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19 February 2026

14 Pages

Effects of Isaria cateniannulata on Enzyme Activities and Chitinase Genes in Tetranychus urticae

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1
Research Center of Buckwheat Industry Technology, College of Life Science, Guizhou Normal University, Guiyang 550025, China
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College of Life Science, Guizhou Normal University, Guiyang 550025, China
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School of International Education, Guizhou Normal University, Guiyang 550025, China
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Author to whom correspondence should be addressed.
J. Fungi2026, 12(2), 151;https://doi.org/10.3390/jof12020151
This article belongs to the Section Fungi in Agriculture and Biotechnology
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Abstract

Tetranychus urticae is a globally important economic pest mite. Isaria cateniannulata can infect the mite through its conidial penetration of the cuticle, ultimately leading to host mortality; however, the immune mechanisms involving enzyme activity systems and chitinase genes of T. urticae during this process remain unclear. In this study, T. urticae infected with I. cateniannulata was used as experimental material to analyze changes in antioxidant and detoxification enzyme activities during infection. In addition, the expression patterns of six chitinase genes were analyzed, and significantly upregulated genes were selected for bioinformatics analysis and functional verification. The results showed that infection with I. cateniannulata enhanced the activity of defense-related enzymes in T. urticae, with Catalase (CAT) and mixed function oxidase (MFO) playing dominant roles. All six chitinase genes were activated, among which TuCHT7 and TuCHT12 were significantly upregulated at 24 h post-infection, and then TuCHT7 gradually declined. Whereas TuCHT12 maintained a sustained and stable induction pattern, TuCHT10 was suppressed, while the other genes exhibited transient expression. Feeding dsTuCHT12 suppressed the expression of this gene within 72 h, with significant suppression observed at 48 h. At this time, the expression levels of TuCHT8, TuCHT9 and TuCHT10 genes were activated, whereas TuCHT7 and TuCHT11 were suppressed. The combined application of dsCHT12 and I. cateniannulata increased the mortality of T. urticae by 3.19-fold and reduced egg production by 52.8%. This study preliminarily revealed the defense response mechanism of T. urticae against I. cateniannulata infection and provides a theoretical basis for eco-friendly pest control based on the combined application of RNAi and arthropodpathogenic fungi.

