A parasitoid regulates 20E synthesis and antibacterial activity of the host for development by inducing host nitric oxide production.

Parasitoids are important components of the natural enemy guild in the biological control of insect pests. They depend on host resources to complete the development of a specific stage or whole life cycle and thus have evolved towards optimal host exploitation strategies. In the present study, we report a specific survival strategy of a fly parasitoid Exorista sorbillans (Diptera: Tachinidae), which is a potential biological control agent for agricultural pests and a pest in sericulture. We found that the expression levels of nitric oxide synthase (NOS) and nitric oxide (NO) production in host Bombyx mori (Lepidoptera: Bombycidae) were increased after E. sorbillans infection. Reducing NOS expression and NO production with an NOS inhibitor (NG-nitro-L-arginine methyl ester hydrochloride) in infected B. mori significantly impeded the growth of E. sorbillans larvae. Moreover, the biosynthesis of 20-hydroxyecdysone (20E) in infected hosts was elevated with increasing NO production, and inhibiting NOS expression lowered 20E biosynthesis. More importantly, induced NO synthesis was required to eliminate intracellular bacterial pathogens that presumably competed for shared host resources. Inhibiting NOS expression down-regulated the transcription of antimicrobial peptide genes and increased the number of bacteria in parasitized hosts. Collectively, this study revealed a new perspective on the role of NO in host-parasitoid interactions and a novel mechanism for parasitoid regulation of host physiology to support its development.

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.

Indoxacarb triggers autophagy and apoptosis through ROS accumulation mediated by oxidative phosphorylation in the midgut of Bombyx mori.

Indoxacarb has been widely utilized in agricultural pest management, posing a significant ecological threat to Bombyx mori, a non-target economic insect. In the present study, short-term exposure to low concentration of indoxacarb significantly suppressed the oxidative phosphorylation pathway, and resulted in an accumulation of reactive oxygen species (ROS) in the midgut of B. mori. While, the ATP content exhibited a declining trend but there was no significant change. Moreover, indoxacarb also significantly altered the transcription levels of six autophagy-related genes, and the transcription levels of ATG2, ATG8 and ATG9 were significantly up-regulated by 2.56-, 1.90-, and 3.36-fold, respectively. The protein levels of ATG8-I and ATG8-II and MDC-stained frozen sections further suggested an increase in autophagy. Furthermore, the protein level and enzyme activity of CASP4 showed a significant increase in accordance with the transcription levels of apoptosis-related genes, indicating the activation of the apoptotic signaling pathway. Meanwhile, the induction of apoptosis signals in the midgut cells triggered by indoxacarb was confirmed through TUNEL staining. These findings suggest that indoxacarb can promote the accumulation of ROS by inhibiting the oxidative phosphorylation pathway, thereby inducing autophagy and apoptosis in the midgut cells of B. mori.

Sublethal chlorantraniliprole exposure induces autophagy and apoptosis through disrupting calcium homeostasis in the silkworm Bombyx mori.

The intensive application of chlorantraniliprole (CAP) leaves residues in the environment, posing a potential threat to non-target organisms. In the present study, we investigated the adverse effects of sublethal CAP exposure on Bombyx mori. Sublethal CAP (0.02 mg/L) was shown to induce the release of intracellular Ca2+ in BmN cells. Meanwhile, Ca2+ -dependent genes were induced in the midgut at 72 h after CAP (0.01 mg/L) exposure, and damaged mitochondria, autophagosomes, nuclear membrane rupture and condensed chromatin were observed. Moreover, the key genes in the oxidative phosphorylation pathway were significantly down-regulated. The transcript levels of autophagy-related genes ATG6 and ATG8 were significantly up-regulated, and the protein levels of LC3-II and ATG7 were significantly increased by 3.72- and 3.33-fold, respectively. Additionally, the transcript levels of the upstream genes in the apoptosis pathway (calpain and Apaf-1) were significantly up-regulated, the protein levels of the downstream gene caspase 3 and its cleaved form were significantly up-regulated by 1.97- and 4.55-fold, respectively, consistent with the elevated caspase 3 activity at 72 h. Collectively, these findings demonstrate that intracellular Ca2+ release induced by sublethal CAP inhibits oxidative phosphorylation pathway, which causes mitochondrial dysfunction, leading to autophagy and apoptosis in the midgut of B. mori.

