# Silencing Chitinase Genes Increases Susceptibility ofTetranychus cinnabarinus (Boisduval) to Scopoletin

Authors: Hong Zhou; Yong-qiang Zhang; Ting Lai; Dan Wang; Jin-lin Liu; Fu-you Guo; Wei Ding
Journal: BioMed Research International
 (2017)

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## Abstract

The carmine spider miteTetranychus cinnabarinusis a major pest of crop and vegetable plants worldwide. Previous studies have shown that scopoletin is a promising acaricidal compound againstTetranychus cinnabarinus.However, the acaricidal mechanism of scopoletin remains unclear. In the present study, 12 full-length cDNAs of chitinase (CHIT) genes fromTetranychus cinnabarinus(designatedTcCHITs) were cloned and characterized. AlthoughTcCHITswere expressed throughout all life stages, their expression levels were significantly upregulated during the larval and nymphal stages.TcCHITswere downregulated 24 h after treatment with scopoletin and upregulated 24 h after treatment with diflubenzuron (DFB, a chitin synthesis inhibitor). Feeding double-stranded RNA effectively silencedTcCHITtranscription inTetranychus cinnabarinus, thus increasing its susceptibility to scopoletin but reducing that to DFB. Meanwhile,TcCHIT silencing in larvae and adult resulted in an extremely low molting rate (7.3%) and high mortality rate (53.3%), respectively, compared with those in the control group. CHIT genes are closely related to arthropod survival, molting, and development inTetranychus cinnabarinus, suggesting that acaricidal mechanisms of scopoletin and DFB may occur by inhibition and activation of CHIT gene expression, respectively.TcCHIT constitutes a possible target of scopoletin and DFB inTetranychus cinnabarinus.


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## Body

## 1. Introduction

Phytophagous mites of the genusTetranychus andPanonychus are major pests on plants worldwide [1, 2]. The carmine spider miteTetranychus cinnabarinus is of particular importance, because this extreme generalist species has been documented on more than 100 plant species, including food and economic crops, ornamental plants, and weeds [3–5]. Cinnabar spider mite is parthenogenic and exhibits strong fecundity and adaptability. This mite is also one of the pests that are most difficult to control because it easily develops resistance to pesticides [6].Control ofTetranychus cinnabarinusin open-field crops primarily relies on synthetic chemical acaricides [2, 7–9]. Chemical acaricides have been extensively used to control mite pests because of their quick and efficient acaricidal effect [10]. However, spider mites rapidly develop resistance to almost all acaricidal agents, presenting a major factor that threatens efficient control of spider mites in agriculture [11, 12]. Furthermore, application of chemical acaricides has led to environmental and human health concerns [13]. Thus, controlling mite pests by traditional chemical acaricides has become challenging. Effective methods for controlling mite pests and environment-friendly acaricides should be developed. Phytogenous acaricides, which present low mammalian toxicity and can be rapidly degraded, are suitable for integrated mite management. Studies have also shown that these naturally occurring products may delay development of pesticide resistance in pests [14, 15].Scopoletin, a coumarin compound, is an important secondary plant metabolite and phytogenous acaricidal compound with excellent contact-killing, systemic, repellent, and oviposition inhibition activities againstTetranychus cinnabarinus[16]. Studies have confirmed that scopoletin manifests growth-regulating, insecticidal, and antibacterial activities [17, 18]. The biological functions influenced by scopoletin are attributed to its various molecular targets, including transcription factors, growth factors, and their receptors, cytokines, enzymes, and genes that regulate cell proliferation and apoptosis [19]. Thus, understanding the mode of action of acaricides is crucial to identifying their molecular targets [20]. Although the acaricidal activity of scopoletin and its possible biochemical mechanism have been investigated, its molecular mechanism or molecular target(s) againstTetranychus cinnabarinus remains unknown.Chitin is a polymer ofβ(1,4)-linked N-acetylglucosamine, which is the second most abundant natural polymer after cellulose. Chitin is extensively distributed as a structural component in arthropods, parasites, and microbes [21, 22]. Chitin is also the principal structural component of arthropod exoskeletons and the peritrophic membrane (PM) that lines the epithelium and envelopes gut contents [23]. New chitin is deposited and synthesized during insect growth and development. However, a part of the old cuticle is degraded. When insects or mites are treated with chitin synthesis inhibitors, such as diflubenzuron (DFB) and allosamidin, symptoms of death include development retardation, dysecdysis, and shrinkage [24, 25]. Findings showed that mites treated with scopoletin also exhibit similar death symptoms (Figure 6(c)) [26].In arthropods, the crucial step of chitin biodegradation pathway is associated with chitinases (CHITs). Insect CHITs, which belong to family 18 of glycosyl hydrolases, mediate digestion of chitin to chitooligosaccharides by hydrolyzing chitin via endo-type cleavage. CHITs are crucial in growth and development of insects and mites and act by hydrolyzing chitin of insect integument and midgut. A total of 16, 22, 20, and 12 CHIT and CHIT-like protein genes have been identified inDrosophila melanogaster,Anopheles gambiae,Tribolium castaneum, andTetranychus urticae, respectively [27]. These genes have been classified into five or more groups on the basis of amino acid sequence similarity and phylogenetic analyses. CHIT and related proteins influence molting, digestion, cell proliferation, and tissue remodeling of mites and insects. Research showed that chitin is a critical component of insect cuticle and PM and that each period of growth and development of insects and mites requires a certain amount of chitin [23]. CHIT is a safe target of novel biological pesticides because chitin is absent in animals and plants [28]. CHIT inhibitors, such as allosamidin, argifin, and argadin, exert insecticidal effects by inhibiting CHIT activity, which interferes with normal growth and development of insects and mites [25, 29, 30]. Scopoletin has been reported for its insecticidal and growth inhibitory effects againstPlutella xylostella,Spilarctia obliqua, andDiabrotica beetles [5, 17, 31].In recent years, experimental evidence has shown that scopoletin inhibits the development ofTetranychus cinnabarinus, indicating that this compound may affect degradation of chitin by regulating expressions of CHIT genes [32].Our laboratory investigated the transcriptomics ofTetranychus cinnabarinusafter treatment with scopoletin or a control solvent (Table S1 and S2, resp.). Interestingly, we observed that the CHIT gene family, which may be involved in the acaricidal mechanism of scopoletin againstTetranychus cinnabarinus, was differentially expressed. Thus, this study aimed to assess the role of differential expression of CHIT mRNA transcripts in acaricidal activity of scopoletin againstTetranychus cinnabarinus using RNA interference (RNAi). RNAi is a common mechanism of gene silencing in eukaryotic organisms. In recent years, this technique has shown considerable potential in controlling insect pests by silencing vital genes. Functional roles of CHIT genes inTribolium castaneumandPanonychus citri were also evaluated using RNAi, and several CHIT genes were found to be essential for insect survival, molting, and development [33, 34], thereby revealing functional specialization among CHIT genes during molting. Thus, developing synthetic inhibitors that target CHITs is important for insect pest management.In the present study, 12 full-length cDNAs of CHIT genes fromTetranychus cinnabarinus were cloned and characterized. Patterns of gene expressions in four developmental stages upon acaricidal treatment were analyzed. We adopted double-stranded RNA (dsRNA) feeding as gene knockdown strategy to investigate the role of CHITs in the acaricidal action of scopoletin and DFB, a chitin synthesis inhibitor, againstTetranychus cinnabarinus. This study demonstrated that suppressingTcCHITtranscription increases susceptibility ofT. cinnabarinus to scopoletin but reduces that to DFB. This study also clarified the role of CHITs in the acaricidal mechanism of scopoletin againstTetranychus cinnabarinus.


