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BACKGROUND Chlorantraniliprole (CAP) is widely used in agriculture and forestry to prevent and control pests. The effects of environmental CAP residue on non‐target insect metamorphosis have not been reported. Our research aimed to investigate the sublethal effect of CAP on larva–pupa transformation in silkworm, and explore the mechanism of sublethal CAP exposure‐mediated pupation metamorphosis defects. RESULT: Sublethal CAP exposure affected the growth and development of silkworm larvae and caused defects in pupation metamorphosis. After CAP exposure, formation the of prepupa procuticle, ecdysial membrane and new epidermis was inhibited. Also, the level of 20‐hydroxyecdysone (20E) and mRNA levels of the 20E signaling pathway‐related genes EcR, USP, E74, E75 and Ftz‐f1 were significantly reduced. Moreover, genes involved in chitin synthesis, such as ChsA, CDA1 and CDA2, were downregulated. Injection of 20E led to the upregulation of chitin synthesis‐related genes and increased formation of new epidermis in CAP‐treated silkworm. However, injection of 20E failed to prevent downregulation of Ftz‐f1 and the defects in pupation metamorphosis. CONCLUSION: Our results suggested that 20E is a target hormone of CAP exposure‐mediated epidermis formation phenotype. Ftz‐f1 was silenced by CAP and might be a direct target gene of sublethal CAP exposure. Our study provided new evidence of the effects of sublethal CAP exposure on insect development and metamorphosis. © 2020 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Silkworm (Bombyx mori) is an important economic insect. Bombyx mori , which is exposed to sublethal doses of pesticides, has a low or no mortality rate, while it is susceptible to infections triggered by foreign pathogens. The immune regulatory mechanism of silkworms caused by trace pesticides still remains unclear. The midgut is the major organ of silkworm for digestion and nutrient absorption, and it plays a critical defensive role against pathogens. In the present study, the silkworm was susceptible to Enterobacter cloacae sp. (E. cloacae) after exposure to sublethal dose of phoxim. The body weight and survival rate of the phoxim- E. cloacae co-treatment group were significantly decreased after 120 h of treatment compared with the phoxim treatment group. The immune responses and expressions of immune-related genes were dysregulated in the midgut of silkworm following exposure to phoxim. Digital gene expression (DGE) analysis revealed that 44 immune response-related and immune defense-related genes were differentially expressed. qRT-PCR results indicated that the transcriptional levels of antimicrobial peptide genes Bmdefensin1 , BmcecA , Bmglv1 , Bmglv2 , Bmmoricin and BmmoricinB3 were down-regulated by 0.77-, 0.37-, 0.05-, 0.19-, 0.34- and 0.54-fold, respectively. The transcriptional levels of Toll signaling pathway genes Bmcactus , Bmspatzle and Bmrel were down-regulated by 0.4-, 0.37- and 0.96-fold, respectively. Peritrophic membrane (PM) protein-related genes BmCBP-02 , BmPM-41 , BmPM-43 and BmCDA7 were down-regulated by 0.18-, 0.02-, 0.66- and 0.16-fold, respectively. The expressions of Toll signaling pathway genes were down-regulated at 48 h and 72 h. Immune deficiency (IMD) and Janus kinase and signal transducer and activator of transcription (JAK/STAT) signaling pathway genes were dysregulated after phoxim exposure. These results indicated that phoxim might cause damage to the PM and reduce the immune response of the silkworm, leading to susceptibility of silkworm to disease and damage from foreign pathogens. Unlabelled Image • Phoxim and Enterobacter cloacae co-treatment enhanced toxic effects on silkworms. • Phoxim dysregulated the expression of Toll, IMD and JAK/STAT signaling pathway genes. • Phoxim damaged immune defense of midgut, inducing silkworm sensitivity to pathogens. [ABSTRACT FROM AUTHOR]