1. Introduction

Tetranychus urticae (Koch) is a globally important economic pest mite that can damage vegetables, fruits, food crops and other plants belonging to 250 families, more than 1400 species [1]. It is considered to be one of the most difficult pests to control because of its short generation cycle, high reproductive capacity, and strong potential for developing resistance [2]. Although chemical control remains the most effective method, its intensive and long-term application has resulted in several critical problems, including the rapid development of resistance in T. urticae, pesticide residues in agricultural products, and adverse effects on non-target organisms and the environment [3], thereby limiting the long-term effectiveness of chemical-based control strategies. In contrast, microbial acaricides can avoid the above issues while effectively controlling mites, making them a current research focus [4]. Among them, I. cateniannulata is an entomopathogenic fungus capable of infecting mites by penetrating the cuticle, proliferating within the host, and disrupting normal physiological processes, ultimately leading to host mortality.
In the research and application of microbial acaricides, entomopathogenic fungi play an important role. Among them, Isaria cateniannulata (Z. Q. Liang) Samson et Hywel-Jones, also known as Cordyceps cateniannulata, belongs to the genus Cordyceps in the family Cordycipitaceae [5] and is considered one of the most potent microbial acaricides. It has been reported to be lethal to various mite species and negatively affects the biological performance, including T. urticae [6], Panonychus citri (McGregor, 1916) [7], and the flat mites Neostigmata tenuicollis (Zhang, 1985) [8]. It primarily colonizes mites and ultimately causes mite mortality through a series of processes: conidial adhesion to the mite cuticle, germination to form infection pegs, and secretion of various hydrolytic enzymes that disrupt the cuticular structure [9].
During infection by entomopathogenic fungi, the mite accumulates high levels of reactive oxygen species (ROS) and harmful metabolites, which cause cellular damage. To counteract such damage, the host typically activates antioxidant and detoxification enzyme systems to remove excess ROS and harmful metabolites [10]. Specifically, catalase (CAT) catalyzes the decomposition of hydrogen peroxide (H2O2) into water and oxygen, preventing the toxic effects of excessive H2O2 on the organism. Peroxidase (POD) degrades excess H2O2, other toxic compounds and catalyzes the oxidative decomposition of peroxide to maintain physiological homeostasis [11]. In addition, detoxification enzymes in mites, glutathione S-transferases (GSTs), can catalyze the conjugation of electrophilic groups of toxic substances with the thiol groups of reduced glutathione, enhancing the hydrophobicity of toxins to facilitate their excretion [12]. Mixed-function oxidases (MFOs) participate in the transformation of exogenous toxins and endogenous metabolites through reactions such as hydroxylation and epoxidation, thereby mitigating damage caused by entomopathogenic fungal infection [13,14].
In addition to antioxidant and detoxification enzymes, chitinases play an important role in innate immune defense by regulating chitin synthesis and degradation in the mite cuticle [15,16,17]. In particular, chitinases have been shown to function in pathogen defense or under environmental stress in various insect orders, including Coleoptera [18], Diptera [19,20], Hemiptera [21,22] and Lepidoptera [23]. However, there is still a lack of systematic research on the changes in enzyme activity and the response characteristics of chitinase genes during the immune process of T. urticae against I. cateniannulata and their functional roles in fungal pathogenesis.
Therefore, this study aims to analyze the changes in the activities of protective enzymes and detoxification enzymes, as well as the expression patterns of six chitinase genes (TuCHT7, TuCHT8, TuCHT9, TuCHT10, TuCHT11, and TuCHT12) in the process of I. cateniannulata infecting T. urticae. The findings will preliminarily elucidate the immune mechanisms of T. urticae against I. cateniannulata, laying a foundation for the development of I. cateniannulata as a biological acaricide.

2. Materials and Methods

2.1. Rearing of Test Mites and Collection of Age-Synchronized Adult Females

Test mites: The test T. urticae mites were reared in the insect laboratory of Buckwheat Engineering Technology Research Center, College of Life Sciences, Guizhou Normal University, China under the following conditions: temperature 25 ± 1 °C, relative humidity 80 ± 5%, and photoperiod L:D = 16:8.
Age-synchronized adult females: In plastic insect-rearing boxes (19 × 14 × 11.5 cm), absorbent cotton soaked in sterile water, double-layered filter paper, and one fresh buckwheat leaf (approximately 3 × 7 cm) was laid flat. Thirty adult female mites were transferred onto each leaf. After oviposition for 24 h, the adult females were removed, leaving only the eggs. The plastic boxes containing the eggs and fresh leaves were then placed in the incubator (temperature 25 ± 1 °C, relative humidity 80 ± 5%, photoperiod L:D = 16:8). Developmental progress was checked daily, and fresh leaves were replaced when the original leaf started to wilt or was consumed. After 10–12 d, newly emerged adult females were collected as age—synchronized individuals for subsequent experiments.

2.2. Culture of the Tested Strains and Preparation of Conidial Suspension

The tested fungal strain 2019BY-4 (preserved in the Buckwheat Research Center of Guizhou Normal University, China) was inoculated into a conical flask containing 150 mL of potato dextrose broth (PDB) [24] and cultured on a constant-temperature shaker at 150 r/min and 25 °C for 5 days. The resulting fungal suspension was filtered through double-layer filter paper to obtain a blastospore suspension. Conidial concentration was determined using a hemocytometer, and the suspension was diluted with sterile water to a final concentration of 1 × 108 conidia/mL for subsequent use.

2.3. Collection of T. urticae Samples After I. cateniannulata Infection

A spraying method was employed to infect mites. Using a throat sprayer (type A), 0.08 mL of I. cateniannulata conidial suspension (1 × 108 conidia/mL) was sprayed onto adult female mites on leaves in the rearing device described in Section 2.1. Samples were collected at 4, 12, 24, 36 and 48 h post-treatment. At each time point, 30 live age-synchronized adult female mites were randomly selected and placed in a 1.5 mL centrifuge tube for subsequent analysis. Control groups were sprayed with sterile water under identical conditions. Three replicates were set at each time point for the treatment and the control groups. All samples were immediately frozen in liquid nitrogen and stored in a refrigerator at −80 °C for use.

