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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 Ca

As a new type of biologically compatible material, TiO2 NPs are widely used in the industry as additives, drug carriers, and components of skin care products. Due to their wide use, residual TiO2 NPs in the environment are a safety concern that has attracted extensive attention. In this study, the ovarian cell line BmN of the model organism Bombyx mori was used to reveal the damaging effects of TiO2 NPs exposure. The results demonstrated that TiO2 NPs exhibited a dose-dependent effect on the relative cell viability, with significant toxic effects being observed above 20 mg/L. Oxidative damage analysis showed that ROS accumulated significantly in BmN cells after exposure to TiO2 NPs (P ≤ 0.05) and induced DNA damage. Further analysis revealed that the transcriptional levels of key superoxide dismutase genes (SOD) decreased significantly, while the transcriptions of key genes of the MAPK/NF-κB signaling pathway (P38, MEK, ERK and REL) and the downstream inflammatory factor genes (IL6 and TNFSF5) were all significantly up-regulated (P ≤ 0.05). Overall, our results indicate that exposure to TiO2 NPs leads to reduced transcription of antioxidant genes, accumulation of peroxides, and inflammation. These findings provide valuable data for the safety evaluation of environmental residues of TiO2 NPs.

Here, we develop a simple, efficient, bacmid-based, selection marker-free method for gene deletion and editing in baculovirus genomes. Specifically, based on pFastbac1, a donor plasmid with long left and right homology arms but without a reporter was constructed for disrupting ie1, an essential baculovirus gene. Instead of ligating with a plasmid, the homology arms were introduced to the polyhedrin locus of BmNPV bacmid using the BmNPV bac-to-bac expression system. Two viruses generated from the modified bacmid and unmodified BmNPV bacmid were then used to co-infect BmN cells in order that recombination takes place at the ie1 locus between them. Finally, without multiple rounds of purification, total cellular DNA was isolated, transformed into Cacl

Mitochondrial genomes (mitogenomes) have been widely used for studies on phylogenetic relationships and molecular evolutionary biology. Here, the complete mitogenome sequence of Spilosoma lubricipedum (Noctuoidea: Erebidae: Arctiinae) was determined (total length 15,375 bp) and phylogenetic analyses S. lubricipedum were inferred from available noctuid sequence data. The mitogenome of S. lubricipedum was found to be highly A + T-biased (81.39%) and exhibited negative AT- and GC-skews. All 13 protein-coding genes (PCGs) were initiated by ATN codons, except for cox1 with CGA. All tRNAs exhibited typical clover-leaf secondary structures, except for trnS1. The gene order of the S. lubricipedum mitogenome was trnM-trnI-trnQ-nad2. The A + T-rich region of S. lubricipedum contained several conservative features common to noctuid insects. Phylogenetic analysis within Noctuoidea was carried out based on mitochondrial data. Results showed that S. lubricipedum belonged to Erebidae and the Noctuoidea insects could be divided into five well-supported families (Notodontidae + (Erebidae + (Nolidae + (Euteliidae + Noctuidae)))).

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 LC

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.

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.

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.

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.

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.

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.

As a new type of biologically compatible material, TiO

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.

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.

The high-temperature stress gene GrpE plays an important role in coping with high-temperature stress. The mutation of key sites of this gene can improve the high-temperature resistance of organisms. In the present study, using complementary DNAs from the silkworm fat body as the template, the open reading frame sequence of the GrpE gene (BmGrpE) was amplified and was found to be 644 bp in length and encode a protein with a predicted molecular weight of 24.1 kDa. The presence of a binding site for the heat shock transcription factor (Hsf1) at -1440 bp upstream of its coding region indicates that BmGrpE may respond to high-temperature stress. BmGrpE was constitutively expressed throughout developmental stages, with the highest level observed in the 5th instar larvae stage. Moreover, in 5th instar larvae (the 3th day), BmGrpE was expressed in all tissues examined, with the highest levels in the fat body, silk gland, and midgut. Interestingly, under high-temperature stress, TiO2 nanoparticle treatment increased the messenger RNA levels of BmGrpE in the fat body and silk gland. After treatment with dsRNA of BmGrpE, the cell viability of BmN cells was significantly decreased under 34°C and H2 O2 stress (p < .05). Mutation of BmGrpE (H163L) enhanced the resistance of BmN cells under high-temperature stress. These results provide new clues for the study of molecular mechanisms of insect resistance to high temperatures.

