Identification and function of the Pax gene Bmgsb in the silk gland of Bombyx mori.

Paired box (Pax) genes are highly conserved throughout evolution, and the Pax protein is an important transcription factor of embryonic development. The Pax gene Bmgsb is expressed in the silk glands of silkworm, but its biological functions remain unclear. This study aimed to investigate the expression pattern of Bmgsb in the silk gland and explore its functions using RNA interference (RNAi). Here, we identified eight Pax genes in Bombyx mori. Phylogenetic analysis showed that the B. mori Pax genes were highly homologous to the Pax genes in other insects and highly evolutionarily conserved. The tissue expression profile showed that Bmgsb was expressed in the anterior silk gland and anterior part of the middle silk gland (AMSG). RNAi of Bmgsb resulted in defective development of the AMSG, and the larvae were mostly unable to cocoon in the wandering stage. RNA-seq analysis showed that the fibroin genes fib-l, fib-h and p25, cellular heat shock response-related genes and phenol oxidase genes were considerably upregulated upon Bmgsb knockdown. Furthermore, quantitative reverse transcription-PCR results showed that the fibroin genes and ubiquitin proteolytic enzyme-related genes were significantly upregulated in the AMSG after Bmgsb knockdown. This study provides a foundation for future research on the biological functions of B. mori Pax genes. In addition, it demonstrates the important roles of Bmgsb in the transcriptional regulation of fibroin genes and silk gland development.

Comparison of Silks from Pseudoips prasinana and Bombyx mori Shows Molecular Convergence in Fibroin Heavy Chains but Large Differences in Other Silk Components.

Many lepidopteran larvae produce silk feeding shelters and cocoons to protect themselves and the developing pupa. As caterpillars evolved, the quality of the silk, shape of the cocoon, and techniques in forming and leaving the cocoon underwent a number of changes. The silk of Pseudoips prasinana has previously been studied using X-ray analysis and classified in the same category as that of Bombyx mori, suggesting that silks of both species have similar properties despite their considerable phylogenetic distance. In the present study, we examined P. prasinana silk using 'omics' technology, including silk gland RNA sequencing (RNA-seq) and a mass spectrometry-based proteomic analysis of cocoon proteins. We found that although the central repetitive amino acid sequences encoding crystalline domains of fibroin heavy chain molecules are almost identical in both species, the resulting fibers exhibit quite different mechanical properties. Our results suggest that these differences are most probably due to the higher content of fibrohexamerin and fibrohexamerin-like molecules in P. prasinana silk. Furthermore, we show that whilst P. prasinana cocoons are predominantly made of silk similar to that of other Lepidoptera, they also contain a second, minor silk type, which is present only at the escape valve.

Proteomic Identification of Bombyx mori Organelles Using the Engineered Ascorbate Peroxidase APEX and Development of Silkworm Organelle Proteome Database (SilkOrganPDB).

Silkworm Bombyx mori is an economically important insect and a lepidopteran model. Organelle proteome is vital to understanding gene functions; however, it remains to be identified in silkworm. Here, using the engineered ascorbate peroxidase APEX, we constructed transgenic B. mori embryo cells (BmE) expressing APEX-NLS, COX4-APEX, APEX-Rev, and APEX-KDEL in nucleus, mitochondrial matrix (MM), cytosol, and endoplasmic reticulum (ER), and isolated the biotin-labeled proteins using streptavidin-affinity purification, respectively. The isolated proteins were determined using LC-MS/MS and annotated by searching B. mori genomes downloaded from GenBank, SilkBase, SilkDB 2.0, and SilkDB 3.0, resulting in 842, 495, 311, and 445 organelle proteins identified, respectively. We mapped the 296 MM proteins annotated in the GenBank data to mitochondrial protein databases of the fly, human, and mouse, and found that 140 (47%) proteins are homologous to 80 fly proteins, and 65 (22%) proteins match to 31 and 29 human and mouse proteins, respectively. Protein orthology was predicted in multiple insects using OrthoMCL, producing 460 families containing 839 proteins we identified. Out of 460 families, 363 were highly conserved and found in all insects, leaving only three proteins without orthology in other insects, indicating that the identified proteins are highly conserved and probably play important roles in insects. A gene ontology enrichment analysis by clusterProfiler revealed that the nucleus proteins significantly enriched in cellular component terms of nucleus and nucleolus, the MM proteins markedly enriched in molecular function terms of nucleotide binding, and the cytosol proteins mainly enriched in biological process terms of small molecule metabolism. To facilitate the usage and analysis of our data, we developed an open-access database, Silkworm Organelle Proteome Database (SilkOrganPDB), which provides multiple modules for searching, browsing, downloading, and analyzing these proteins, including BLAST, HMMER, Organelle Proteins, Protein Locations, Sequences, Gene Ontology, Homologs, and Phylogeny. In summary, our work revealed the protein composition of silkworm BmE organelles and provided a database resource helpful for understanding the functions and evolution of these proteins.