2.4. Determination of the Enzyme Activity in T. urticae

Samples treated with I. cateniannulata and sterile water were collected at 4, 12, 24, 36 and 48 h as described in Section 2.3, with thirty live adult female mites used per sample. The enzyme extract buffer (Suzhou Keming Biotechnology Co., Ltd., Suzhou, China) was added at a mass-to-volume ratio of 1:100, and the samples were fully homogenized under ice-bath conditions. The homogenates were centrifuged at 4 °C and 8000× g for 10 min, and the supernatants were used for enzyme activity assays. All enzyme activities were determined using commercial assay kits (Suzhou Keming Biotechnology Co., Ltd., Suzhou, China) according to the manufacturer’s instructions. Protein content was determined using a commercial protein assay kit (Suzhou Keming Biotechnology Co, Ltd., Suzhou, China). Enzyme activities were expressed per unit protein to standardize comparisons among samples and treatments. POD activity was determined based on changes in absorbance at 470 nm in the presence of H2O2. CAT activity was determined according to the decomposition rate of H2O2 at 240 nm. GST activity was calculated by detecting the rate of increase in absorbance at 340 nm of the conjugate product formed by the binding of GSH and CDNB. MFO activity was evaluated by measuring changes in absorbance at 400 nm associated with the formation of p-nitrophenol from p-nitroanisole.

2.5. RNA Extraction from T. urticae

Samples treated with I. cateniannulata or sterile water at 4, 12, 24, 36, and 48 h were collected as described in Section 2.3, with thirty live adult female mites used per sample. Total RNA was extracted from T. urticae using TaKaRa MiniBEST Universal RNA Extraction Kit (Tiangen, Beijing, China). The integrity of RNA was detected by 1% agarose gel electrophoresis, and RNA concentration and purity were determined by micro-spectrophotometer. Only RNA samples with an OD260/280 ratio between 1.80 and 2.20 were used for subsequent experiments.

2.6. Analysis of Chitinase Gene Expression in T. urticae

Total RNA obtained in Section 2.5 was reverse-transcribed into cDNA using the PrimeScript RT Master Mix Perfect Real Time Kit (Tiangen, Beijing, China). Gene-specific primers for RT-qPCR were designed using Primer5 software based on gene sequences obtained from the T. urticae whole genome database (https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_000239435.1, accessed on 8 November 2025) (Table 1). Gene expression levels were normalized using reference genes validated by Yang [25]. RT-qPCR was performed using TB Green Premix Ex Taq TM II (Tli RNase H Plus) (Takara, Dalian, China) on the Bio-Rad CFX Connect Real-Time PCR system. The specificity and amplification efficiency of PCR primers were analyzed by the melting curve. All reactions were conducted with three biological replicates. The results of real-time quantitative PCR were analyzed by the 2−ΔΔct method [26] to evaluate the gene expression level.
Table 1. Primer sequences for qPCR and dsRNA experiments in Tetranychus urticae.

2.7. Cloning of Highly Expressed Genes

Based on the sequence of highly expressed genes obtained in Section 2.6, the coding sequence (CDS) fragments of target genes were cloned. Gene-specific primers (see Table 1) were designed using Primer 5 software. PCR amplification was performed using the first-strand cDNA in Section 2.4 as a template and TaKaRa Premix Taq enzyme (RR902Q) (Tiangen, Beijing, China). The PCR products, verified by 1% agarose gel electrophoresis, were subjected to gel extraction using SanPrep Column DNA Gel Extraction Kit (B518131-0050) (Sangon, Shanghai, China) and ligated into the pGM-T vector (Tiangen, Beijing, China). The recombinant vector was transformed into DH5α competent cells (Tiangen, Beijing, China) and positive clones were sent to Sangon Biotech Co., Ltd. (Shanghai, China) for sequencing. The obtained cloned gene sequences were aligned with the T. urticae reference genome (GenBank:GCA_000239435.1) to verify the correctness of the cloning results.

