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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.

This study investigated the transcriptional characteristics of acetylcholinesterase gene (AChE) in domesticated silkworm ( Bombyx mori ) after pesticide phomix induction. We first determined the LC 50 (Lethal Concentration 50) of phomix in 3-day old, 5th instar B. mori , then fed the B. mori with low dose of phoxim and examined the transcription level of two acetylcholinesterase genes ( Bm-AChE-1 and Bm-AChE-2 ) in brain, fat body, midgut and silk gland at 24, 48, 72 and 96 h later using real-time quantitative PCR. The results showed that LC 50 of phoxim on B. mori was 7.86 μg/mL (24 h). After feeding with 4.00 μg/mL phoxim, transcription levels of Bm-AChE-1 and Bm-AChE-2 in brain, fat body and silk gland showed a trend of first increasing and then decreasing. At 24 h, transcription of Bm-AChE-1 and Bm-AChE-2 increased in the brain by 16.22 and 68.71-fold, respectively, while that of Bm-AChE-1 and Bm-AChE-2 in the fat body and silk glands only increased slightly. Moreover, the increase of Bm-AChE-1 was greater than that of Bm-AChE-2 . By contrast, in the midgut, transcription of Bm-AChE-1 and Bm-AChE-2 showed a trend of first decreasing and then increasing to around their initial levels at 96 h. In addition, the results showed that transcription of Bm-AChE-1 was different from that of Bm-AChE-2 in different tissues of B. mori during the metabolism process of phoxim, suggesting that during metabolic process of pesticides, Bm-AChE-1 plays an important role in the fat body, while Bm-AChE-2 plays an important role in the brain. This study provides a scientific rationale for studying the functional differentiation of the two AChE genes.

In order to investigate the gene expression of domesticated silkworm (Bombyx mori) exposed to phoxim, we used a whole-genome oligonucleotide microarray to survey the B. mori larvae induced by phoxim [O,O-diethyl O-(alpha-cyanobenzylideneamino) phosphorothioate] after 24 h, and examined the gene expression profile. The results showed that, 6177 genes expressed in the tested B. mori larvae, and 247 genes were differentially transcribed in the phoxim-induced group compared to the control group. Among these differentially expressed genes, 68 genes were up-regulated and 179 genes were down-regulated after phoxim-induced and the highest changes were 26-fold up-regulated and 29-fold down-regulated, respectively. Analysis result of the differentially expressed genes indicates that their functions could be divided into 10 categories. The up-regulated genes are mainly involved in glycometabolism, fat metabolism, protein hydrolysis electron transfer process and the down-regulated genes are mainly involved in cell division, transcriptional regulation, intracellular signaling cascade. In this study, we firstly reported the whole-genome transcribed expression profile of the B. mori larvae induced by phomix and provided the gene source for studying the metabolic detoxicification mechanisms to organophosphorus by Bombyx mandarina and other Lepidoptera insects.

To investigate the genotyping criteria for the insect acetylcholinesterase gene (ace), we cloned two types of ace genes in domestic (Bombyx mori) and wild silkworm (Bombyx mandarina) through RT-PCR. The cloned genes were named Bm-ace1, Bm-ace2, Bmm-ace1 and Bmm-ace2, respectively. The ORFs of Bm-ace1 and Bmm-ace1 contained 2025 base pairs, encoding 683 amino acid residues (AA’s). The predicted protein has a molecular weight (MW) of 76.955 kDa and an isoelectric point (pI) of 6.36. The Bm-ace2 and Bm-ace2 genes contained 1917 bp nucleotides, encoding 638 AA’s. The predicted protein has a MW of 71.675 kDa and a pI of 5.49. Both ace1 and ace2 contain signature domains of acetylcholinesterases. Homology analysis of 18 NCBI downloaded insect AChEs peptide sequences and the 4 AChEs deducted in this study revealed that type 1 and type 2 insect AChEs had significant differences. Type 2 sequence is more conserved than type 1. Near the active centers of both types of AChEs, 48 strictly conserved AA’s (336–384) are present, and homology of these two peptide fragments was only 54.16%. Meanwhile, at AA positions 280–297, type 1 and type 2 AChEs both have conserved sequences with the similarity of the two being 52.94%. In type 2 AChEs, a uniquely conserved peptide sequence is found at positions 226–239 (QHLRVRHHQDKPL). We propose to use the above mentioned three conserved regions as criteria for insect acetylcholinesterases gene genotyping. This will benefit the genotyping of other acetylcholinesterase genes and the study of their functions.

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.