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Silkworm is an important economic insect. Abuse of organophosphorus pesticides in recent years often leads to poisoning of silkworms, which significantly affects sericulture development by reducing silk production. Previous studies have shown that TiO2 NPs can effectively mitigate the damages caused by organophosphorus pesticides in silk glands and nerve tissues. The fat body is an important metabolic detoxification organ of silkworms, but it is unknown whether TiO2 NPs affect pesticide metabolism in fat body. In this study, we characterized the transcription of antioxidant genes and enzyme activity in fat body after TiO2 NPs and phoxim treatments using transcriptome sequencing, real-time PCR, and enzyme activity assay. Transcriptome sequencing detected 10 720, 10 641, 10 403, and 10 489 genes for control group, TiO2 NPs group, phoxim group, and TiO2 NPs+phoxim group, respectively. The TiO2 NPs+phoxim group had 705 genes with significantly differential expression (FDR<0.001), among which the antioxidant genes thioredoxin reductase 1 and glutathione S-transferase omega 3 were significantly upregulated. In phoxim group, the expression levels of superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase delta (GSTd), and thioredoxin peroxidase (TPx) were increased by 1.365 -fold, 1.335 -fold, 1.642 -fold, and 1.765 -fold, respectively. The level changes of SOD, CAT, GSTd, and TPx were validated by real time PCR. The contents of reactive oxygen species (ROS), malondialdehyde (MDA), and hydrogen peroxide (H2O2) were increased by 1.598 -fold, 1.946 -fold, and 1.506 -fold, respectively, indicating that TiO2 NPs treatment can relieve phoxim-induced oxidative stress. To clarify the mechanism of TiO2 NPs's effect, the transcription levels of P450 gene family were measured for the TiO2 NPs+phoxim group; the expression levels of CYP4M5, CYP6AB4, CYP6A8, and CYP9G3 were elevated by 2.784 -fold, 3.047 -fold, 2.254 -fold, and 4.253 -fold, respectively, suggesting that high expression of P450 family genes can enhance the metabolism of phoxim in the fat body. The results of this study indicated that TiO2 NPs treatment promoted the transcriptional expression of the P450 family genes to improve the fat body's ability to metabolize phoxim and reduce phoxim-induced oxidative stress. This may be the main mechanism of TiO2 NPs' mitigation of phoxim-induced damages in the fat body., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The silkworm, Bombyx mori, is an economically important insect. However, poisoning of silkworms by organophosphate pesticides causes tremendous loss to the sericulture. The fat body is the major tissue involved in detoxification and produces antimicrobial peptides and regulates hormones. In this study, a microarray system comprising 22,987 oligonucluotide 70-mer probes was employed to examine differentially expressed genes in the fat body of B. mori exposed to phoxim insecticide. The results showed that a total of 774 genes were differentially expressed upon phoxim exposure, including 500 up-regulated genes and 274 down-regulated genes. The expression levels of eight detoxification-related genes were up-regulated upon phoxim exposure, including six cytochrome P450s and two glutathione-S-transferases. It was firstly found that eight antimicrobial peptide genes were down-regulated, which might provide important references for studying the larvae of B. mori become more susceptible to microbial infections after phoxim treatment. In addition, we firstly detected the expression level of metamorphosis-related genes after phoxim exposure, which may lead to impacted reproduction. Our results may facilitate the overall understanding of the molecular mechanism of multiple pathways following exposure to phoxim insecticide in the fat body of B. mori., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Silkworm (Bombyx mori), a model Lepidoptera insect, is an important economic insect. Its silk gland is the important organ for silk protein synthesis and secretion. Phoxim exposure causes deficient cocooning of silkworm and has become one of the major negative factors for the silk industry. To study the impact of phoxim exposure on silk gland, using gene chip technology, we examined differentially expressed genes in silk gland after silkworms were exposed to phoxim (4.0μg/mL) for 24h. Functional annotation, classification and KEGG signaling pathway analysis were performed. The results showed that out of 3206 genes detected in silk gland after phoxim exposure, 270 were differentially expressed significantly, including 249 up-regulated genes and 21 down-regulated genes. These differentially expressed genes related to apoptosis, detoxification and protein degradation were selected. Using qRT-PCR, the expression levels of 9 genes involved in apoptosis, detoxification and protein degradation were validated. In addition, the expression profiles of three related fibroin synthesis genes (Fib-H, Fib-L and P25) were analyzed. Our results showed that phoxim exposure induced apoptosis of silk gland cells and inhibition of fibroin synthesis. This may be the cause of deficient silkworm cocooning., (Copyright © 2013. Published by Elsevier Inc.)

Silkworm (Bombyx mori) is an economically important insect. However, non-cocoon caused by chemical insecticide poisoning has largely hindered the development of sericulture. To explore the roles of detoxification enzymes in B. mori after insecticide poisoning, we monitored the activity changes of cytochrome P450 monooxygenase, glutathione-S-transferase, and carboxylesterase in B. mori midgut and fatbody after phoxim feeding. At the same time, the expression levels of detoxification enzyme-related genes were also determined by real-time quantitative PCR. Compare to the control levels, the activity of P450 in the midgut and fatbody was increased to 1.72 and 6.72 folds; the activity of GST was no change in midgut, and in fatbody increased to 1.11 folds; the activity of carboxylesterase in the midgut was decreased to 0.69 folds, and in fatbody increased to 1.13 folds. Correspondingly, the expression levels of detoxifying enzyme genes CYP6ae22, CYP9a21, GSTo1 and Bmcce were increased to 15.99, 3.32, 1.86 and 2.30 folds in the midgut and to 3.58, 1.84, 2.14 and 4.21 folds in the fatbody after phoxim treatment. These results demonstrated the important roles of detoxification enzymes in phoxim metabolism. In addition, the detected activities of such enzymes were generally lower than those in cotton bollworms (Helicoverpa armigera), which may contribute to the high susceptibility of B. mori to insecticides. Our findings laid the foundation for further investigations of the molecular mechanisms of organophosphorus pesticide metabolism in B. mori., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Silkworm (Bombyx mori) is not only an economically important insect but also a model system for lepidoptera. As a vital organ of digestion and nutrient absorption, the midgut of insects also serves as the first physiological barrier to chemical pesticides. In this study, microarray was performed to profile the gene expression changes in the midgut of silkworms exposed to phoxim. After 24h of phoxim exposure (4.0μg/mL), 266 genes displayed at least 2.0-fold changes in expression levels. Among them, 192 genes were up-regulated, and 74 genes were down-regulated. The most significant changes were 14.88-fold up-regulation and 23.36-fold down-regulation. According to gene ontology annotation and pathway analysis, differentially expressed genes were mainly classified into different groups based on their potential involvements in detoxification, immunne response, stress response, energy metabolism and transport. Particularly, the transcription levels of detoxification-related genes were up-regulated, such as cytochrome P450s, esterases and glutathione-S-transferase (GST), indicating increased detoxification activity in the midgut. Our study provides new insights into the molecular mechanism of pesticide metabolism in the midgut of insects, which may promote the development of highly efficient insecticides., (Copyright © 2012 Elsevier Inc. All rights reserved.)