2.8. Bioinformatics Analysis of Highly Expressed Genes

The physicochemical properties of the proteins, including amino acid composition, molecular weight and isoelectric point, were calculated by the ExPASy ProtParam tool. The open reading frames (ORFs) of the target genes were analyzed using DNAMAN 8.0 software, and each ORF was translated into a protein sequence for multiple sequence alignment. The phylogenetic tree was constructed using MEGA7 software based on the Neighbor-Joining (NJ) method, and the reliability of each branch was evaluated by Bootstrap analyses with 1000 replicates.

2.9. RNAi of Highly Expressed Genes and Its Effects on Physiological Parameters of T. urticae

According to the cloned sequence of the highly expressed genes, gene-specific primers containing T7 RNA polymerase promoter sequences were designed using Primer5 (Table 1). Target fragments were amplified by PCR, and purified products were used as templates to synthesize double-stranded RNA (dsRNA) using the TranscriptAid T7 High Yield Transcription Kit (Thermo Scientific, Vilnius, Lithuania). Buckwheat leaves were cut into leaf disks (3 cm × 7 cm), and dried at 60 °C for 1 min. Each leaf disk was pipetted with 100 μL of dsRNA suspension (200 ng/μL) on the adaxial surface and allowed to absorb for 4 h. The disks were then placed on a rearing device described in Section 2.1, with the abaxial surface facing down, to maintain humidity.
Thirty adult female mites starved at room temperature for 24 h were transferred per leaf. Four experimental groups were established: (1) RNAi treatment group (dsRNA + I. cateniannulata sprayed at 1 × 108 conidia/mL), (2) RNAi control group (dsGFP + I. cateniannulata sprayed at 1 × 108 conidia/mL), (3) conidial treatment group (I. cateniannulata sprayed at 1 × 108 conidia/mL) and (4) blank control group (sterile water). The mortality of mites and the number of eggs laid were recorded daily for 5 consecutive days. Each treatment consisted of three biological replicates.

2.10. Statistical Analysis

Basic data processing and graphing were performed using Excel 2010. Statistical analyses were conducted using SPSS 24.0 software. Differences between the two groups were analyzed using independent-samples t-tests and comparisons among multiple groups were performed using Tukey’s test. Differences were considered statistically significant at p < 0.05 and highly significant at p < 0.01.

3. Results and Analysis

3.1. Effects of I. cateniannulata Infection on Enzyme Activities in T. urticae

After I. cateniannulata infection, significant changes in enzyme activities were observed in T. urticae. Both CAT (Figure 1A) and MFO activities (Figure 1D) were higher than those in the control group. CAT (Figure 1A) and GST activities (Figure 1C) both showed a trend of first increasing and then decreasing with time, reaching the highest levels at 12 h and 36 h, which were 3-fold and 1.3-fold higher than those in the control group, respectively. POD activity (Figure 1B) decreased gradually with time and was lower than that in the control group after 24 h. MFO activity (Figure 1D) decreased first and then increased with time, and was significantly higher than that in the control group at 4 h, 12 h and 48 h.
Figure 1. Effects of Tetranychus urticae enzyme activities after infection with Isaria cateniannulata at a final concentration of 1 × 108 conidia/mL. Note: EF represents Isaria cateniannulata, CK represents sterile water control (A) CAT, (B) POD, (C) GST, (D) MFO.

