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.

Glycogen Phosphorylase and Glycogen Synthase: Gene Cloning and Expression Analysis Reveal Their Role in Trehalose Metabolism in the Brown Planthopper, Nilaparvata lugens Stål (Hemiptera: Delphacidae).

RNA interference has been used to study insects' gene function and regulation. Glycogen synthase (GS) and glycogen phosphorylase (GP) are two key enzymes in carbohydrates' conversion in insects. Glycogen content and GP and GS gene expression in several tissues and developmental stages of the Brown planthopper Nilaparvata lugens Stål (Hemiptera: Delphacidae) were analyzed in the present study, using quantitative reverse-transcription polymerase chain reaction to determine their response to double-stranded trehalases (dsTREs), trehalose-6-phosphate synthases (dsTPSs), and validamycin injection. The highest expression of both genes was detected in the wing bud, followed by leg and head tissues, and different expression patterns were shown across the developmental stages analyzed. Glycogen content significantly decreased 48 and 72 h after dsTPSs injection and 48 h after dsTREs injection. GP expression increased 48 h after dsTREs and dsTPSs injection and significantly decreased 72 h after dsTPSs, dsTRE1-1, and dsTRE1-2 injection. GS expression significantly decreased 48 h after dsTPS2 and dsTRE2 injection and 72 h after dsTRE1-1 and dsTRE1-2 injection. GP and GS expression and glycogen content significantly decreased 48 h after validamycin injection. The GP activity significantly decreased 48 h after validamycin injection, while GS activities of dsTPS1 and dsTRE2 injection groups were significantly higher than that of double-stranded GFP (dsGFP) 48 h after injection, respectively. Thus, glycogen is synthesized, released, and degraded across several insect tissues according to the need to maintain stable trehalose levels.

Suppressing the activity of trehalase with validamycin disrupts the trehalose and chitin biosynthesis pathways in the rice brown planthopper, Nilaparvata lugens.

Trehalase (TRE) is a key enzyme in trehalose degradation and has important functions in insect growth and chitin synthesis. Though validamycin has the potential for pest control by suppressing TRE activities, it is not known whether validamycin acts on both trehalose and chitin metabolism. TRE1 and TRE2 activities and glucose and glycogen contents decreased significantly after the injection of different doses of validamycin solution compared with the control group, while the trehalose content increased significantly. Overall, it showed that about 13 to 38% insects was appeared abnormal phenotypes, and 10 to 57% of insects died 48h after injection of solutions with different concentrations of validamycin; the chitin content also decreased significantly. Validamycin altered the relative expression levels of trehalose, glycogen and chitin metabolism-related genes by suppressing the activities of two TREs. We showed that the expression levels of three TRE and two trehalose-6-phosphate synthase (TPS) genes increased, while the expression levels of GP; CHS1 and its two transcripts, CHS1a, CHS1b; six chitinases, including Cht3, Cht4, Cht5, Cht6, Cht7, Cht9; and the HK, G6PI2, GFAT, GNPNA, PAGM1, UAP, VVL, CI and AP genes decreased significantly 48h after the injection of any validamycin concentration compared with the control group. These results demonstrate that by inhibiting the activities of two TREs, validamycin alters N. lugens chitin synthesis and degradation and affects trehalose and chitin metabolism-related gene expression. The development of TRE inhibitors may provide effective pest control in the future.

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.

Transcriptome analysis and identification of induced genes in the response of Harmonia axyridis to cold hardiness.

Harmonia axyridis is an important predatory lady beetle that is a natural enemy of agricultural and forestry pests. In this research, the cold hardiness induced genes and their expression changes in H. axyridis were screened and detected by the way of the transcriptome and qualitative real-time PCR under normal and low temperatures, using high-throughput transcriptome and digital gene-expression-tag technologies. We obtained a 10Gb transcriptome and an 8Mb gene expression tag pool using Illumina deep sequencing technology and RNA-Seq analysis (accession number SRX540102). Of the 46,980 non-redundant unigenes identified, 28,037 (59.7%) were matched to known genes in GenBank, 21,604 (46.0%) in Swiss-Prot, 19,482 (41.5%) in Kyoto Encyclopedia of Genes and Genomes and 13,193 (28.1%) in Gene Ontology databases. Seventy-five percent of the unigene sequences had top matches with gene sequences from Tribolium castaneum. Results indicated that 60 genes regulated the entire cold-acclimation response, and, of these, seven genes were always up-regulated and five genes always down-regulated. Further screening revealed that six cold-resistant genes, E3 ubiquitin-protein ligase, transketolase, trehalase, serine/arginine repetitive matrix protein 2, glycerol kinase and sugar transporter SWEET1-like, play key roles in the response. Expression from a number of the differentially expressed genes was confirmed with quantitative real-time PCR (HaCS_Trans). The paper attempted to identify cold-resistance response genes, and study the potential mechanism by which cold acclimation enhances the insect's cold endurance. Information on these cold-resistance response genes will improve the development of low-temperature storage technology of natural enemy insects for future use in biological control.