## 2. Materials and Methods

### 2.1. Mite Rearing

TheTetranychus cinnabarinuscolony used in this study was collected from cowpeas in Beibei, Chongqing, China, and has been maintained for more than 16 years without exposure to any pesticides [35]. The mites were reared on potted cowpea seedlings(Vigna unguiculata) in the insectary at 25±1°C, 50% ± 5% RH, and 14 : 10 h (L : D) photoperiod.


### 2.2. Bioassays and RNA-seq Data

Food and Agriculture Organization-recommended slip-dip method was used to measure scopoletin (purity, 95%; Southwest University, Beibei, Chongqing, China) and DFB (purity, 98.5%; Taitan, Shanghai, China) (FigureS1) toxicity against adult femaleTetranychus cinnabarinus[36]. We adopted the bioassay procedure described by Ding et al. [35]. A total of 30 adult female mites (3–5 days old) were briefly placed on their backs on double-sided tape on glass. The mites were then dipped into each test solution for 5 s. Each dose was performed in triplicate. The use of sterile distilled water with 0.1% (v/v) Tween 80 and 3% (v/v) acetone was designated as control treatment. The mites were observed under an anatomical microscope after 48 h of rearing under controlled growth conditions as described in mite rearing. Mites that exhibited immobility or irregular trembling of legs were considered dead. Lethal and sublethal concentrations for subsequent experiments were determined on the basis of log-probit analysis of concentration–mortality data. RNA-seq was employed to analyze transcriptome changes inTetranychus cinnabarinustreated with median lethal concentration (LC50) of scopoletin and the solvent againstTetranychus cinnabarinus for 24 and 48 h, respectively. For scopoletin treatment, more than 300 female adults were transferred onto three freshly potted cowpea leaves, which were placed in a small Petri dish containing water. The leaves were sprayed with scopoletin solution with the abovementioned concentration. Sterile distilled water with 0.1% Tween 80 and 3% acetone was used as solvent for the control group. Three Petri dishes from one independent experiment comprised a replicate, and two biological replicates were used for RNA purification and sequencing. All sequencing data were submitted to the GEO website (https://www.ncbi.nlm.nih.gov/geo/) with the accession number GSE92959 (unpublished data).


### 2.3. Total RNA Extraction, cDNA Synthesis, andTcCHIT Cloning

Total RNA was extracted from 300 adult (3–5 days old)Tetranychus cinnabarinus females. Extraction was performed by using RNeasy® Plus Micro Kit (Tiangen, Beijing, China). To determine RNA quantity, the absorbance at 260 nm and absorbance ratio of OD260/280 were measured by using a Nanovue UV-Vis spectrophotometer (GE Healthcare, Fairfield, CT). RNA integrity was further confirmed by 1% agarose gel electrophoresis. Reverse transcription was performed by using a PrimeScript® 1st Strand cDNA Synthesis Kit (Takara, Dalian, China). Synthesized cDNA was stored at −20°C. To obtain full-lengthTcCHITs, specific primers were designed and synthesized (Table S3) on the basis of complete genomic sequences from sister speciesTetranychus urticae (http://bioinformatics.psb.ugent.be/orcae/overview/Teur). Specific polymerase chain reactions (PCRs) were performed in a C1000™ Thermal Cycler (BIO-RAD, Hercules, CA, USA). PCRs were performed with a 25 μL reaction volume with 2.5 μL 10x PCR buffer (Mg2+-free), 2.0 μL dNTPs (2.5 mM), 2.5 μL MgCl2 (25 mM), 1 μL cDNA templates, 1 μL each primer (10 mM), 0.2 μL rTaq™ polymerase (TaKaRa), and 14.8 μL ddH2O. PCR program was 94°C for 3 min, followed by 35 cycles of 94°C for 30 s, 48°C to 60°C (based on primer annealing temperatures) for 30 s, 72°C extension for 1 min to 2 min (based on the predicted length of amplified products), and a final extension of 10 min at 72°C. Amplified PCR fragments were gel-purified by using a gel extraction mini kit (Tiangen, Beijing, China), ligated into pMD™ 19-T vector (Takara, Dalian, China) and then transformed into Trans5α competent cells ofEscherichia coli(Tiangen, Beijing, China). Recombinant plasmids were sequenced at the Beijing Genomics Institute (Beijing, China).


