Chitin deacetylase 1 and 2 are indispensable for larval–pupal and pupal–adult molts in Heortia vitessoides (Lepidoptera: Crambidae)

Highlights

• CDAs are involved in the enzymatic alteration of chitin via deacetylation.

• We identified and characterized Group I CDAs from H. vitessoides.

• 20E increased the expression levels of HvCDA1 and HvCDA2.

• Deformed pupae and adults were observed after HvCDA1 and HvCDA2 silencing.

• The survival rates decreased during the larval–pupal and pupal–adult transitions.

Abstract

Heortia vitessoides Moore is a notorious defoliator of Aquilaria sinensis (Lour.) Gilg trees. Chitin deacetylases (CDAs) catalyze the N-deacetylation of chitin, which is a crucial process for chitin modification. Here, we identified and characterized HvCDA1 and HvCDA2 from H. vitessoides. HvCDA1 and HvCDA2 possess typical domain structures of CDAs and belong to the Group I CDAs. HvCDA1 and HvCDA2 were highly expressed before and after the larval–larval molt. In addition, both exhibited relatively high mRNA expression levels during the larval–pupal molt, the pupal stage, and the pupal–adult molt. HvCDA1 and HvCDA2 transcript expression levels were highest in the body wall and relatively high in the larval head. Significant increases in the HvCDA1 and HvCDA2 transcript expression levels were observed in the larvae upon exposure to 20-hydroxyecdysone. RNA interference-mediated HvCDA1 and HvCDA2 silencing significantly inhibited HvCDA1 and HvCDA2 expression, with abnormal or nonviable phenotypes being observed. Post injection survival rates of the larvae injected with dsHvCDA1 and dsHvCDA2 were 66.7% and 46.7% (larval–pupal) during development and 23.0% and 6.7% (pupal–adult), respectively. These rates were significantly lower than those of the control group insects. Our results suggest that HvCDA1 and HvCDA2 play important roles in the larval–pupal and pupal–adult transitions and represent potential targets for the management of H. vitessoides.

Introduction

Aquilaria sinensis (Lour.) Gilg (Malvales: Thymelaeaceae), an economically important evergreen tree, is well distributed in southeast Asia and southern China, including the Hainan, Guangdong, Guangxi, Fujian, Yunnan, and Taiwan provinces (Qiao et al., 2018; Cheng et al., 2018a). A. sinensis is listed as a rare and endangered Chinese plant and produces valuable agarwood, which is extensively used in traditional medicine, the incense industry, and in religious ceremonies (Jin et al., 2016; Cheng et al., 2017). Heortia vitessoides Moore (Lepidoptera: Crambidae) is a notorious defoliator of A. sinensis. The pest annually produces up to seven or eight overlapping generations in southern China and completely defoliates A. sinensis, causing severe economic losses (Qiao et al., 2012). Currently employed strategies to control H. vitessoides primarily involve spraying with conventional insecticides, such as avermectin, spinosad, and trichlorfon (Liang et al., 2019). However, overreliance on chemical control has led to the pest developing resistance to various insecticides (Cheng et al., 2018b). To date, no study has reported sustainable and effective methods for controlling H. vitessoides.

Chitin, a high-molecular-weight N-acetylglucosamine polymer, is a major component of the exoskeletons of insects as well as the lining of the trachea and peritrophic matrix, which is located in the midgut (Merzendorfer and Zimoch, 2003; Merzendorfer, 2006). In some insect species, chitin accounts for approximately 60% of the dry weight, thereby highlighting its importance in the growth, development, and survival processes (Doucet and Retnakaran, 2012). The growth and development of insects relies on rigorous control of chitin metabolism (Cohen, 2001). In contrast, chitin is absent in vertebrates (Wu et al., 2019a). Therefore, enzymes involved in chitin synthesis, degradation, and modification are attractive targets for disrupting insect growth because this approach would be safe for vertebrates. In insects, enzymes that catalyze chitin degradation can be classified into two major categories; chitinases (CHTs) (which hydrolyze chitin into various small N-acetylglucosamine oligomers) and N-acetylglucosaminidases (NAGs) (which catalyze the nonreducing end of chitin into N-acetylglucosamine monomers) (Kramer and Muthukrishnan, 2005). Chitin deacetylases (CDAs) are involved in the enzymatic alteration of chitin via deacetylation (Liu et al., 2019; Yu et al., 2019; Zhang et al., 2019a, Zhang et al., 2019b).

CDAs (EC 3.5.1.41) convert chitin into chitosan, a polymer comprising β-1,4-linked D-glucosamine residues. CDAs belong to the carbohydrate esterase family 4 (CE-4) in the carbohydrate-active enzymes database (http://www.cazy.org) (Tsigos et al., 2000). Insect CDAs have been assigned to five groups (Groups I–V) based on their sequence homology and domain organization. Group I comprises CDA1 and CDA2, which contain a chitin-binding peritrophin-A domain (ChBD), a low-density lipoprotein receptor class A (LDLa) domain, and a polysaccharide deacetylase-like catalytic domain with five conserved motifs (Dixit et al., 2008). Group I CDAs have been studied in several insect species. In Drosophila melanogaster, mutations in CDA1 (serpentine, serp) or CDA2 (vermiform, verm) lead to limited embryonic tracheal tube elongation (Luschnig et al., 2006; Wang et al., 2006) and affect larval cuticle development (Gangishetti et al., 2012). Double-stranded RNA (dsRNA)-mediated knockdown of Group I CDA genes can lead to: developmental disturbances and nonviable phenotypes in Tribolium castaneum (Arakane et al., 2009); delayed pupation in Bombyx mori (Zhang et al., 2019a, Zhang et al., 2019b); molt failure, dysplasia, and increased mortality in Nilaparvata lugens (Xi et al., 2014), Locusta migratoria (Yu et al., 2016a), Leptinotarsa decemlineata (Wu et al., 2019a; Wu et al., 2019b), Choristoneura fumiferana (Quan et al., 2013), and Stegobium paniceum (Yang et al., 2018). These results suggest that Group I CDAs are potential targets for RNA interference (RNAi)-mediated and environmentally friendly control strategies.

In the present study, we only focused on Group I CDAs in H. vitessoides. We first identified HvCDA1 and HvCDA2 and evaluated their transcript expression levels in several developmental stages and larval tissues. Then, we examined their transcription patterns in response to treatment with 20-hydroxyecdysone (20E). Subsequently, we knocked out HvCDA1 and HvCDA2 using RNAi to investigate the in vivo effects of HvCDA1 and HvCDA2 on larval–pupal and pupal–adult molting in H. vitessoides.