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The silkworm Bombyx mori L. is a model organism of the order Lepidoptera. Understanding the mechanism of pesticide resistance in silkworms is valuable for Lepidopteran pest control. In this study, comparative metabolomics was used to analyze the metabolites of 2 silkworm strains with different pesticide resistance levels at 6, 12, and 24 h after feeding with fenpropathrin. Twenty-six of 27 metabolites showed significant differences after fenpropathrin treatment and were classified into 6 metabolic pathways: glycerophospholipid metabolism, sulfur metabolism, glycolysis, amino acid metabolism, the urea cycle, and the tricarboxylic acid (TCA) cycle. After analyzing the percentage changes in the metabolic pathways at the 3 time points, sulfur metabolism, glycolysis, and the TCA cycle showed significant responses to fenpropathrin. Confirmatory experiments were performed by feeding silkworms with key metabolites of the 3 pathways. The combination of iron(II) fumarate + folic acid (IF-FA) enhanced fenpropathrin resistance in silkworms 6.38 fold, indicating that the TCA cycle is the core pathway associated with resistance. Furthermore, the disruption of several energy-related metabolic pathways caused by fenpropathrin was shown to be recovered by IF-FA in vitro. Therefore, IF-FA may have a role in boosting silkworm pesticide resistance by modulating the equilibrium between the TCA cycle and its related metabolic pathways.

Background Camptothecin (CPT) and matrine (MAT) have potential as botanical pesticides against several pest species. However, the mechanisms of metabolic and physiological changes in pests induced by CPT and MAT are unknown. In this study, a toxicological test, an NMR-based metabolomic study, an enzymatic test, and an RT quantitative PCR (RT-qPCR) experiment were all conducted to examine the effect of CPT and MAT on Spodoptera litura. Results CPT (0.5-1%) exerted high toxicity against larvae of S. litura and caused growth stagnation and high mortality of larvae. A variety of metabolites were significantly influenced by 0.5% CPT, including several energy-related metabolites such as trehalose, lactate, succinate, citrate, malate, and fumarate. In contrast, MAT showed low toxicity against larvae and induced almost no changes in hemolymph metabolites of S. litura. Enzymatic tests showed that trehalase activity was significantly decreased in larvae after feeding with 0.5% CPT. RT-qPCR showed that the transcription levels of alanine aminotransferase, malate dehydrogenase, and isocitrate dehydrogenase were decreased while lactate dehydrogenase was increased in the 0.5% CPT-treated group. Conclusions These data indicate that one of the important mechanisms of CPT against S. litura larvae is via the inhibition of trehalose hydrolysis and glycolysis. Our findings also suggest that CPT exhibits a stronger toxicological effect than MAT against S. litura, which provides basic information for the application of CPT in the control of S. litura or other lepidoptera pests.

Validamycin, as a broadly applied antibiotic, has been used to control rice sheath blight disease. Furthermore, validamycin was considered as an insecticide to control agricultural pests. Insight into the mechanism of validamycin's action on insects can provide molecular targets for the control of agricultural pests. In this study, a toxicological test analysis revealed that Spodoptera litura larval growth and development was significantly inhibited and the pupation rate was significantly reduced with the increase of the concentration of validamycin. According to the nuclear magnetic resonance (NMR)-based metabolomic analysis, a total of 15 metabolites involved in glycolysis and tricarboxylic acid cycle (TCA) pathways were identified. Additionally, trehalase activities, glucose and chitin contents were significantly down-regulated, but the trehalose content was up-regulated after exposure to validamycin. Reverse transcription-quantitative PCR (RT-qPCR) analysis revealed that the expression level of genes involved in glycolysis, TCA and chitin synthesis were up-regulated after treating with validamycin. Further chitin staining also confirmed that chitin content was downregulated at 12 h after validamycin treatment. Our results indicated that validamycin worked via two different molecular mechanisms, one through inhibiting glycometabolism and the other by inhibiting chitin synthesis in S. litura. The information lay a theoretical foundation for further control of S. litura. This article is protected by copyright. All rights reserved.

