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Article

Identification of a Chitin Synthase Gene from Arma chinensis (Hemiptera: Pentatomidae) Under Temperature Stress

by
Dianyu Liu
1,2,
Zhihan Su
1,2,
Changjin Lin
2,
Wenyan Xu
2,
Xiaoyu Yan
1,2,
Yu Chen
1,2,
Yichen Wang
2,
Xiaolin Dong
1,* and
Chenxi Liu
2,*
1
College of Agriculture, Yangtze University, No. 1 Nanhuan Road, Jingzhou 434025, China
2
Sino-American Biological Control Laboratory, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, No. 2 Yuanmingyuan West Road, Haidian District, Beijing 100193, China
*
Authors to whom correspondence should be addressed.
Agronomy 2025, 15(9), 2157; https://doi.org/10.3390/agronomy15092157
Submission received: 5 August 2025 / Revised: 6 September 2025 / Accepted: 8 September 2025 / Published: 9 September 2025
(This article belongs to the Section Pest and Disease Management)
Browse Figures
Versions Notes

Abstract

Chitin synthase (CHS) is essential for maintaining exoskeletal integrity and environmental adaptability in insects. CHS genes are categorized into two types, CHS1 and CHS2. Hemipteran insects possess only the CHS1 gene due to the absence of a peritrophic matrix (PM) in their midgut. However, the identification and functional characterization of the CHS1 gene in Pentatomidae species have not been reported. This study reports the first identification of a CHS gene, ArmaCHS1, from the predatory stink bug, Arma chinensis, and investigates its role in response to temperature stress. The ArmaCHS1 open reading frame spans 4407 bp, encoding a protein of 1468 amino acids, with 14 transmembrane helices and seven N-glycosylation sites. Phylogenetic analysis confirmed its classification within the CHS1 clade, closely related to CHS1 from Halyomorpha halys. qRT-PCR analysis revealed that ArmaCHS1 is predominantly expressed in the exoskeleton and displays developmentally regulated expression (lowest in eggs, highest in adults). Temperature stress experiments demonstrated that ArmaCHS1 expression was significantly upregulated at low temperatures (12 °C, 19 °C) and markedly downregulated at high temperatures (33 °C, 40 °C). These findings indicate that ArmaCHS1 likely contributes to thermal adaptation in A. chinensis by modulating chitin biosynthesis, providing new insights into the environmental stress responses of beneficial predatory insects.

