Engineering a complex, multiple enzyme-mediated synthesis of natural plant pigments in the silkworm, Bombyx mori.

Plants produce various pigments that not only appear as attractive colors but also provide valuable resources in applications in daily life and scientific research. Biosynthesis pathways for these natural plant pigments are well studied, and most have multiple enzymes that vary among plant species. However, adapting these pathways to animals remains a challenge. Here, we describe successful biosynthesis of betalains, water-soluble pigments found only in a single plant order, Caryophyllales, in transgenic silkworms by coexpressing three betalain synthesis genes, cytochrome P450 enzyme CYP76AD1, DOPA 4,5-dioxygenase, and betanidin 5-O-glucosyltransferase. Betalains can be synthesized in various tissues under the control of the ubiquitous IE1 promoter but accumulate mainly in the hemolymph with yields as high as 274 µg/ml. Additionally, transformed larvae and pupae show a strong red color easily distinguishable from wild-type animals. In experiments in which expression is controlled by the promoter of silk gland-specific gene, fibroin heavy-chain, betalains are found predominantly in the silk glands and can be secreted into cocoons through spinning. Betalains in transformed cocoons are easily recovered from cocoon shells in water with average yields reaching 14.4 µg/mg. These data provide evidence that insects can synthesize natural plant pigments through a complex, multiple enzyme-mediated synthesis pathway. Such pigments also can serve as dominant visible markers in insect transgenesis applications. This study provides an approach to producing valuable plant-derived compounds by using genetically engineered silkworms as a bioreactor.

Identification of genes associated with the silk gland size using multi-omics in silkworm (Bombyx mori).

Silk gland size in silkworms (Bombyx mori) affects silk output. However, the molecular mechanisms by which genes regulate silk gland size remain unclear. In this study, silk glands from three pure silkworm strains (A798, A306 and XH) with different silk gland weight phenotypes were compared using transcriptomics and proteomics to identify differentially expressed genes (DEGs) and proteins (DEPs). When comparing A798 to A306 and A798 to XH, 830 and 469 DEGs were up-regulated, respectively. These genes were related to the gene ontology terms, metabolic process, transport activity and biosynthesis process. In addition, 372 and 302 up-regulated differentially expressed proteins were detected in A798 to A306 and A798 to XH, respectively, related to the gene ontology terms, ribosome and protein export, ribosome and polypeptide biosynthesis processes. Moreover, combined transcriptomics, proteomics and weighted correlation network analyses showed that five genes (BGIBMGA002524, BGIBMGA002629, BGIBMGA005659, BGIBMGA005711 and BGIBMGA010889) were significantly associated with the silk gland weight. Reverse Transcription-quantitative real-time Polymerase Chain Reaction (RT-qPCR) and Enzyme linked immunosorbent assay (ELISA) were used to verify the mRNA and protein expression of five genes in the silk glands and tissues of 18 silkworm strains. The results showed that four genes have higher expression levels in heavier silk glands. These genes are associated with glycogen metabolism, fatty acid synthesis and branched chain amino acid metabolism, thus potentially promoting growth and silk protein synthesis. These findings provide valuable insights into the molecular mechanisms underlying the relationship between silk gland weight and silk yield in silkworms.

The silk gland proteome of Stenopsyche angustata provides insights into the underwater silk secretion.

Caddisworms (Trichoptera) spin adhesive silks to construct a variety of underwater composite structures. Many studies have focused on the fibroin heavy chain of caddisworm silk and found that it contains heavy phosphorylation to maintain a stable secondary structure. Besides fibroins, recent studies have also identified some new silk proteins within caddisworm silk. To better understand the silk composition and its secretion process, this study reports the silk gland proteome of a retreat-building caddisworm, Stenopsyche angustata Martynov (Trichoptera, Stenopsychidae). Using liquid chromatography tandem mass spectrometry (LC-MS/MS), 2389 proteins were identified in the silk gland of S. angustata, among which 192 were predicted as secreted silk proteins. Twenty-nine proteins were found to be enriched in the front silk gland, whereas 109 proteins were enriched in the caudal silk gland. The fibroin heavy chain and nine uncharacterized silk proteins were identified as phosphorylated proteins. By analysing the sequence of the fibroin heavy chain, we found that it contains 13 Gly/Thr/Pro-rich regions, 12 Val/Ser/Arg-rich regions and a Gly/Arg/Thr-rich region. Three uncharacterized proteins were identified as sericin-like proteins due to their larger molecular weights, signal peptides and repetitive motifs rich in serine. This study provides valuable information for further clarifying the secretion and adhesion of underwater caddisworm silk.