1-Deoxynojirimycin (DNJ) is a natural d -glucose analogue from mulberry with promising physiological activity in vivo. Up to the present, the antidiabetic effects of DNJ on lowering blood sugar and accelerating lipid metabolism in mammals were broadly reported, but the specific character of DNJ against insects was vastly ignored. In this study, a toxicological test of DNJ againgst eri-silkworm, Samia cynthia ricini was carried out to investigate the potential of DNJ in insect management. Further, a method of nuclear magnetic resonance (NMR) metabonomics and real-time qPCR (RT-qPCR) were performed to analyze the alteration in midgut of eri-silkworm caused by DNJ. The result of toxicology showed that 5% and 10% DNJ could significantly inhibit the development of third-instar larvae on day 1–5, and mass deaths happened in DNJ groups on day 3–5. The quantitative analysis of 1H NMR in fifth-instar larvae showed that trehalose level increased in midgut of 0, 6 and 12 h DNJ groups, while the concentrations of glucose, lactate, alanine, pyruvate, α-ketoglutarate and fumarate were reduced in varying degrees. Meanwhile, principal component analysis (PCA) indicated that there were significant differences in the metabolic profiles among 12 h DNJ groups and the control group. In addition, RT-qPCR results displayed that four genes coding α-glucosidase, trehalase (THL) and lactate dehydrogenase (LDH) were lowered in expression of 12 h DNJ groups. Simultaneously, THL activity was significantly lowerd in 12 h DNJ groups. These mutually corroborated results indicated that the backbone pathways of energy metabolism, including hydrolysis of trehalose and glycogens, glycolysis and tricarboxylic acid (TCA) cycle were significantly inhibited by DNJ. Thus, the specific mechanism of DNJ efficiently suppressing the growth and energy metabolism of eri-silkworm was explored in this study, providing the potential of DNJ as to the production of botanical insecticide.

In digital micromirror device (DMD)-based projection photolithography, the throughput largely depends on the effectiveness of the laser energy utilization, which is directly correlated to the diffraction efficiency of DMD. Here, to optimize the DMD diffraction efficiency and thus the laser energy utilization, we calculate the diffraction efficiencies Ediffraction of DMD with various pitch sizes at wavelengths ranging from 200 nm to 800 nm, using the two-dimensional blazed grating diffraction theory. Specifically, the light incident angle is optimized for 343 nm laser and 7.56 μm pitch-size DMD, and the maximum single-order diffraction efficiency Ediffraction is increased from 40% to 96%. Experimentally, we use the effective energy utilization ηeff = Ediffraction,(m,n)/Σ[Ediffraction,(m,n)] at the entrance pupil plane of the objective to verify the effectiveness of the optimized illumination angle in a lithography illumination system with parallel beams of two wavelengths (343 nm and 515 nm). The ηeff of a “blaze” order at a 34° angle of incidence can be optimized up to 88%. The experimental results are consistent with the tendency of the calculated results, indicating that this optimization model can be used to improve the energy utilization of projection lithography with the arbitrarily designable wavelengths and the DMD’s pitch size.

This study was designed to investigate the metabolic and transcriptional alterations in seminal fluid caused by asthenozoospermia (AS). To address these issues, a method of metabonomics based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and real-time quantitative PCR (RT-qPCR) was performed to identify some crucial biomarkers and transcription levels of the enzymes in seminal fluid. Seminal fluid samples were collected from 87 AS patients and 73 healthy males with normozoospermia. The quantitative analysis by UPLC-MS/MS showed that 19 metabolites in seminal plasma were associated with AS, and they were involved in several metabolic pathways, such as energy metabolism, purine metabolism, methionine cycle, and branched chain amino acid metabolism. Among these metabolites, the levels of citric acid, malic acid, succinic acid, and pyruvic acid, which are related to energy metabolism, were collectively reduced in the AS group, whereas the lactic acid level was enhanced. These results indicated that lesser energy source (adenosine triphosphate) was produced through the anaerobic glycolysis pathway rather than via aerobic catabolism of suger and tricarboxylic acid cycle, resulting in reduced power of sperms. Meanwhile, partial least squares discriminant analysis showed significant differences in metabolic profiles between the AS and control groups. In addition, RT-qPCR results revealed that the expression levels of four genes encoding fructokinase citrate synthase, succinate dehydrogenase, and spermine synthase, which were related to energy metabolism, were decreased in the AS group. The 23 descriptors with differential expression in AS may be valuable for the diagnosis and sequential study on AS. These results will help highlight the role of sperm inactivity in AS pathogenesis.

Sanguinarine (Sang) is a natural alkaloid and distributed in several plants of Papaveraceae. The antitumor, antioxidant, antimicrobial and anti-inflammatory effects of Sang were extensively reported, but its speciality and mechanism against Lepidoptera insects were still unknown. In this study, detailed toxicological parameters of Sang against silkworms, Bombyx mori (B. mori), were determined by a toxicological test. Then, a nuclear magnetic resonance-based (NMR) metabolomics method was adopted to analyze the changes in hemolymph metabolites of silkworms after feeding Sang. The growth of fourth-instar larvae was significantly ceased by the oral administration of 0.05–0.3% Sang and vast deaths appeared in 0.3% Sang group on Day 4 and Day 5. The quantitative analysis of metabolites indicated that trehalose and citrate levels in hemolymph were increased after 24 h of feeding 0.3% Sang, whereas the concentrations of pyruvate, succinate, malate and fumarate were decreased. In addition, the enzymatic determination and reverse transcription quantitative PCR (RT-qPCR) showed that the trehalase (THL) activity and the transcriptional level of one gene coding THL were uniformly weakened by 0.3% Sang. One of the important mechanisms of Sang against silkworms might be interpreted as follows. Sang impaired trehalose hydrolysis, reduced THL activity and transcription, and led to the inhibition of energy metabolism, consequent antigrowth and high lethality in larvae of B. mori. Our findings offered new insights into the insecticidal effect of Sang from the perspective of energy metabolism and provided the basis for the application of Sang in the control of Lepidoptera pests.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a primary silkworm pathogen, and the molecular mechanism of silkworm defense to BmNPV infection is still unclear. Herein, comparative metabolomics was adopted to analyze the variations in the hemolymph metabolites of different resistant silkworm strains following BmNPV inoculation using a