### 2.4. Gene Characterization and Phylogenetic Analysis

Nucleotide sequences ofTcCHITswere edited by DNAMAN 5.2.2. The deduced amino acid sequences of 12 CHIT proteins were aligned with ClustalW program [31, 37]. Molecular weight and isoelectric point of the proteins were calculated by using ExPASy Proteomics Server (http://cn.expasy.org/tools/pi_tool.html) [38]. The signal peptide was predicted by using SignalP 4.1 (http://www.cbs.dtu.dk/service/SignalP/) [39], and the transmembrane region was analyzed by using TMHMM Server (v.2.0) (http://www.cbs.dtu.dk/services/TMHMM/) [40].N-glycosylation sites were predicted by using NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/) [41]. The phylogenetic tree was constructed by using MEGA 5.0 via neighbor-joining method with 1000 bootstrap replicates [42].


### 2.5. dsRNA Synthesis, dsRNA Feeding, and Knockdown ofTcCHIT Expression by RNAi

A set of T7 RNA polymerase promoter primers (TableS3) were designed to amplify 160–600 bp lengths of target genes to generate PCR products for in vitro transcription and dsRNA production (Table S3).TcCHITsand green fluorescent protein(GFP) (ACY56286) genes were amplified by PCR. The PCR program was as described in Section 2.3. Recombinant plasmids were used as templates. TheGFP gene was used as negative control. Amplified segments were gel-purified and used in application of TranscriptAid T7 High Yield Transcription Kit (Thermo Scientific, Lithuania, EU). dsRNAs were further purified by using GeneJET RNA Purification Kit (Thermo Scientific, Lithuania, EU). Size of dsRNA products was determined by 1% agarose gel electrophoresis. Concentration of dsRNAs was determined by using a spectrophotometer. dsRNAs were stored at −70°C. Systemic delivery ofTcCHIT dsRNAs via leaf-disc feeding was used to knock downTcCHIT expression. In this study, to investigate whether knock down of expression of target genes affects transcript levels of nontarget genes via leaf-disc feeding method, the chitin metabolic pathways related to two chitin synthetase genes (tetur03g08510 and tetur08g00170, designated CHS) were detected when a mixture of 12TcCHIT dsRNAs was applied. The mites were fed with a mixture of 12 differentTcCHIT dsRNAs for 48 h. Figure S2 shows the schematic diagram of artificial feeding of dsRNA. Briefly, cowpea leaves were cut to a feeding arena (2 cm in diameter) and dehydrated via incubation at 60°C for 3–5 min. The leaves were then treated with diethylpyrocarbonate- (DEPC-) water, dsRNA-GFP, orTcCHIT dsRNAs (1000 ng/μl) for 3-4 h at room temperature. After complete absorption of liquids, the leaves were placed on wet filter paper. The leaf discs were then placed on water-saturated sponges. Thirty female adults (3–5 days old and starved for 24 h) were placed on each pretreated leaf-disc. The leaf discs were then placed upside down on Petri dishes (7 cm in diameter) to prevent mites from escaping. The dsRNA-treated leaf discs, which were infested withTetranychus cinnabarinus, were placed under controlled growth conditions as described in Section 2.1. The mites were finally collected for subsequent experiments 48 h after feeding.


### 2.6. Quantitative Real-Time PCR (qPCR)

To detectTcCHITexpression throughout the different life stages of mites, approximately 2000 eggs, 1500 larvae, 800 nymphs, and 200 adults were collected per sample in triplicate. To quantifyTcCHITexpression in response to scopoletin and DFB exposure, we collected 200 female adults per sample in triplicate. To examine the effects of scopoletin and DFB exposure onTcCHITexpression, female adults were treated with scopoletin or DFB, with 0.1% (v/v) Tween 80 and 3% (v/v) acetone as surfactant. As in the slip-dip assay, LC10, LC30, and LC50 of scopoletin and LC50 of DFB corresponded to 0.099, 0.374, 0.938, and 0.477 mg/mL, respectively. For scopoletin and DFB exposure experiment, we adopted a slightly modified version of the leaf-disc dipping method described by Michel et al. [43]. More than 200 female adults (3–5 days old) were briefly transferred to three freshly potted cowpea leaves in a small Petri dish with water. Each detached cowpea leaf was dipped for 5 s in the test solution with the abovementioned concentration. When the liquid had dried around the mites, they were subjected again under the abovementioned conditions. Sterile distilled water with 0.1% Tween 80 and 3% acetone was then used as the control treatment (CK). After a 24 h interval, only surviving female adult mites from the treated and control groups were collected and frozen at −80°C for RNA extraction. Each experiment was performed at least in triplicate and utilized independent biological samples. To examine the effectiveness of RNAi, approximately 200 female adult mites were collected per sample 48 h after dsRNA feeding. Samples were prepared in triplicate. The specific primers used for qPCR ofTcCHITswere designed by using Primer 3.0 (http://frodo.wi.mit.edu/) (Table S3) [44].RPS18(FJ608659) was used as the stable reference gene for all qPCR assays (Table S3) [45]. qPCR was performed by using a Mx3000P thermal cycler (Agilent Technologies, Inc., Wilmington, NC, USA) on 20 μL reaction mixtures containing 1 μL cDNA template (200 ng/μL), 10 μL iQ™ SYBR® Green Supermix (BIO-RAD, Hercules, CA, USA), 1 μL of each gene-specific primer (0.2 mM), and 7 μL ddH2O. The optimized qPCR protocol used for amplification was 95°C for 2 min, followed by 40 cycles of denaturation at 95°C for 15 s, 60°C for 30 s, and elongation at 72°C for 30 s. Melt curve analyses (from 60°C to 95°C) were performed to ensure consistency of amplified products. Quantification of expression level was analyzed using 2-ΔΔCt method [46].


### 2.7. Susceptibility Test ofTetranychus cinnabarinus to Acaricides after RNAi ofTcCHITs

Sublethal doses of scopoletin and DFB (LC30 and LC50 of scopoletin and DFB, resp.) were applied in bioassays. We also adopted the slip-dip method described above and the detailed bioassay procedure described by Ding et al. [35]. LC30 and LC50 values of two acaricides were used as diagnostic doses to compare changes in susceptibility to acaricides inTetranychus cinnabarinus48 h after feeding ofTcCHITdsRNAs.


