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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.

Aminoacyl-tRNA synthetases are the key enzymes for protein synthesis. Glycine, alanine, serine and tyrosine are the major amino acids composing fibroin of silkworm. Among them, the genes of alanyl-tRNA synthetase (AlaRS) and glycyl-tRNA synthetase (GlyRS) have been cloned. In this study, the seryl-tRNA synthetase (SerRS) and tyrosyl-tRNA synthetase (TyrRS) genes from silkworm were cloned. Their full length are 1709 bp and 1868 bp and contain open reading frame (ORF) of 1485 bp and 1575 bp, respectively. RT-PCR examination showed that the transcription levels of SerRS, TyrRS, AlaRS and GlyRS are significantly higher in silk gland than in other tissues. In addition, their transcription levels are much higher in middle and posterior silk gland than in anterior silk gland. Moreover, treatment of silkworms with phoxim, an inhibitor of silk protein synthesis, but not TiO2 NP, an enhancer of silk protein synthesis, significantly reduced the transcription levels of aaRS and content of free amino acids in posterior silk gland, therefore affecting silk protein synthesis, which may be the mechanism of phoxim-silking disorders. Furthermore, low concentration of TiO2 NPs showed no effect on the transcription of aaRS and content of free amino acids, suggesting that TiO2 NPs promotes silk protein synthesis possibly by increasing the activity of fibroin synthase in silkworm.

Silk gland is a silkworm organ where silk proteins are synthesized and secreted. Dietary supplement of TiO

Insect fat body is an important intermediate metabolic organ that plays an important role in protein metabolism and detoxification. In order to study the effects of TiO2 NPs and phoxim on fat body protein synthesis through MAPK and PI3K/Akt signaling pathways in silkworms, we determined the effects of TiO2 NPs and phoxim, alone and in combination, on fat body protein content of silkworms, analyzed the gene expression profile of the fat body, and verified the expression of characteristic genes. We found that TiO2 NPs and phoxim alone increased the total protein content of the fat body, and up-regulated MAPK and PI3K/Akt signaling pathway genes. TiO2 NPs up-regulated the expression of two growth and development-related genes-insulin-like peptide and neuropeptide receptor B-by 5.17 and 3.89-fold, respectively. Phoxim up-regulated the expression of detoxification genes-P450, GST, and CarE2. Pretreatment with TiO2 NPs could reduce phoxim-increased total protein content and up-regulated MAPK and PI3K/Akt signaling pathway genes and detoxification genes; the activities of detoxification enzymes were consistent with the gene expression pattern. Our results showed that MAPK and PI3K/Akt signaling pathways both regulate fat body protein synthesis in silkworms, but the target proteins induced to express were different under different inducing factors. Our finding may provide a reference for investigating the mechanism of protein synthesis regulation through MAPK and PI3K/Akt signaling pathways.

The acetylcholinesterase of Lepidoptera insects is encoded by two genes, ace1 and ace2. The expression of the ace1 gene is significantly higher than that of the ace2 gene, and mutations in ace1 are one of the major reasons for pesticide resistance in insects. In order to investigate the effects of the mutations in ace1's characteristic sites on pesticide resistance, we generated mutations for three amino acids using site-directed mutagenesis, which were Ala(GCG)303Ser(TCG), Gly(GGA)329Ala(GCA) and Leu (TCT)554Ser(TTC). The Baculovirus expression system was used for the eukaryotic expression of the wild type ace1 (wace1) and the mutant ace1 (mace1). SDS-PAGE and Western blotting were used to detect the targeting proteins with expected sizeof about 76 kDa. The expression products were purified for the determination of AChE activity and the inhibitory effects of physostigmine and phoxim. We observed no significant differences in the overall activity of the wild type and mutant AChEs. However, with 10 min of physostigmine (10 μM) inhibition, the remaining activity of the wild type AChE was significantly lower than that of the mutant AChE. Ten min inhibition with 33.4 μM phoxim also resulted in significantly lower remaining activity of the wild type AChE than that of the mutant AChE. These results indicated that mutations for the three amino acids reduced the sensitivity of AChE to physostigmine and phoxim, which laid the foundation for future in vivo studies on AChE's roles in pesticide resistance.