Effects of poly (ADP-ribose) polymerase 1 (PARP1) on silk proteins in the silkworm, Bombyx mori.

Animal silk is economically important, while silk secretion is a complex and subtle mechanism regulated by many genes. We identified the poly (ADP-ribose) polymerase (PARP1) gene of the silkworm and successfully cloned its coding sequence (CDS) sequence. Using clustered regularly interspaced short palindromic repeat (CRISPR/Cas9) technology, we screened single guide RNA (sgRNA) with high knockout efficiency by cellular experiments and obtained PARP1 mutants by knocking out the PARP1 gene of the silkworm at the individual level. We found that the mutants mainly exhibited phenotypes such as smaller cocoon size and reduced cocoon shell rate than the wild type. We also detected the expression of silk protein genes in the mutant by quantitative real-time PCR (qPCR) and found that the expression of some silk protein genes was slightly down-regulated. Meanwhile, together with the results of transcriptomic analysis, we hypothesized that PARP1 may affect the synthesis of silk proteins, resulting in their failure to function properly. Our study may provide an important reference for future in-depth refinement of the molecular mechanism of silk protein expression in silk-producing animals, as well as a potential idea for future development of molecular breeding lines of silkworms to improve silk production.

Mechanism of programmed cell death in the posterior silk gland of the silkworm, Bombyx mori, during pupation based on Ca2+ homeostasis.

The silkworm, Bombyx mori, is a complete metamorphosed economic insect, and the silk gland is a significant organ for silk protein synthesis and secretion. The silk gland completely degenerates during pupation, but the regulatory mechanism of programmed cell death (PCD) has not yet been understood. In the present study, we investigated the non-genetic pathway of 20E-induced PCD in the posterior silk gland (PSG) based on intracellular Ca2+ levels. Silk gland morphology and silk gland index indicated rapid degeneration of silk gland during metamorphosis from mature silkworm (MS) to pupal day 1 (P1), and Ca2+ levels within the PSG were found to peak during the pre-pupal day 1 (PP1) stage. Moreover, the results of autophagy and apoptosis levels within the PSG showed that autophagy was significantly increased in MS-PP1 periods, and significantly decreased in PP2 and P1 periods. Apoptosis was almost absent in MS-PP1 periods and significantly increased in PP2 and P1 periods. Additionally, western blotting results showed that autophagy preceded apoptosis, and the autophagy-promoting ATG5 was cleaved by calpain to the autophagy-inhibiting and apoptosis-promoting NtATG5 since PP1 period, while decreased autophagy was accompanied by increased apoptosis. Collectively, these findings suggest that Ca2+ is a key factor in the shift from autophagy to apoptosis.

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.

Juvenile hormone regulates silk gene expression by m6A RNA methylation.