1. Introduction

Insects are the most abundant and widely distributed arthropods on Earth [1]. Chitin biosynthesis is essential for insect survival [2] as chitin forms the core scaffolding material for the exoskeleton [3], the tracheal cuticle, and the peritrophic membrane (PM) in the midgut [4]. These chitinous structures provide not only morphological and mechanical support, but also serve as protective barriers for maintaining physiological integrity and enhancing resistance against predation, pathogens, and physical injury.
Chitin is a linear aminopolysaccharide composed of β-1,4-linked N-acetyl-d-glucosamine (GlcNAc) and is the second most abundant biopolymer in nature, following cellulose. It is widely distributed in various organisms, including algae, fungi, nematodes, and arthropods [5,6]. In arthropods, chitin biosynthesis typically proceeds through three stages: conversion of trehalose to the amino sugar GlcNAc, activation of GlcNAc to UDP-GlcNAc, and polymerization into chitin [7]. This biosynthetic pathway involves a series of enzymes, including trehalase (TRE), hexokinase (HK), glucosamine-6-phosphate isomerase (G6PI), glutamine-fructose-6-phosphate aminotransferase (GFAT), glucosamine-6-phosphate N-acetyltransferase (GNA), phosphoacetylglucosamine mutase (PGM), UDP-N-acetylglucosamine pyrophosphorylase (UAP), and chitin synthase (CHS) [7,8].
Chitin synthase (CHS; UDP-GlcNAc: chitin 4-β-N-acetylglucosaminyltransferase, EC 2.4.1.16) is a glycosyltransferase belonging to the hexosyltransferase family [9,10]. It catalyzes the final step of chitin synthesis, converting UDP-N-acetyl-D-glucosamine into chitin and UDP [8]. In insects, CHS is classified into two isoforms: CHS1 and CHS2 [11]. CHS1 is expressed primarily in the epidermis underlying the cuticular exoskeleton and associated ectodermal cells, including those lining the tracheal [12,13]. In contrast, CHS2 is specifically expressed in midgut epithelium, where it synthesizes PM-associated chitin [14,15]. However, only one CHS gene (CHS1) has been identified in hemipteran insects, which lack a PM [16,17,18].
CHS plays critical roles in insect development and physiology, particularly in molting, diapause, and resistance to insecticides and fungal pathogens. Multiple studies have shown that CHS1 knockout significantly reduces chitin content in larvae and the exoskeleton, severely disrupting molting and adult emergence [11,19,20,21]. For instance, SfCHS1 is highly expressed in the cuticle of Spodoptera frugiperda (J.E. Smith, 1797), and silencing this gene via SfCHS1-siRNA nanocomplexes prevents most individuals from molting or pupating [22]. Similarly, silencing MysCHS1 in Mythimna separata (Walker, 1865) through dsRNA injection generates lethal larval phenotypes [23], while RNAi targeting AiCHS1 in Agrotis ipsilon (Hufnagel, 1766) causes abnormal pupation and mortality [11]. In Acyrthosiphon pisum (Harris, 1776) CHS1 interference significantly increases nymphal mortality and deformities [24]. Moreover, HvCHS1 knockdown impairs tracheal system development, resulting in thinner taenidial folds [13].
Recent studies reveal that insect CHS genes enhance resistance to chemical and fungal stressors. In S. frugiperda, CHS is regulated by miR-9a and miR-10482-5p increase tolerance to fipronil [25]. TRE1 and CHS1 upregulation in Culex pipiens subsp. pallens (Coquillett, 1898) enhances deltamethrin resistance [26]. Conversely, RNAi-mediated CHS1 knockdown in Nilaparvata lugens (Stål, 1854) significantly increases susceptibility to Cordyceps javanica and two insecticides (nitenpyram and dinotefuran) [27]. In addition, CHS1 suppression in Tribolium castaneum (Herbst, 1797) severely compromises host defense against fungal infection [28]. While the insect exoskeleton protects against environmental stress, few studies address chitin synthase gene expression under thermal stress. Research on Cacopsylla chinensis (Yang & Li, 1981) morph transition (summer-morph to winter-morph) reveals that CHS1 upregulation enhances host tolerance to low temperatures [29].
The family Pentatomidae (true bugs) exhibits remarkable ecological diversity, encompassing phytophagous, zoophagous (predatory), and omnivorous species distributed worldwide across various habitats [30]. Within this family, the genus Arma is particularly noteworthy for its members being exclusively zoophagous and serving as crucial natural enemies in agricultural and forest ecosystems [31]. Arma chinensis (Fallou, 1881) (Hemiptera: Pentatomidae) is an economically important generalist predator of over ten insect orders, including key Lepidopteran and Coleopteran pests, throughout its nymphal and adult stages. It is widely distributed across East Asia, including China, Mongolia, and the Korean Peninsula [31,32]. As a hemimetabolous insect, A. chinensis develops through three primary life stages (egg, nymph, and adult) with five nymphal instars. This predator is notably effective against various agricultural and forest pests [31,33] and exhibits high tolerance to environmental stressors such as elevated temperatures [34], starvation [35], and desiccation [36]. Furthermore, its comparative resilience to pyrethroid insecticides suggests potential compatibility with integrated pest management programs [37].
Despite extensive research on its biocontrol applications and physiological traits, the chitin synthase (CHS) genes in A. chinensis remain entirely uncharacterized. Given the critical role of the chitin-based exoskeleton in environmental stress resistance, we hypothesized that CHS expression modulates cuticular integrity under thermal stress. To test this, we conducted the first genome-wide identification and characterization of CHS genes in A. chinensis, representing the first such analysis within the family Pentatomidae, and investigated their expression patterns under varying temperature conditions. Our findings provide novel insights into the molecular mechanisms underlying thermal adaptation in this ecologically significant predator.

2. Materials and Methods

2.1. Experimental Insects

The Arma chinensis individuals used in this study were derived from a stable colony reared for over 70 generations in the Sino-American Biological Control Laboratory at the Institute of Plant Protection, Chinese Academy of Agricultural Sciences. Antheraea pernyi (Guérin-Méneville, 1855) pupae, which serve as the exclusive diet for all instars of A. chinensis, were obtained from a long-term supplier and stored at 4 °C to preserve freshness. The predators were cultured under controlled environmental conditions: temperature 26 ± 1 °C, relative humidity 65 ± 5% RH, and photoperiod 16L:8D. Visually screened healthy individuals exhibiting uniform body size and no morphological deformities were selected for bioassays.

