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.

Roles of the PTP61F Gene in Regulating Energy Metabolism of Tribolium castaneum (Coleoptera: Tenebrionidae).

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator in the insulin signaling pathway. It belongs to a class of non-receptor phosphatases of protein tyrosine phosphatase and can catalyze the dephosphorylation of tyrosine to regulate cell differentiation, growth, and metabolism. However, few studies have focused on the role of PTP1B in regulating energy metabolism of insects. In this study, we investigated the expression profiles and the functions of a PTP1B gene (designated TcPTP61F) in the red flour beetle Tribolium castaneum. Quantitative real-time PCR analyzed showed that TcPTP61F was highly expressed in the pupal and adult stages. In adult tissues, TcPTP61F was prominently expressed in the tarsus and head. RNA interference-mediated silencing of TcPTP61F reduced the expression of eight genes in trehalose metabolic and glycolytic pathways. TcPTP61F depletion also caused a significant change in the distribution of trehalose, glucose, and glycogen. Additionally, knockdown of TcPTP61F inhibited the pyruvate kinase (PK) activity and significantly decreased the adenosine triphosphate (ATP) level. The results suggest that TcPTP61F is indispensible for trehalose and energy metabolism of T. castaneum.

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.

Evaluation of the Expression and Function of the TRE2-like and TRE2 Genes in Ecdysis of Harmonia axyridis.

Harmonia axyridis is an important predatory insect and widely used in biological control of agricultural and forestry pests. Trehalose is directly involved in the energy storage of the H. axyridis and in the oxidative function of various physiological activities thereby providing an energy source for its growth and development. The aim of this study was to explore the potential function of membrane-bound-like trehalase (TRE2-like) and membrane-bound trehalase (TRE2) genes in H. axyridis by RNAi. In addition, the activity of soluble and membrane-bound trehalase and the expression of genes related to trehalose and glycogen metabolism were determined in the larvae injected with dsTRE2-like or dsTRE2. The results showed that wing abnormality and mortality appeared in adults, as well as the activity of soluble trehalase and glycogen contents increased when interfering with TRE2-like gene. However, the activity of membrane-bound trehalase, trehalose and glucose contents in the larvae decreased. The expression of glycogen synthase (GS) and glycogen phosphorylase (GP) genes were decreased after RNAi in the ecdysis stage. The expression of chitin synthase gene A (CHSA), chitin synthase gene B (CHSB), and trehalose-6-phosphate synthase genes (TPS) were decreased significantly after RNAi, especially in the ecdysis stage. These results indicated that RNA interference is capable of knocking down gene expression of TRE2-like and TRE2, thereby disrupting trehalose metabolism which affects the chitin synthesis pathway in turn and also leads to developmental defects, such as wing deformities. This study could provide some theoretical guidance for the function of TRE2 gene in other insects.

Glucose Utilization in the Regulation of Chitin Synthesis in Brown Planthopper.

Glucose-6-phosphatase (G6Pase) and hexokinase (HK) are two key enzymes in the glycolysis and gluconeogenesis pathways, which catalyze the synthesis and degradation of glucose in insects, respectively. G6Pase and HK play an important role in insect growth by regulating the metabolism of glucose, leading to the efficient metabolism of other macromolecules. However, it is unclear whether these genes could be investigated for pest control through their actions on chitin metabolism. We studied the potential functions of G6Pase and HK genes in the regulation of chitin metabolism pathways by RNAi technology. Interference with G6Pase expression did not affect trehalose and chitin metabolism pathways in brown planthopper, Nilaparvata lugens (Stål). However, knockdown of the HK gene resulted in a significant decrease of expression of genes associated with the trehalose metabolic pathway but had no significant effect on trehalase activity, trehalose content, or glucogen content. Additionally, HK knockdown resulting in downregulation of the genes involved in chitin metabolism in the brown planthopper. These insects also showed wing deformities and difficulty in molting to varying degrees. We suggest that the silencing of HK expression directly inhibited the decomposition of glucose, leading to impaired chitin synthesis.