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.A sublethal dose of phoxim combined with Bombyx mori nucleopolyhedrovirus (BmNPV) elevated the silkworm mortality at 96 h. We found a higher content of HOur 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 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

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) (B. mori) is an economically important insect and a model species for Lepidoptera. It has been reported that feeding of low concentrations of titanium dioxide nanoparticles (TiO2 NPs) can improve feed efficiency and increase cocoon mass, cocoon shell mass, and the ratio of cocoon shell. However, high concentrations of TiO2 NPs are toxic. In this study, we fed B. mori with different concentrations of TiO2 NPs (5, 10, 20, 40, 80, and 160 mg/L) and investigated B. mori growth, feed efficiency, and cocoon quality. We found that low concentrations of TiO2 NPs (5 and 10 mg/L) were more effective for weight gains, with significant weight gain being obtained at 72 h (P < 0.05). TiO2 NPs at 20 mg/L or higher had certain inhibitory effects, with significant inhibition to B. mori growth being observed at 48 h. The feed efficiency was significantly improved at low concentrations of 5 and 10 mg/L for 14.6 and 13.1 %, respectively (P < 0.05). All B. mori fed with TiO2 NPs showed increased cocoon mass and cocoon shell mass; at 5 and 10 mg/L TiO2 NPs, cocoon mass was significantly increased by 8.29 and 9.39 %, respectively (P < 0.05). We also found that low concentrations (5 and 10 mg/L) of TiO2 NPs promoted B. mori growth and development, improved feed efficiency, and increased cocoon production, while high concentrations (20 mg/L or higher) of TiO2 NPs showed inhibitory effect to the B. mori. Consecutive feeding of high concentrations of TiO2 NPs led to some degrees of adaptability. This study provides a reference for the research on TiO2 NPs toxicity and the basis for the development of TiO2 NPs as a feed additive for B. mori.

CeCl3 can reduce the damage caused by OP pesticides, in this study we used the brain of silkworms to investigate the mechanism of CeCl3 effects on pesticide resistance. The results showed that phoxim treatments led to brain damages, swelling and death of neurons, chromatin condensation and mitochondrial damage. Normal nerve conduction was severely affected by phoxim treatments, as revealed by: increases in the contents of neurotransmitters Glu, NO and ACh by 63.65%, 61.14% and 98.54%, respectively; decreases in the contents of 5-HT and DA by 53.19% and 43.71%, respectively; reductions in the activities of Na+/K+-ATPase, Ca2+/Mg2+-ATPase and AChE by 85.27%, 85.63% and 85.63%, respectively; and increase in the activity of TNOS by 22.33%. CeCl3 pretreatment can significantly reduce such damages. Results of DGE and qRT-PCR indicated that CeCl3 treatments significantly upregulated the expression levels of CYP4G23, cyt-b5, GSTs-σ1, ace1, esterase-FE4 and β-esterase 2. Overall, phoxim treatments cause nerve tissue lesions, neuron death and nerve conduction hindrance, but CeCl3 pretreatments can promote the expression of phoxim resistance-related genes in silkworm brains to reduce phoxim-induced damages. Our study provides a potential new method to improve the resistance of silkworms against OP pesticides.

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.

Silkworm (Bombyx mori) is an important economic insect and the model insect of Lepidoptera. Because of its high fecundity and short reproduction cycle, it has been widely used in reproduction and development research. The high concentrations of titanium dioxide nanoparticles (TiO2 NPs) show reproductive toxicity, while low concentrations of TiO2 NPs have been used as feed additive and demonstrated significant biological activities. However, whether the low concentrations of TiO2 NPs affect the reproduction of B. mori has not been reported. In this study, the growth and development of gonad of B. mori fed with a low concentration of TiO2 NPs (5 mg/L) were investigated by assessing egg production and expression of reproduction-related genes. The results showed that the low concentration of TiO2 NPs resulted in faster development of the ovaries and testes and more gamete differentiation and formation, with an average increase of 51 eggs per insect and 0.34 × 10(-4) g per egg after the feeding. The expressions of several reproduction-related genes were upregulated, such as the yolk-development-related genes Ovo-781 and vitellogenin (Vg) were increased by 5.33- and 6.77-folds, respectively. This study shows that TiO2 NPs feeding at low concentration can enhance the reproduction of B. mori, and these results are useful in developing new methods to improve fecundity in B. mori and providing new clues for its broad biological applications.