Silkworm (Bombyx mori) is an economic insect of the Lepidoptera. Chlorantraniliprole (CAP) exposure results in reduced growth and development of B. mori and failure in cocooning, seriously affecting the development of sericulture. To study the mechanisms underlying the damage to silkworm caused by sublethal doses of CAP, we examined the oxidative damage, the activities of digestive enzymes in midgut, and the expressions of midgut-related genes at the mRNA level. We found that CAP exposure inhibited the growth of silkworm, decreased the body mass and caused the accumulation of reactive oxygen species (ROS) [the levels of O2−, H2O2 and lipid peroxidation (MDA) were increased by 1.62-, 1.87- and 1.46-fold, respectively]. Moreover, we also found that the midgut cells were disintegrated, microvilli disappeared, the stroma became thinner, and the chromatin of nucleus became aggregated after CAP exposure by the analysis of transmission electron microscopy (TEM). In addition, the activities of digestive enzymes were dysregulated in midgut (the activities of α-amylase and trypsin were decreased 0.69- and 0.20-fold, respectively). Furthermore, digital gene expression (DGE) profiling analysis revealed that the expressions of oxidative phosphorylation pathway and antioxidant defense system related genes in midgut were decreased, indicating that it was the oxidative damage in midgut caused by CAP that mainly affected the growth of silkworm, rather than the toxicological effects of CAP. Collectively, this study provided valuable insights into the toxic effects of CAP on insects. [ABSTRACT FROM AUTHOR]

Acetamiprid is widely used for agricultural pest control. However, it remains poorly understood whether the environmental residues of acetamiprid have the potential effects on economic insect. In this study, we evaluated the effects of acetamiprid on silkworm growth and development. The exposure to trace amounts of acetamiprid significantly decreased body weight, viability, and spinning ability. In addition, the activity of trypsin in the midgut was decreased after exposure. DGE and KEGG pathway enrichment analysis revealed that the significantly differentially expressed genes were mainly involved in nutrient metabolism, stress responses, and inflammation pathways. These results, in combination with hematoxylin‐eosin staining and transmission electron microscopy, indicated that acetamiprid could cause oxidative damage to midgut, lead to inflammatory responses, and affect the activities of midgut digestive enzymes, thus resulting in abnormal growth and development. Our findings greatly contributed to the evaluation of the effects of acetamiprid residues on other nontarget beneficial insect. [ABSTRACT FROM AUTHOR]

Chlorantraniliprole (CAP) can induce excessive calcium release from muscle of insects, causing muscle paralysis until death, and its residues in farmland can cause poisoning in Bombyx mori (B. mori), resulting in the failure of cocooning. No reports have investigated the effects of CAP exposure on detoxification enzyme activities and detoxification-related gene expression in B. mori. In the present study, we treated mulberry leaves with CAP by the leaf-dipping method, and then B. mori larvae were continuously fed with the polluted mulberry leaves. Moreover, the detoxification enzyme activities and the expressions of detoxification-related genes in the fat body of B. mori were examined. The results showed that at 24 h after CAP exposure, the activities of P450 and GST enzymes were all significantly increased, with P450 enzymes responding fastest. CarE enzyme activity was up-regulated in 24 h, and then it was decreased compared with the control group. Furthermore, the expressions of the key genes in the PI3K/Akt/CncC signaling pathway (PI3K , PDK , Akt , CncC and Keap1) at the mRNA were significantly increased. Western blotting analysis revealed that Akt was inhibited at the protein level, resulting in decreased expression of Keap1 and increased expression of CncC. These results indicated that the PI3K/Akt/CncC signaling pathway in the fat body of B. mori responded to CAP exposure and regulated the expressions of downstream detoxification enzymes, thus enhancing the detoxifying capability of B. mori. Image 1 • Chlorantraniliprole causes Bombyx mori poisoning, reduced the body weight. • Chlorantraniliprole affected detoxifying enzymatic activity and transcription of detoxification-related genes. • Chlorantraniliprole increased the transcriptional levels and protein levels of CncC. • PI3K/Akt/CncC pathway mediates the regulation of metabolic detoxification of Chlorantraniliprole. [ABSTRACT FROM AUTHOR]