2.2. Identification and Physicochemical Characterization of Chitin Synthase Genes in Arma chinensis

To identify CHS gene family members in Arma chinensis, annotated protein sequences of Halyomorpha halys (Stål, 1855) were retrieved from the National Center for Biotechnology Information (NCBI) database (Supplementary Table S1). These sequences served as queries for BLAST 2.11.0+ searches against a custom database of predicted A. chinensis proteins (GenBank accession: PRJNA660864), using an E-value cutoff < 1 × 10−5. Genomic coordinates (start/end positions) and chromosome lengths were extracted from the reference genome, and chromosomal localization maps were generated using TBtools-II (Toolbox for Biology, v1.108). Subsequently, protein domains were annotated using HMMER (v2.41.2) against the Pfam database. Motif analysis was conducted using the MEME web service (https://meme-suite.org/, accessed on 15 February 2025). All putative A. chinensis protein sequences were aligned with CHS sequences from nine representative insect species using ClustalW with default parameters. A maximum-likelihood phylogenetic tree was constructed in MEGA11 with 1000 bootstrap replicates (Table S2).
Physicochemical properties of Arma chinensis chitin synthase (ArmaCHS1), including molecular weight and isoelectric point, were determined using the ProtParam tool on the ExPASy server (https://www.expasy.org/, accessed on 20 February 2025). Hydrophobicity profiles were generated with ProtScale tool on the ExPASy server. Transmembrane helix predictions were performed with TMHMM-2.0, while signal peptide identification was conducted using SignalP-5.0 (https://services.healthtech.dtu.dk/, accessed on 24 February 2025). Glycosylation sites were analyzed using NetNGlyc 1.0 (https://services.healthtech.dtu.dk/service.php?NetNGlyc-1.0, accessed on 3 March 2025).

2.3. Sequence Alignment and Phylogenetic Analysis

Representative CHS protein sequences from six insect orders (Hemiptera, Lepidoptera, Hymenoptera, Diptera, Coleoptera, and Orthoptera) were retrieved from the NCBI database. To infer phylogenetic relationship, putative Arma chinensis CHS protein sequences were aligned with these orthologous sequences using MUSCLE under default parameters. A neighbor-joining phylogenetic tree was subsequently constructed in MEGA11 with 1000 bootstrap replicates (Table S3).

2.4. Analysis of Protein Three-Dimensional Structures

Homology modeling was employed to predict the three-dimensional structures of target genes. Structural models were generated using the SwissModel web server (https://swissmodel.expasy.org/, accessed on 7 March 2025) for CHS1 proteins from Arma chinensis, Halyomorpha halys, Nilaparvata lugens, Plutella xylostella (Linnaeus, 1767), and Bombyx mori (Linnaeus, 1758). The optimal predicted model for each target was selected based on confidence metrics, sequence identity, and query coverage. Structural conservation was evaluated using Visual Molecular Dynamics (VMD), with residue conservation quantified through root mean square deviation (RMSD) calculations.

2.5. Total RNA Extraction, Reverse Transcription, and qRT-PCR Validation

The expression levels of chitin synthase genes in A. chinensis were quantified using quantitative real-time PCR (qRT-PCR). For adult samples, 60 three-day-old post-eclosion females and 60 males were separately collected and divided into three biological replicates, with each replicate consisting of 20 insects of the same sex. After anesthesia with 75% ethanol, the exoskeleton (from both the anterior and posterior abdominal regions), midgut, fat body, and head (including antennae and mouthparts) were rapidly dissected under a stereomicroscope. All tissues were immediately placed in RNA stabilization reagent for preservation.
Additionally, for developmental stage analysis, each replicate consisted of 30 eggs, 10 individuals each of first- to fifth-instar nymphs, and 5 male and 5 female adults, with three independent replicates performed per stage. All samples were snap-frozen in liquid nitrogen and stored at −80 °C until RNA extraction.
Total RNA was extracted from all collected samples using the LanEasy Total RNA Kit. RNA quality was assessed via a Nano-300 Microspectrophotometer (Thermo Scientific, Waltham, MA, USA), with integrity verified by agarose gel electrophoresis. First-strand cDNA synthesis was performed by reverse transcription PCR (RT-PCR) using 5× SynScript™ II RT SuperMix (Tsingke Biotechnology Co., Beijing, China). Transcript-specific primers were designed using Primer 3 (v0.4.0, http://fokker.wi.mit.edu/primer3/, accessed on 26 April 2025) (Table S4). qRT-PCR was conducted on an Applied Biosystems QuantStudio™ 5 system with 20 μL reactions containing 2 μL cDNA, 10 μL 2× T5 Fast qPCR Mix (YEASEN, Shanghai, China), 0.4 μL forward/reverse primers each, and 7.2 μL nuclease-free water. The thermal cycling conditions were initial denaturation at 95 °C for 1 min; 40 cycles of 95 °C for 30 s, 60 °C for 3 s, and 72 °C for 10 s. Each assay included three technical replicates and four independent biological replicates, with dissociation curve analysis monitoring potential primer-dimer formation. Relative mRNA expression was calculated using the 2−ΔΔCt method with A. chinensis GAPDH used as the endogenous reference [38].

