Novel compounds ZK-PI-5 and ZK-PI-9 regulate the reproduction of Spodoptera frugiperda (Lepidoptera: Noctuidae), with insecticide potential.

Trehalase inhibitors prevent trehalase from breaking down trehalose to provide energy. Chitinase inhibitors inhibit chitinase activity affecting insect growth and development. This is an important tool for the investigation of regulation of trehalose metabolism and chitin metabolism in insect reproduction. There are few studies on trehalase or chitinase inhibitors' regulation of insect reproduction. In this study, ZK-PI-5 and ZK-PI-9 were shown to have a significant inhibitory effect on the trehalase, and ZK-PI-9 significantly inhibited chitinase activity in female pupae. We investigated the reproduction regulation of Spodoptera frugiperda using these new inhibitors and evaluated their potential as new insecticides. Compounds ZK-PI-5 and ZK-PI-9 were injected into the female pupae, and the control group was injected with solvent (2% DMSO). The results showed that the emergence failure rate for pupae treated with inhibitors increased dramatically and aberrant phenotypes such as difficulty in wings spreading occurred. The oviposition period and longevity of female adults in the treated group were significantly shorter than those in the control group, and the ovaries developed more slowly and shrank earlier. The egg hatching rate was significantly reduced by treatment with the inhibitor. These results showed that the two new compounds had a significant impact on the physiological indicators related to reproduction of S. frugiperda, and have pest control potential. This study investigated the effect of trehalase and chitin inhibitors on insect reproduction and should promote the development of green and efficient insecticides.

Roles of GFAT and PFK genes in energy metabolism of brown planthopper, Nilaparvata lugens.

Glutamine:fructose-6-phosphate aminotransferases (GFATs) and phosphofructokinase (PFKs) are the principal rate-limiting enzymes involved in hexosamine biosynthesis pathway (HBP) and glycolysis pathway, respectively. In this study, the NlGFAT and NlPFK were knocked down through RNA interference (RNAi) in Nilaparvata lugens, the notorious brown planthopper (BPH), and the changes in energy metabolism were determined. Knockdown of either NlGFAT or NlPFK substantially reduced gene expression related to trehalose, glucose, and glycogen metabolism pathways. Moreover, trehalose content rose significantly at 72 h after dsGFAT injection, and glycogen content increased significantly at 48 h after injection. Glucose content remained unchanged throughout the experiment. Conversely, dsPFK injection did not significantly alter trehalose, but caused an extreme increase in glucose and glycogen content at 72 h after injection. The Knockdown of NlGFAT or NlPFK significantly downregulated the genes in the glycolytic pathway, as well as caused a considerable and significant decrease in pyruvate kinase (PK) activity after 48 h and 72 h of inhibition. After dsGFAT injection, most of genes in TCA cycle pathway were upregulated, but after dsNlPFK injection, they were downregulated. Correspondingly, ATP content substantially increased at 48 h after NlGFAT knockdown but decreased to an extreme extent by 72 h. In contrast, ATP content decreased significantly after NlPFK was knocked down and returned. The results have suggested the knockdown of either NlGFAT or NlPFK resulted in metabolism disorders in BPHs, highlighting the difference in the impact of those two enzyme genes on energy metabolism. Given their influence on BPHs energy metabolism, developing enzyme inhibitors or activators may provide a biological control for BPHs.

Potential inhibitory effects of compounds ZK-PI-5 and ZK-PI-9 on trehalose and chitin metabolism in Spodoptera frugiperda (J. E. Smith).

Introduction: Spodoptera frugiperda is an omnivorous agricultural pest which is great dangerous for grain output. Methods: In order to investigate the effects of potential trehalase inhibitors, ZK-PI-5 and ZK-PI-9, on the growth and development of S. frugiperda, and to identify new avenues for S. frugiperda control, we measured the content of the trehalose, glucose, glycogen and chitin, enzyme activity, and gene expression levels in trehalose and chitin metabolism of S. frugiperda. Besides, their growth and development were also observed. Results: The results showed that ZK-PI-9 significantly reduced trehalase activity and ZK-PI-5 significantly reduced membraned-bound trehalase activity. Moreover, ZK-PI-5 inhibited the expression of SfTRE2, SfCHS2, and SfCHT, thus affecting the chitin metabolism. In addition, the mortality of S. frugiperda in pupal stage and eclosion stage increased significantly after treatment with ZK-PI-5 and ZK-PI-9, which affected their development stage and caused death phenotype (abnormal pupation and difficulty in breaking pupa). Discussion: These results have provided a theoretical basis for the application of trehalase inhibitors in the control of agricultural pests to promote future global grain yield.

