Regulatory networks of the F-box protein FBX206 and OVATE family proteins modulate brassinosteroid biosynthesis to regulate grain size and yield in rice.

F-box proteins participate in the regulation of many processes, including cell division, development, and plant hormone responses. Brassinosteroids (BRs) regulate plant growth and development by activating core transcriptional and other multiple factors. In rice, OVATE family proteins (OFPs) participate in BR signalling and regulate grain size. Here we identified an F-box E3 ubiquitin ligase, FBX206, that acts as a negative factor in BR signalling and regulates grain size and yield in rice. Suppressed expression of FBX206 by RNAi leads to promoted plant growth and increased grain yield. Molecular analyses showed that the expression levels of BR biosynthetic genes were up-regulated, whereas those of BR catabolic genes were down-regulated in FBX206-RNAi plants, resulting in the accumulation of 28-homoBL, one of the bioactive BRs. FBX206 interacted with OsOFP8, a positive regulator in BR signalling, and OsOFP19, a negative regulator in BR signalling. SCFFBX206 mediated the degradation of OsOFP8 but suppressed OsOFP19 degradation. OsOFP8 interacted with OsOFP19, and the reciprocal regulation between OsOFP8 and OsOFP19 required the presence of FBX206. FBX206 itself was ubiquitinated and degraded, but interactions of OsOFP8 and OsOFP19 synergistically suppressed the degradation of FBX206. Genetic interactions indicated an additive effect between FBX206 and OsOFP8 and epistatic effects of OsOFP19 on FBX206 and OsOFP8. Our study reveals the regulatory networks of FBX206, OsOFP8, and OsOFP19 in BR signalling that regulate grain size and yield in rice.

The promoting effects of pyriproxyfen on autophagy and apoptosis in silk glands of non-target insect silkworm, Bombyx mori.

Pyriproxyfen is a juvenile hormone analogue. The physiological effects of its low-concentration drift during the process of controlling agricultural and forestry pests on non-target organisms in the ecological environment are unpredictable, especially the effects on organs that play a key role in biological function are worthy of attention. The silk gland is an important organ for silk-secreting insects. Herein, we studied the effects of trace pyriproxyfen on autophagy and apoptosis of the silk gland in the lepidopteran model insect, Bombyx mori (silkworm). After treating fifth instar silkworm larvae with pyriproxyfen for 24 h, we found significant shrinkage, vacuolization, and fragmentation in the posterior silk gland (PSG). In addition, the results of autophagy-related genes of ATG8 and TUNEL assay also demonstrated that autophagy and apoptosis in the PSG of the silkworm was induced by pyriproxyfen. RNA-Seq results showed that pyriproxyfen treatment resulted in the activation of juvenile hormone signaling pathway genes and inhibition of 20-hydroxyecdysone (20E) signaling pathway genes. Among the 1808 significantly differentially expressed genes, 796 were upregulated and 1012 were downregulated. Among them, 30 genes were identified for autophagy-related signaling pathways, such as NOD-like receptor signaling pathway and mTOR signaling pathway, and 30 genes were identified for apoptosis-related signaling pathways, such as P53 signaling pathway and TNF signaling pathway. Further qRT-PCR and in vitro gland culture studies showed that the autophagy-related genes Atg5, Atg6, Atg12, Atg16 and the apoptosis-related genes Aif, Dronc, Dredd, and Caspase1 were responsive to the treatment of pyriproxyfen, with transcription levels up-regulated from 24 to 72 h. In addition, ATG5, ATG6, and Dronc genes had a more direct response to pyriproxyfen treatment. These results suggested that pyriproxyfen treatment could disrupt the hormone regulation in silkworms, promoting autophagy and apoptosis in the PSG. This study provides more evidence for the research on the damage of juvenile hormone analogues to non-target organisms or organs in the environment, and provides reference information for the scientific and rational use of juvenile hormone pesticides.

Combined analysis of transcriptomics and metabolomics on the cumulative effect of nano-titanium dioxide on mulberry seedlings.

Introduction: Titanium dioxide nanoparticles (TiO2 NPs) are among the most widely used inorganic nanomaterials in industry, medicine and food additives. There are increasing concerns regarding their potential risks to plants and the environment. Mulberry trees are widely grown in China due to their high survival rate and ability to aid ecological recovery. Methods: Herein, the effects of TiO2 NPs with different concentrations (100, 200, 400 and 800 mg/L) on the growth and physiology of the mulberry tree were systematically evaluated in aspects of physiology, transcriptomics and metabolomics. Results: Results showed that TiO2 NPs could be absorbed by the mulberry sapling root system and be transferred to the plant shoot. This results in the destruction of mulberry sapling root and leaf tissue. Furthermore, the number of chloroplasts and their pigment contents were reduced and the homeostasis of metal ions was disrupted. The toxic effects of TiO2 NPs attenuated the mulberry sapling's stress resistance, the contents of malondialdehyde in 100 mg/L, 200 mg/L 400 mg/L and 800 mg/L treatment groups increased by 87.70%, 91.36%, 96.57% and 192.19% respectively compared with the control group. The transcriptomic data showed that TiO2 NPs treatment mainly affected the expression of genes related to energy synthesis and transport, protein metabolism, and response to stress. Meanwhile, the results of metabolomics showed that 42 metabolites produced significant differences in mulberry, of which 26 differential metabolites were up-regulated in expression and 16 differential metabolites were down-regulated, mainly including metabolic pathways such as secondary metabolite biosynthesis, citric acid cycle, and tricarboxylic acid cycle, and was not conducive to the seed germination and or growth of the mulberry sapling. Discussion: This study enriches the understanding of the effects of TiO2 NPs on plants and provides a reference for the comprehensive scientific assessment of the potential risks of nanomaterials on plants.