2.6. Statistical Analysis

All data are presented as the mean ± standard error of the mean (SEM). Differences among treatment groups were assessed for statistical significance using one-way analysis of variance (ANOVA). If a significant effect was found, multiple comparisons were performed using Tukey’s honestly significant difference (HSD) post hoc test. A value of p < 0.05 was considered statistically significant. The statistical analyses were performed using GraphPad Prism v8.0.1 (GraphPad Software, San Diego, CA, USA) and SPSS 24 (IBM Corp., Chicago, IL, USA). Graphs were constructed using GraphPad Prism.

3. Results

3.1. Identification and Physicochemical Characterization of CHS Genes in Arma chinensis

A CHS gene was identified from the A. chinensis genome and designated ArmaCHS1 based on its sequence similarity to known insect CHS1 genes (see below). The gene contains an open reading frame (ORF) of 4407 bp, encoding a polypeptide of 1468 amino acids. The predicted molecular weight of the encoded protein is 168.74 kDa, with an isoelectric point (pI) of 7.48. ArmaCHS1 has a grand average of hydropathicity of −0.061, indicating it is a hydrophilic protein. Chromosomal mapping localized ArmaCHS1 to chromosome 1 (chr1) (Figure 1). TMHMM-2.0 results showed that the ArmaCHS1 protein contains 14 transmembrane helices. Glycosylation site prediction revealed seven N-linked glycosylation sites in ArmaCHS1. No signal peptide was predicted (Figure S1).

3.2. Sequence Conservation and Phylogenetic Analysis of A. chinensis CHS

A maximum-likelihood phylogenetic tree was reconstructed using amino acid sequences of A. chinensis CHS alongside 19 orthologous CHS proteins from nine insect species: Halyomorpha halys, Nilaparvata lugens, Bombyx mori, Plutella xylostella, Locusta migratoria (Linnaeus, 1758), Apis cerana subsp. cerana, Tribolium castaneum, Drosophila melanogaster (Meigen, 1830), and Aedes aegypti (Linnaeus, 1762). Concurrently, comparative analysis of conserved domains and motifs, based on Pfam annotation, across these 10 insect CHS sequences revealed that A. chinensis CHS shares the conserved chitin-synth-2 domain and characteristic sequence motifs with these orthologs (Figure 1).
Multiple sequence alignment of A. chinensis CHS with 72 CHS sequences from 41 insect species across six orders was performed using MUSCLE, followed by the construction of a neighbor-joining phylogenetic tree encompassing all 42 species. The analysis revealed distinct evolutionary segregation into CHS1 and CHS2 clades. ArmaCHS1 was phylogenetically classified as a CHS1 isoform, demonstrating the highest sequence similarity to Halyomorpha halys CHS1. Notably, no CHS2 ortholog was identified in A. chinensis (Figure 2).

3.3. Three-Dimensional Structure Prediction and Conservation Analysis of ArmaCHS1

Given that protein function is often conserved through structural similarity, we investigated the 3D structures of CHS1 orthologs from Arma chinensis, Halyomorpha halys, Nilaparvata lugens, Plutella xylostella, and Bombyx mori. Predictions revealed a conserved N-terminal glycosyltransferase domain (GTD) architecture and multiple transmembrane helices, demonstrating analogous spatial organization of constituent elements across species (Figure 3A). Structural alignment showed root mean square deviation (RMSD) values ranging from 0.006 to 0.116 Å between orthologous CHS domains, confirming high structural conservation throughout evolution (Figure 3B).

3.4. Tissue-, Stage-, and Sex-Specific Expression Profiles of ArmaCHS1

The expression profile of ArmaCHS1 was examined across four developmental stages: eggs, nymphs, adult males, and adult females. The results showed that ArmaCHS1 expression was lowest in the egg stage, remained relatively low prior to the 3rd instar nymph stage, increased markedly from the 4th instar nymph, and was highest in both male and female adults (Figure 4A). We further determined the expression level of ArmaCHS1 in the midgut, thoracic exoskeleton, fat body, and head (including antennae and mouthparts) of male and female A. chinensis adults. The results indicate that ArmaCHS1 expression was highest in the exoskeleton, followed by the head, while expression in the fat body and midgut was relatively low (Figure 4B).

3.5. Temperature Stress Response of ArmaCHS1

To evaluate the thermal response of ArmaCHS1, adult A. chinensis were exposed to two low-temperature treatments (12 °C and 19 °C) and two high-temperature treatments (33 °C and 40 °C) in 7 °C increments, with 26 °C serving as the control. Insects were individually housed without access to food/water for 24 h, using three biological replicates per temperature (5 adults per replicate). The results show progressive downregulation of ArmaCHS1 expression with increasing temperature. Significantly reduced transcript levels (p < 0.05) were observed at 33 °C and 40 °C compared to controls. Conversely, both 12 °C and 19 °C exposures triggered significant upregulation of ArmaCHS1 (Figure 5).