### 2.8. Statistical Analysis

All experiments included at least three biological replicates. Differences in expression levels ofTcCHITs during the four developmental stages and mortality rates were analyzed by one-way analysis of variance, followed by Duncan’s multiple tests in SPSS (v.16.0, SPSS Inc., Chicago, IL, USA), at alpha = 0.05.


## 2.1. Mite Rearing

TheTetranychus cinnabarinuscolony used in this study was collected from cowpeas in Beibei, Chongqing, China, and has been maintained for more than 16 years without exposure to any pesticides [35]. The mites were reared on potted cowpea seedlings(Vigna unguiculata) in the insectary at 25±1°C, 50% ± 5% RH, and 14 : 10 h (L : D) photoperiod.


## 2.2. Bioassays and RNA-seq Data

Food and Agriculture Organization-recommended slip-dip method was used to measure scopoletin (purity, 95%; Southwest University, Beibei, Chongqing, China) and DFB (purity, 98.5%; Taitan, Shanghai, China) (FigureS1) toxicity against adult femaleTetranychus cinnabarinus[36]. We adopted the bioassay procedure described by Ding et al. [35]. A total of 30 adult female mites (3–5 days old) were briefly placed on their backs on double-sided tape on glass. The mites were then dipped into each test solution for 5 s. Each dose was performed in triplicate. The use of sterile distilled water with 0.1% (v/v) Tween 80 and 3% (v/v) acetone was designated as control treatment. The mites were observed under an anatomical microscope after 48 h of rearing under controlled growth conditions as described in mite rearing. Mites that exhibited immobility or irregular trembling of legs were considered dead. Lethal and sublethal concentrations for subsequent experiments were determined on the basis of log-probit analysis of concentration–mortality data. RNA-seq was employed to analyze transcriptome changes inTetranychus cinnabarinustreated with median lethal concentration (LC50) of scopoletin and the solvent againstTetranychus cinnabarinus for 24 and 48 h, respectively. For scopoletin treatment, more than 300 female adults were transferred onto three freshly potted cowpea leaves, which were placed in a small Petri dish containing water. The leaves were sprayed with scopoletin solution with the abovementioned concentration. Sterile distilled water with 0.1% Tween 80 and 3% acetone was used as solvent for the control group. Three Petri dishes from one independent experiment comprised a replicate, and two biological replicates were used for RNA purification and sequencing. All sequencing data were submitted to the GEO website (https://www.ncbi.nlm.nih.gov/geo/) with the accession number GSE92959 (unpublished data).


## 2.3. Total RNA Extraction, cDNA Synthesis, andTcCHIT Cloning

Total RNA was extracted from 300 adult (3–5 days old)Tetranychus cinnabarinus females. Extraction was performed by using RNeasy® Plus Micro Kit (Tiangen, Beijing, China). To determine RNA quantity, the absorbance at 260 nm and absorbance ratio of OD260/280 were measured by using a Nanovue UV-Vis spectrophotometer (GE Healthcare, Fairfield, CT). RNA integrity was further confirmed by 1% agarose gel electrophoresis. Reverse transcription was performed by using a PrimeScript® 1st Strand cDNA Synthesis Kit (Takara, Dalian, China). Synthesized cDNA was stored at −20°C. To obtain full-lengthTcCHITs, specific primers were designed and synthesized (Table S3) on the basis of complete genomic sequences from sister speciesTetranychus urticae (http://bioinformatics.psb.ugent.be/orcae/overview/Teur). Specific polymerase chain reactions (PCRs) were performed in a C1000™ Thermal Cycler (BIO-RAD, Hercules, CA, USA). PCRs were performed with a 25 μL reaction volume with 2.5 μL 10x PCR buffer (Mg2+-free), 2.0 μL dNTPs (2.5 mM), 2.5 μL MgCl2 (25 mM), 1 μL cDNA templates, 1 μL each primer (10 mM), 0.2 μL rTaq™ polymerase (TaKaRa), and 14.8 μL ddH2O. PCR program was 94°C for 3 min, followed by 35 cycles of 94°C for 30 s, 48°C to 60°C (based on primer annealing temperatures) for 30 s, 72°C extension for 1 min to 2 min (based on the predicted length of amplified products), and a final extension of 10 min at 72°C. Amplified PCR fragments were gel-purified by using a gel extraction mini kit (Tiangen, Beijing, China), ligated into pMD™ 19-T vector (Takara, Dalian, China) and then transformed into Trans5α competent cells ofEscherichia coli(Tiangen, Beijing, China). Recombinant plasmids were sequenced at the Beijing Genomics Institute (Beijing, China).


## 2.4. Gene Characterization and Phylogenetic Analysis

Nucleotide sequences ofTcCHITswere edited by DNAMAN 5.2.2. The deduced amino acid sequences of 12 CHIT proteins were aligned with ClustalW program [31, 37]. Molecular weight and isoelectric point of the proteins were calculated by using ExPASy Proteomics Server (http://cn.expasy.org/tools/pi_tool.html) [38]. The signal peptide was predicted by using SignalP 4.1 (http://www.cbs.dtu.dk/service/SignalP/) [39], and the transmembrane region was analyzed by using TMHMM Server (v.2.0) (http://www.cbs.dtu.dk/services/TMHMM/) [40].N-glycosylation sites were predicted by using NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/) [41]. The phylogenetic tree was constructed by using MEGA 5.0 via neighbor-joining method with 1000 bootstrap replicates [42].