Regulatory function of the trehalose-6-phosphate synthase gene TPS3 on chitin metabolism in brown planthopper, Nilaparvata lugens.

Brown planthopper (Nilaparvata lugens) is one of the important pests that damage rice. Trehalose-6-phosphate synthase (TPS) is a key enzyme responsible for catalysing the biosynthesis of trehalose, which is the energy substance of insects. In this study, combined with the reported N. lugens TPS1, TPS2 and newly discovered TPS3, we studied the regulation of TPS in chitin metabolism by RNA interference. Firstly, we found that the relative expression levels of TRE1-1, TRE1-2 and TRE2 increased significantly after 48 h of dsTPS3 injection, and the activity of TRE1 enhanced significantly. Secondly, abnormal and lethal phenotypes were observed after dsTPS3 and dsTPSs injection. The relative expression levels of PGM2, G6PI2, Cht1-4, Cht6-10 and IDGF decreased significantly after 48 h of dsTPS3 injection. At 72 h after injection of dsTPS3, the relative expression levels of CHS1, Cht2, Cht4, Cht7 and Cht8 reduced significantly, but the expression levels of G6PI1, Cht5 and ENGase increased significantly. The relative expression levels of GFAT, UAP, PGM2, G6PI2, CHS1, CHS1a, CHS1b, Cht2, Cht4, Cht8, Cht9 and Cht10 decreased significantly after 48 h of dsTPSs injection. However, at 72 h after the injection of dsTPSs, the expression levels of GNPNA, UAP, PGM1, G6PI1, HK, CHS1, CHS1a, CHS1b, Cht3, Cht5, Cht7 and ENGase increased significantly. Finally, the chitin content decreased in dsTPS1, dsTPS2 and dsTPSs treatments. In conclusion, the inhibition of TPS expression affected the metabolism of trehalose and chitin in N. lugens. The related research results provide a theoretical basis for pest control.

GFAT and PFK genes show contrasting regulation of chitin metabolism in Nilaparvata lugens.

Glutamine:fructose-6-phosphate aminotransferase (GFAT) and phosphofructokinase (PFK) are enzymes related to chitin metabolism. RNA interference (RNAi) technology was used to explore the role of these two enzyme genes in chitin metabolism. In this study, we found that GFAT and PFK were highly expressed in the wing bud of Nilaparvata lugens and were increased significantly during molting. RNAi of GFAT and PFK both caused severe malformation rates and mortality rates in N. lugens. GFAT inhibition also downregulated GFAT, GNPNA, PGM1, PGM2, UAP, CHS1, CHS1a, CHS1b, Cht1-10, and ENGase. PFK inhibition significantly downregulated GFAT; upregulated GNPNA, PGM2, UAP, Cht2-4, Cht6-7 at 48 h and then downregulated them at 72 h; upregulated Cht5, Cht8, Cht10, and ENGase; downregulated Cht9 at 48 h and then upregulated it at 72 h; and upregulated CHS1, CHS1a, and CHS1b. In conclusion, GFAT and PFK regulated chitin degradation and remodeling by regulating the expression of genes related to the chitin metabolism and exert opposite effects on these genes. These results may be beneficial to develop new chitin synthesis inhibitors for pest control.

Regulatory Functions of Nilaparvata lugens GSK-3 in Energy and Chitin Metabolism.