4. Discussion

4.1. Gene Identification and Phylogenetic Analysis

In this study, we identified and characterized the CHS 1 gene (ArmaCHS1) from A. chinensis for the first time. The gene contains an ORF of 4407 bp, encoding a 1468-amino acid polypeptide, which is a hydrophilic protein. Phylogenetic analysis revealed that, consistent with other hemipteran insects, A. chinensis possesses only the CHS1 isoform and lacks the CHS2 one. ArmaCHS1 clusters closely with Halyomorpha halys CHS1 (Figure 2), a finding potentially linked to the absence of a PM in the hemipteran gut [14,16]. Conservation analysis showed that ArmaCHS1 contains the conserved chitin_synth_2 domain and typical CHS signature motifs, including EDR and QRRRW (Figure 1), which are considered essential for catalytic function [11,21]. Secondary structure prediction indicated it primarily consists of α-helices, extended strands, and random coils. ArmaCHS1 was predicted to have 14 transmembrane helices and seven N-glycosylation sites (Figure S1).
Similarly, comparable transmembrane topologies have been reported in other insect species. For example, 14 transmembrane regions were found in CHS1 of Diaphorina citri (Kuwayama, 1908) [39], while Agrotis ipsilon AiCHS1 has 16–17 transmembrane domains, and AiCHS2 has 16 [11]. Both CHS isoforms in Glyphodes pyloalis (Walker, 1859) possess 13 transmembrane regions [40], and Spodoptera litura (Fabricius, 1775) CHS contains 16 [21]. These transmembrane structures and glycosylation sites are likely involved in UDP-GlcNAc substrate recognition and transmembrane chitin transport in A. chinensis. Studies indicate that CHS protein tertiary structure comprises three parts: a large cytoplasmic soluble region, a transmembrane domain, and cytoplasmic N- and C-termini. Following the flexible N-terminus is the glycosyltransferase domain (GTD), which adopts a conserved GT-A fold characterized by a 10-stranded β-sheet flanked by several α-helices and additional β-sheets [41,42]. This architecture was also reflected in our 3D prediction results (Figure 3A).

4.2. Spatiotemporal Expression Patterns of ArmaCHS1

To elucidate the role of ArmaCHS1 in the growth and development of A. chinensis, we analyzed its spatiotemporal expression patterns using qRT-PCR. The results showed that ArmaCHS1 is expressed across all developmental stages. Temporally, its expression level gradually increased throughout development, with the lowest expression observed in the egg stage and the highest in the adult stage. During the nymphal stages, expression remained relatively low until the 3rd instar, after which it increased significantly in the 4th and 5th instars (Figure 4A). This developmental upregulation pattern is consistent with the CHS1 genes in Diaphorina citri and Locusta migratoria manilensis (Meyen, 1835) [16,43]. However, unlike D. citri, A. chinensis exhibited peak expression during the adult stage, which may be related to the highly sclerotized exoskeleton characteristic of heteropteran adults. Insect development and growth are marked by periodic molting, during which the cuticle is shed and replaced to accommodate internal growth. This process requires a substantial increase in chitin biosynthesis to form a new exoskeleton [13,44], likely explaining the progressive rise in ArmaCHS1 expression over time. Regarding tissue-specific expression, ArmaCHS1 showed the highest expression in the exoskeleton and the lowest in the gut and fat body, with head expression ranking second only to the exoskeleton (Figure 4B)—a pattern also observed in other insect species [11,21]. In most insects, the two CHS genes function separately: one synthesizes cuticular chitin, while the other produces chitin for the PM in the midgut. Due to the presence of heavily sclerotized cuticle and the absence of a PM in A. chinensis, ArmaCHS1 expression was minimal in the midgut but markedly elevated in the exoskeleton.