Effects of artificial diet breeding on intestinal microbial populations at the young stage of silkworm (Bombyx mori).

The silkworm (Bombyx mori) is an economically important insect and serves as a model organism for Lepidoptera. To investigate the effects of the intestinal microbial population on the growth and development of larvae fed an artificial diet (AD) during the young stages, we analyzed the characteristics of the intestinal microbial population using 16S rRNA gene sequencing technology. Our results revealed that the intestinal flora of the AD group tended to be simple by the 3rd-instar, which Lactobacillus accounting for 14.85% and leading to a decreased pH in the intestinal fluid. In contrast, the intestinal flora of silkworms in the mulberry leaf (ML) group showed continuous growth of diversity, with Proteobacteria accounting for 37.10%, Firmicutes accounting for 21.44%, and Actinobacteria accounting for 17.36%. Additionally, we detected the activity of intestinal digestive enzymes at different instars and found that the activity of digestive enzymes in the AD group increased by larval instar. Protease activity in the AD group was lower during the 1st- to 3rd-instars compared to the ML group, while α-amylase and lipase activities were significantly higher in the AD group during the 2nd- and 3rd-instar compared to the ML group. Furthermore, our experimental results indicated that changes in the intestinal population decreased the pH and affected the activity of proteases, which might contribute to the slower growth and development of larvae in the AD group. In summary, this study provides a reference for investigating the relationship between artificial diet and intestinal flora balance.

Tachinid parasitoid Exorista japonica affects the utilization of diet by changing gut microbial composition in the silkworm, Bombyx mori.

Changes in both intake and digestion of feed have been demonstrated in the host following parasitization. However, its regulatory mechanism has not been clarified. In this study, silkworms and Exorista japonica were used as research objects to analyze the effect of parasitism on the midgut immune system of the silkworm. After being parasitized, the expressions of antimicrobial peptide (AMP) genes of silkworms showed a fluctuating trend of first upregulation and then downregulation, while phenoloxidase and lysozyme activities were inhibited. To study the possible impact of the downregulation of AMP genes on intestinal microorganisms, the characteristics of the intestinal microbial population of silkworms on the third day of parasitism were analyzed. The relative abundance of Firmicutes, Proteobacteria, and Bacteroidota decreased, while that of Actinobacteriota increased. The increased abundance of conditionally pathogenic bacteria Serratia and Staphylococcus might lead to a decrease in the amount of silkworm ingestion. Meanwhile, the abundance of Acinetobacter, Bacillus, Pseudomonas, and Enterobacter promotes an increase in the digestion of nutrients. This study indicated that the imbalance of intestinal microbial homeostasis caused by parasitism may affect the absorption and digestion of nutrients by the host. Collectively, our findings provided a new clue for further exploring the mechanism of nutrient transport among the host, parasitoid, and intestinal microorganisms.

Low concentration of indoxacarb interferes with the growth and development of silkworm by damaging the structure of midgut cells.