Juvenile hormone (JH) is an indispensable insect hormone that is critical in regulating insect development and physiology. N6-methyladenosine (m6A) is the most abundant modification of RNA that regulates RNA fate in eukaryotic organisms. However, the relationship between m6A and JH remains largely unknown. Here, we found that the application of a Juvenile hormone analog (JHA) extended the larval period of Bombyx mori and increased the weight and thickness of the cocoon. Interestingly, global transcriptional patterns revealed that m6A-related genes are specifically regulated by JHA in the posterior silk gland (PSG) that synthesizes the major component of cocoon silk. By transcriptome and m6A sequencing data conjointly, we discovered that JHA significantly regulated the m6A modification in the PSG of B. mori and many m6A-containing genes are related to nucleic acid binding, nucleus, and nucleobase-containing compound metabolism. Notably, 547 genes were significantly regulated by JHA at both the m6A modification and expression levels, especially 16 silk-associated genes, including sericin2, seroin1, Serine protease inhibitors 4 (BmSPI4), Serine protease inhibitors 5 (BmSPI5), and LIM domain-binding protein 2 (Ldb). Among them, 11 silk associated genes were significantly affected by METTL3 knockdown, validating that these genes are targets of m6A modification. Furthermore, we confirm that JHA directly regulates the expression of BmSPI4 and BmSPI5 through m6A modification of CDS regions. These results demonstrate the essential role of m6A methylation regulated by JH in PSG, and elucidate a novel mechanism by which JH affects silk gland development via m6A methylation. This study uncovers that m6A modification is a critical factor mediating the effect of JH in insects.

CRISPR/Cas9-Mediated Editing of BmEcKL1 Gene Sequence Affected Silk Gland Development of Silkworms (Bombyx mori).

The silkworm (Bombyx mori) has served humankind through silk protein production. However, traditional sericulture and the silk industry have encountered considerable bottlenecks and must rely on major technological breakthroughs to keep up with the current rapid developments. The adoption of gene editing technology has nevertheless brought new hope to traditional sericulture and the silk industry. The long period and low efficiency of traditional genetic breeding methods to obtain high silk-yielding silkworm strains have hindered the development of the sericulture industry; the use of gene editing technology to specifically control the expression of genes related to silk gland development or silk protein synthesis is beneficial for obtaining silkworm strains with excellent traits. In this study, BmEcKL1 was specifically knocked out in the middle (MSGs) and posterior (PSGs) silk glands using CRISPR/Cas9 technology, and ΔBmEcKL1-MSG and ΔBmEcKL1-PSG strains with improved MSGs and PSGs and increased silk production were obtained. This work identifies and proves that BmEcKL1 directly or indirectly participates in silk gland development and silk protein synthesis, providing new perspectives for investigating silk gland development and silk protein synthesis mechanisms in silkworms, which is of great significance for selecting and breeding high silk-yielding silkworm varieties.

Geminin is essential for DNA re-replication in the silk gland cells of silkworms.

Endoreplication, known as endocycles or endoreduplication, is a cell cycle variant in which the genomic DNA is re-replicated without mitosis leading to polyploidy. Endoreplication is essential for the development and functioning of the different organs in animals and plants. Deletion of Geminin, a DNA replication licensing inhibitor, causes DNA re-replication or damage. However, the role of Geminin in endoreplication is still unclear. Here, we studied the role of Geminin in the endoreplication of the silk gland cells of silkworms by constructing two transgenic silkworm strains, including BmGeminin1-overexpression and BmGeminin1-RNA interference. Interference of BmGeminin1 led to body weight gain, increased silk gland volume, increased DNA content, and enhanced DNA re-replication activity relative to wild-type Dazao. Meanwhile, overexpression of BmGeminin1 showed an opposite phenotype compared to the BmGem1-RNAi strain. Furthermore, RNA-sequencing of the transgenic strains was carried out to explore how BmGeminin1 regulates DNA re-replication. Our data demonstrated a vital role of Geminin in the regulation of endoreplication in the silk gland of silkworms.

The promoting effects of pyriproxyfen on autophagy and apoptosis in silk glands of non-target insect silkworm, Bombyx mori.