To determine the systematic status of family Limacodidae within Lepidoptera, the complete mitochondrial genome (mitogenome) of Thosea sinensis (Lepidoptera: Zygaenoidea: Limacodidae) was sequenced. The genome is 15,544 base pairs (bp), including 13 protein-coding genes (PCGs), two ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs), and an AT-rich region. These characteristics are similar to of other lepidopterans. The gene order of T. sinensis is identical to that of Ditrysia lepidopterans. The nucleotide composition of the T. sinensis mitochondrial genome is highly biased toward A + T nucleotides (81.1%) and exhibits negative AT and GC skew. All the other 13 PCGs except cox1 are initiated by ATN codons. All tRNA genes are folded into the typical cloverleaf secondary structure, except for trnS1, which lacked the dihydrouridine (DHU) stem. There are 20 intergenic spacer regions ranging from 1 to 56 bp in length, and two gene overlap regions throughout the entire genome. The AT-rich region includes the ATAGA motif, followed by a 19-bp poly T stretch, a microsatellite-like (AT)10, and a poly-A element. Analysis of phylogenetic relationships indicated that T. sinensis belongs to the Limacodidae, and the monophyly of each lepidopteran family was well supported.

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.

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 LC50 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 LC50, 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.

Silkworm, Bombyx mori (L.; Lepidoptera: Bombycidae), is an economically important insect, which is sensitive to the environment and susceptible to oxidative damages at high temperature. Low concentrations of TiO2 NPs (titanium dioxide nanoparticles) can scavenge reactive oxygen species (ROS) produced by oxidative damages in vivo. To explore whether TiO2 NPs could alleviate oxidative damages of high temperature, the effects of TiO2 NPs treatment on silkworm growth, the levels of ROS and H2O2, as well as the transcription level of antioxidant-related genes were studied at 30°C. These results showed that TiO2 NPs treatment increased silkworm body weight by 6.0% and reduced the occurrence of irregular cocoon at 30°C. TiO2 NPs treatment at 30°C decreased ROS levels in fat body and increased expression of Hsp70, SOD by 5.70-fold at 48 h, TPx by 1.61-fold, CAT by 1.81-fold. These results indicated that TiO2 NPs treatment at 30°C could promote the expression of antioxidant genes and reduce oxidative stress and provide a new method to alleviate high-temperature induced oxidative stress to silkworm.

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.

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.

The widespread use of nanomaterials has raised concerns about the potential impact of nanoparticles on human health and the natural environment. Although high doses of TiO

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.

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.

Silk is widely used in the biomedical field (e.g., surgical sutures) for its excellent mechanical properties and biocompatibility. The properties of silk can be further enhanced by a multitude of methods, including nano particle feeding, which is convenient and green. Generally, the filament length of a silkworm cocoon ranges from 1300 to 1700 m. Despite the fact that the filament size, a key factor affecting the mechanical properties of silk, varies along the length, evaluation of strengthened silk by segment and the specific distribution along the length has not been reported. Therefore, in the present study, we fed silkworms with graphene oxide-sprayed mulberry leaves and evaluated the silk properties segment by segment. The silk's strength and elongation were significantly enhanced, with more α-helical/random coils and thicker mesophase regions. Specifically, the silk from 2‰ GO-treated group had higher strength in the first 60% of the length, whereas the silk from 1‰ GO-treated group was stronger in the last 40% of the length. Notably, the silk from 1‰ GO-treated group had the highest strength and Young's modulus in the last 20% of the length, indicating that this segment is more suitable for use as a surgical suture. Our findings demonstrate that different silk segments offer a great range of desirable assets, and the feasibility to select a specific segment with the desired properties for a specific application.

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.

The main mechanism of toxicity of organophosphate (OP) and carbamate (CB) insecticides is their irreversible binding and inhibition of acetylcholinestrase (AChE), encoded by ace1 (acetylcholinestrase gene 1), leading to eventual death of insects. Mutations in AChE may significantly reduce insects susceptibility to these pesticides. Bombyx mori is an important beneficial insect, and no OP- or CB-resistant strains have been generated. In this study, wild-type ace1 (wace1) and mutant ace1 (mace1) were introduced into BmN cells, confirmed by screening and identification. The expression of wace1 and mace1 in the cells was confirmed by Western blot and their expression levels were about 21-fold higher than the endogenous ace1 level. The activities of AChE in wace1 and mace1 transgenic cells were 10.6 and 20.2% higher compared to control cells, respectively. mace1 transgenic cells had higher remaining activity than wace1 transgenic cells under the treatment of physostigmine (a reversible cholinesterase inhibitor) and phoxim (an OP acaricide). The results showed that ace1 transgene can significantly improve ace1 expression, and ace1 mutation at a specific site can reduce the sensitivity to AChE inhibitors. Our study provides a new direction for the exploration of the relationship between AChE mutations and drug resistance.