As an important economic insect, Bombyx mori plays an essential role in the development of the agricultural economy. Indoxacarb, a novel sodium channel blocker insecticide, has been widely used for the control of various pests in agriculture and forestry, and its environmental pollution caused by flight control operations has seriously affected the safe production of sericulture in recent years. However, the lethal toxicity and adverse effects of indoxacarb on silkworm remain largely unknown. In this study, the toxicity of indoxacarb on the 5th instar larvae of silkworm was determined, with an LC50 (72 h) of 2.07 mg/L. Short-term exposure (24 h) to a low concentration of indoxacarb (1/2 LC50) showed significantly reduced body weight and survival rate of silkworm larvae. In addition, indoxacarb also led to decreased cocoon weight and cocoon shell weight, but had no significant effects on pupation, adult eclosion, and oviposition. Histopathological and ultrastructural analysis indicated that indoxacarb could severely damage the structure of the midgut epithelial cells, and lead to physiological impairment of the midgut. A total of 3883 differentially expressed genes (DEGs) were identified by midgut transcriptome sequencing and functionally annotated using GO and KEGG. Furthermore, the transcription level and enzyme activity of the detoxification related genes were determined, and our results suggested that esterases (ESTs) might play a major role in metabolism of indoxacarb in the midgut of B. mori. Future studies to examine the detoxification or biotransformation function of candidate genes will greatly enhance our understanding of indoxacarb metabolism in B. mori. The results of this study provide a theoretical basis for elucidating the mechanism of toxic effects of indoxacarb on silkworm by interfering with the normal physiological functions of the midgut.

Disruption of peritrophic matrix chitin metabolism and gut immune by chlorantraniliprole results in pathogenic bacterial infection in Bombyx mori.

Chlorantraniliprole (CAP) is widely used in pest control, and its environmental residues affect the disease resistance of non-target insect silkworms. Studies have demonstrated that changes in gut microbial communities of insects are associated with susceptibility to pathogens. In the present study, we examined the effects of CAP exposure on the immune system and gut microbial community structure of silkworms. The results showed that after 96 h of exposure to low-concentration CAP, the peritrophic matrix (PM) of silkworm larvae was disrupted, and pathogenic bacteria invaded hemolymph. The trehalase activity in the midgut was significantly decreased, while the activities of chitinase, ß-N-acetylglucosaminidase, and chitin deacetylase were increased considerably, resulting in decreased chitin content in PM. In addition, exposure to CAP reduced the expressions of key genes in the Toll, IMD, and JAK/STAT pathways, ultimately leading to the downregulation of antimicrobial peptides (AMPs) genes and alterations in the structure of the gut microbial community. Therefore, after infection with the conditional pathogen Enterobacter cloacae (E. cloacae), CAP-exposed individuals exhibited significantly lower body weight and higher mortality. These findings showed that exposure to low-concentration CAP impacted the biological defense system of silkworms, changed the gut microbial community structure, and increased silkworms' susceptibility to bacterial diseases. Collectively, these findings provided a new perspective for the safety evaluation of low-concentration CAP exposure in sericulture.

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.

Characterization of the sublethal toxicity and transcriptome-wide biological changes induced by λ-cyhalothrin in Bombyx mori.

λ-Cyhalothrin (λ-cyh) is widely used in agricultural production and has been reported to cause damages to numerous nontarget insects. As an important economic and model insect of Lepidoptera, Bombyx mori was extremely sensitive to λ-cyh, and pesticide drift often leads to silkworm poisoning. However, little is known about the persistence of sublethal effects or the potential recovery from short-term exposure to sublethal doses of pesticides. In this study, we estimated the sublethal effects caused by short-term exposure (24 h) of λ-cyh LC1 , LC10 , LC25 , and LC50 , respectively, and investigated the persistent negative effects on the growth, survival, and pupal metamorphosis of silkworm larvae. Silkworm growth was mostly retarded after λ-cyh exposure, with dose-dependent recovery observed at delayed time points. Relative to the control, the treatment groups showed significantly higher larval mortalities and abnormal pupa rates. Additionally, transcriptome sequencing was conducted to investigate the effects of λ-cyh LC10 on the normal physiological functions in the midgut of B. mori. A total of 2697 differentially expressed genes were identified, and 57.1% of DEGs were down-regulated. Gene ontology and Kyoto encyclopedia of genes and genomes enrichment analysis further revealed that energy and nutrient metabolisms were negatively affected. Moreover, we demonstrated that sublethal λ-cyh inhibited the oxidative phosphorylation pathway by reducing the expression of mitochondrial electron transport chain complex genes and consequently the synthesis of ATP. This study has provided useful transcriptome-wide expression resources to facilitate the overall knowledge of the molecular basis of sublethal toxicity caused by λ-cyh in the midgut of B. mori.