Silkworm (Bombyx mori), a model Lepidoptera insect, is economically important. Its growth and development are regulated by endogenous hormones. During the process of transition from larvae to pupae, 20-hydroxyecdysone (20E) plays an important role. The recent surge in consumer products and applications using metallic nanoparticles has increased the possibility of human or ecosystem exposure due to their unintentional release into the environment. We investigated the effects of exposure to titanium dioxide nanoparticles (TiO2 NPs) on the action of 20E in B. mori. Titanium dioxide nanoparticle treatment shortened the molting duration by 8 hr and prolonged the molting peak period by 10 %. Solexa sequencing profiled the changes in gene expression in the brain of fifth-instar B. mori in response to TiO2NPS exposure for 72 hr, to address the effects on hormone metabolism and regulation. Thirty one genes were differentially expressed. The transcriptional levels of pi3k and P70S6K, which are involved in the target of the rapamycin (TOR) signaling pathway, were up-regulated. Transcriptional levels of four cytochrome P450 genes, which are involved in 20E biosynthesis, at different developmental stages (48, 96, 144, and 192 hr) at 5th instars of all displayed trends of increasing expression. Simultaneously, the ecdysterone receptors, also displayed increasing trends. The 20E titers at four developmental stages during the 5th instar were 1.26, 1.23, 1.72, and 2.16 fold higher, respectively, than the control group. These results indicate that feeding B. mori with TiO2 NPs stimulates 20E biosynthesis, shortens the developmental progression, and reduces the duration of molting. Thus, application of TiO2 NPs is of high significance for saving the labor force in sericulture, and our research provides a reference for the ecological problems in the field of Lepidoptera exposured to titanium dioxide nanoparticles.

This study aimed to investigate the effects of titanium dioxide nanoparticles (TiO2 NPs) on feed efficiency of silkworm (Bombyx mori) (B. mori). The results showed that the amount of ingested food was increased by 3.31%, the percentage of ingested food was increased by 16.36%, the instar was prolonged by 6.77 h, the body mass of mature silkworm was increased by 11.41%, the cocoon mass was increased by 5.47%, the cocoon shell mass was increased by 9.80%, and the ratio of cocoon shell was increased by 3.54%. Average cocoon filament length, reelability, and neatness were increased by 3.15, 17.57, and 1.72%, respectively, whereas the cocoon filament size was decreased by 0.19%. The gene expression profiles were also examined with digital gene expression (DGE). The results showed that among 4,011 genes detected, 127 genes were differentially expressed including 57 genes whose expression was upregulated and 70 genes whose expression was downregulated. The expression levels of five genes related to metabolism were verified with quantitative real-time PCR, and the results were consistent with the DGE data. The activities of three major digestive enzymes in the midgut were also assayed; the results showed that the activities of trehalase, protease, and lipase in TiO2 NP-fed group were increased by 42.55, 78.13, and 33.33%, respectively. These results indicate that feeding B. mori with TiO2 NPs can stimulate the transcriptional level of genes related to digestion and absorption of nutrients in the midgut and increase the activities of trehalase, protease, and lipase and, thus, increase the feed efficiency of B. mori.

It was predicted that the genome of silkworm, Bombyx mori, has at least 79 P450 genes; however, P450 genes that are related to the catabolism of exogenous compounds were not reported. In this study we cloned two CYP4 (named CPY4M5 and CYP4M9) and four CYP6 (named CYP6AB5, CYP6AE9, CYP6AE22 and CYP6AU1) genes by using both bioinformatics and RT-PCR approaches. Sequence analysis showed that these genes contained conserved P450 gene sequence regions and one conserved intron. CYP4M5 and CYP4M9 genes were clustered together in a mode of "head-to-tail" possibly due to gene duplication. Blast analysis showed that these P450 genes shared significant similarity with CYP4 and CYP6 genes that are involved in the catabolism and detoxification of exogenous compounds in other insect species. RT-PCR results showed that these P450 genes were highly expressed in the midgut and fat body of B. mori. As the instar age increased, these P450 genes exhibit different expression patterns. When B. mori was exposed to 1.75 × 10(-5)% of cypermethrin, 3.5 × 10(-6)% of cypermethrin and 0.1% of rutin, expression of CYP6AB5 was increased by 2.3-fold, 2.2-fold and 1.9-fold, respectively. Exposure of B. mori to 0.1% quercetin does not change the expression of CYP6AB5. In contrast, expression of the other five P450 genes was inhibited after exposed to these compounds.

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.

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.

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.