Background Silkworm (Bombyx mori) is an economically important insect. It is relatively less resistant to certain chemicals and environment exposures such as pesticides and pathogens. After pesticide exposures, the silkworms are more susceptible to microbial infections. The mechanism underlying the susceptibility might be related to immune response and oxidative stress. Results A sublethal dose of phoxim combined with Bombyx mori nucleopolyhedrovirus (BmNPV) elevated the silkworm mortality at 96 h. We found a higher content of H2 O2 and increased levels of genes related to oxidative stress and immune response after treatment with a sublethal dose of phoxim for 24 h or 48 h. However, such response decreased with longer pesticide treatment. Mortality increased by 44% when B. mori was exposed to combined treatment with BmNPV and phoxim rather than BmNPV alone. The level of examined immune-related and oxidative-stress-related genes significantly decreased in the combined treatment group compared with the BmNPV group. Our results indicated that, with long-term exposure to pesticides such as OPs, even at sublethal dose, the oxidative stress response and immune responses in silkworm were inhibited, which may lead to further immune impairment and accumulation of oxidative stress, resulting in susceptibility to the virus and harm to the silkworm. Conclusion Our study provided insights for understanding the susceptibility to pathogen after pesticide exposures, which may promote the development of better pesticide controls to avoid significant economic losses. © 2016 Society of Chemical Industry.

Silkworm (Bombyx mori) is an important economic insect with a fat body that plays a crucial role in the storage and transfer of nutrients. It is also known that TiO2 nanoparticles (NPs) can improve feed efficiency and promote silk protein synthesis in the silkworm. In this study, we profiled gene expression in the silkworm fat body after TiO2 NP treatment, validated the major RNA-seq findings, and determined the contents of trehalose and triglyceride, the activity of lipase, and the amount of total proteins. RNA-seq analysis revealed that TiO2 NP treatment caused significant expression changes in 341 genes (P≤0.01), 138 of which were upregulated while the other 203 were downregulated. The expression levels of two target genes in the insulin signaling pathway and two protein metabolism-related target genes, three lipid metabolism-associated target genes, two carbohydrate metabolism related target genes and expression levels of seven heat shock protein genes were increased, and that of threonine dehydratase gene and fatty acid transport protein gene were decreased. The RNA-seq results of 16 genes were validated by quantitative real-time PCR. The lipase activity, content of trehalose, and amount of total proteins were elevated by 3.86-fold, 1.34-fold, and 1.21-fold, respectively, and the content of triglyceride was decreased by 0.94-fold after TiO2 NP treatment. These results indicated that TiO2 NPs activated the insulin signaling pathway, promoted the metabolism of protein, fat, and carbohydrate, and improved nutrition metabolism. Our study provides new support for the understanding of the beneficial effect of TiO2 NPs on silkworm nutrient metabolism., Summary: Our study indicates that TiO2 nanoparticles promote nutrient metabolism in the fat body of silkworms, and provide a reference for studies investigating the biological function of TiO2 nanoparticles.

AChE is the target of organophosphate (OP) and carbamate (CB) pesticides, and mutations in the gene can significantly reduce insects' sensitivity to these pesticides. Bombyx mori is highly sensitive to pesticides. To investigate the effects of mutations on AChE1 structure and function, we used a prokaryotic system to express B.mori wild type AChE1 (wAChE1) and mutant AChE1 (mAChE1) in this study. Active AChE1 proteins were obtained after refolding and purification, and wAChE1 and mAChE1 had similar activities. After incubation with 10(-6)M physostigmine and 10(-3)mg/mL phoxim, the remaining enzyme activity of mAChE1 was 4.42% and 8.86% higher than that of wAChE1's, respectively. Three-dimensional analysis of mutation AChE1 (mAChE1) revealed that the Ser and Ala side chains extended toward the central part of S285 with distances of just 2.80Å and 3.68Å, respectively, which changed the spatial structure of the active center and reduced its sensitivity to pesticides. These results indicated that the mutations altered the 3D structure of AChE1, which may affect the binding of physostigmine and phoxim to the serine residue at the active center, leading to reduced sensitivity. Our study helps understand the relationship between AChE1 mutations and pesticide resistance and provides a new direction for the cultivation of new pesticide-resistant varieties of B.mori.