Homeostatic Regulation of the Duox-ROS Defense System: Revelations Based on the Diversity of Gut Bacteria in Silkworms (Bombyx mori).

The Duox-ROS defense system plays an important role in insect intestinal immunity. To investigate the role of intestinal microbiota in Duox-ROS regulation herein, 16S rRNA sequencing technology was utilized to compare the characteristics of bacterial populations in the midgut of silkworm after different time-periods of treatment with three feeding methods: 1-4 instars artificial diet (AD), 1-4 instars mulberry leaf (ML) and 1-3 instars artificial diet + 4 instar mulberry leaf (TM). The results revealed simple intestinal microbiota in the AD group whilst microbiota were abundant and variable in the ML and TM silkworms. By analyzing the relationship among intestinal pH, reactive oxygen species (ROS) content and microorganism composition, it was identified that an acidic intestinal environment inhibited the growth of intestinal microbiota of silkworms, observed concurrently with low ROS content and a high activity of antioxidant enzymes (SOD, TPX, CAT). Gene expression associated with the Duox-ROS defense system was detected using RT-qPCR and identified to be low in the AD group and significantly higher in the TM group of silkworms. This study provides a new reference for the future improvement of the artificial diet feeding of silkworm and a systematic indicator for the further study of the relationship between changes in the intestinal environment and intestinal microbiota balance caused by dietary alterations.

Resistance to RNA interference by plant-derived double-stranded RNAs but not plant-derived short interfering RNAs in Helicoverpa armigera.

Plant-mediated RNA interference (RNAi) has emerged as a promising technology for pest control through expression of double-stranded RNAs (dsRNAs) targeted against essential insect genes. However, little is known about the underlying molecular mechanisms and whether long dsRNA or short interfering RNAs (siRNAs) are the effective triggers of the RNAi response. Here we generated transplastomic and nuclear transgenic tobacco plants expressing dsRNA against the Helicoverpa armigera ATPaseH gene. We showed that expression of long dsRNA of HaATPaseH was at least three orders of magnitude higher in transplastomic plants than in transgenic plants. HaATPaseH-derived siRNAs are absent from transplastomic plants, while they are abundant in transgenic plants. Feeding transgenic plants to H. armigera larvae reduced gene expression of HaATPaseH and delayed growth. Surprisingly, no effect of transplastomic plants on insect growth was observed, despite efficient dsRNA expression in plastids. Furthermore, we found that dsRNA ingested by H. armigera feeding on transplastomic plants was rapidly degraded in the intestinal fluid. In contrast, siRNAs are relatively stable in the digestive system. These results suggest that plant-derived siRNAs may be more effective triggers of RNAi in Lepidoptera than dsRNAs, which will aid the optimization of the strategies for plant-mediated RNAi to pest control.

A novel system for the generation of baculoviruses mutant for an essential gene.

BACKGROUND: Currently, to delete an essential gene from a baculovirus genome, a cell line stably expressing the gene to be knocked-out should be first generated, which is time-consuming. Alternatively, essential genes can be deleted in E. coli using the λ Red recombination system, which requires an electroporation system. Here, based on homologous recombination in insect cells, we develop an alternative efficient system that requires neither generation of a cell line nor an electroporation system. METHODS AND RESULTS: Using puc19-based inverse PCR, a transfer vector for deleting BmNPV orf92 (Bm92, an essential gene) was efficiently constructed. A copy of Bm92 was introduced into the polyhedrin locus of BmNPV bacmid. The transfer vector was then co-transfected into BmN cell with the modified bacmid to enable homologous recombination at the Bm92 locus. An agarose-free approach was developed for the purification of Bm92-disrupted bacmid viruses in insect cells. Subsequently, BmN cells were co-infected with purified Bm92-disrupted bacmid viruses and unmodified bacmid viruses to allow recombination at the Tn7 insertion site between the two viruses. Finally, bacmid DNA extracted from BmN cells was transformed into chemically-treated competent DH10B cells, and blue colonies containing Bm92-disrupted bacmid were selected using PCR. CONCLUSIONS: For its efficiency and convenience, the system has great potential to be used for the generation of baculovirus knockout mutants.

