Trehalose-6-phosphate synthase regulates chitin synthesis in Mythimna separata

Abstract

Trehalose is a substrate for the chitin synthesis pathway in insects. Thus, it directly affects chitin synthesis and metabolism. Trehalose-6-phosphate synthase (TPS) is a crucial enzyme in the trehalose synthesis pathway in insects, but its functions in Mythimna separata remain unclear. In this study, a TPS-encoding sequence in M. separata (MsTPS) was cloned and characterized. Its expression patterns at different developmental stages and in diverse tissues were investigated. The results indicated that MsTPS was expressed at all analyzed developmental stages, with peak expression levels in the pupal stage. Moreover, MsTPS was expressed in the foregut, midgut, hindgut, fat body, salivary gland, Malpighian tubules, and integument, with the highest expression levels in the fat body. The inhibition of MsTPS expression via RNA interference (RNAi) resulted in significant decreases in the trehalose content and TPS activity. It also resulted in significant changes in Chitin synthase (MsCHSA and MsCHSB) expression, and significantly decrease the chitin content in the midgut and integument of M. separata. Additionally, the silencing of MsTPS was associated with a significant decrease in M. separata weight, larval feed intake, and ability to utilize food. It also induced abnormal phenotypic changes and increased the M. separata mortality and malformation rates. Hence, MsTPS is important for M. separata chitin synthesis. The results of this study also suggest RNAi technology may be useful for enhancing the methods used to control M. separata infestations.

Keywords: Mythimna separata; RNAi; chitin; trehalose; trehalose-6-phosphate synthase.

FIGURE 1

Spatiotemporal MsTPS expression pattern in different developmental stages (A) and in diverse tissues (B) of M. separata. The qRT-PCR data for MsTPS were analyzed using the 2^−ΔΔCT method to determine relative expression levels, which were calculated as the mean ± SE. The expression data were normalized on the basis of the geometric mean of the expression of two reference genes (encoding β-actin and glyceraldehyde-3-phosphate dehydrogenase). The GraphPad Prism software was used for the data analysis. Different letters indicate significant differences (p < 0.05) according to the Tukey’s test.

FIGURE 2

Expression of MsTPS at different post-RNAi time-points. The relative MsTPS expression levels were determined by qRT-PCR and the 2^−ΔΔCT data analysis method. The expression data are provided as the mean ± SE. Data were normalized against the expression data for two reference genes (encoding β-actin and glyceraldehyde-3-phosphate dehydrogenase). Statistical analyses were performed using the GraphPad Prism software. Statistically significant differences by t-test at same treatment time shown as asterisks (*p < 0.05, **p < 0.01, ***p < 0.001, ns > 0.05).

FIGURE 3

M. separata TPS activity (A) and trehalose content (B) at different time-points after the MsTPS RNAi treatment. Data are presented as the mean ± SE. Statistical analyses were performed using the GraphPad Prism software. Statistically significant differences by t-test at same treatment time shown as asterisks (*p < 0.05, **p < 0.01, ***p < 0.001, ns > 0.05).

FIGURE 4

Effect of the silencing of MsTPS on chitin synthase gene expression and the chitin content. (A) Expression of MsCHSA at different time-points after the MsTPS RNAi treatment. (B) Expression of MsCHSB at different time-points after the MsTPS RNAi treatment. (C) Chitin content in the M. separata integument at different time-points after the MsTPS RNAi treatment. (D) Chitin content in the M. separata midgut at different time-points after the MsTPS RNAi treatment. Relative MsTPS expression levels were determined by qRT-PCR and the 2^−ΔΔCT data analysis method. Expression data are provided as the mean ± SE. Data were normalized against the expression data for two reference genes (encoding β-actin and glyceraldehyde-3-phosphate dehydrogenase). Statistical analyses were performed using the GraphPad Prism software. Statistically significant differences by t-test at same treatment time shown as asterisks (*p < 0.05, **p < 0.01, ***p < 0.001, ns > 0.05).

FIGURE 5

Effect of the silencing of MsTPS on M. separata mortality (A), developmental time (B), and weight (C). Data are presented as the mean ± SE. Statistically significant differences by t-test at same treatment time shown as asterisks (*p < 0.05, **p < 0.01, ***p < 0.001, ns > 0.05).

FIGURE 6

Effects of the silencing of MsTPS on M. separata molting (A) and pupation (B). (A) 1, 3, and 5: larvae were unable to molt normally before dying; 2: larvae retained the cuticle from the fourth instar larvae; 4: larvae were too small after molting. (B) 1: larvae failed to pupate; 2: pupae were too small.