Silkworm (Bombyx mori) is an important economic insect. Each year, poisoning caused by phoxim pesticide leads to huge economic losses in sericulture in China. Silkworm midgut is the major organ for food digestion and nutrient absorption. In this study, we found that the activity and expression of nutrition metabolism-related enzymes were dysregulated in midgut by phoxim exposure. DGE analysis revealed that 40 nutrition metabolism-related genes were differentially expressed. qRT-PCR results indicated that the expression levels of insulin/insulin growth factor signaling (IIS) pathway genes Akt, PI3K, PI3K60, PI3K110, IRS and PDK were reduced, whereas PTEN's expression was significantly increased in the midgut at 24 h after phoxim treatment. However, the transcription levels of Akt, PI3K60, PI3K110, IRS, InR and PDK were elevated and reached the peaks at 48 h, which were 1.48-, 1.35-, 1.21-, 2.24-, 2.89-, and 1.44-fold of those of the control, respectively. At 72 h, the transcription of these genes was reduced. Akt phosphorylation level was increasing along with the growth of silkworms in the control group. However, phoxim treatment led to increased Akt phosphorylation that surged at 24 h but gradually decreased at 48 h and 72 h. The results indicated that phoxim dysregulated the expression of IIS pathway genes and induced abnormal nutrient metabolism in silkworm midgut, which may be the reason of the slow growth of silkworms.

Organophosphate pesticides are applied widely in the world for agricultural purposes, and their exposures often resulted in non-cocooning of Bombyx mori in China. Silkworm midgut is the major organ for digestion and nutrient absorption, importantly it is also a barrier against foreign substances and chemical pesticides. The purpose of this study was to determine the mechanism of oxidative injury in silkworm midgut with phoxim induction. The results showed that the transcription level of oxidative phosphorylation signaling pathway genes of midgut under phoxim stress. Digital gene expression (DGE) analysis revealed that 24 electron transport chain (ETC)-related genes were upregulated. Quantitative real time polymerase chain reaction results indicated that the ETC the genes encoding NADH-CoQ1, Succinic-Q, cyt c reductase-S, cyt c oxidase-S, cytochrome c oxidase polypeptide IV, ATP synthase, and vacuolar H+ ATP synthase were all significantly up-regulated by 1.50-, 1.31-, 1.42-, 1.44-, 1.70-, 2.03- and 1.43-fold, respectively. Phoxim induction enhanced the activity of ETC complex in mitochondria, and induced the accumulation of ROS in midgut. These results indicated that trace phoxim enhanced respiration in midgut, and the imbalance between the activity changes of ETC may led to reactive oxygen species accumulation. The ETC of mitochondria may be potential biomarkers of midgut toxicity in B. mori caused by phoxim exposure. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 167-175, 2017.

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

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 H2O-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.

The widespread use of nanomaterials has raised concerns about the potential impact of nanoparticles on human health and the natural environment. Although high doses of TiO2 nanoparticles (NPs) are toxic to animals, low doses of TiO2 NPs have been shown to benefit their growth and immune functions. Intestinal microorganisms are critical in maintaining the normal life activities and ensuring the health of their host. The intestinal microorganisms of lepidopteran insects can promote growth and development, foster insecticide resistance, and improve resilience against diseases. However, to date, there is no report on the effects of TiO2 NPs on the intestinal microbiota of lepidopteran insects. In this work, we examined the effects of a low dose of TiO2 NPs (5 mg/L) on the intestinal microbiota of silkworm (Bombyx mori). The results showed that the exposure to TiO2 NPs did not alter the dominant species of intestinal microbiota significantly, but changed the abundance of individual species of intestinal microorganisms. Specifically, exposure to TiO2 NPs increased the uniformity of intestinal microorganisms. The abundance of Lachnospiraceae_NK4A136_group, involved in the metabolism of nutrients, as well as the abundance of Pseudomonas and Sphingomonas, both involved in detoxification and disease resistance, was increased. Meanwhile, among the non-dominant species, the conditional pathogenic bacteria Serratia exhibited decreased abundance. In addition, exposure to TiO2 NPs also increased the abundance of norank_f_Bacteroidales_S24-7_group, which could help relieve inflammation and regulate immune functions. The current study is the first to report the effects of TiO2 NPs on the intestinal microbiota of lepidopteran insects. The results demonstrated that TiO2 NPs could alter the composition of the intestinal microbiota of B. mori, and thus promote its growth and development, regulate its immune functions, and enhance its resistance to insecticide.