4.3. The Role of ArmaCHS1 Under Thermal Stress

The functional roles of CHS genes in insect development—including molting, diapause, and responses to insecticides and fungal immunity—have been extensively documented. Studies across multiple insect species indicate that knockdown of CHS1 reduces chitin levels throughout the larva and exoskeleton, severely impairing molting and eclosion [11,19,20,21]. The immune-related functions of CHS genes are primarily evidenced in pesticide resistance and antifungal responses [25,26,27,28]. However, their roles under temperature stress remain poorly explored. This study presents the first characterization of A. chinensis CHS gene expression under varying temperatures. Our results demonstrate that ArmaCHS1 expression gradually decreases as temperature rises, showing significantly lower levels under high-temperature conditions compared to controls, while exhibiting significantly higher expression under low-temperature conditions (Figure 5). We propose that under low-temperature conditions, Arma chinensis enhances chitin synthesis efficiency through elevated expression of chitin synthase, leading to increased cuticular thickness and improved cold tolerance. This pattern of upregulated CHS expression in response to cold is consistent with observations in Cacopsylla chinensis, where enhanced CHS expression under low temperatures correlated with thickening of the chitin-based exoskeleton [29]. In contrast, under high-temperature stress, insects often exhibit severe phenotypic impairments, such as cuticular lightening and exoskeletal softening [45], which are closely associated with cuticular integrity. These observations align with our results, and we speculate that reduced CHS expression leads to decreased chitin biosynthesis, thereby compromising exoskeleton formation. However, this hypothesized causal relationship between thermal suppression of CHS, reduced chitin content, and the observed phenotypic defects requires further validation through functional assays, such as RNA interference (RNAi). Furthermore, chitin metabolism is dynamically regulated by both synthesis and degradation. Notably, the expression pattern of chitinase—a key enzyme in chitin catabolism—shows an opposite trend to that of chitin synthase under thermal stress. For instance, ApCht10 in the Acyrthosiphon pisum (Harris, 1776) is significantly upregulated at 30 °C [46], and chitinase in Spodoptera frugiperda also exhibits increased expression under high temperatures [47]. The combined effect of reduced chitin synthesis and enhanced degradation may lead to a net loss of chitin, which could partly explain the reduced fitness of insects under high-temperature stress.
In recent years, RNAi has been widely employed to investigate CHS function across various insect species [13,20,24]. Knockdown of chitin synthase genes leads to notable phenotypic defects, including molting delay [24], eclosion failure [21], reduced pesticide resistance [26], and even mortality [39,48], demonstrating the crucial role of CHS in insect development and immunity. Unfortunately, this study did not utilize RNAi to validate the function of CHS1 under high-temperature stress, a limitation that remains to be addressed in future work. Furthermore, given the vital function of chitin synthase, chitin synthase inhibitors (CSIs)—such as nucleoside-peptides, benzoylureas (BPUs), oxazolines, and thiadiazines—have been extensively applied in pest control [49,50,51]. However, most of these inhibitors are broad-spectrum and may adversely affect non-target predatory insects, a risk that must be seriously considered as it could compromise the efficacy of biological control using natural enemies.

5. Conclusions

In this study, we conducted an in-depth analysis of the chitin synthase (CHS) gene in Arma chinensis. It is noteworthy that this research represents the first identification and characterization of the CHS gene in this species. ArmaCHS1 exhibited evolutionary conservation, and its expression increased gradually during development, with the highest levels detected in the exoskeleton and lower levels in the midgut and fat body. Furthermore, we investigated the expression patterns of ArmaCHS1 under high- and low-temperature stress, revealing a gradual decrease in its expression as the temperature rose. These expression patterns pave the way for future functional studies, such as employing RNAi knockdown to validate the specific role of CHS1 under temperature stress or utilizing proteomic approaches to confirm alterations in chitin biosynthesis. Additionally, our findings provide a theoretical foundation for the potential application of gene editing technologies to overexpress CHS1 for selecting heat-tolerant strains of A. chinensis.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy15092157/s1, Table S1: The probe sequences for the chitin synthase of Halyomorpha halys and the chitin synthase sequences of Arma chinensis; Table S2: Insect chitin synthase genes used for conservation analysis; Table S3: Insect chitin synthase genes for phylogenetic analysis; Table S4: The primer sequences for qRT-PCR; Figure S1: Prediction results for the ArmaCHS1 protein from Arma chinensis.

Author Contributions

Conceptualization: X.D. and C.L. (Chenxi Liu); Methodology: X.D. and D.L.; Validation: D.L. and C.L. (Changjin Lin); Formal analysis: D.L.; Investigation: D.L., C.L. (Changjin Lin), Z.S., W.X., X.Y., Y.W. and Y.C.; Data curation: D.L., C.L. (Changjin Lin), Z.S., W.X., X.Y., Y.W. and Y.C.; Writing—original draft preparation: D.L.; Writing—review and editing: X.D. and C.L. (Chenxi Liu); Funding acquisition: C.L. (Chenxi Liu); Supervision: X.D. and C.L. (Chenxi Liu); Project administration: C.L. (Chenxi Liu); Visualization: D.L. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the Commonwealth Scientific and Industrial Research Organization [CSIRO grant number 2024082216]. The sponsor played no role in any aspect of this study.