Effects of artificial diet rearing during all instars on silk secretion and gene transcription in Bombyx mori (Lepidoptera: Bombycidae).

Silkworms (Bombyx mori) reared on artificial diets during all instars have the advantages of simplicity and efficiency, year-round production, and reduced risk of poisoning. However, low silk yield remains a challenge, limiting its industrial application. To address this issue, the spinning behavior, nutrient absorption, and transcriptomics of silkworms were investigated. Compared with silkworms reared on mulberry leaves during all instars, those fed with artificial diets showed significantly lower cocoon weight, cocoon shell weight, cocoon shell rate, and silk gland tissue somatic index at the end of the fifth instar (P < 0.01). The spinning duration and crawling distance of silkworms reared on artificial diets were also significantly lower than those reared on mulberry leaves (P < 0.01). Regarding nutrient absorption, the dietary efficiency indexes of silkworms fed with artificial diets were significantly lower than those fed with mulberry leaves, except for the efficiency conversion of digesta to cocoon (P < 0.01). Further RNA-Seq analysis revealed 386 differentially transcribed genes between the 2 groups, with 242 upregulated and 144 downregulated genes. GO enrichment analysis showed that differential transcriptional genes were mainly enriched in organic acid metabolism, oxidation-reduction, and drug catabolism. KEGG enrichment analysis showed that differential transcriptional genes were mainly enriched in genetic information processing and metabolism pathways. Our findings provide new insights into the silk secretion and can serve as a reference for future research and application of silkworms fed with artificial diets.

Toxicological effects of trace amounts of pyriproxyfen on the midgut of non-target insect silkworm.

Pyriproxyfen is an insect growth regulator that is widely used in public health and pest control in agriculture. Our previous studies have shown that trace amounts of pyriproxyfen in the environment can cause serious toxic effects in the non-target insect silkworm, including failing to pupate, metamorphose and spin cocoons. However, it is unknown why pyriproxyfen not only has no lethal effects on fifth instar larvae but also tend to increase their body weight. The midgut is the main digestive organs of the silkworm, our results showed that the residual of pyriproxyfen in the silkworm at 24 h after 1 × 10-4 mg/L pyriproxyfen treatment caused severe damage to the midgut microvilli, goblet cells, and nuclei of the silkworm, but body weight and digestibility of the larval were both increased. In addition, pyriproxyfen significantly (p < 0.05) increased the activities of digestive enzymes (α-amylase, trehalase, trypsin and lipase) in the midgut of silkworm. However, it caused down-regulation of ecdysone synthesis-related genes at the end of the fifth instar silkworm, decreased ecdysone titer, and prolonged larval instar. At the same time, pyriproxyfen also activated transcription of detoxification enzymes-related genes such as the cytochrome P450 enzyme genes Cyp9a22 and Cyp15C1, the carboxylesterase genes CarE-8 and CarE-11, and the glutathione S-transferase gene GSTo2. This study elucidated a novel toxicological effect of pyriproxyfen to insects, which not only expands the understanding of the effects of juvenile hormone pesticides on lepidopteran insects but also provides a reference for exploring the ecological security of non-target organisms.

The JH-Met2-Kr-h1 pathway is involved in pyriproxyfen-induced defects of metamorphosis and silk protein synthesis in silkworms, Bombyx mori.

Environmental residues of pryriproxyfen, a juvenile hormone analogue (JHA) type pesticide, may have on unintended consequences on non-target insects. However, the mechanism of pyriproxyfen action and silk protein synthesis in silkworms has not been reported. In the present study, we treated the silkworms with trace pyriproxyfen (1 × 10-4 mg/L) and found that the silkworm larvae showed no obvious poisoning symptoms, while the development of silk glands and cocoon-forming function were both seriously damaged due to the accumulation of pyriproxyfen in posterior silk gland (PSG). The titer of the juvenile hormone (JH) was increased, whereas the content of 20-hydroxyecdysone (20E) was reduced in pyriproxyfen-exposed hemolymph. Met2 is a component of the JH receptor complex and JH can promote its phosphorylation. We found Met2 and SRC were up-regulated in the larval stage after pyriproxyfen exposure, the JH-Met2/SRC complex led to the up-regulation of downstream genes Kr-h1, and Dimm, and then specifically inhibited the transcription of Fib-H. Meanwhile, the transcription of ecdysone inducible transcription factor Br-C Z4 was also inhibited by pyriproxyfen and resulted in the defects of metamorphosis. In conclusion, the trace pyriproxyfen could affect the metamorphosis and silk protein synthesis through the Met2-mediated pathway. Our study provided new evidence that Met2 might be a potential target gene of JHA in Lepidoptera.

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.

Effect of oxidative phosphorylation signaling pathway on silkworm midgut following exposure to phoxim.

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.

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.

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.

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.

Effects of the biosynthesis and signaling pathway of ecdysterone on silkworm (Bombyx mori) following exposure to titanium dioxide nanoparticles.

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.

Effects of feeding silkworm with nanoparticulate anatase TiO2 (TiO2 NPs) on its feed efficiency.

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.