## 2.5. dsRNA Synthesis, dsRNA Feeding, and Knockdown ofTcCHIT Expression by RNAi

A set of T7 RNA polymerase promoter primers (TableS3) were designed to amplify 160–600 bp lengths of target genes to generate PCR products for in vitro transcription and dsRNA production (Table S3).TcCHITsand green fluorescent protein(GFP) (ACY56286) genes were amplified by PCR. The PCR program was as described in Section 2.3. Recombinant plasmids were used as templates. TheGFP gene was used as negative control. Amplified segments were gel-purified and used in application of TranscriptAid T7 High Yield Transcription Kit (Thermo Scientific, Lithuania, EU). dsRNAs were further purified by using GeneJET RNA Purification Kit (Thermo Scientific, Lithuania, EU). Size of dsRNA products was determined by 1% agarose gel electrophoresis. Concentration of dsRNAs was determined by using a spectrophotometer. dsRNAs were stored at −70°C. Systemic delivery ofTcCHIT dsRNAs via leaf-disc feeding was used to knock downTcCHIT expression. In this study, to investigate whether knock down of expression of target genes affects transcript levels of nontarget genes via leaf-disc feeding method, the chitin metabolic pathways related to two chitin synthetase genes (tetur03g08510 and tetur08g00170, designated CHS) were detected when a mixture of 12TcCHIT dsRNAs was applied. The mites were fed with a mixture of 12 differentTcCHIT dsRNAs for 48 h. Figure S2 shows the schematic diagram of artificial feeding of dsRNA. Briefly, cowpea leaves were cut to a feeding arena (2 cm in diameter) and dehydrated via incubation at 60°C for 3–5 min. The leaves were then treated with diethylpyrocarbonate- (DEPC-) water, dsRNA-GFP, orTcCHIT dsRNAs (1000 ng/μl) for 3-4 h at room temperature. After complete absorption of liquids, the leaves were placed on wet filter paper. The leaf discs were then placed on water-saturated sponges. Thirty female adults (3–5 days old and starved for 24 h) were placed on each pretreated leaf-disc. The leaf discs were then placed upside down on Petri dishes (7 cm in diameter) to prevent mites from escaping. The dsRNA-treated leaf discs, which were infested withTetranychus cinnabarinus, were placed under controlled growth conditions as described in Section 2.1. The mites were finally collected for subsequent experiments 48 h after feeding.


## 2.6. Quantitative Real-Time PCR (qPCR)

To detectTcCHITexpression throughout the different life stages of mites, approximately 2000 eggs, 1500 larvae, 800 nymphs, and 200 adults were collected per sample in triplicate. To quantifyTcCHITexpression in response to scopoletin and DFB exposure, we collected 200 female adults per sample in triplicate. To examine the effects of scopoletin and DFB exposure onTcCHITexpression, female adults were treated with scopoletin or DFB, with 0.1% (v/v) Tween 80 and 3% (v/v) acetone as surfactant. As in the slip-dip assay, LC10, LC30, and LC50 of scopoletin and LC50 of DFB corresponded to 0.099, 0.374, 0.938, and 0.477 mg/mL, respectively. For scopoletin and DFB exposure experiment, we adopted a slightly modified version of the leaf-disc dipping method described by Michel et al. [43]. More than 200 female adults (3–5 days old) were briefly transferred to three freshly potted cowpea leaves in a small Petri dish with water. Each detached cowpea leaf was dipped for 5 s in the test solution with the abovementioned concentration. When the liquid had dried around the mites, they were subjected again under the abovementioned conditions. Sterile distilled water with 0.1% Tween 80 and 3% acetone was then used as the control treatment (CK). After a 24 h interval, only surviving female adult mites from the treated and control groups were collected and frozen at −80°C for RNA extraction. Each experiment was performed at least in triplicate and utilized independent biological samples. To examine the effectiveness of RNAi, approximately 200 female adult mites were collected per sample 48 h after dsRNA feeding. Samples were prepared in triplicate. The specific primers used for qPCR ofTcCHITswere designed by using Primer 3.0 (http://frodo.wi.mit.edu/) (Table S3) [44].RPS18(FJ608659) was used as the stable reference gene for all qPCR assays (Table S3) [45]. qPCR was performed by using a Mx3000P thermal cycler (Agilent Technologies, Inc., Wilmington, NC, USA) on 20 μL reaction mixtures containing 1 μL cDNA template (200 ng/μL), 10 μL iQ™ SYBR® Green Supermix (BIO-RAD, Hercules, CA, USA), 1 μL of each gene-specific primer (0.2 mM), and 7 μL ddH2O. The optimized qPCR protocol used for amplification was 95°C for 2 min, followed by 40 cycles of denaturation at 95°C for 15 s, 60°C for 30 s, and elongation at 72°C for 30 s. Melt curve analyses (from 60°C to 95°C) were performed to ensure consistency of amplified products. Quantification of expression level was analyzed using 2-ΔΔCt method [46].


## 2.7. Susceptibility Test ofTetranychus cinnabarinus to Acaricides after RNAi ofTcCHITs

Sublethal doses of scopoletin and DFB (LC30 and LC50 of scopoletin and DFB, resp.) were applied in bioassays. We also adopted the slip-dip method described above and the detailed bioassay procedure described by Ding et al. [35]. LC30 and LC50 values of two acaricides were used as diagnostic doses to compare changes in susceptibility to acaricides inTetranychus cinnabarinus48 h after feeding ofTcCHITdsRNAs.


## 2.8. Statistical Analysis

All experiments included at least three biological replicates. Differences in expression levels ofTcCHITs during the four developmental stages and mortality rates were analyzed by one-way analysis of variance, followed by Duncan’s multiple tests in SPSS (v.16.0, SPSS Inc., Chicago, IL, USA), at alpha = 0.05.


## 3. Results

### 3.1. Analysis of Acaricidal Toxicity

Table1 presents the LC50 values calculated for the two acaricides against adultTetranychus cinnabarinus. Estimated LC50 values of scopoletin and DFB reached 0.938 and 0.477 mg/mL, respectively. These results showed that DFB exhibited more significant acaricidal efficiency compared with scopoletin. However, LC50 of scopoletin indicated its excellent toxic effects as a botanical acaricide.Table 1
Toxicity of acaricides against adultT. cinnabarinusafter 48 h of exposure time.
Acaricide
N
LC50 (mg⋅mL−1)a 95% CIb
Slope (±SE)
χ
2
c
P
Scopoletin
540
0.938 (0.576~2.292)
1.314 (±0.15)
6.321
0.097
DFB
540
0.477 (0.118~0.902)
2.254 (±0.24)
5.939
0.051
aLC50: median lethal concentration. bCI: 95% confidence interval. cChi-square testing linearity, alpha = 0.05.