The neonicotinoid insecticide acetamiprid is widely applied for pest control in agriculture production, and its exposure often results in adverse effects on a non-target insect, Bombyx mori. However, only few studies have investigated the effects of exposure to sublethal doses of neonicotinoid insecticides on gut microbiota and susceptibility to pathogenic bacteria. In this study, we aimed to explore the possible mechanisms underlying the acetamiprid-induced compositional changes in gut microbiota of silkworm and reduced host resistance against detrimental microbes. This study indicated that sublethal dose of acetamiprid activated the dual oxidase-reactive oxygen species (Duox-ROS) system and induced ROS accumulation, leading to dysregulation of intestinal immune signaling pathways. The evenness and structure of bacterial community were altered. Moreover, after 96 h of exposure to sublethal dose of acetamiprid, several bacteria, such as Pseudomonas sp (Biotype A, DOP-1a, XW34) and Staphylococcus sp (RCB1054, RCB314, X302), invaded the silkworm hemolymph. The survival rate and bodyweight of the acetamiprid treated silkworm larvae inoculated with Enterobacter cloacae (E. cloacae) were significantly lower than the acetamiprid treatment group, suggesting that acetamiprid reduced silkworm resistance against pathogens. These findings indicated that acetamiprid disturbed gut microbial homeostasis of Bombyx mori , resulting in changes in gut microbial community and susceptibility to detrimental microbes. [Display omitted] • Sublethal doses of acetamiprid activated the Duox-ROS system and induced ROS production. • Sublethal doses of acetamiprid dysregulated intestinal IMD immune signaling pathways. • Acetamiprid perturbs intestinal microbial homeostasis and changed microbial community. • Acetamiprid reduces silkworm resistance against harmful microbes. [ABSTRACT FROM AUTHOR]

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. ga1 • GONPs affects the reproduction of B. mori in a dose dependent manner with a safe dosage < 25 mg/L. • GONPs exposure causes oxidative stress in BmN cells and Bombyx mori , resulting in oxidative damage to DNA. • GONPs exposure damages ovarian tissue of B. mori. • GONPs exposure leads to reduced oviposition of B. mori. [ABSTRACT FROM AUTHOR]

Acetamiprid is a new neonicotinoid insecticide widely used in the prevention and control of pests in agriculture. However, its residues in the environment affect the cocooning of the silkworm, Bombyx mori (B. mori), a non-target insect. To investigate the mechanism of damage, B. mori larvae were fed with trace amounts of acetamiprid (0.15 mg/L). At 96 h after exposure, the larvae showed signs of poisoning and decreased body weight, resulting in reduced survival and ratio of cocoon shell. At 48 h and 96 h after exposure, the residues in the posterior silk gland (PSG), which is responsible for synthesizing silk fibroin, were 0.72 μg/mg and 1.21 μg/mg, respectively, as measured by high performance liquid chromatography, indicating that acetamiprid can accumulate in the PSG. Moreover, pathological sections and transmission electron microscopy also demonstrate the damage of the PSG by acetamiprid. Digital gene expression (DGE) and KEGG pathway enrichment analysis revealed that genes related to metabolism, stress responses and inflammation were significantly up-regulated after exposure. Quantitative RT-PCR analysis showed that the transcript levels of FMBP-1 and FTZ-F1 (transcription factors for synthesizing silk protein) were up-regulated by 2.55-and 1.56-fold, respectively, and the transcript levels of fibroin heavy chain (Fib-H) , fibroin light chain (Fib-L) , P25, Bmsage and Bmdimm were down-regulated by 0.75-, 0.76-, 0.65-, 0.44- and 0.40-fold, respectively. The results indicate that accumulated acetamiprid causes damage to the PSG and leads to reduced expression of genes responsible for synthesizing silk fibroin. Our data provide reference for evaluating the safety of acetamiprid residues in the environment for non-target insects. Unlabelled Image • Trace amounts of acetamiprid could cause damage to the PSG of Bombyx mori. • Acetamiprid exposure could affect the transcript levels of genes responsible for synthesizing silk fibroin. • Accumulated acetamiprid could cause oxidative stress and inflammation to the PSG. [ABSTRACT FROM AUTHOR]