Simultaneous sex and species classification of silkworm pupae by NIR spectroscopy combined with chemometric analysis.

BACKGROUND: Most studies only focus on the sex discrimination of silkworm pupae. However, species differentiation of silkworm pupae is also needed in sericulture. To classify the sex and species at the same time, the present study adopts near infrared (NIR) spectroscopy combined with multivariate analysis. RESULTS: First, spectra samples were acquired using an NIR sensor, comprising female and male silkworm pupae from three species. Second, three different variables selection approaches were used, including a successive projections algorithm, competitive adaptive reweighted sampling (CARS) and interval partial least squares (iPLS). Third, identification models were built based on random forest and partial least squares discriminant analysis (PLSDA). The experimental results show that iPLS-PLSDA model (95.24%) gives a high performance when using the one of the three variable selection methods alone. To further increase the performance, the variable selection methods are optimized. The accuracy of the iPLS-CARS-PLSDA model is as high as 98.41%. CONCLUSION: The present study demonstrates that the optimized variable selection method in combination with NIR spectroscopy represents a suitable strategy for sex and species identification of silkworm pupae. © 2020 Society of Chemical Industry.

Characterization and potential application of an α-amylase (BmAmy1) selected during silkworm domestication.

Efficient resource utilization plays a central role in the high productivity of domesticated plants and animals. Whether artificial selection acts on digestive enzymes in the domesticated silkworm (Bombyx mori), which is larger than its wild ancestor, Bombyx mandarina (B. mandarina), remains unknown. In this study, we present the characteristics of a novel alpha-amylase, BmAmy1, in B. mori. The activity of recombinant BmAmy1 was maximal at 35 °C and pH 9.0, and could be suppressed by amylase inhibitors from mulberry, the exclusive food source of silkworms. Three different transposable element fragments, which were independently inserted in the 5'-upstream regulatory region, might be responsible for the enhanced expression of BmAmy1 in different domesticated silkworm strains as revealed by dual-luciferase reporter assay. The BmAmy1 overexpression increased the weight of female and male B. mori by 11.9% and 6.8%, respectively, compared with non-transgenic controls. Our results emphasize that, by exploring the genetic mechanisms of human-selected traits, the domestication process could be further accelerated through genetic engineering and targeted breeding.

Scavenger receptor B8 improves survivability by mediating innate immunity in silkworm, Bombyx mori.

Scavenger receptor class B (SR-B) is an extracellular transmembrane glycoprotein that plays a vital role in innate immunity. Although SR-Bs have been widely studied in vertebrates, their functions remained to elucidate in insects. Here, we identified and characterized a scavenger receptor class B member from the silkworm, Bombyx mori (designated as BmSCRB8). BmSCRB8 is broadly expressed in various immune tissues/organs, including fat body, gut, and hemocyte. Its expression is dramatically enhanced after challenge with different types of bacteria or pathogen-associated molecular patterns (PAMPs). The recombinant BmSCRB8 protein can detect different types of bacteria by directly binding to PAMPs and significantly improve the bacterial clearance in vivo. After knockdown of BmSCRB8, the pathogenic bacterial clearance was strongly impaired, and several AMP genes were down-regulated following E. coli challenge. Moreover, pathogenic bacteria's treatment following the depletion of BmSCRB8 remarkably decreased silkworm larvae's survival rate. Taken together, these results demonstrate that BmSCRB8 acts as a pattern recognition protein and plays an essential role in silkworm innate immunity by enhancing bacterial clearance and contributing to the production of AMPs in vivo.

Identification of the Vo domain of V-ATPase in Bombyx mori silkworm.

