Identification, Characterization, and Functional Analysis of Chitin Synthase Genes in Glyphodes pyloalis Walker (Lepidoptera: Pyralidae).

Zuo-Min Shao,Fu-An Wu,Sheng Sheng,Xiao-Rui Zhang,Zhi-Xiang Liu,Yi-Jiangcheng Li,Jian-Hao Ding,Jun Wang

doi:10.3390/ijms21134656

Abstract

Glyphodes pyloalis Walker (G. pyloalis) causes significant damage to mulberry every year, and we currently lack effective and environmentally friendly ways to control the pest. Chitin synthase (CHS) is a critical regulatory enzyme related to chitin biosynthesis, which plays a vital role in the growth and development of insects. The function of CHS in G. pyloalis, however, has not been studied. In this study, two chitin synthase genes (GpCHSA and GpCHSB) were screened from our previously created transcriptome database. The complete coding sequences of the two genes are 5,955 bp and 5,896 bp, respectively. Expression of GpCHSA and GpCHSB could be detected throughout all developmental stages. Relatively high expression levels of GpCHSA occurred in the head and integument and GpCHSB was most highly expressed in the midgut. Moreover, silencing of GpCHSA and GpCHSB using dsRNA reduced expression of downstream chitin metabolism pathway genes and resulted in abnormal development and wings stretching, but did not affect normal pupating of larvae. Furthermore, the inhibitor of chitin synthesis diflubenzuron (DFB) was used to further validate the RNAi result. DFB treatment significantly improved expression of GpCHSA, except GpCHSB, and their downstream genes, and also effected G. Pyloali molting at 48 h (62% mortality rate) and 72 h (90% mortality rate), respectively. These results show that GpCHSA and GpCHSB play critical roles in the development and wing stretching in G. pyloalis adults, indicating that the genes are attractive potential pest control targets.

Highlights

Glyphodes pyloalis Walker is an important mulberry pest which is widely distributed throughout major mulberry growing areas of China, India, Korea, Japan, Pakistan, and Burma
Our study preliminarily clarifies the function of GpCHSA and GpCHSB in the chitin metabolism pathway and may facilitate the finding of new tools for managing G. pyloalis
GpCHSA and GpCHSB cDNA sequences have been uploaded to NCBI GenBank, and accession numbers are MN915086 and MN915087, respectively

Summary

Introduction

Glyphodes pyloalis Walker is an important mulberry pest which is widely distributed throughout major mulberry growing areas of China, India, Korea, Japan, Pakistan, and Burma This insect damages sericulture by feeding on mulberry and by transmitting viruses to the silkworm [1]. CHS plays an important role in insect growing development by regulating molting and cuticle regeneration. RNA interference (RNAi) is a useful tool used to analyze gene function in different organisms by delivering gene-specific, double-stranded RNA (dsRNA). It is known as a promising approach for pest control [24]. Clarification of the function of CHS genes in G. pyloalis may contribute to identify candidate molecular targets for control of the pest. Our study preliminarily clarifies the function of GpCHSA and GpCHSB in the chitin metabolism pathway and may facilitate the finding of new tools for managing G. pyloalis

Characterization of the GpCHSA and GpCHSB Sequences

Spatio-Temporal Expression Patterns of GpCHSA and GpCHSB

Materials and Methods

Leaf Dip Bioassay