Silkworm (Bombyx mori) is an economically important insect. However, the survival of silkworms has been significantly affected by the assault of chemical pesticides on mulberry trees through aerial application and water currents. Phoxim is a broad-spectrum organophosphorus insecticide widely used in China. Currently, very little is known about the non-neuronal effects of sublethal exposure to phoxim. The purpose of this study was to investigate the non-neuronal effects of sublethal phoxim exposure in the silkworm midgut, with a focus on nutrient metabolism. After phoxim treatment, lipase activity in the silkworm was shown to be up-regulated at 24 h before a decreasing trend was seen. Meanwhile, α-amylase activity showed the opposite trend. The expression levels of mitochondrial respiratory chain-related genes were all up-regulated at 24 h before falling continuously. To ensure that the effects of phoxim on nutrient metabolism were not simply a consequence of a decrease in mulberry consumption, the silkworms were treated with a reduced-food diet before the digestive enzyme activities and the transcription levels of mitochondrial respiratory chain-related genes were analyzed. Our results showed that the patterns in the reduced-diet and phoxim-exposed silkworm were markedly different, suggesting the alterations in the phoxim-exposed silkworm cannot readily be explained by nutrient deprivation Unlabelled Image • Phoxim exposure alters the mRNA levels of glucose and lipid metabolism-related genes. • Lipase activity showed the opposite trend to α-amylase activity after exposure. • The patterns in the reduced-diet and phoxim-exposed silkworm were markedly different. [ABSTRACT FROM AUTHOR]

Organophosphate pesticides are widely applied worldwide for agricultural purposes, and their exposures often result in adverse effects on Bombyx mori. The insect gut is a complicated ecosystem inhabited by a large number of microbes that play important roles in insect physiology and behavior. Recent studies have reported that alteration of their microbiota due to stressful conditions or environmental changes has been linked to a compromised health status and a susceptibility to diseases. In the present study, we aimed to assess the effects of phoxim exposure on intestinal microbes in silkworms. The results showed that phoxim exposure increased the bacterial community evenness and altered the structure of gut microbiota in silkworm larvae. The abundances of several genera, such as Methylobacterium and Aurantimonadaceae , in phoxim-treated larval guts were significantly reduced compared with the H 2 O-treated group, whereas the abundances of non-dominant bacteria, such as Staphylococcus, were significantly increased. Moreover, phoxim inhibited the expressions of antimicrobial peptides (AMPs) at the mRNA level and enhanced the pathogenesis of Enterobacter cloacae (E. cloacae) against silkworm larvae, suggesting that the immune system was inhibited after phoxim exposure. Therefore, the gut microbial community shifts were apparent after phoxim exposure. The compositional and structural changes of intestinal microbes caused by phoxim exposure might affect the normal function of the intestinal tract of silkworm. These results highlighted the importance of the gut bacterial community when investigating the mechanisms of midgut injury after pesticide exposure in Bombyx mori. Image 1 • Low-level phoxim inhibited the mRNA expressions of antimicrobial peptides. • Low-level phoxim enhanced the pathogenesis of E. cloacae against silkworm larvae. • The gut microbial community shifts were apparent after phoxim exposure. • The structural changes of gut microbes might affect normal function of silkworm gut. [ABSTRACT FROM AUTHOR]

Bombyx mori is an important economic insect. However, the environmental pollution caused by the widespread use of neonicotinoid insecticides has significantly affected the safe production of sericulture. In this paper, we determined the LC 50 of acetamiprid, a kind of neonicotinoid insecticides, to 5th instar silkworm larvae, examined its residues in hemolymph and midgut of silkworm after continuous exposure to low-dose of acetamiprid, and investigated the transcription level of detoxifying-related genes and the activity of detoxifying enzymes. The results showed that acetamiprid was highly toxic (24-h LC 50 , 1.50 mg/L) to silkworm larvae. After continuous exposure to low-dose of acetamiprid (0.15 mg/L), the acetamiprid residue concentrations in hemolymph and midgut were 0.90 and 0.58 μg/mg, respectively, at 48 h, but all decreased at 96 h. At 24 h of acetamiprid exposure, the transcription levels of CYP4M5 and CYP6AB4 and the P450 enzyme activity were significantly enhanced. However, the transcription levels of CarE and CarE-11 and the activity of CarE enzymes were both inhibited by acetamiprid exposure. After 24 h–72 h of acetamiprid exposure, the transcription levels of GSTe3 and GSTd1 were significantly up-regulated, and the GST enzyme activity was also significantly elevated from 48 h to 96 h. Furthermore, the expression levels of FoxO , CncC and Keap1 , the key upstream genes of detoxification enzymes, showed a similar trend as the GST genes. These results indicated that acetamiprid was reduced in midgut and the expression of GSTs was upregulated may via FoxO/CncC/Keap1 signaling pathway, which plays a key role in detoxification responses. Image 1 • The 24 h LC 50 of acetamiprid to Bombyx mori was 1.50 mg/L. • Acetamiprid triggered the transcription and activity of detoxification enzymes. • GSTs played a key role in scavenging acetamiprid toxicity in B. mori. • FoxO/CncC/Keap1 pathway mediated detoxification in midgut of B. mori. [ABSTRACT FROM AUTHOR]