Glucose metabolism is a biologically important metabolic process. Glycogen synthase kinase (GSK-3) is a key enzyme located in the middle of the sugar metabolism pathway that can regulate the energy metabolism process in the body through insulin signaling. This paper mainly explores the regulatory effect of glycogen synthase kinase on the metabolism of glycogen and trehalose in the brown planthopper (Nilaparvata lugens) by RNA interference. In this paper, microinjection of the target double-stranded GSK-3 (dsGSK-3) effectively inhibited the expression of target genes in N. lugens. GSK-3 gene silencing can effectively inhibit the expression of target genes (glycogen phosphorylase gene, glycogen synthase gene, trehalose-6-phosphate synthase 1 gene, and trehalose-6-phosphate synthase 2 gene) in N. lugens and trehalase activity, thereby reducing glycogen and glucose content, increasing trehalose content, and regulating insect trehalose balance. GSK-3 can regulate the genes chitin synthase gene and glucose-6-phosphate isomerase gene involved in the chitin biosynthetic pathway of N. lugens. GSK-3 gene silencing can inhibit the synthesis of chitin N. lugens, resulting in abnormal phenotypes and increased mortality. These results indicated that a low expression of GSK-3 in N. lugens can regulate the metabolism of glycogen and trehalose through the insulin signal pathway and energy metabolism pathway, and can regulate the biosynthesis of chitin, which affects molting and wing formation. The relevant research results will help us to more comprehensively explore the molecular mechanism of the regulation of energy and chitin metabolism of insect glycogen synthase kinases in species such as N. lugens.

Regulation of Carbohydrate Metabolism by Trehalose-6-Phosphate Synthase 3 in the Brown Planthopper, Nilaparvata lugens.

Nilaparvata lugens (Stål) (Hemiptera: Delphacidae) is one of the pests that harm rice. In this paper, a new trehalose-6-phosphate synthase gene, TPS3, was identified by transcriptome sequencing and gene cloning. To explore its role in the energy metabolism of N. lugens we examined the carbohydrate contents at different stages of development, the tissue expression of TPS, and some physiological and biochemical indicators by injecting dsTPS3 and dsTPSs (a proportional mixture of dsTPS1, dsTPS2, and dsTPS3). The glucose content at the fifth instar was significantly higher than that in the fourth instar and the adult stages. The trehalose and glycogen contents before molting were higher than those after molting. TPS1, TPS2, and TPS3 were expressed in the head, leg, wing bud, and cuticle, with the highest expression in the wing bud. In addition, compared with the control group, the glucose content increased significantly at 48 h after RNA interference, and the trehalose content decreased significantly after 72 h. qRT-PCR showed that the expression level of UGPase decreased significantly at 48 h after injection, whereas GS expression increased significantly at 48 h after injecting dsTPS3. After dsTPS injection, the expression levels of PPGM2, UGPase, GP, and GS increased significantly at 72 h. After interfering with the expression of TPS3 gene alone, UGPase expression decreased significantly at 48 h, and GS expression increased significantly at 72 h. Finally, combined with the digital gene expression and pathway analysis, 1439 and 1346 genes were upregulated, and 2127 and 1927 genes were downregulated in the dsTPS3 and dsTPSs groups, respectively. The function of most differential genes was concentrated in sugar metabolism, lipid metabolism, and amino acid metabolism. The results indicated that TPS3 plays a key role in the energy metabolism of N. lugens and confirmed that TPS3 is a feasible target gene for RNA interference in N. lugens. Simultaneously, they provide a theoretical basis for the development and utilization of TPS3 to control pests.

Role of phosphoglucomutase in regulating trehalose metabolism in Nilaparvata lugens.

Phosphoglucomutase (PGM) is a key enzyme in glycolysis and gluconeogenesis, regulating both glycogen and trehalose metabolism in insects. In this study, we explored the potential function of phosphoglucomutase (PGM) using RNA interference technology in Nilaparvata lugens, the brown planthopper. PGM1 and PGM2 were found highly expressed in the midgut of brown planthoppers, with different expression levels in different instar nymphs. The glycogen, glucose, and trehalose levels were also significantly increased after brown planthoppers were injected with dsRNA targeting PGM1 (dsPGM1) or PGM2 (dsPGM2). In addition, injection of dsPGM1 or dsPGM2 resulted in increased membrane-bound trehalase activity but not soluble trehalase activity. Furthermore, the expression of genes related to trehalose and glycogen metabolism decreased significantly after injection with dsPGM1 and dsPGM2. The expression levels of genes involved in chitin metabolism in the brown planthopper were also significantly decreased and the insects showed wing deformities and difficulty molting following RNAi. We suggest that silencing of PGM1 and PGM2 expression directly inhibits trehalose metabolism, leading to impaired chitin synthesis.