Effects of glyphosate on the growth, development, and physiological functions of silkworm, Bombyx mori.

Glyphosate is an herbicide widely used worldwide, but whether it is safe to nontarget organisms is controversial. In this study, the lepidopteran model insect silkworm was used to investigate the effects of glyphosate residues. The LC50 (72 h) of glyphosate on silkworm was determined to be 14875.98 mg/L, and after exposure to glyphosate at 2975.20 mg/L (a concentration comparable to that used for weed control in mulberry fields), silkworm growth was inhibited by 9.00%, total cocoon weight was lowered by 10.53%, feed digestibility was decreased by 7.56%, and the activities of alpha-amylase and trypsin were reduced by 10.41% and 21.32%, respectively. Pathological analysis revealed that glyphosate exposure led to significantly damaged midgut, along with thinner basal layer, shedding microvilli, blurred cytoplasmic membrane, and appearance of vacuoles. Exposure to glyphosate also led to accumulation of peroxides in the intestinal tissue; the messenger RNA transcription of SOD, Cu/Zn-SOD, and Mn-SOD was all significantly upregulated by glyphosate treatment for 24 h, while CAT transcription was increased at 24, 48, and 72 h. The activity of SOD was increased significantly at 24 h, while significant activity changes were observed for CAT at 72 and 96 h. These results indicated that exposure to glyphosate caused oxidative stress in the midgut of silkworm and affected the midgut's physiological function. This study provides important insights in evaluating the impact of glyphosate residues in the environment on nontarget organisms.

Evaluation of tolerance to λ-cyhalothrin and response of detoxification enzymes in silkworms reared on artificial diet.

A representative silkworm rearing mode of Ⅰ-Ⅲ instars reared on artificial diet and Ⅳ-Ⅴ instars reared on fresh mulberry leaves has been recognized in some sericultural areas of China. Under this rearing mode, silkworms are prone to be poisoned by pesticide residues on mulberry leaves at the Ⅳ and Ⅴ instar stages. As one of the most widely applied insecticides, λ-cyhalothrin was used to study the insecticide tolerance of silkworm reared on artificial diet (referred as the AD group). Our results showed that the newly ecdysized Ⅳ instar larvae in the AD group were less tolerant to λ-cyhalothrin compared to the mulberry leaves reared group (referred as the ML group). After continuous exposure to trace λ-cyhalothrin, the weight gain and the survival rate of silkworms were significantly lower in the AD group than those in the ML group, even though compensatory growth was observed in the control of the AD group. Histopathology and ultrastructure of fat body showed that λ-cyhalothrin induced more severe cell injuries in the AD group, such as shrunken nucleus, dilatation of endoplasmic reticulum, and mitochondrial swelling. The transcription levels of detoxification related genes (CYP4M5, CYP6AB4, CarE2, CarE5, GSTe1 and GSTe3) and the enzyme activities of P450s, CarEs and GSTs were inducible by trace λ-cyhalothrin in a time-specific manner, and the data showed that the response of P450 enzyme activity was retarded in the AD group, indicating a potential reason for a higher sensitivity to λ-cyhalothrin. Our results provided a new clue for the study of the relationship between feed nutrition and detoxification ability, and also provided an important reference for the development of modern silkworm rearing mode.

Impact of sublethal chlorantraniliprole on epidermis of Bombyx mori during prepupal-pupal transition.