Data Availability Statement

The data reported in this study are available in the article and Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
PMthe peritrophic membrane
CHSChitin synthase
GlcNAcβ-1,4-linked N-acetyl-d-glucosamine
UDP-GlcNAcChitin 4-β-N-acetylglucosaminyltransferase
TRETrehalase
HKHexokinase
G6PIGlucosamine-6-phosphate isomerase
GFATGlutamine-fructose-6-phosphate aminotransferase
GNAGlucosamine-6-phosphate aminotransferase
PGMPhosphoacetylglucosamine mutase
UAPUDP-N-acetylglucosamine pyrophosphorylase
NCBINational Center for Biotechnology Information
RMSDRoot mean square deviation
SEMStandard error of the mean
qRT-PCRQuantitative Reverse-Transcription PCR
ORFOpen reading frame
RNAiRNA interference

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Figure 1. Conservation analysis and genomic distribution of chitin synthase genes in Arma chinensis. An unrooted phylogenetic tree was constructed using the maximum likelihood method in MEGA 11, with 1000 bootstrap replicates. Colored shading demarcates different CHS gene subtypes in insects. Conserved protein domains for each insect were annotated using Pfam via the NCBI database and are represented by green stripes. All motifs were determined using the MEME Suite based on the full-length amino acid sequences and are displayed as sequence logos, where a column corresponds to the information content of all amino acids at that position; within each column, the conservation of each residue is visualized by the relative height of the symbol representing the amino acid. The arrow points to the specific gene locus representing ArmaCHS1. The color gradient of the chromosomes, from light to dark, indicates increasing chromosomal gene density.
Figure 1. Conservation analysis and genomic distribution of chitin synthase genes in Arma chinensis. An unrooted phylogenetic tree was constructed using the maximum likelihood method in MEGA 11, with 1000 bootstrap replicates. Colored shading demarcates different CHS gene subtypes in insects. Conserved protein domains for each insect were annotated using Pfam via the NCBI database and are represented by green stripes. All motifs were determined using the MEME Suite based on the full-length amino acid sequences and are displayed as sequence logos, where a column corresponds to the information content of all amino acids at that position; within each column, the conservation of each residue is visualized by the relative height of the symbol representing the amino acid. The arrow points to the specific gene locus representing ArmaCHS1. The color gradient of the chromosomes, from light to dark, indicates increasing chromosomal gene density.
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Figure 2. Phylogenetic analysis of chitin synthase in Arma chinensis and insects from various orders. The unrooted phylogenetic tree was constructed using the neighbor-joining method in MEGA11 with 1000 bootstrap replicates. Clades are colored according to different chitin synthase subtypes. Labels are color-coded to represent insect orders. The right panel displays insects included in the phylogeny, arranged by order, along with their Latin names; the color labels correspond to those used in the phylogenetic tree. Image credits: the photo of Arma chinensis was obtained from our laboratory; Cotesia typhae was sourced from Passion entomologie (https://passion-entomologie.fr/cotesia-typhae-lutte-biologique1-2/, accessed on 2 September 2025); Holotrichia oblita was acquired from the National Animal Collection Resource Center (http://museum.ioz.ac.cn/species_detail.aspx?id=32349, accessed on 2 September 2025; images of all other species were retrieved from GBIF (https://www.gbif.org/, accessed on 2 September 2025).
Figure 2. Phylogenetic analysis of chitin synthase in Arma chinensis and insects from various orders. The unrooted phylogenetic tree was constructed using the neighbor-joining method in MEGA11 with 1000 bootstrap replicates. Clades are colored according to different chitin synthase subtypes. Labels are color-coded to represent insect orders. The right panel displays insects included in the phylogeny, arranged by order, along with their Latin names; the color labels correspond to those used in the phylogenetic tree. Image credits: the photo of Arma chinensis was obtained from our laboratory; Cotesia typhae was sourced from Passion entomologie (https://passion-entomologie.fr/cotesia-typhae-lutte-biologique1-2/, accessed on 2 September 2025); Holotrichia oblita was acquired from the National Animal Collection Resource Center (http://museum.ioz.ac.cn/species_detail.aspx?id=32349, accessed on 2 September 2025; images of all other species were retrieved from GBIF (https://www.gbif.org/, accessed on 2 September 2025).