Pyriproxyfen is a juvenile hormone analogue. The physiological effects of its low-concentration drift during the process of controlling agricultural and forestry pests on non-target organisms in the ecological environment are unpredictable, especially the effects on organs that play a key role in biological function are worthy of attention. The silk gland is an important organ for silk-secreting insects. Herein, we studied the effects of trace pyriproxyfen on autophagy and apoptosis of the silk gland in the lepidopteran model insect, Bombyx mori (silkworm). After treating fifth instar silkworm larvae with pyriproxyfen for 24 h, we found significant shrinkage, vacuolization, and fragmentation in the posterior silk gland (PSG). In addition, the results of autophagy-related genes of ATG8 and TUNEL assay also demonstrated that autophagy and apoptosis in the PSG of the silkworm was induced by pyriproxyfen. RNA-Seq results showed that pyriproxyfen treatment resulted in the activation of juvenile hormone signaling pathway genes and inhibition of 20-hydroxyecdysone (20E) signaling pathway genes. Among the 1808 significantly differentially expressed genes, 796 were upregulated and 1012 were downregulated. Among them, 30 genes were identified for autophagy-related signaling pathways, such as NOD-like receptor signaling pathway and mTOR signaling pathway, and 30 genes were identified for apoptosis-related signaling pathways, such as P53 signaling pathway and TNF signaling pathway. Further qRT-PCR and in vitro gland culture studies showed that the autophagy-related genes Atg5, Atg6, Atg12, Atg16 and the apoptosis-related genes Aif, Dronc, Dredd, and Caspase1 were responsive to the treatment of pyriproxyfen, with transcription levels up-regulated from 24 to 72 h. In addition, ATG5, ATG6, and Dronc genes had a more direct response to pyriproxyfen treatment. These results suggested that pyriproxyfen treatment could disrupt the hormone regulation in silkworms, promoting autophagy and apoptosis in the PSG. This study provides more evidence for the research on the damage of juvenile hormone analogues to non-target organisms or organs in the environment, and provides reference information for the scientific and rational use of juvenile hormone pesticides.

Low concentration chlorantraniliprole-promoted Ca2+ release drives a shift from autophagy to apoptosis in the silk gland of Bombyx mori.

The novel pesticide chlorantraniliprole (CAP) is widely used for pest control in agriculture, and the safety for non-target organisms of trace residues in the environment has received widespread attention. In the present study, exposure to low concentrations of CAP resulted in abnormal silk gland development in the B. mori, and induced the release of intracellular Ca2+ in addition to the triggering of Ca2+-dependent gene transcription. Moreover, the CAP treatment group exhibited down-regulation of oxidative phosphorylation and antioxidant enzyme-related genes in the silk gland, resulting in peroxide accumulation. Furthermore, transcript levels of autophagy-related genes were significantly up-regulated and protein levels of LC3-I and LC3-II were up-regulated, indicating an increase in autophagy. The protein levels of ATG5 and NtATG5 were also significantly up-regulated. While the protein levels of caspase3 and active caspase3 were significantly up-regulated consistent with the transcript levels of key genes in the apoptotic signaling pathway, ultimately affecting silk protein synthesis. Overall, these findings indicate that low concentration CAP induced abnormal development in the silk gland of B. mori by causing intracellular Ca2+ overload, which inhibits oxidative phosphorylation pathway and the removal of reactive oxygen species, leading to a driving a shift from autophagy to apoptosis. The findings herein provided a basis for evaluating the safety of CAP environmental residues on non-target organisms.

Dynamic Changes and Characterization of the Metal Ions in the Silk Glands and Silk Fibers of Silkworm.

Metal ions are involved in the conformational transition of silk fibroin and influence the structure and mechanical properties of silk fibers. However, the dynamic characteristics of metal ions during the formation of silk fibers remain unclear. In this study, we found that the silk glands of silkworms contain various metal elements, with varying levels of the metal elements in different zones of the glands and higher levels in the anterior silk glands. Additionally, the content of various metallic elements in the silk glands varied greatly before and after spinning, similar to their content in different cocoon layers, thus, indicating that the anterior silk glands maintain a certain metal ion environment for the transport and conformational transformation of the silk proteins. Most of the metallic elements located in fibroin were confirmed using degumming experiments. For the first time, a scanning electron microscope energy spectrometry system was used to characterize the metal elements in the cross-section of silk and cocoons. These findings have deepened our understanding of the relationship between the overall metal ion environment and silk fiber formation and help us further conceptualize the utilization of metal ions as targets to improve the mechanical properties of the silk fibers.