Vacuolar H+-ATPase (V-ATPase) is very important for eukaryotes and consists of a conserved V1 domain and slightly variable Vo domain. However, the Vo domain has not been systematically identified in the silkworm Bombyx mori. In this study, 11 Vo domain subunit members were identified throughout the genome of B. mori, including four isoforms of subunit a (BmVoa1-4), two isoforms of subunit e (BmVoe1-2), one each of subunit c″ (BmVob), subunit c (BmVoc), and subunit d (BmVod), and two accessory subunits (BmVoap1 and BmVoap2). Further analysis revealed BmVoa3 and BmVoa4 were located on the same chromosome and had similar molecular weights and isoelectric points, but separated to different small branches on the phylogenetic tree. Reverse transcription polymerase chain reaction results indicated that most Vo domain subunits were expressed during all silkworm developmental stages. Quantitative polymerase chain rection (qPCR) showed BmVoa1 was hemocyte-specific and BmVoe1 was testis-specific. BmVoa2 was not expressed in the midgut, while the other members were specifically or highly expressed in the midgut and Malpighian tubules. Further qPCR analysis indicated BmVoa4 in the midgut and BmVoa3 in BmE cells were significantly induced by B. mori nucleopolyhedrovirus (BmNPV), suggesting that these two genes may be involved in BmNPV infection.

Toxicology study of fraxinellone as ovicidal agents against Mythimna separata Walker and Bombyx mori Linaeus.

Fraxinellone is a naturally occurring degraded limonoid isolated from many species of plants in Meliaceae and Rutaceae. Besides structural modification of the lead compounds, the toxicology study of the lead compounds is also a very important procedure to develop insecticidal agents. Herein the toxicology study of fraxinellone was carried out as the ovicidal agent against the eggs of two lepidopteran insects Mythimna separata Walker and Bombyx mori Linaeus. Fraxinellone selectively exhibited an ovicidal activity against the eggs of M. separata. After treatment with fraxinellone, the eggshells of M. separata were shrinked, whereas those of B. mori had no obvious change. The dynamic process of M. separata embryo development demonstrated that the distinct difference between the treated eggs and the control ones was obvious at the second day after treatment, especially, the control embryo finished blastokinesis, whereas the treated ones were still laid at pre-reversion status and a lot of yolk can be seen around the embryo. It ultimately resulted in the eggshell withered and the egg hatching inhibited.

A Multi-Sensor System for Silkworm Cocoon Gender Classification via Image Processing and Support Vector Machine.

Sericulture is traditionally a labor-intensive rural-based industry. In modern contexts, the development of process automation faces new challenges related to quality and efficiency. During the silkworm farming life cycle, a common issue is represented by the gender classification of the cocoons. Improper cocoon separation negatively affects quantity and quality of the yield resulting in disruptive bottlenecks for the productivity. To tackle this issue, this paper proposes a multi sensor system for silkworm cocoons gender classification and separation. Utilizing a load sensor and a digital camera, the system acquires weight and digital images from individual silkworm cocoons. An image processing procedure is then applied to extract significant shape-related features from each image instance, which, combined with the weight data, are provided as inputs to train a Support Vector Machine-based pattern classifier for gender classification. Subsequently, an air blower mechanism and a conveyor system sort the cocoons into their respective bins. The developed system was trained and tested on two different types of silkworm cocoons breeds, respectively CSR2 and Pure Mysore. The system performances are finally discussed in terms of accuracy, robustness and computation time.

Unprecedented reorganization of holocentric chromosomes provides insights into the enigma of lepidopteran chromosome evolution.

Chromosome evolution presents an enigma in the mega-diverse Lepidoptera. Most species exhibit constrained chromosome evolution with nearly identical haploid chromosome counts and chromosome-level gene collinearity among species more than 140 million years divergent. However, a few species possess radically inflated chromosomal counts due to extensive fission and fusion events. To address this enigma of constraint in the face of an exceptional ability to change, we investigated an unprecedented reorganization of the standard lepidopteran chromosome structure in the green-veined white butterfly (Pieris napi). We find that gene content in P. napi has been extensively rearranged in large collinear blocks, which until now have been masked by a haploid chromosome number close to the lepidopteran average. We observe that ancient chromosome ends have been maintained and collinear blocks are enriched for functionally related genes suggesting both a mechanism and a possible role for selection in determining the boundaries of these genome-wide rearrangements.

Genetic diversity and phylogeny analysis of Antheraea assamensis Helfer (Lepidoptera: Saturniidae) based on mitochondrial DNA sequences.