Global warming is known to affect the growth, development and reproduction of insects. In this study, the larvae developmental process and endogenous hormone levels under high temperature (36 °C) stress were investigated in the lepidopteran model insect Bombyx mori (B. mori). After high temperature treatment, the duration of 5th instar larvae was shortened by 28 ± 2 h, the content of 20-hydroxyecdysone(20E) in hemolymph was significantly increased, and the transcription levels of the 20E response genes E93, Br–C, USP and E75 were up-regulated by 1.35-, 1.25-, 1.28-, and 1.27-fold, respectively. High temperature treatment also elevated the phosphorylation level of Akt and activated the downstream BmCncC/keap1 pathway, and the transcription levels of the 20E synthesis-related genes cyp302a1, cyp306a1, cyp314a1 and cyp315a1 were up-regulated by 1.12-, 1.51-, 2.17- and 1.23-fold, respectively. The transcription levels of cyp302a1 and cyp306a1 were significantly decreased in BmN cells after treatment with the double stranded RNA of BmCncC (dsBmCncC), whereas their transcription levels were significantly increased (2.15- and 1.31-fold, respectively) after treatment with the CncC agonist Curcumin. These results demonstrated that high temperature treatment promoted the metamorphosis and the BmCncC/keap1 pathway played a role in the metamorphosis of B. mori. Our results provided clues for understanding the CncC/keap1 pathway-mediated regulation of metamorphosis of Lepidopteran insects. • High temperature stress promotes the larvae development and changes endogenous hormone levels. • The activation of BmCncC/keap1 pathway is one of the reasons for the increase of 20E level. • BmCncC mediates ecdysone synthesis via Cyp302a1 and Cyp306a1. [ABSTRACT FROM AUTHOR]

Sublethal doses of chlorantraniliprole (CAP) disrupt spinning disorder in the silkworm Bombyx mori (B. mori) and cause reduced cocoon production. In the present study, we investigated the effects of trace amounts of CAP on morphology and gene expression of the B. mori silk gland, found the posterior silk gland cells were possessed of disintegrated Endoplasmic reticulum (ER), unevenly distributed chromatin after exposure to CAP (0.01 mg/L). Gene expression analysis revealed that IRE1 and ATF6 ER stress-signaling pathways were inhibited, the PERK/CncC pathway was activated. Digital gene expression (DGE) analysis showed that detoxification-related genes, antioxidant genes and genes involved in ER protein processing pathway were expressed differentially in CAP-treated silkworm larvae. Notably, the transcript levels of the detoxification-related genes (CYP4M5 , CYP6AB4 , GSTD3 and GSTS1) and the antioxidant genes (CAT , TPX and SOD) were significantly increased, and the expression of ER protein processing-related genes (Sec61β , Sec61γ , Sec23α and ERGIC-53) was significantly decreased after CAP exposure. The results showed that sublethal doses of CAP exposure caused ER stress, oxidative damage to the silk gland and the perturbation of protein processing in ER, thereby probably leading to abnormal growth of the silk gland and triggering the spinning failure in silkworm. Unlabelled Image • Silk gland cells were possessed of swelling ER exposed to sublethal doses of CAP. • Sublethal doses of CAP activated ER stress in silk gland. • The activation of PERK/CncC might induced detoxification and antioxidant responses. [ABSTRACT FROM AUTHOR]