Small heat shock proteins are a molecular chaperone and implicated in various physiological and stress processes in animals. However, the immunological functions of Hsp genes remain to elucidate in the crustaceans, particularly in red swamp crayfish, Procambarus clarkii. Here we report the cloning of heat shock protein 21 from the P. clarkii (hereafter Pc-Hsp21). The open reading frame of Pc-Hsp21 was 555 base pairs, encoding a protein of 184 amino acid residues with an alpha-crystallin family domain. Quantitative real-time PCR (qRT-PCR) analysis revealed a constitutive transcript expression of Pc-Hsp21 in the tested tissue, with the highest in hepatopancreas. The transcript abundance for this gene enhanced in hepatopancreas following immune challenge with the lipopolysaccharide, peptidoglycan, and poly I:C compared to the control group. The depletion of Pc-Hsp21 by double-stranded RNA altered transcript expression profiles of several genes in hepatopancreas, genes involved in the crucial immunological pathways of P. clarkii. These results suggest that Pc-Hsp21 plays an essential biological role in the microbial stress response by modulating the expression of immune-related genes in P. clarkii.

Samia cynthia ricini (Lepidoptera: Saturniidae) is an important commercial silk-producing insect; however, in contrast to the silkworm, mulberry leaves are toxic to this insect because the leaves contain the component 1-deoxynojirimycin (DNJ). A transcriptomic analysis of eri-silkworm haemolymph was conducted to examine the genes related to different metabolic pathways and to elucidate the molecular mechanism underlying eri-silkworm haemolymph responses to DNJ. Eight hundred sixty-five differentially expressed genes (DEGs) were identified, among which 577 DEGs were up-regulated and 288 DEGs were down-regulated in the 2% DNJ group compared to control (ddH2O) after 12h. Based on the results of the functional analysis, these DEGs were associated with ribosomes, glycolysis, N-glycan biosynthesis, and oxidative phosphorylation. In particular, according to the KEGG analysis, 138 DEGs were involved in energy metabolism, glycometabolism and lipid metabolism, and the changes in the expression of nine DEGs were confirmed by reverse transcription quantitative PCR (RT-qPCR). Thus, DNJ induced significant metabolic alterations in eri-silkworm haemolymph. These results will lay the foundation for research into the toxic effects of DNJ on eri-silkworm as a model and provide a reference for the exploitation of new drugs in humans.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a primary silkworm pathogen, and the molecular mechanism of silkworm defense to BmNPV infection is still unclear. Herein, comparative metabolomics was adopted to analyze the variations in the hemolymph metabolites of different resistant silkworm strains following BmNPV inoculation using a 1H NMR method. Trehalose, as an instant source of energy, plays a crucial role in the response to pathogen infections in insects. The level of trehalose was persistently upregulated in the hemolymph of the resistant silkworm strain YeA following infection with BmNPV, compared to that of the susceptible strain YeB, indicating that trehalose metabolism plays a vital role in the response to BmNPV infection. The significant upregulation of TCA cycle relevant metabolites, including malate, fumarate, citrate, succinate, and α-ketoglutarate, was identified at 0 h, 12 h, 48 h, and 96 h post-infection in YeA hemolymph, whereas a significant upregulation in YeB hemolymph was only detected at an early stage of infection (0 h–24 h). The expression level of selected key metabolic enzymes, determined using RT-qPCR, validated the differences in trehalose and TCA cycle relevant metabolite levels. The variations in branched-chain amino acid (BCAA) pathway relevant metabolites in resistant silkworm strains following BmNPV infection showed a regular undulation at different times after infection. A significant accumulation of phenylalanine and tyrosine was observed in YeA following BmNPV infection compared to YeB. The glycolysis and gluconeogenesis pathways showed a relatively low activity in YeA following BmNPV infection. Moreover, the levels of other metabolites related to fat metabolism, transamination, energy metabolism, and glycometabolism, such as glycine, threonine, glutamine, and glutamate, were unstable in the two silkworm strains following BmNPV infection. Thus, our study provides an overview of the metabolic response of the silkworm in response to BmNPV infection, which lays the foundation for clarifying the mechanism of silkworm resistance to BmNPV infection.