Titanium nanoparticles influence the Akt/Tor signal pathway in the silkworm, Bombyx mori, silk gland.

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.

TiO2 NPs Alleviates High-Temperature Induced Oxidative Stress in Silkworms.

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.

Apoptosis of posterior silk gland of Bombyx mori during spinning period and the role of PI3K/Akt pathway.

Bombyx mori is an economic insect of the Lepidoptera. Its posterior silk gland (PSG) is an important organ for fibroin synthesis. In order to study the occurrence of apoptosis in PSG and the role of PI3K/Akt signaling pathway during spinning period, changes in morphology of silk gland, expressions of fibroin components Fib-H, Fib-L and P25 and Akt, TOR2, P70S6K and S6 in PI3K/Akt pathway, expressions of apoptosis related genes caspase-3, caspase-9 and activity of caspase-3 were explored. The results showed that the morphology of silk gland dramatically degenerated; transcription of Fib-H, Fib-L, and P25 gradually declined with time; and Fib-L protein level reduced by 0.6-fold at 72 h. Moreover, the transcription levels of Akt, TOR2, P70S6K, and S6 also decreased by 0.3-, 0.8-, 0.7-, and 0.1-fold, respectively, indicating that the downregulation of PI3K/Akt signaling pathway could lead to reduction in fibroin synthesis. In addition, the transcription levels of caspase-3 and caspase-9 increased by 1.3- and 3.6-fold, respectively, and the enzyme activity of caspase-3 grew at a maximum of 1.6-fold. The results showed the occurrence of apoptosis in PSG during spinning period. In conclusion, the present study indicated that both the decline in fibroin components and the increase in apoptosis-related genes were regulated by PI3K/Akt signaling pathway during spinning period, which shed new light on the functions of PI3K/Akt signaling pathway.

Differentially expressed genes in the silk gland of silkworm (Bombyx mori) treated with TiO2 NPs.

Silk gland is a silkworm organ where silk proteins are synthesized and secreted. Dietary supplement of TiO2 nanoparticles (NPs) promotes silk protein synthesis in silkworms. In this study, digital gene expression (DGE) tag was used to analyze the gene expression profile of the posterior silk gland of silkworms that were fed with TiO2 NPs. In total, 5,702,823 and 6,150,719 clean tags, 55,096 and 74,715 distinct tags were detected in TiO2 NPs treated and control groups, respectively. Compared with the control, TiO2 NPs treated silkworms showed 306 differentially expressed genes, including 137 upregulated genes and 169 downregulated genes. Of these differentially expressed genes, 106 genes were related to silk protein synthesis, among which 97 genes were upregulated and 9 genes were downregulated. Pathway mapping using the Kyoto Encyclopedia of Genes and Genomes (KEGG) showed that 20 pathways were significantly enriched in TiO2 NPs treated silkworms, and the metabolic pathway-related genes were the most significantly enriched. The DGE results were verified by qRT-PCR analysis of eight differentially expressed genes. The DGE and qRT-PCR results were consistent for all three upregulated genes and three of the five downregulated genes, but the expression trends of the remaining two genes were different between qRT-PCR and DGE analysis. This study enhances our understanding of the mechanism of TiO2 NPs promoted silk protein synthesis.

Clone and functional analysis of Seryl-tRNA synthetase and Tyrosyl-tRNA synthetase from silkworm, Bombyx 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.

Sublethal dose of phoxim and Bombyx mori nucleopolyhedrovirus interact to elevate silkworm mortality.

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.

EXPRESSION AND EFFECTS OF MUTANT Bombyx mori ACETYLCHOLINESTRASE1 IN BmN CELLS.

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.

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 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.

Effects of TiO2 NPs on Silkworm Growth and Feed Efficiency.

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.

Molecular Signatures of Reduced Nerve Toxicity by CeCl3 in Phoxim-exposed Silkworm Brains.

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, Ca(2+)/Mg(2+)-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 ß-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.

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 72 h after phoxim exposure. The expression levels of silkworm molting hormone synthesis-related genes started to change 24 h 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 CYP9A22's 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 72 h 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.

Effects of Titanium Dioxide Nanoparticles on the Synthesis of Fibroin in Silkworm (Bombyx mori).

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.

Effect of TiO2 nanoparticles on the reproduction of silkworm.

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.