Overexpression of Ultrabithorax Changes the Development of Silk Gland and the Expression of Fibroin Genes in Bombyx mori.

Ultrabithorax (Ubx) is a member of the Hox gene group involved in cell fate decisions, cell proliferation and organ identity. Its function has been extensively researched in Drosophila melanogaster but little is known about it in Lepidoptera. To uncover the function of Ubx in the development of lepidopterans, we constructed the Ubx overexpression (UbxOE) strain based on the Nistari strain of Bombyx mori. The UbxOE strain showed a small body size, transparent intersegmental membrane and abnormal posterior silk gland (PSG). In the current study, we focused on the effect of Ubx overexpression on the posterior silk gland. As the major protein product of PSG, the mRNA expression of fibroin heavy chain (Fib-H) and fibroin light chain (Fib-L) was upregulated three times in UbxOE, but the protein expression of Fib-H and Fib-L was not significantly different. We speculated that the overexpression of Ubx downregulated the expression of Myc and further caused abnormal synthesis of the spliceosome and ribosome. Abnormalities of the spliceosome and ribosome affected the synthesis of protein in the PSG and changed its morphology.

[Quality status of Bombyx Batryticatus and suggestions for Chinese Pharmacopoeia (2025)].

To understand the current quality status and rearing situation of Bombyx Batryticatus, the authors collected 102 batches of Bombyx Batryticatus from different main producing areas and five major Chinese medicine markets from 2016 to 2018, and measured the properties and quality of the silk gland, to clarify the quality status of Bombyx Batryticatus from different producing areas and markets. In addition, 35 batches of Bombyx Batryticatus from 2019 to 2022 were used to verify the silk gland after revision. Moreover, Beauveria Bassiana was inoculated in the silkworm of 4-5 instars, and standardized rearing was carried out until they die. The death rate and the quality of Bombyx Batryticatus were measured to determine the differences in Bombyx Batryticatus of different instars, and explore the rationality of the infection age of Bombyx Batryticatus in Chinese Pharmacopoeia(2020). The results revealed that in the 102 batches of Bombyx Batryticatus, the qualification rate of silk gland was low; the content of total ash far exceeded the standard; the content of beauvericin varied greatly. The qualification rate of the silk gland of the 35 batches of Bombyx Batryticatus was only 47.49%, which could be increased to 73.00% if the number of silk gland was 2 to 4. The death rate of Bombyx Batryticatus at different infection ages was quite different, with uneven quality. Generally, the yield of Bombyx Batryticatus inoculated on the first day of the fifth instar was high with good quality. Therefore, in combination with the quality and actual production of Bombyx Batryticatus, the following suggestions were proposed for revision of Bombyx Batryticatus in Chinese Pharmacopoeia(2025): The number of silk gland should be revised as 2-4 bright brown or bright black silk glands, after which, the quality of Bombyx Batryticatus could be guaranteed, and the "quality identification based on character" could also be reflected scientifically; the content determination index that the content of beauvericin shall not be less than 0.017% should be added to better control the quality of Bombyx Batryticatus; the infection age should be revised as the first day of the fifth instar to narrow the age span, which could better fit the actual production and ensure the quality of Bombyx Batryticatus.

Comparative transcriptome and proteome reveal synergistic functions of differentially expressed genes and proteins implicated in an over-dominant silkworm heterosis of increased silk yield.