3.2. Effects of I. cateniannulata Infection on the Expression Patterns of Chitinase Genes in T. urticae

After I. cateniannulata infection, significant changes in the transcription levels of chitinase genes were observed in T. urticae (Figure 2). The expression trends of TuCHT7 and TuCHT12 were generally similar, both increased first and then decreased with time. Their expression levels were higher than those in the control group after 24 h, reaching the peak values at 24 h and 36 h, which were 1.58 ± 0.43-fold and 1.29 ± 0.04-fold higher than those in the control group, respectively. After reaching the peak, TuCHT7 decreased rapidly, while TuCHT12 maintained a continuous and stable induction pattern. TuCHT8 and TuCHT9 genes showed transient induction characteristics, with transient expression peaks at 48 h and 12 h, which were 2.33 ± 0.02-fold and 2.24 ± 0.7-fold higher than those in the control group, respectively. In contrast, TuCHT11 showed an extremely significant upregulation at 36 h, while TuCHT10 was continuously downregulated throughout the infection process. Overall, despite some variability at specific time points (TuCHT9 at 12 h and TuCHT11 at 48 h), the expression trends clearly indicate that chitinase genes are induced following I. cateniannulata infection.
Figure 2. Expression patterns of chitinase genes in T. urticae after I. cateniannulata infection. Note: the expression level of the control group was set as 1 (dashed line in the figure), and the relative expression levels of the treatment group were compared with it for graphing. Tukey’s test was used to analyze the significant differences among multiple sets of data at the 0.05 level. Data are presented as mean ± standard error (SEM) (n = 3). * indicates p < 0.05, ** indicates p < 0.01.

3.3. Cloning and Sequence Analysis of TuCHT12 Gene

In the above process, the expression level of TuCHT12 remained stable after reaching the peak, suggesting that it may play a key regulatory role in the immune defense process of mites against entomopathogenic fungal infection. Therefore, TuCHT12 was selected as the candidate gene for functional verification.
By cloning the coding sequence of TuCHT12 and conducting bioinformatics analysis on it, it was found that the ORF of the gene was 1020 bp in length, which encodes 339 amino acids. The molecular weight of the protein is 48.69 kDa, and the theoretical isoelectric point is 6.50. These results suggest that the protein encoded by TuCHT12 is a putative secreted chitinase (Figure 3A, Table 2). By constructing a phylogenetic tree (Figure 3B), it can be seen that the protein encoded by TuCHT12 is clustered with the chitinase 10 of Drosophila melanogaster within the same clade, and the amino acid sequence identity between the two is as high as 80%. It is known that chitinase 10 of Drosophila melanogaster belongs to type II chitinase, which is mainly involved in cuticle remodeling and the molting process [27]. Based on this, it is speculated that the protein encoded by TuCHT12 in T. urticae in this study is also a type II chitinase and may exert similar functions. Multiple sequence alignment results further showed that the protein encoded by TuCHT12 had the highest sequence identity of 70.23% with the homologous protein of T. cinnabarinus, while the sequence identity with chitinases of other arthropods, such as horseshoe crabs, beetles, mosquitoes and various fruit flies ranged from 25.82% to 28.78% (Figure 3C). This cross-species sequence conservation strongly supports that the protein encoded by TuCHT12 performs important and conserved physiological functions in arthropods.
Figure 3. Molecular characterization analysis of chitinase gene (TuCHT12) from Tetranychus urticae. Note: (A) Cloning of the TuCHT12 gene of Tetranychus urticae 1: TuCHT12, M: Marker; (B) Phylogenetic tree based on the amino acid sequence of TuCHT12 from Tetranychus urticae. The red box highlights the TuCHT12 gene. Numbers at the nodes indicate bootstrap support values; (C) Multiple sequence alignment of TuCHT12 from Tetranychus urticae with that from other species.
Table 2. Physicochemical property.

3.4. Gene Silencing Efficiency of TuCHT12 and Other Chitinases

After feeding T. urticae with dsTuCHT12, the expression of TuCHT12 was examined at 24 h, 48 h and 72 h (Figure 4A). The results revealed that TuCHT12 expression was consistently downregulated within 72 h, with significant down-regulation observed at 48 h, at which point the silencing efficiency was 33.8%. It is speculated that feeding with dsTuCHT12 effectively induced gene silencing, with the strongest inhibitory effect occurring at 48 h.
Figure 4. Detection of gene silencing efficiency in Tetranychus urticae. Note: The expression level of the control group was set as 1 (dashed line in the figure), and the relative expression levels of the treatment group were compared with it for graphing. Tukey’s test was used to analyze the significant differences among multiple sets of data at the 0.05 level. * indicates p < 0.05, (A) Gene expression level of TuCHT12, (B) Gene expression of other genes in Tetranychus urticae.
After 48 h of dsTuCHT12 RNAi treatment, the expression levels of other chitinase genes in T. urticae showed significant alterations (Figure 4B). The expression levels of TuCHT8, TuCHT9 and TuCHT10 genes were upregulated, among which TuCHT9 exhibited a significant increase, suggesting its predominant involvement in chitin metabolism regulation under disrupted chitin homeostasis. In contrast, the expression levels of TuCHT7 and TuCHT11 were downregulated, with TuCHT11 showing the most significant decrease. It can be attributed to the fact that TuCHT11 and TuCHT12 both belong to type II chitinases, which are likely subject to co-directional regulation.