Antheraea assamensis Helfer, popularly known as Muga silkworm, the golden silk producer of northeast India is economically important and unique among the Saturniid silkworms. In this study, the genetic diversity and phylogeny of semidomesticated and wild morphs of Muga silkwormcollected from different geographical locations of northeast India were investigated based on the sequences of five mitochondrial loci, i.e. 12S rRNA, 16S rRNA, CoxI, Cytb and CR. All the five mitochondrial loci showed a strong bias towards higher 'A' and 'T' contents. Transitional substitutions were found to be more than the transversional substitutions. The rate of nucleotide substitution and average genetic divergence were found to be highest in CR sequences and lowest in 12S rRNA gene sequences among the morphs of Muga silkworm. The morphs collected from same geographical area had identical 12S rRNA, 16S rRNA, CoxI and Cytb gene sequences. Moreover, the 12S rRNA and 16S rRNA gene sequences of somesemi-domesticated and wild morphs collected from different geographical locations were also found to be similar. In the phylogenetic trees generated based on themitochondrial loci, mixing of semi-domesticated and wild morphs was observed as they shared the same group. The information generated in this study will help in formulating strategies to conserve the natural biodiversity present among these unique silkworms in northeast India. In addition, this will be useful in identifying diverse morphs of Muga silkworm, which will help in effective breeding programmes to improve its productivity.

Expression profile of several genes on ecdysteroidogenic pathway related to diapause in pupal stage of Bombyx mori bivoltine strain.

Ecdysone is involved in regulation of embryonic diapause in the silkworm, Bombyx mori. However, its mechanism still remains unclear. To explore the role of ecdysteroidogenic pathway (EP) genes in diapause process of bivoltine B. mori, the eggs of "Qiufeng", a bivoltine strain, were used as the study materials and arranged into diapause eggs producers (DEPs) and non-diapause eggs producers (NDEPs), respectively. The differential expression of EP genes between two groups was analysed during the early pupal stage. The expression of Shadow was significantly increased in the NDEPs in day-3 pupae and reached the peak simultaneously, indicating that Shadow was in coincidence with diapause process. To validate this hypothesis, a repression of Shadow by RNA interference was performed in day-2 pupae of NDEPs. The expression of Shadow was downregulated by RNAi, and ßFtz-F1, a downstream gene of EP, was also decreased. Furthermore, the genes encoding the kynurenine-synthetase were upregulated in the ovary, and Brown, AdenoK which link Shadow to the kynurenine-synthase gene were also upregulated in the fat body. The progeny eggs appeared a light purple colour at 48 h after oviposition, revealing a certain tendency to diapause. We speculate that inhibition of Shadow upregulates 3-hydroxy-kynurenine synthesis by increasing the expression of Brown and AdenoK. In addition, Shadow was cloned, and expressed in E. coli for further functional study of Shadow protein. Our study provided insight into the role of EP genes in the process of diapause of B. mori.

Genome-Wide Identification and Expression Profiling of Wnt Family Genes in the Silkworm, Bombyx mori.

Wnt is a family of conserved glycoproteins that participate in a variety of important biological processes including embryo development, cell proliferation and differentiation, and tissue regeneration. The Wnt family is a metazoan novelty found in all animal phyla. Studies have revealed that the number of Wnt genes varies among species, presumably due to reproduction and loss of genes during evolution. However, a comprehensive inventory of Wnt genes in Lepidoptera is lacking. In this study, we identified the repertoire of Wnt genes in the silkworm and seven other species of Lepidoptera and obtained eight Wnt genes (Wnt1, Wnt5⁻Wnt7, Wnt9⁻Wnt11, and WntA) in each species. Four of these Wnt genes are clustered in two orientations (5'-Wnt9-Wnt1-Wnt6-Wnt10-3' and 5'-Wnt10-Wnt6-Wnt1-Wnt9-3') in both moths and butterflies. Transcript analysis of Wnt in silkworm embryonic stages showed that each BmWnt gene had a unique expression pattern during embryological development. Analysis of a larval stage revealed differential expression of Wnt family members in diverse tissues. Our study provides an overview of the Wnt family in Lepidoptera and will inspire further functional study of the Wnt genes in the silkworm.

Transcriptomic analysis of the prothoracic gland from two lepidopteran insects, domesticated silkmoth Bombyx mori and wild silkmoth Antheraea pernyi.