Various nanoparticles, such as silver nanoparticles (AgNPs) and titanium nanoparticles (TiO2 NPs) are increasingly used in industrial processes. Because they are released into the environment, research into their influence on the biosphere is necessary. Among its other effects, dietary TiO2 NPs promotes silk protein synthesis in silkworms, which prompted our hypothesis that TiO2 NPs influence protein kinase B (Akt)/Target of rapamycin (Tor) signaling pathway (Akt/Tor) signaling in their silk glands. The Akt/Tor signaling pathway is a principle connector integrating cellular reactions to growth factors, metabolites, nutrients, protein synthesis, and stress. We tested our hypothesis by determining the influence of dietary TiO2 NPs (for 72 h) and, separately, of two Akt/Tor pathway inhibitors (LY294002 and rapamycin) on expression of Akt/Tor signaling pathway genes and proteins in the silk glands. TiO2 NPs treatments led to increased accumulation of mRNAs for Akt, Tor1 and Tor2 by 1.6-, 12.1-, and 4.8-fold. Dietary inhibitors led to 2.6- to 4-fold increases in mRNAs encoding Akt and substantial decreases in mRNAs encoding Tor1 and Tor2. Western blot analysis showed that dietary TiO2 NPs increased the phosphorylation of Akt and its downstream proteins. LY294002 treatments led to inhibition of Akt phosphorylation and its downstream proteins and rapamycin treatments similarly inhibited the phosphorylation of Tor-linked downstream proteins. These findings support our hypothesis that TiO2 NPs influence Akt/Tor signaling in silk glands. The significance of this work is identification of specific sites of TiO2 NPs actions.

Silkworm (Bombyx mori) is an economically important insect, and its silk production capacity largely depends on its ability to synthesize fibroin. While breeding of B. mori varieties has been a key strategy to improve silk production, little improvement of B. mori silk production has been achieved to date. As a result, the development of sericulture economy has not progressed well, pointing to the need of new ways for improvement of B. mori silk production. Titanium dioxide nanoparticles (TiO2 NPs), a food additive widely used for livestock, have been shown to promote animal growth and increase the protein synthesis in animals. However, no studies on effect of TiO2 NPs on fibroin synthesis in B. mori have been available. In this study, the differential expression profiles of genes and proteins in the silk gland of B. mori fed without or with TiO2 NPs (5 μg ml(-1)) were analyzed and compared using digital gene expression (DGE), reverse transcription quantitative polymerase chain reaction (RT-qPCR), semi-qPCR, and Western blot analysis. The effects of TiO2 NPs feeding on the activity of proteases in the midgut and the synthesis and transportation of amino acids in hemolymph were also investigated. DGE analyses showed that among a total of 4,741 genes detected, 306 genes were differentially expressed after the TiO2 NPs feeding, of which 137 genes were upregulated whereas 169 genes were downregulated. 106 genes were shown to be involved in fibroin synthesis, of which 97 genes, including those encoding cuticular protein glycine-rich 10, serine protease inhibitor 28, aspartate aminotransferase, lysyl-tRNA synthetase, and splicing factor arginine/serine-rich 6, and silk gland factor-1 (SGF-1), were upregulated with the maximum induction of 8.52-folds, whereas nine genes, including those encoding aspartylglucosaminidase, the cathepsin L in Tribolium castaneum, and similar to SPRY domain-containing SOCS box protein 3, were downregulated with the maximum reduction of 8.11-folds. Transcription levels of nine genes were further verified by RT-qPCR, and the results were consistent with those with DGE. Transcription and expression levels of fibroin light chain (Fib-L) gene were increased after TiO2 NPs feeding, indicating that TiO2 NPs improves fibroin synthesis. Compared with that of control, the mean protease activity was increased by 56.67% in the B. mori fed with TiO2 NPs, and the transport of four key amino acids used for fibroin synthesis in hemolymph was also increased. These findings indicated that TiO2 NPs feeding can improve the absorption and utilization of amino acids from the feed and could be a new way to increase the fibroin synthesis in B. mori.