Effect of glycogen synthase and glycogen phosphorylase knockdown on the expression of glycogen- and insulin-related genes in the rice brown planthopper Nilaparvata lugens.

Nilaparvata lugens is a serious threat to rice growth. Glycogen metabolism is one of the important physiological processes of insects, which is mainly regulated by glycogen synthase (GS) and glycogen phosphorylase (GP). In the present study, trehalose content was significantly reduced at 72 h after NlGP and NlGS knockdown, whereas glucose content was significantly increased at both 48 h and 72 h after GS knockdown. RNAi combined with RNA-Seq was used to identify NlGP- and NlGS-related pathways and genes in N. lugens. A total of 593 genes were up-regulated and 5969 genes were down-regulated after NlGP and NlGS knockdown, respectively. Moreover, the NlGS-knockdown group was mapped to 10,967 pathways, whereas the NlGP-knockdown group was mapped to 7948 pathways, and the greatest differences between the groups were associated with carbohydrate, lipid, amino acid and energy metabolism. Meanwhile, 1800, 1217, and 1211 transcripts in the NlGP-knockdown group and 2511, 1666, and 1727 transcripts in the NlGS-knockdown group were involved in bioprocess, cellular ingredients and molecular function, respectively. Almost all these genes were down-regulated by either NlGP or NlGS knockdown, with significant down-regulation of the 6-trehalose phosphate synthase (TPS), trehalase (TRE), GS, GP, phosphoacetylglucosamine mutase (PGM, n = 2), Insulin receptors (InRs) and insulin-like peptides (Ilps) genes. These results have demonstrated that RNAi-mediated NlGP and NlGS knockdown could lead to content of trehalose and glucose out of balance, but have no obvious effect on glycogen content, and have suggested that GS plays more complex role in other metabolism pathway of N. lugens.

Molecular Cloning and Induced Expression of Six Small Heat Shock Proteins Mediating Cold-Hardiness in Harmonia axyridis (Coleoptera: Coccinellidae).

The main function of small heat shock proteins (sHSPs) as molecular chaperones is to protect proteins from denaturation under adverse conditions. Molecular and physiological data were used to examine the sHSPs underlying cold-hardiness in Harmonia axyridis. Complementary DNA sequences were obtained for six H. axyridis sHSPs based on its transcriptome, and the expression of the genes coding for these sHSPs was evaluated by quantitative real-time PCR (qRT-PCR) in several developmental stages, under short-term cooling or heating conditions, and in black and yellow females of experimental and overwintering populations under low-temperature storage. In addition, we measured water content and the super cooling and freezing points (SCP and FP, respectively) of H. axyridis individuals from experimental and overwintering populations. The average water content was not significantly different between adults of both populations, but the SCP and FP of the overwintering population were significantly lower than that of the experimental population. Overall, the six sHSPs genes showed different expression patterns among developmental stages. In the short-term cooling treatment, Hsp16.25 and Hsp21.00 expressions first increased and then decreased, while Hsp10.87 and Hsp21.56 expressions increased during the entire process. Under short-term heating, the expressions of Hsp21.00, Hsp21.62, Hsp10.87, and Hsp16.25 showed an increasing trend, whereas Hsp36.77 first decreased and then increased. Under low-temperature storage conditions, the expression of Hsp36.77 decreased, while the expressions of Hsp21.00 and Hsp21.62 were higher than that of the control group in the experimental population. The expression of Hsp36.77 first increased and then decreased, whereas Hsp21.56 expression was always higher than that of the control group in the overwintering population. Thus, differences in sHSPs gene expression were correlated with the H. axyridis forms, suggesting that the mechanism of cold resistance might differ among them. Although, Hsp36.77, Hsp16.25, Hsp21.00, and Hsp21.62 regulated cold- hardiness, the only significant differences between overwintering and experimental populations were found for Hsp16.25 and Hsp21.00.