B.B. Wang

Changes in the activity and the expression of detoxification enzymes in silkworms (Bombyx mori) after phoxim feeding

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.

The adverse effects of phoxim exposure in the midgut of silkworm, Bombyx mori

The silkworm is an important economic insect. Poisoning of silkworms by organophosphate pesticides causes tremendous loss to the sericulture. In this study, Solexa sequencing technology was performed to profile the gene expression changes in the midgut of silkworms in response to 24h of phoxim exposure and the impact on detoxification, apoptosis and immune defense were addressed. The results showed that 254 genes displayed at least 2.0-fold changes in expression levels, with 148 genes up-regulated and 106 genes down-regulated. Cytochrome P450 played an important role in detoxification. Histopathology examination and transmission electron microscope revealed swollen mitochondria and disappearance of the cristae of mitochondria, which are the important features in insect apoptotic cells. Cytochrome C release from mitochondria into the cytoplasm was confirmed. In addition, the Toll and immune deficiency (IMD) signal pathways were all inhibited using qRT-PCR. Our results could help better understand the impact of phoxim exposure on silkworm.

Molecular mechanisms of reduced nerve toxicity by titanium dioxide nanoparticles in the phoxim-exposed brain of Bombyx mori.

Bombyx mori (B. mori), silkworm, is one of the most important economic insects in the world, while phoxim, an organophosphorus (OP) pesticide, impact its economic benefits seriously. Phoxim exposure can damage the brain, fatbody, midgut and haemolymph of B. mori. However the metabolism of proteins and carbohydrates in phoxim-exposed B. mori can be improved by Titanium dioxide nanoparticles (TiO2 NPs). In this study, we explored whether TiO2 NPs treatment can reduce the phoxim-induced brain damage of the 5th larval instar of B. mori. We observed that TiO2 NPs pretreatments significantly reduced the mortality of phoxim-exposed larva and relieved severe brain damage and oxidative stress under phoxim exposure in the brain. The treatments also relieved the phoxim-induced increases in the contents of acetylcholine (Ach), glutamate (Glu) and nitric oxide (NO) and the phoxim-induced decreases in the contents of norepinephrine (NE), Dopamine (DA), and 5-hydroxytryptamine (5-HT), and reduced the inhibition of acetylcholinesterase (AChE), Na+/K+-ATPase, Ca2+-ATPase, and Ca2+/Mg2+-ATPase activities and the activation of total nitric oxide synthase (TNOS) in the brain. Furthermore, digital gene expression profile (DGE) analysis and real time quantitative PCR (qRT-PCR) assay revealed that TiO2 NPs pretreatment inhibited the up-regulated expression of ace1, cytochrome c, caspase-9, caspase-3, Bm109 and down-regulated expression of BmIap caused by phoxim; these genes are involved in nerve conduction, oxidative stress and apoptosis. TiO2 NPs pretreatment also inhibited the down-regulated expression of H+ transporting ATP synthase and vacuolar ATP synthase under phoxim exposure, which are involved in ion transport and energy metabolism. These results indicate that TiO2 NPs pretreatment reduced the phoxim-induced nerve toxicity in the brain of B. mori.

Differentially expressed genes in the fat body of Bombyx mori in response to phoxim insecticide

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.

Transcriptional characteristics of gene expression in the midgut of domestic silkworms (Bombyx mori) exposed to phoxim

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.

Mechanisms of TiO2 NPs-induced phoxim metabolism in silkworm (Bombyx mori) fat body.

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 NPss 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 bodys 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.

Expression profile analysis of silkworm P450 family genes after phoxim induction

Silkworm (Bombyx mori) is an important economic insect and a model species for Lepidopteran. Each year, O,O-diethyl O-(alpha-cyanobenzylideneamino) phosphorothioate (phoxim) pesticide poisoning in China results in huge economic losses in sericulture. Silkworm fat body is the main organ for nutrient storage, energy supply, intermediary metabolism, and detoxification. Microarray analysis of silkworm Cytochrome P450 detoxification enzyme genes revealed that all tested P450 4 (CYP4) family genes are expressed in the fat body. Quantitative Real-time PCR (QRT-PCR) was used to detect the expression of CYP4 family genes in silkworm fat body 0, 24, 48, and 72u2009h after phoxim exposure. The expression levels of silkworm molting hormone synthesis-related genes started to change 24u2009h after phoxim exposure, with those of CYP302A1, CYP306A1, and CYP314A1 being elevated by 1.38-, 1.33-, and 2.10-fold, respectively. The CYP18A1 gene that participates in steroid hormone inactivation and the CYP15C1 gene that participates in the epoxidation during the synthesis of juvenile hormone (JH) from methyl farnesoate (MF) were increased by 3.85- and 7.82-fold, respectively. Phylogenetic analysis indicated that these endogenous hormone metabolism-related genes belong to CYP mito clan and clan 2, and that phoxim exposure may affect silkworm development and metamorphosis. The CYP4, CYP6, and CYP9 families all showed some degrees of increases in gene expression; among them, CYP49A1, CYP4L6, CYP6AB4, CYP9G3, CYP9A19, and CYP9A22s transcription levels were significantly upregulated to 12.77-, 2.64-, 2.42-, 4.06-, 3.32-, and 2.98-fold, respectively, of the control levels. In the fat body, CYP49A1, CYP6AB4, CYP9A19, and CYP9A22 were constantly expressed at high levels after 24, 48, and 72u2009h of phoxim treatments; according to phylogenetic analysis, these genes belong to detoxification-related clan 3 and clan 4 CYP families. These genes may participate in the metabolism of phoxim in silkworm fat body. The results obtained in this study provide a basis for future in-depth investigations of insect P450 family genes in metabolic detoxification.

Characteristics of phoxim-exposed gene transcription in the silk gland of silkworms

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.

Effects of phoxim on nutrient metabolism and insulin signaling pathway in silkworm midgut

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 PTENs 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.

Mechanism of Enhanced Bombyx mori Nucleopolyhedrovirus-Resistance by Titanium Dioxide Nanoparticles in Silkworm

The infection of Bombyx mori nucleopolyhedrovirus (BmNPV) in silkworms is often lethal. It is difficult to prevent, and its lethality is correlated with both viral particle characteristics and silkworm strains. Low doses of titanium dioxide nanoparticles (TiO2 NPs) can promote silkworm growth and improve its resistance to organophosphate pesticides. In this study, TiO2 NPs’ effect on BmNPV resistance was investigated by analyzing the characteristics of BmNPV proliferation and transcriptional differences in silkworm midgut and the transcriptional changes of immunity related genes after feeding with TiO2 NPs. We found that low doses of TiO2 NPs improved the resistance of silkworm against BmNPV by 14.88-fold, with the mortalities of the experimental group and control group being 0.56% and 8.33% at 144 h, respectively. The proliferation of BmNPV in the midgut was significantly increased 72 h after infection in both experimental and control groups; the control group reached the peak at 120 h, while the experimental group took 24 more hours to reach the maximal value that was 12.63 times lower than the control, indicating that TiO2 NPs can inhibit BmNPV proliferation in the midgut. Consistently, the expression of the BmNPV-resistant gene Bmlipase-1 had the same increase pattern as the proliferation changes. Immune signaling pathway analysis revealed that TiO2 NPs inhibited the proliferation of silkworm BmNPV to reduce the activation levels of janus kinase/signal transducer and activator of transcription (JAK/STAT) and phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, while promoting the expression of Bmakt to improve the immunity. Overall, our results demonstrate that TiO2 NPs increase silkworm resistance against BmNPV by inhibiting virus proliferation and improving immunity in silkworms.