The prothoracic gland (PG) is an important endocrine organ of synthesis and secretion of ecdysteroids that play critical roles in insects. Here, we used a comparative transcriptomic approach to characterize some common features of PGs from two lepidopteran species Bombyx mori and Antheraea pernyi. Functional and pathway annotations revealed an overall similarity in gene profile between the two PG transcriptomes. As expected, almost all steroid hormone biosynthesis genes and the prothoracicitropic hormone receptor gene (Torso) were well represented in the two PGs. Impressively, two ecdysone receptor genes, eleven juvenile hormone related genes, more than 10 chemosensory protein genes, and a set of genes involved in circadian clock were also presented in the two PGs. Quantitative real time -PCR (qRT-PCR) validated the expression of 8 juvenile hormone and 12 clock related genes in B. mori PG, and revealed a different expression pattern during development in whole fifth larval instar. This contribution to insect PG transcriptome data will extend our understanding of the function and regulation of this important organ.

Comparative mitochondrial genomes provide new insights into the true wild progenitor and origin of domestic silkworm Bombyx mori.

The domestication of domestic silkworm Bombyx mori, the only truly domesticated insect, is a distinctive event in agricultural history. The domestication and origin of domestic silkworm remains unclear, although it has connected with human for ~5500 years. In the present study, we would like to highlight our evidence from whole mitochondrial genome for the presence of two genetically distinctive subtypes in Chinese B. mandarina populations, corresponding to northern Chinese B. mandarina and southern Chinese B. mandarina, respectively. The mitochondrial genomes and mitochondrial phylogenetic tree provide a solid molecular evidence that the true wild ancestor of domestic silkworm is northern Chinese B. mandarina, rather than southern Chinese B. mandarina, thus implying that the early domestication event may have occurred in northern China. Our finding provides new insights into the origin and evolution of domestic silkworm.

Species Identification of Silks from Bombyx mori, Eri Silkworm and Chestnut Silkworm Using Western Blot and Proteomics Analyses.

Species identification is of key significance for exploring the origin and transmission of ancient silks. In this study, two novel methods, i.e. western blot (WB) and proteomics analyses, were proposed and established to identify the differences between silks from Bombyx mori (B. mori) and two other distinctive species (Eri silkworm and Chestnut silkworm). Three diagnostic antibodies, a polyclonal anti-silk fibroin (anti-SF) antibody (pAb), a polyclonal anti-SF-specific peptide antibody (pAsb), and a monoclonal anti-SF antibody (mAb) were designed and prepared to distinguish silk species using the antibody-based WB technique. Proteomics analysis by liquid chromatography-tandem mass spectrometry was performed to further identify silk species at the protein level. WB results indicated that the three antibodies showed high specificity and affinity and could discern B. mori silk from Eri and Chestnut silks. Biomarkers for each SF were obtained using proteomics analysis, and they have the potential to serve as standards for identifying silk species. Thus, combining WB and proteomics analyses with conventional methods can provide more accurate silk information and may be suitable for identifying other proteinaceous materials in archaeological field.

Silkworms suppress the release of green leaf volatiles by mulberry leaves with an enzyme from their spinnerets.

In response to herbivory, plants emit a blend of volatile organic compounds that includes green leaf volatiles (GLVs) and terpenoids. These volatiles are known to attract natural enemies of herbivores and are therefore considered to function as an indirect defense. Selection should favor herbivores that are able to suppress these volatile emissions, and thereby make themselves less conspicuous to natural enemies. We tested this possibility for silkworms, which were observed to leave secretions from their spinnerets while feeding on mulberry leaves. When we ablated the spinnerets of silkworms, no secretions were observed. Leaves infested by intact silkworms released smaller amounts of GLVs than leaves infested by ablated silkworms, indicating that the spinneret secretion suppressed GLV production. This difference in GLV emissions was also reflected in the behavioral response of Zenillia dolosa (Tachinidae), a parasitoid fly of silkworms. The flies laid fewer eggs when exposed to the volatiles from intact silkworm-infested leaves than when exposed to the volatiles from ablated silkworm-infested leaves. We identified a novel enzyme in the secretion from the spinneret that is responsible for the GLV suppression. The enzyme converted 13(S)-hydroperoxy-(9Z,11E,15Z)-octadecatrienoic acid, an intermediate in the biosynthetic pathway of GLVs, into its keto-derivative in a stereospecific manner. Taken together, this study shows that silkworms are able to feed on mulberry in a stealthy manner by suppressing GLV production with an enzyme in secretions of their spinnerets, which might be a countermeasure against induced indirect defense by mulberry plants.