3.5. Effects of Combined Application of RNAi with I. cateniannulata Treatment on T. urticae

The combined application of RNAi and I. cateniannulata significantly affected the mortality of T. urticae showed that the mortality of T. urticae gradually increased with time, and the mortality rates in all experimental groups were higher than those in the control group (Figure 5A). By day 5, the mortality rate reached 81.63% in the dsEGFP + XS08-1 group and 82.17% in the dsCHT12-only group. Notably, the mortality rate of dsCHT12 + XS08-1 reached 98.13%, while the mortality rate in the sterile water group was only 30.7%. Compared with the single treatment groups, the combined application of dsCHT12 and I. cateniannulata resulted in a higher mortality rate of T. urticae, which was 3.19-fold that of the sterile water group. This indicated that the sensitivity of adult female T. urticae to I. cateniannulata was enhanced after dsCHT12 treatment.
Figure 5. Changes in mortality and egg production of T. urticae after RNAi and I. cateniannulata treatments. Note: XS08-1: I. cateniannulata. (A) mortality of T. urticae; (B) number of eggs laid by T. urticae.
The total egg production reported here was based on a defined number of adult female T. urticae at the start of the experiment. The effect of combined RNAi and I. cateniannulata treatment on the egg production of adult female T. urticae (Figure 5B) showed that the number of eggs laid by T. urticae increased gradually over time in all groups. On day 5, females in the dsEGFP + XS08-1 group laid 298 eggs, whereas those in the dsCHT12-only group laid 260 eggs. The number of eggs laid in the dsCHT12 + XS08-1 group was 140, and that in the sterile water group was 298. The results indicate that the combined treatment of dsCHT12 interference and I. cateniannulata could inhibit the egg production of T. urticae, which could reduce the number of eggs by 52.8% compared with the control group.