### 3.2. cDNA Cloning and Characterization ofTcCHITs

The deduced amino acid sequences and full-length cDNAs of 12TcCHITs, which contained open reading frames (ORFs), were deposited in GenBank under the accession numbers indicated in Table 2. Table 2 summarizes the lengths of deduced amino acid sequences, predicted protein molecular weights, and theoretical isoelectric points. A signal peptide was detected at the N-terminal end ofTcCHIT1,TcCHIT2,TcCHIT3,TcCHIT4,TcCHIT7,TcCHIT8,TcCHIT9,TcCHIT10,TcCHIT11, andTcCHIT12(Figure 1). Meanwhile,TcCHIT1,TcCHIT2,TcCHIT3,TcCHIT6,TcCHIT7,TcCHIT8,TcCHIT9,TcCHIT10,TcCHIT11, andTcCHIT12were predicted to contain a chitin-binding domain (Figure 1).TcCHIT1,TcCHIT2,TcCHIT4,TcCHIT5,TcCHIT6,TcCHIT7,TcCHIT8, andTcCHIT9were predicted to include one catalytic domain;TcCHIIT10comprised three catalytic domains; andTcCHIT3, TcCHIT11, andTcCHIT12comprised two catalytic domains. Furthermore,TcCHT3,TcCHIT5, andTcCHIT6were predicted to contain one transmembrane span domain. These genes, except forTcCHIT7, were also observed to possess potentialN-glycosylation sites (Figure 1).Table 2
Complete sequence information of the 12 CHIT genes ofT. cinnabarinus.
Gene
Accession numbers
Coding sequence (bp)
Deduced full-length of amino acid
Calculated full-length of molecular (kDa)
Isoelectric point
TcCHIT1
KT956964
1632
543
60.8
5.44
TcCHIT2
KT956965
1887
628
69.03
8.65
TcCHIT3
KT956966
2793
930
104.32
6.23
TcCHT4
KT956967
1593
530
59.75
6.07
TcCHT5
KT956968
1194
397
45.63
6.12
TcCHT6
KT956969
1272
423
49.05
8.27
TcCHIT7
KY084261
1233
410
47.34
9.19
TcCHIT8
KY084262
1260
419
48.51
9.64
TcCHIT9
KY084263
834
277
32.02
8.92
TcCHT10
KY084264
1881
627
69.55
6.65
TcCHIT11
KY084265
2748
915
99.42
6.46
TcCHT12
KY084266
1293
430
48.75
6.98Figure 1
Domain architecture of putative chitinases of T. cinnabarinus. Blue boxes: signal peptide; yellow boxes: catalytic domain; green boxes: chitin-binding domain; red boxes: transmembrane span; red circles: theN-glycosylation sites; lines: linker regions.


### 3.3. Phylogenetic Analysis ofTcCHITs

Phylogenetic analysis was performed by using MEGA 5.0 with the maximum likelihood method on the basis of deduced amino acid sequences ofTcCHITsand other known CHIT proteins, including orthologs from the family ofTetranychus urticae,Anopheles gambiae, andDrosophila melanogaster. All CHIT sequences, which possess complete ORFs, were obtained from theTetranychus urticaegenome and the National Center for Biotechnology Information (Bethesda, MD) (https://www.ncbi.nlm.nih.gov/) (Table S4). Results of phylogenetic analysis revealed that CHIT genes forTetranychus cinnabarinus can be divided into four groups (Figure 2):TcCHIT4,TcCHIT5, TcCHIT6, TcCHIT7, TcCHIT8, andTcCHIT9under Group I CHITs;TcCHT1,TcCHIT11in Group II CHITs;TcCHIT3, TcCHIT12under Group III CHITs;TcCHIT2, TcCHIT10 under Group IV CHITs. CHIT genes fromTetranychus cinnabarinusandTetranychus urticaeclustered into the CHIT family and shared a single clade (Figure 2). This result suggests thatTcCHITsandTuCHITs are evolutionarily related and possibly share similar physiological functions.Figure 2
Phylogenic analysis of TcCHITs. Maximum likelihood tree constructed by MEGA 5.0. Phylogeny testing was conducted via the bootstrap method with 1000 replications. Sequences used for constructing the tree are listed in Supplementary Table S4.


