1 H NMR-based metabonomic evaluation of the pesticides camptothecin and matrine against larvae of Spodoptera litura.

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.

Metabolic and transcriptional changes in seminal plasma of asthenozoospermia patients.

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 caused larval lethality and growth inhibition by suppressing energy metabolism in silkworms, Bombyx mori.

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.

A toxicological, metabonomic and transcriptional analysis to investigate the property of mulberry 1-deoxynojirimycin against the growth of Samia cynthia ricini.

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.

Metabolic changes in the midgut of Eri silkworm after Oral administration of 1-deoxynojirimycin: A 1H-NMR-based metabonomic study.

1-deoxynojirimycin (DNJ) is a natural D-glucose analogue and has a strong physiological activity in inhibiting α-glucosidase in vivo. The antidiabetic effects of DNJ in mice or other mammals were extensively explored, but the physiological and toxic roles of DNJ in insects was seldom reported. In this study, the biological effects of DNJ were examined in midgut extracts of fourth-instar larvae of Eri silkworm (Samia cynthia ricini, Saturniidae). Based on nuclear magnetic resonance (NMR) metabonomics technology, we analyzed the alterations of glycometabolism, lipids, and energy metabolism pathways in the midgut of S. cynthia ricini caused by DNJ. Pattern recognition analysis (partial least square-discriminant analysis, PLS-DA) showed that four groups of latex, 0.25% DNJ, 0.5% DNJ and the mixture of 0.5% DNJ and latex (1:1) were distinctly different from the control group. Moreover, several metabolic pathways of DNJ-mediated modulation in the midgut were identified. Compared with the control group, alanine, succinate, glutamate, and fumarate concentrations decreased in three groups of 0.5% DNJ, latex, and the mixture, choline levels increased in two DNJ groups, and trehalose levels increased in all experimental groups. Therefore, these results suggest that DNJ modulated lipid metabolism by limiting the hydrolysis pathways of phospholipids metabolism. Additionally, DNJ has a potent negative effect on energy metabolism by inhibiting the hydrolysis of trehalose, glycolysis and the tricarboxylic acid (TCA) cycle. Overall, DNJ, as a single-ingredient, is an efficient substance for modulating lipid metabolism and inhibiting energy metabolism.

An Evaluation of 1-Deoxynojirimycin Oral Administration in Eri Silkworm through Fat Body Metabolomics Based on (1) H Nuclear Magnetic Resonance.

1-Deoxynojirimycin (DNJ), the main hypoglycemic constituent in mulberry (Morus alba) latex, has been extensively researched. Although there is considerable interest in the biological effects of DNJ, the roles of 1-deoxynojirimycin (DNJ) in glycometabolism and energy metabolism in insects have received little attention. In this paper, (1)H nuclear magnetic resonance ((1)H NMR) based metabonomic was performed to study the effects of the oral supplementation of 0.25% DNJ, 0.5% DNJ, latex, and the mixture of 0.5% DNJ and latex (1 : 1) on the fat body glycometabolism and energy metabolism of the fourth-instar larvae of Eri silkworms, Samia cynthia ricini. Metabolic pattern recognition analysis (partial least square-discriminant analysis, PLS-DA) of fat body extracts indicated that the groups of 0.25% DNJ, 0.5% DNJ, latex, and the mixture of 0.5% DNJ and latex (1 : 1) were significantly different from the control group. Further, compared to the control group, the metabolites levels of lactate, trehalose, succinate, malate, and fumarate were remarkably changed in experimental groups, which were involved in glycolysis, hydrolysis of trehalose, and tricarboxylic acid (TCA) cycle. Our results indicate that DNJ has a positive impact on the reverse energy metabolism of Eri silkworms and metabonomic analysis based on NMR can be used as a tool to identify potential biomarkers.