Knockdown of two trehalose-6-phosphate synthases severely affects chitin metabolism gene expression in the brown planthopper Nilaparvata lugens.

BACKGROUND: RNA interference combined with digital gene expression (DGE) analysis can be used to study gene function. Trehalose-6-phosphate synthase (TPS) plays a key role in the synthesis of trehalose and insect development. RESULTS: DGE analysis revealed that the expression of nine or four chitinase genes was reduced significantly 48 h after NlTPS1 and NlTPS2 knockdown by RNAi, respectively. Additionally, abnormal phenotypes were noted, and approximately 30% of insects died. HK and G6PI2 expression decreased significantly whereas GFAT, GNPNA and UAP expression increased significantly 72 h after NlTPS1 and NlTPS2 knockdown. PGM1 expression decreased significantly after TPS2 knockdown, whereas PGM2 expression increased significantly and the expression of three CHS genes decreased 48 h after TPS1 knockdown. The mRNA expression of all 12 chitin degradation genes decreased 48 h after NlTPS1 and NlTPS2 treatment, and Cht2, Cht3, Cht6, Cht7, Cht10 and ENGase levels remained significantly decreased up to 72 h after NlTPS1 and NlTPS2 knockdown. CONCLUSIONS: These results demonstrate that silencing of TPS genes can lead to increased moulting deformities and mortality rates owing to the misregulation of genes involved in chitin metabolism, and TPS genes are potential pest control targets in the future. © 2016 Society of Chemical Industry.

Knockdown of five trehalase genes using RNA interference regulates the gene expression of the chitin biosynthesis pathway in Tribolium castaneum.

BACKGROUND: RNA interference is a very effective approach for studies on gene function and may be an efficient method for controlling pests. Trehalase is a key gene in the chitin biosynthesis pathway in insects. Five trehalase genes have been cloned in Tribolium castaneum, though it is not known whether the detailed functions of these trehalases can be targeted for pest control. RESULTS: The functions of all five trehalase genes were studied using RNAi, and the most important results showed that the expression of all 12 genes decreased significantly from 12 to 72 h compared with the control groups, except GP1 at 72 h, when the expression of the TcTre2 gene was suppressed. The results also revealed different abnormal phenotypes, and the observed mortality rates ranged from 17 to 42 %. The qRT-PCR results showed that the expression of TPS, GS, two GP, CHS1a and CHS1b genes decreased significantly, while that of the CHS2 gene decreased or increased after RNAi after the five trehalases were silenced at 48 h. In addition, TPS gene expression decreased from 12 to 72 h after dsTcTre injection. CONCLUSIONS: These results demonstrate that silencing of any individual trehalase gene, especially Tre1-4 and Tre2 gene can lead to moulting deformities and a high mortality rate through the regulation of gene expression in the chitin biosynthesis pathway and may be a potential approach for pest control in the future.

Functional characterization of three trehalase genes regulating the chitin metabolism pathway in rice brown planthopper using RNA interference.

RNA interference (RNAi) is an effective gene-silencing tool, and double stranded RNA (dsRNA) is considered a powerful strategy for gene function studies in insects. In the present study, we aimed to investigate the function of trehalase (TRE) genes (TRE 1-1, TRE 1-2, and TRE-2) isolated from the brown planthopper Nilaparvata lugens, a typical piercing-sucking insect in rice, and investigate their regulating roles in chitin synthesis by injecting larvae with dsRNA. The results showed that TRE1 and TRE2 had compensatory function, and the expression of each increased when the other was silenced. The total rate of insects with phenotypic deformities ranged from 19.83 to 24.36% after dsTRE injection, whereas the mortality rate ranged from 14.16 to 31.78%. The mRNA levels of genes involved in the chitin metabolism pathway in RNA-Seq and DGEP, namely hexokinase (HK), glucose-6-phosphate isomerase (G6PI) and chitinase (Cht), decreased significantly at 72 h after single dsTREs injection, whereas two transcripts of chitin synthase (CHS) genes decreased at 72 h after dsTRE1-1 and dsTREs injection. These results demonstrated that TRE silencing could affect the regulation of chitin biosynthesis and degradation, causing moulting deformities. Therefore, expression inhibitors of TREs might be effective tools for the control of planthoppers in rice.