### 3.4. Expression Patterns ofTcCHITs in Different Developmental Stages and upon Acaricide Treatment

qPCR was performed to evaluateTcCHITgene expression levels during different developmental stages (egg, larva, nymph, and adult) and upon acaricidal treatment. Results showed that the 12 CHIT genes(TcCHIT1 to -12) were expressed throughout all life stages, suggesting the involvement ofTcCHITsin biological processes in all developmental and growth stages. Specifically,TcCHITswere highly expressed during the larval and nymphal stages compared to in other developmental stages;TcCHITexpression levels were the lowest during the egg stage (Figure 3). Statistical analysis suggests that relative expression levels ofTcCHITstotaled 0.012, 0.056, 0.038, 0.027, 0.514, 0.254, 0.029, 0.004, 0.019, 0.120, 0.126, and 0.004 during the egg stage; 1.009, 0.845, 0.677, 1.009, 1.982, 1.006, 6.434, 1.318, 2.648, 5.192, 6.243, and 0.650 during the larval stage; 0.745, 2.130, 1.583, 1.024, 0.834, 0.672, 1.730, 0.078, 14.873, 2.902, 0.981, and 0.181 during the nymphal stage; and 0.029, 0.006, 0.175, 0.002, 0.275, 0.416, 1.504, 0.057, 0.051, 1.000, 1.000, and 0.129 during the adult stage (Figure 3).Figure 3
RT-qPCR evaluation of the developmentally specific expression patterns of the 12 CHIT genes in T. cinnabarinus. The following life stages were analyzed: egg, larvae, nymph, and adult. Error bars represent the standard error of the calculated mean based on three biological replicates. Different letters on the error bars indicate significant difference according to Duncan’s multiple tests (alpha = 0.05), that is, no statistical difference between “A” and “A”; significant difference among “A,” “B,” “C,” and “D.”RPS18 was used as the reference gene. (a) to (l) wereTcCHIT1~12, respectively.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
(k)
(l)Results of scopoletin treatment experiment showed that, compared with the genes in the control group, all 12 CHIT genes(TcCHIT1 to -12) were downregulated after 24 h of exposure to scopoletin (Figure 4). Statistical analysis suggests that compared with expression levels of the control (CK), relative expression levels ofTcCHITswere 1.5-, 1.1-, 1.3-, 3.5-, 1.7-, 2.9-, 1.3-, 0.9-, 1.3-, 2.3-, 1.6-, and 1.7-fold lower at LC10 doses of scopoletin; 1.8-, 4.8-, 2.3-, 6.0-, 1.3-, 12.4-, 2.3-, 7.7-, 5.2-, 6.7-, 3.7-, and 5.2-fold lower at LC30 doses of scopoletin; and 1.8-, 1.4-, 1.1-, 1.7-, 1.2-, 3.2-, 1.1-, 1.0-, 1.7-, 2.9-, 0.9-, and 2.2-fold lower at LC50 doses of scopoletin. However, relative expression levels of all the 12 CHIT genes were upregulated and were 1.1-, 1.2-, 1.1-, 1.3-, 1.1-, 1.2-, 1.3-, 1.7-, 1.6- 1.3-, 1.5, and 1.1-fold higher than those in the control (CK) after treatment with DFB at LC50.Figure 4
Expression profiles of TcCHITs transcripts after scopoletin treatment for 24 h at three different concentrations. Error bars represent the standard error of the calculated mean based on three biological replicates. Water containing 0.1% Tween-80 was used as the control treatment (CK). LC50 of DFB was used as the positive control. Asterisk (∗) on the error bar indicates a significant difference between the treatment and group (CK) according tot-tests, (p∗<0.05) or (p∗∗<0.01).RPS18 was used as the reference gene. (a) to (l) wereTcCHIT1~12, respectively.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
(k)
(l)


### 3.5. RNAi via dsRNA Knockdown

To validate existence of offsite effects, expressions of all 12 CHIT genes and 2 CHS genes were detected when a mixture of 12TcCHIT dsRNAs was applied (Figure 5). mRNA expressions ofTcCHITs significantly decreased but not those ofTcCHSs whenTcCHIT dsRNAs were applied at the adult stage in mites (Figure 5). Results showed that transcript levels ofTcCHITs significantly decreased to 57.30%, 53.87%, 32.23%, 60.37%, 72.32%, 61.78%, 55.49%, 70.14%, 81.16%, 51.02%, 77.57%, and 35.50% compared with transcript levels ofTcCHITs after DEPC-water treatment (Figure 5). No significant difference in transcript efficiency existed between the two controls (water and dsGFP) (Figure 5). These results indicate the absence of offsite effect of RNAi experiments in this study. At 48 h after feeding ofTcCHITdsRNAs, 53.3% of mites died because of integument shrinkage or damage (Figures 6(b) and 7). By contrast, the mites fed with dsGFP showed 4.7% mortality. These results reveal successful knockdown ofTcCHITtranscripts by RNAi inTetranychus cinnabarinus.Figure 5
Quantitative PCR detection of target gene expression after dsRNA-TcCHITs feeding relative to expression levels after DEPC-water treatment. dsGFP was adopted as negative control.RPS18 was used as the reference gene. Asterisk (∗) on the error bar indicates a significant difference between the treatment and group (DEPC-water) according tot-tests, p<0.05.Figure 6
The phenotypes of T. cinnabarinus after DEPC-water exposure (a), dsRNA-TcCHITs feeding (b), scopoletin (c), and DFB (d) exposure for 48 h at the adult stage. Adult mites were treated with the LC50 of scopoletin and DFB. Note that dsRNA-TcCHITsfeeding (b), scopoletin (c), and DFB (d) exposure exhibited similar death symptoms: the adult individuals integument shrinkage or damage.
(a)
(b)
(c)
(d)Figure 7
Mortality rate of T. cinnabarinus after being artificially fed with dsGFP or dsRNA-TcCHITs at the adult stage after 48 h. Means were compared byt-tests, p<0.05 (n=3, including 180 mites).To explore the biological functions ofTcCHITs, RNAi method was applied to knock downTcCHITsexpression at the larval stage. Consequently, at 48 h after dsRNA feeding, 92.7% of mites died because of failure to molt or to undergo dysecdysis (Figures 8 and 9, resp.). By contrast, the mites fed with dsGFP exhibited only 1.4% mortality. At 72 h after dsRNA feeding, the remaining mites still failed to molt after treatment withTcCHITdsRNAs, whereas all the mites in the control group successfully turned into nymphs. At 72 h after dsRNA feeding, molting rate reached 7.3% during treatment ofTcCHITs dsRNAs and totaled 98.6% in dsGFP treatment (Figure 9).Figure 8
The phenotypes of T. cinnabarinus after being artificially fed with dsGFP or dsRNA-TcCHITs at the larval stage after 72 h. The artificial feeding of dsTcCHITs resulted in a lethal phenotype: the larva individuals failed molting or undergoing dysecdysis, whereas mites fed with dsGFP developed normally.Figure 9
Molting rate of T. cinnabarinus after being artificially fed with dsGFP or dsRNA-TcCHITs at the larval stage after 72 h. Means were compared byt-tests, p<0.05.


