants. The knockouts affected two circadian behavioral activities: adult emergence and embryo hatching rhythms. Quantitative real-time polymerase chain reaction results confirmed that tim-knockouts induced relative reductions in the expression levels, and thereby the oscillation amplitudes, of Bmper and Bmclk messenger RNAs during both the photophase and scotophase. Additionally, the daily rhythmic expression of Bmdbt was upregulated in the photophase and downregulated in the scotophase in a tim-knockout. Our study reveals that tim is integral to the B. mori circadian clock and may be involved in regulating eclosion and hatching rhythms.

Bmapaf-1 is Involved in the Response against BmNPV Infection by the Mitochondrial Apoptosis Pathway.

Discovery of the anti-BmNPV (Bombyx mori nuclearpolyhedrovirus) silkworm strain suggests that some kind of antiviral molecular mechanism does exist but is still unclear. Apoptosis, as an innate part of the immune system, plays an important role in the response against pathogen infections and may be involved in the anti-BmNPV infection. Several candidate genes involved in the mitochondrial apoptosis pathway were identified from our previous study. Bombyx mori apoptosis protease-activating factor-1 (Bmapaf-1) was one of them, but the antiviral mechanism is still unclear. In this study, sequences of BmApaf-1 were characterized. It was found to contain a unique transposase_1 functional domain and share high CARD and NB-ARC domains with other species. Relatively high expression levels of Bmapaf-1 were found at key moments of embryonic development, metamorphosis, and reproductive development. Further, the significant difference in expression of Bmapaf-1 in different tissues following virus infection indicated its close relationship with BmNPV, which was further validated by RNAi and overexpression in BmN cells. Briefly, infection of budded virus with enhanced green fluorescent protein (BV-EGFP) was significantly inhibited at 72 h after overexpression of Bmapaf-1, which was confirmed after knockdown of Bmapaf-1 with siRNA. Moreover, the downstream genes of Bmapaf-1, including Bmnedd2-like caspase (BmNc) and Bmcaspase-1 (Bmcas-1), were upregulated after overexpression of Bmapaf-1 in BmN cells, which was consistent with the RNAi results. Furthermore, the phenomenon of Bmapaf-1 in response to BmNPV infection was determined to be related to apoptosis using the apoptosis inducer NSC348884 and inhibitor Z-DEVD-FMK. Therefore, Bmapaf-1 is involved in the response against BmNPV infection by the mitochondrial apoptosis pathway. This result provides valuable data for clarifying the anti-BmNPV mechanism of silkworms and breeding of resistant silkworm strains.

BmSd gene regulates the silkworm wing size by affecting the Hippo pathway.

Insect wings are developed from the wing disc during metamorphosis. Bombyx mori, a model lepidopteran insect, loses flight ability after long-term domestication from the wild silkworm, Bombyx mandarina. The mw mutant (u11 strain) shows minute wings compared to wild type (e.g., p50 strain) wings. RNA sequencing analysis previously revealed differential Hippo-pathway-related gene expression between the u11 and p50 strains. The Hippo pathway is an evolutionarily conserved signaling cascade that controls organ size during development in animals. In this study, the function of BmSd which has been characterized as one of the Hippo-pathway-related genes was analyzed for silkworm wing development. We found that mats, warts, and hippo expression levels were higher in u11 compared to p50 wing discs. BmSd (scalloped) expression, which encodes a prominent transcriptional partner to Yorkie (Yki), gradually decreased during the wandering stage in u11, but exhibited the opposite expression pattern in p50. When BmSd was knocked down by small interfering RNA during the wandering stage in the p50 strain, 57.9% of the individuals showed minute wings. Additionally, ex, kibra, and wingless expression levels decreased in the BmSd knockdown mutant. Further, BmSd deletion mediated by clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein 9 induced 50% of individuals with minute wings, a phenotype similar to the mw mutant. This result demonstrates that BmSd plays pivotal roles in silkworm wing development. Our results show that the Hippo signaling pathway participates and plays crucial roles in the regulation of silkworm wing development, and our findings provide a basis for further research on B. mori wing development.

A palmitoyltransferase Approximated gene Bm-app regulates wing development in Bombyx mori.

The silkworm Bombyx mori is an important lepidopteran model insect in which many kinds of natural mutants have been identified. However, molecular mechanisms of most of these mutants remain to be explored. Here we report the identification of a gene Bm-app is responsible for the silkworm minute wing (mw) mutation which exhibits exceedingly small wings during pupal and adult stages. Compared with the wild type silkworm, relative messenger RNA expression of Bm-app is significantly decreased in the u11 mutant strain which shows mw phenotype. A 10 bp insertion in the putative promoter region of the Bm-app gene in mw mutant strain was identified and the dual luciferase assay revealed that this insertion decreased Bm-app promoter activity. Furthermore, clustered regularly interspaced short palindromic repeats/RNA-guided Cas9 nucleases-mediated depletion of the Bm-app induced similar wing defects which appeared in the mw mutant, demonstrating that Bm-app controls wing development in B. mori. Bm-app encodes a palmitoyltransferase and is responsible for the palmitoylation of selected cytoplasmic proteins, indicating that it is required for cell mitosis and growth during wing development. We also discuss the possibility that Bm-app regulates wing development through the Hippo signaling pathway in B. mori.