The silkworm Bombyx mori, an economically important insect with a long domestication history, exhibits high sensitivity to chemical pesticides. Extensive application of chlorantraniliprole (CAP) in control of pests of agricultural crops and mulberry plants causes residue toxicity to silkworm. We have demonstrated that sublethal concentration of CAP exposure causes defects in the formation of new epidermis and incomplete shedding of old epidermis during prepupal-pupal transition of B. mori. However, the underlying mechanism still remains unclear. Here, we investigated the transcriptional responses of the epidermis of B. mori on day 2 at prepupal stage to sublethal CAP exposure using digital gene expression (DGE) profiling sequencing. We identified 5823 differentially expressed genes (DEGs), with 4830 genes up-regulated and 993 genes down-regulated. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that CAP exposure induced disruption of energy homeostasis, oxidative stress, autophagy and apoptosis in the epidermis of B. mori. Meanwhile, trehalose content was increased while most of the genes involved in trehalose metabolism were down-regulated. In addition, chitin contents in CAP-exposed silkworms were decreased. Taken together, these results reveal that sublethal concentration of CAP probably targets trehalose metabolism to impair chitin synthesis, leading to perturbation of pupation metamorphosis in B. mori.

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.

Cloning and functional analysis of autophagy-related gene 7 in Bombyx mori, silkworm.

Silkworm (Bombyx mori) is an important economic insect and an attractive model system. A series of autophagy-related genes (Atgs) are involved in the autophagic process, and these Atgs have been proved to play important roles in the development. Atg7 stands at the hub of two ubiquitin-like systems involving Atg8 and Atg12 in the autophagic vesicle. In the present study, we cloned and characterized a BmAtg7 gene in Bombyx mori. The open reading frame (ORF) of BmAtg7 was 1908 bp in length, and it encoded a polypeptide of 635 amino acids. BmAtg7 was highly expressed in the posterior silk gland, fatbody, and epidermis. The expression profile of BmAtg7 in the fatbody showed an increasing tendency from day 1 of the 5th instar to the prepupal stage. After chlorantraniliprole (CAP) exposure, the transcriptional level of BmAtg7 was continuously decreased. After depletion of BmAtg7 by RNAi, the expressions of BmAtg7, BmAtg8, and BmEcr were all downregulated, while the expression of BmJHBP2 was upregulated. However, depletion of BmAtg7 did not prevent the metamorphosis of silkworm from larvae to pupae, while the occurrence of such process was delayed. After the 20-hydroxyecdysone (20E) treatment, the expression characteristics of these four genes (BmAtg7, BmAtg8, BmEcr and BmJHBP2) were contrary to the results after depletion of BmAtg7. Our results suggested that although CAP exposure could significantly inhibit the expression of BmAtg7 continuously, the changes of BmAtg7 was not the key factor in CAP-induced metamorphosis defects.

An assessment of the reproductive toxicity of GONPs exposure to Bombyx mori.

This study aimed to explore the toxicity of environmental residues of graphene oxide nanoparticles (GONPs) to reproduction of Lepidopteron insects using both ovary cell line (BmN) and individual female Bombyx mori as the research subjects. The results showed that GONPs dose dependently affect BmN cells. At higher concentrations (>25 mg/L), GONPs led to oxidative stress, ROS accumulation and DNA damage in BmN cells and significantly reduced their survival rate (p ≤ 0.05). Moreover, feeding female B. mori larvae with mulberry leaves treated with 25 mg/L GONPs significantly decreased their gonadosomatic index (GSI) by 40.84%, and increased oxidation levels and antioxidant enzyme activity in silkworm ovary tissues. Pathological analysis found that exposure to GONPs decreased the numbers of both oogonia and oocytes in ovarian tissues, increased the formation of peroxisome and vacuoles in follicle cells, reduced the transcription of genes (Vg, Ovo, Sxl-s, Sxl-l, and Otu) related to ovarian development in B. mori by 0.61, 0.65, 0.75, 0.72, and 0.42-fold, respectively, and lowered the amount of spawning by 52.25%. Overall, these results revealed that GONPs exposure is toxic to the reproduction of B. mori. The underlying mechanism is that oxidative stress due to GONPs causes oxidative damage to DNA, damages ovarian tissues, as well as hinders B. mori development and spawning. Thus, this study provides important experimental data for safety evaluation of reproductive toxicity due to GONPs exposure.

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.