Isolation and functional characterization of the pheromone biosynthesis activating neuropeptide receptor of Chinese oak silkworm, Antheraea pernyi.

Insect pheromone biosynthesis activating neuropeptide (PBAN) controls the synthesis and actuating of sex pheromones of female adult. In the current examination, the full-length cDNA encoding the PBAN receptor was cloned from the pheromone gland (PG) of Antheraea pernyi (AntpePBANR). The AntpePBANR displayed the characteristic seven transmembrane areas of the G protein-coupled receptor (GPCR) and was closely related to the PBANR from Bombyx mori and Manduca sexta in the phylogenetic tree. The AntpePBANR expressed in mammalian cell lines were enacted by AntpePBAN in a concentration-dependent manner. AntpePBANR activation resulted in the calcium mobilization but did not activate the cAMP elevation pathway. Cells expressing AntpePBANR were profoundly responsive to Antpe-γ-SGNP (suboesophageal ganglion neuropeptides) and Antpe-DH (diapause hormone), different individuals from FXPRLamide (X = T, S or V) family in A. pernyi. Deletion of residues in the C-terminal hexapeptide (FSPRLamide) proved that P, R and L played the key parts in initiating the AntpePBANR, the amination to the last C terminal residues which can also likewise impact the activation of AntpePBAN receptor altogether. The mRNA of the AntpePBANR gene demonstrated the most noteworthy transcript levels in pheromone gland followed by fat body.

Identification of a novel host protein SINAL10 interacting with GP64 and its role in Bombyx mori nucleopolyhedrovirus infection.

Bombyx mori nucleopolyhedrovirus (BmNPV) is the most important pathogen of Bombyx mori, silkworm and causes severe losses in the silk industry. During the virus infectious cycle, budded virus (BVs) and occlusion-derived virus (ODVs) particles, which have identical genetic content but different phenotypes, are produced. The envelope glycoprotein GP64, specific in BVs, is involved in host cell receptor binding and is sufficient to mediate membrane fusion during the viral entry. However, the host cell factors, interacting with GP64 to mediate BVs infection, are still unknown. In this study, a cDNA library of Bombyx mori cells (BmN) was constructed and yeast two-hybrid screening was used to identify the host cell factors interacting with GP64. One of the eight candidate proteins encoded the E3 ubiquitin-protein ligase SINA-like 10 (SINAL10), was further confirmed through coimmunoprecipitation assays as novel GP64 binding protein. Moreover, overexpression of SINAL10 significantly enhances viral reproduction, and conversely, silencing its expression by small interfering RNAs showed significant inhibitory effects. Collectively, we demonstrated that SINAL10 is a novel GP64-binding protein that stimulates BmNPV proliferation.

Cloning and Functional Analysis of CncC and Keap1 Genes in Silkworm.

CncC/keap1-ARE is an important signaling pathway for detoxification and antioxidation in Diptera and Coleoptera insects. However, such a signaling pathway has not been studied in Bombyx mori. In this study, BmCncC and Bmkeap1 genes were cloned, their amino acid sequences were analyzed, and each functional domain was mapped. Through phylogenetic analysis and sequence comparison among multiple species, we found that the Neh1 motif of CncC was highly conserved and the DLG motif was replaced by the DMG motif in Neh2. Conformational analysis showed that Neh1 of BmCncC forms a hairpin structure to bind DNA. The DGR region of Bmkeap1 contained abundant ß sheets, which was involved in the recognition of Neh2. The transcription and expression analyses showed that both BmCncC and Bmkeap1 were highly expressed in the first instar larvae, and these two genes were expressed at a high level in the reproductive gland, fat body, and head. The transcriptional and expression levels of Akt and BmCncC in the fat body were significantly upregulated, and the expression of Bmkeap1 was downregulated after the phoxim treatment in silkworm. The transcriptional levels of CncC-regulated detoxification enzymes GST, cyp4M5, cyp6AE2, and cyp9G3 were increased by 4.026-, 5.246-, 3.821-, and 9.787-fold, respectively, while the activities of GST and CYP450 were increased by 1.521- and 1.231-fold, respectively, after phoxim treatment. These results indicated that the BmCncC/Bmkeap1 signaling pathway was activated by phoxim, leading to the expression of downstream detoxifying enzymes and detoxification of phoxim in silkworm.