4. Discussions

During the infection by entomopathogenic fungi, host antioxidant and detoxification enzyme systems constitute essential physiological defenses that affect host immune survival. Previous studies have shown that, after infection by entomopathogenic fungi such as Isaria javanica, Beauveria bassiana, and Acremonium spp., antioxidant enzyme activities in Hyphantria cunea, Bombyx mori, and T. urticae typically exhibited a pattern of “first increase and then decline” [28,29,30]. In this study, after infection with I. cateniannulata, CAT activity in T. urticae similarly first increased and subsequently declined over time. This pattern indicates that mites initially mitigate the accumulation of ROS by enhancing antioxidant enzyme activity. However, as fungal hyphae proliferate within the host, the mites may be unable to sustain high antioxidant capacity. The subsequent decline in enzyme activity induces oxidative imbalance, which may ultimately contribute to mite mortality. These results highlight the timing of host defense responses and suggest that optimizing fungal application according to these dynamics could improve biocontrol efficiency and economic feasibility.
In addition to the antioxidant response, detoxification enzyme systems, GST and MFO, also play crucial roles in coping with exogenous toxins and fungal metabolites. Previous studies have reported that in Galleria mellonella, Spodoptera frugiperda, Aphis gossypii (Hemiptera:Aphididae), detoxification enzyme activities typically exhibit a pattern of initial increase followed by a subsequent decline under fungal stress caused by B. bassiana and related entomopathogenic fungi [31,32,33]. Consistent with these findings, the present study demonstrated that GST and MFO activities increased in the early stage of I. cateniannulata infection and then decreased gradually. This pattern likely reflects an adaptive enhancement of detoxification metabolism to alleviate the toxic pressure imposed by fungal infection. However, as the fungus rapidly proliferates within the hemocoel, it becomes difficult for the mite to maintain its detoxification system at high activity, ultimately resulting in detoxification failure and promoting host mortality. Compared with other pest models, I. cateniannulata induced similar detoxification patterns but achieved higher early-stage enzyme activation, indicating strong potential for rapid host suppression. This information can guide dosing and timing strategies to enhance both efficacy and economic viability of fungal biocontrol.
Chitinase genes in arthropods, including insects and mites, undergo changes due to entomopathogenic fungal infection. In insects such as Manduca sexta (tobacco hornworm) and Bombyx mori (silkworm), chitinase expression is induced under immune stimulation [34,35]; in T. cinnabarinus (carmine spider mite), curcumin treatment can also significantly alter chitinase expression patterns [36]. Moreover, during the development of M. alternatus (pine sawyer beetle), chitinase genes display periodic expression profiles, with significant upregulation at the prepupal and pupal stages [37]. In this study, the expression trends of TuCHT7 and TuCHT12 were significantly up-regulated during I. cateniannulata infection, suggesting their involvement in host defense responses. In contrast, TuCHT8, TuCHT9, and TuCHT11 exhibited transient expression patterns, which may be associated with functions such as local chitin degradation or initial immune signal transmission, and further research is required [38].
Chitinase-related genes play pivotal roles in host resistance to entomopathogenic fungi. Silencing CHTs in S. frugiperda significantly reduced the survival rate of larvae following the treatment with B. bassiana [39]. Similarly, in Drosophila, RNAi-mediated reduction in DmCht2 and DmIDGF6 significantly weakened the host’s defense capability against pathogenic infection [40]. In Chilo suppressalis, silencing Cht6 combined with B. bassiana treatment accelerated host mortality [41,42]. There are many examples of the combined application of entomopathogenic fungi and dsRAN for pest control. The achieved results can be found in Table 3. In agreement with these studies, the present results demonstrated that silencing TuCHT12 followed by infection with I. cateniannulata (strain XS08-1) produced the highest mortality rate against T. urticae, which was 3.19-fold that of the control group. These findings highlight the potential for integrating TuCHT12-targeted strategies with optimized fungal application to enhance biocontrol efficacy, economic viability, and environmental sustainability in mite management programs.
Table 3. Effects of entomopathogenic fungi combined with dsRNA on the mortality of pests.

5. Conclusions

Infection of T.urticae by I. cateniannulata increased the activities of defense enzymes, with CAT and MFO playing dominant roles. Fungal infection induced dynamic expression changes in six chitinase genes. Among them, TuCHT7 and TuCHT12 genes were significantly upregulated after 24 h; TuCHT7 subsequently declined, whereas TuCHT12 maintained a sustained and stable induction pattern. In contrast, the TuCHT10 gene was suppressed, while the other chitinase genes exhibited transient expression patterns. Feeding dsTuCHT12 inhibited the expression of TuCHT12 within 72 h, with significant suppression observed at 48 h. At this time point, the expression of TuCHT8, TuCHT9, and TuCHT10 genes was upregulated, whereas that of TuCHT7 and TuCHT11 was downregulated. Notably, the combined application of dsCHT12 and I. cateniannulata resulted in a 3.19-fold increase in the mortality of T. urticae and a 52.8% reduction in egg production. Overall, these findings provide a theoretical basis for the development of eco-friendly pest control strategies based on the combined application of RNAi and I. cateniannulata.

Author Contributions

L.G. and X.Y. conceived and designed the experiments and wrote the manuscript. K.T. and Y.R. analyzed data and drafted the manuscript. C.L., W.Y. and Q.C. carried out the review and editing. X.Z. contributed project administration and funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

Guizhou Province High Value Core Patent Cultivation Center Funding Project (Qian Zhi Yun Pei [2025] 02).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author and the first author upon reasonable request.

Conflicts of Interest

The authors declare no conflicts of interest.

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