We previously observed an over-dominant silkworm heterosis of increased yield in a cross of Bombyx mori nuclear polyhydrosis virus-resistant strain NB with a susceptible strain 306. In the present study, we found that heterosis also exists in crosses of NB with other susceptible strains, indicating it is a more general phenomenon. We performed comparative transcriptome and proteome and identified 1624 differentially expressed genes (DEGs) and 298 differentially expressed proteins (DEPs) in silk glands between parents and F1 hybrids, of which 24 DEGs/DEPs showed consistent expression at mRNA and protein levels revealed by Venn joint analysis. Their expressions are completely non-additive, mainly transgressive and under low-parent, suggesting recombination of parental genomes may be the major genetic mechanism for the heterosis. GO and KEGG analyses revealed that they may function in generally similar but distinctive aspects of metabolisms and processes with signal transduction and translation being most affected. Notably, they may not only up-regulate biosynthesis and transport of silk proteins but also down-regulate other unrelated processes, synergistically and globally remodelling the silk gland to increase yield and cause the heterosis. Our findings contribute insights into the understanding of silkworm heterosis and silk gland development and provide targets for transgenic manipulation to further increase the silk yield.

Mechanism of autophagy induced by low concentrations of chlorantraniliprole in silk gland, Bombyx mori.

Chlorantraniliprole (CAP) is widely used in the control of agricultural pests, and its residues can affect the formation of silkworm (Bombyx. mori) cocoon easily. To accurately evaluate the toxicity of CAP to silkworms and clarify the mechanism of its effect on silk gland function, we proposed a novel toxicity evaluation method based on the body weight changes after CAP exposure. We also analyzed the Ca2+-related ATPase activity, characterized energy metabolism and transcriptional changes about the autophagy key genes on the downstream signaling pathways. The results showed that after a low concentration of CAP exposed for 96 h, there were CAP residues in the silk glands of B. mori, the activities of Ca2+-ATPase and Ca2+-Mg2+-ATPase decreased significantly (P ≤ 0.01), and the activation of AMPK-related genes AMPK-α and AMPK-ß were up-regulated by 6.39 ± 0.02-fold and 12.33 ± 1.06-fold, respectively, reaching a significant level (P ≤ 0.01)). In addition, the autophagy-related genes Atg1, Atg6, Atg5, Atg7, and Atg8 downstream AMPK were significantly up-regulated at 96 h (P ≤ 0.05). The results of immunohistochemistry and protein expression assay for autophagy marker Atg8 further confirmed the occurrence of autophagy. Overall, our results indicate that CAP exposure leads to autophagy in the silk gland of B. mori and affects their physiological functions, which provides guidance for the evaluation of toxicity of low concentration environmental CAP residues to insects.

Gene mapping reveals the association between tyrosine protein kinase Abl1 and the silk yield of Bombyx mori.

The Z chromosome of the silkworm contains a major gene that influences silk yield. This major locus on chromosome Z accounts for 35.10% of the phenotypic variance. The location and identification of the gene have been a focus of silkworm genetics research. Unfortunately, identification of this gene has been difficult. We used extreme phenotype subpopulations and selected from a backcross population, BC1 M, which was obtained using the high-yield strain 872B and the low-yield strain IS-Dazao as parents, for mapping the gene on the chromosome Z. The candidate region was narrowed down to 134 kb at the tip of the chromosome. BmAbl1 in this region correlated with silk gland development by spatiotemporal expression analysis. This gene was differentially expressed in the posterior silk glands of the high- and low-yield strains. In BmAbl1, an insertion-deletion (indel) within the 10th exonic region and an SNP within the 6th intronic region were detected and shown to be associated with cocoon shell weight in 84 Bombyx mori strains with different yields. Nucleotide diversity analysis of BmAbl1 and its 50 kb flanking regions indicated that BmAbl1 has experienced strong artificial selection during silkworm domestication. This study is the first to identify the genes controlling silk yield in the major QTL of the Z chromosome using forward genetics.

Low concentration acetamiprid-induced oxidative stress hinders the growth and development of silkworm posterior silk glands.