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Figure 3. Three-dimensional structures of insect chitin synthase CHS1 gene and root mean square deviation (RMSD) matrix. (A) Protein three-dimensional structures of insect CHS1 genes. I: Arma chinensis; II, III: Halyomorpha halys; IV, V: Nilaparvata lugens; VI: Plutella xylostella; VII, VIII: Bombyx mori. IX: Structural alignment of chitin synthase domains across insect species; colors correspond to panels I–VIII, respectively. (B) Root mean square deviation (RMSD) matrix of CHS1 three-dimensional structures from different insects. The numerical values within the matrix indicate the RMSD values. The color gradient of the blocks on the left represents the relative structural similarity of CHS1 between the corresponding species, ranging from strong to weak.
Figure 3. Three-dimensional structures of insect chitin synthase CHS1 gene and root mean square deviation (RMSD) matrix. (A) Protein three-dimensional structures of insect CHS1 genes. I: Arma chinensis; II, III: Halyomorpha halys; IV, V: Nilaparvata lugens; VI: Plutella xylostella; VII, VIII: Bombyx mori. IX: Structural alignment of chitin synthase domains across insect species; colors correspond to panels I–VIII, respectively. (B) Root mean square deviation (RMSD) matrix of CHS1 three-dimensional structures from different insects. The numerical values within the matrix indicate the RMSD values. The color gradient of the blocks on the left represents the relative structural similarity of CHS1 between the corresponding species, ranging from strong to weak.
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Figure 4. Spatiotemporal expression profiles of ArmaCHS genes in Arma chinensis analyzed by quantitative real-time polymerase chain reaction (qPCR). (A) Expression of ArmaCHS genes across developmental stages. (B) Expression of ArmaCHS genes across different body tissues. The y-axis values in (B) are presented on a log10 scale. Different colors represent different developmental stages and tissues. The y-axis indicates expression levels. Data shown represent the mean relative expression levels ± standard error (SE). Data were analyzed by one-way ANOVA (p < 0.05), followed by Tukey HSD post-hoc test (n = 3). The Tukey HSD test was performed to compare all groups within the entire figure. Different lowercase letters above the error bars indicate statistically significant differences.
Figure 4. Spatiotemporal expression profiles of ArmaCHS genes in Arma chinensis analyzed by quantitative real-time polymerase chain reaction (qPCR). (A) Expression of ArmaCHS genes across developmental stages. (B) Expression of ArmaCHS genes across different body tissues. The y-axis values in (B) are presented on a log10 scale. Different colors represent different developmental stages and tissues. The y-axis indicates expression levels. Data shown represent the mean relative expression levels ± standard error (SE). Data were analyzed by one-way ANOVA (p < 0.05), followed by Tukey HSD post-hoc test (n = 3). The Tukey HSD test was performed to compare all groups within the entire figure. Different lowercase letters above the error bars indicate statistically significant differences.
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Figure 5. qPCR analysis of ArmaCHS gene expression profiles in Arma chinensis under different temperature treatments. (♀) Female, (♂) Male. Insects were exposed to each temperature for 24 h. Expression levels are shown on the y-axis. Data represent mean relative expression levels ± standard error (SE). Data were analyzed by one-way ANOVA (p < 0.05), followed by Tukey HSD post-hoc test (n = 3). Different lowercase letters above the error bars indicate statistically significant differences.
Figure 5. qPCR analysis of ArmaCHS gene expression profiles in Arma chinensis under different temperature treatments. (♀) Female, (♂) Male. Insects were exposed to each temperature for 24 h. Expression levels are shown on the y-axis. Data represent mean relative expression levels ± standard error (SE). Data were analyzed by one-way ANOVA (p < 0.05), followed by Tukey HSD post-hoc test (n = 3). Different lowercase letters above the error bars indicate statistically significant differences.
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Liu, D.; Su, Z.; Lin, C.; Xu, W.; Yan, X.; Chen, Y.; Wang, Y.; Dong, X.; Liu, C. Identification of a Chitin Synthase Gene from Arma chinensis (Hemiptera: Pentatomidae) Under Temperature Stress. Agronomy 2025, 15, 2157. https://doi.org/10.3390/agronomy15092157

AMA Style

Liu D, Su Z, Lin C, Xu W, Yan X, Chen Y, Wang Y, Dong X, Liu C. Identification of a Chitin Synthase Gene from Arma chinensis (Hemiptera: Pentatomidae) Under Temperature Stress. Agronomy. 2025; 15(9):2157. https://doi.org/10.3390/agronomy15092157

Chicago/Turabian Style

Liu, Dianyu, Zhihan Su, Changjin Lin, Wenyan Xu, Xiaoyu Yan, Yu Chen, Yichen Wang, Xiaolin Dong, and Chenxi Liu. 2025. "Identification of a Chitin Synthase Gene from Arma chinensis (Hemiptera: Pentatomidae) Under Temperature Stress" Agronomy 15, no. 9: 2157. https://doi.org/10.3390/agronomy15092157

APA Style

Liu, D., Su, Z., Lin, C., Xu, W., Yan, X., Chen, Y., Wang, Y., Dong, X., & Liu, C. (2025). Identification of a Chitin Synthase Gene from Arma chinensis (Hemiptera: Pentatomidae) Under Temperature Stress. Agronomy, 15(9), 2157. https://doi.org/10.3390/agronomy15092157

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