Acetamiprid is a new type of nicotinic insecticide that is widely used in pest control. Its environmental residues may cause silkworm cocooning disorder. In this study, silkworms that received continuous feeding of low concentration acetamiprid (0.15 mg/L) showed significantly decreased silk gland index and cocooning rate. Gene expression profiling of posterior silk glands (PSGs) revealed that the differentially expressed genes were significantly enriched in oxidative stress-related signal pathways with significant up-regulation. The contents of both H2O2 and MDA were increased, along with significantly elevated SOD and CAT activities, all of which reached maximal values at 48 h when H2O2 and MDA's contents were 10.46 and 7.98 nmol/mgprot, respectively, and SOD and CAT activities were 5.51 U/mgprot and 33.48 U/gprot, respectively. The transcription levels of antioxidant enzyme-related genes SOD, Mn-SOD, CuZn-SOD, CAT, TPX and GPX were all up-regulated, indicating that exposure to low concentration acetamiprid led to antioxidant response in silkworm PSG. The key genes in the FoxO/CncC/Keap1 signaling pathway that regulates antioxidant enzyme activity, FoxO, CncC, Keap1, NQO1, HO-1 and sMaf were all up-regulated during the whole process of treatment, with maximal values being reached at 72 h with 2.91, 1.46, 1.82, 2.52, 2.32 and 4.01 times of increases, respectively. These results demonstrate that exposure to low concentration acetamiprid causes oxidative stress in silkworm PSG, which may be the cause of cocooning disorder in silkworm. Our study provides a reference for the safety evaluation of environmental residues of acetamiprid on non-target insects.

The JH-Met2-Kr-h1 pathway is involved in pyriproxyfen-induced defects of metamorphosis and silk protein synthesis in silkworms, Bombyx mori.

Environmental residues of pryriproxyfen, a juvenile hormone analogue (JHA) type pesticide, may have on unintended consequences on non-target insects. However, the mechanism of pyriproxyfen action and silk protein synthesis in silkworms has not been reported. In the present study, we treated the silkworms with trace pyriproxyfen (1 × 10-4 mg/L) and found that the silkworm larvae showed no obvious poisoning symptoms, while the development of silk glands and cocoon-forming function were both seriously damaged due to the accumulation of pyriproxyfen in posterior silk gland (PSG). The titer of the juvenile hormone (JH) was increased, whereas the content of 20-hydroxyecdysone (20E) was reduced in pyriproxyfen-exposed hemolymph. Met2 is a component of the JH receptor complex and JH can promote its phosphorylation. We found Met2 and SRC were up-regulated in the larval stage after pyriproxyfen exposure, the JH-Met2/SRC complex led to the up-regulation of downstream genes Kr-h1, and Dimm, and then specifically inhibited the transcription of Fib-H. Meanwhile, the transcription of ecdysone inducible transcription factor Br-C Z4 was also inhibited by pyriproxyfen and resulted in the defects of metamorphosis. In conclusion, the trace pyriproxyfen could affect the metamorphosis and silk protein synthesis through the Met2-mediated pathway. Our study provided new evidence that Met2 might be a potential target gene of JHA in Lepidoptera.

Insights into regulatory characteristics of the promoters of Sericin 1 and Sericin 3 in transgenic silkworms.

Sericin, produced in the middle silk gland (MSG) of silkworms, is a group of glue proteins that coat and cement silk fibers. Several genes are known to encode sericin, but their spatiotemporal regulation has yet to be fully elucidated. Here, we report in detail the expression profiles of the promoters of two major sericin-coding genes, Sericin 1 (Ser1)and Sericin 3 (Ser3), by analyzing Gal4/UAS transgenic silkworms. We found that UAS-linked EGFP fluorescence in transgenic silkworms driven by Ser1-Gal4was detected in only the R3, R4 and R5 regions of MSG starting inday-3 fifth-instar larvae and was continuously expressed until silk gland degradation. In transgenic silkworms driven by Ser3-Gal4, EGFP fluorescence was detected at a low level in the R2 region of MSG since the last day of fifth-instar larvae, and the expression increased during the wandering stages and was continuously detected until silk gland degradation. The molecular detection of EGFP expression in each of the Gal4/UAS transgenic silkworms was consistent with fluorescence observations. These findings reveal clear differences in the regulatory characteristics of the promoters of Ser1and Ser3 and provide new insights into the regulatory mechanism of the expression of sericin-coding genes.