### 3.6. Susceptibility Test ofTetranychus cinnabarinus to Acaricides after RNAi ofTcCHITs

Susceptibilities to the two acaricides at 48 h afterTcCHITsdsRNA feeding at the adult stage were detected by the slip-dip method.TcCHITstranscripts in the LC50 and LC30 assays of scopoletin were knocked down by RNAi inTetranychus cinnabarinus. Mortality significantly increased to 16.92% and 20.12% in the mites fed withTcCHITsdsRNAs compared with mites treated with DEPC-water (Figure 10). Contradictory results were generated whenTcCHITstranscripts in the LC50 and LC30 assays of DFB were knocked down by RNAi inTetranychus cinnabarinus. Mortality significantly decreased to 18.43% and 14.88% in the mites fed withTcCHITsdsRNAs compared with those treated with DEPC-water (Figure 10). No significant difference in mortality existed between DEPC-water and dsRNA-GFPtreatments (Figure 10). These results demonstrate that RNAi of CHIT genes increased susceptibility ofTetranychus cinnabarinusto scopoletin but reduced that to DFB. These results indicate that CHIT genes may play a crucial role in the acaricidal effects of scopoletin and DFB.Figure 10
Knockdown of TcCHITs expressions increased susceptibility to scopoletin and reduced susceptibility to DFB in mites. (a, b) Mortality ofTcCHITs-silencedT. cinnabarinusto scopoletin at LC50 and LC30, respectively. (c, d) Mortality ofTcCHITs-silencedT. cinnabarinusto DFB at LC50 and LC30, respectively. dsGFP was adopted as negative control. Error bars represent the standard error of the calculated mean based on three biological replicates. Different letters on the error bars indicate significant difference according to Duncan’s multiple tests (alpha = 0.05) that is, no statistical difference between “A” and “A”; significant difference between “A” and “B”.
(a)
(b)
(c)
(d)


## 3.1. Analysis of Acaricidal Toxicity

Table1 presents the LC50 values calculated for the two acaricides against adultTetranychus cinnabarinus. Estimated LC50 values of scopoletin and DFB reached 0.938 and 0.477 mg/mL, respectively. These results showed that DFB exhibited more significant acaricidal efficiency compared with scopoletin. However, LC50 of scopoletin indicated its excellent toxic effects as a botanical acaricide.Table 1
Toxicity of acaricides against adultT. cinnabarinusafter 48 h of exposure time.
Acaricide
N
LC50 (mg⋅mL−1)a 95% CIb
Slope (±SE)
χ
2
c
P
Scopoletin
540
0.938 (0.576~2.292)
1.314 (±0.15)
6.321
0.097
DFB
540
0.477 (0.118~0.902)
2.254 (±0.24)
5.939
0.051
aLC50: median lethal concentration. bCI: 95% confidence interval. cChi-square testing linearity, alpha = 0.05.


## 3.2. cDNA Cloning and Characterization ofTcCHITs

The deduced amino acid sequences and full-length cDNAs of 12TcCHITs, which contained open reading frames (ORFs), were deposited in GenBank under the accession numbers indicated in Table 2. Table 2 summarizes the lengths of deduced amino acid sequences, predicted protein molecular weights, and theoretical isoelectric points. A signal peptide was detected at the N-terminal end ofTcCHIT1,TcCHIT2,TcCHIT3,TcCHIT4,TcCHIT7,TcCHIT8,TcCHIT9,TcCHIT10,TcCHIT11, andTcCHIT12(Figure 1). Meanwhile,TcCHIT1,TcCHIT2,TcCHIT3,TcCHIT6,TcCHIT7,TcCHIT8,TcCHIT9,TcCHIT10,TcCHIT11, andTcCHIT12were predicted to contain a chitin-binding domain (Figure 1).TcCHIT1,TcCHIT2,TcCHIT4,TcCHIT5,TcCHIT6,TcCHIT7,TcCHIT8, andTcCHIT9were predicted to include one catalytic domain;TcCHIIT10comprised three catalytic domains; andTcCHIT3, TcCHIT11, andTcCHIT12comprised two catalytic domains. Furthermore,TcCHT3,TcCHIT5, andTcCHIT6were predicted to contain one transmembrane span domain. These genes, except forTcCHIT7, were also observed to possess potentialN-glycosylation sites (Figure 1).Table 2
Complete sequence information of the 12 CHIT genes ofT. cinnabarinus.
Gene
Accession numbers
Coding sequence (bp)
Deduced full-length of amino acid
Calculated full-length of molecular (kDa)
Isoelectric point
TcCHIT1
KT956964
1632
543
60.8
5.44
TcCHIT2
KT956965
1887
628
69.03
8.65
TcCHIT3
KT956966
2793
930
104.32
6.23
TcCHT4
KT956967
1593
530
59.75
6.07
TcCHT5
KT956968
1194
397
45.63
6.12
TcCHT6
KT956969
1272
423
49.05
8.27
TcCHIT7
KY084261
1233
410
47.34
9.19
TcCHIT8
KY084262
1260
419
48.51
9.64
TcCHIT9
KY084263
834
277
32.02
8.92
TcCHT10
KY084264
1881
627
69.55
6.65
TcCHIT11
KY084265
2748
915
99.42
6.46
TcCHT12
KY084266
1293
430
48.75
6.98Figure 1
Domain architecture of putative chitinases of T. cinnabarinus. Blue boxes: signal peptide; yellow boxes: catalytic domain; green boxes: chitin-binding domain; red boxes: transmembrane span; red circles: theN-glycosylation sites; lines: linker regions.


## 3.3. Phylogenetic Analysis ofTcCHITs

Phylogenetic analysis was performed by using MEGA 5.0 with the maximum likelihood method on the basis of deduced amino acid sequences ofTcCHITsand other known CHIT proteins, including orthologs from the family ofTetranychus urticae,Anopheles gambiae, andDrosophila melanogaster. All CHIT sequences, which possess complete ORFs, were obtained from theTetranychus urticaegenome and the National Center for Biotechnology Information (Bethesda, MD) (https://www.ncbi.nlm.nih.gov/) (Table S4). Results of phylogenetic analysis revealed that CHIT genes forTetranychus cinnabarinus can be divided into four groups (Figure 2):TcCHIT4,TcCHIT5, TcCHIT6, TcCHIT7, TcCHIT8, andTcCHIT9under Group I CHITs;TcCHT1,TcCHIT11in Group II CHITs;TcCHIT3, TcCHIT12under Group III CHITs;TcCHIT2, TcCHIT10 under Group IV CHITs. CHIT genes fromTetranychus cinnabarinusandTetranychus urticaeclustered into the CHIT family and shared a single clade (Figure 2). This result suggests thatTcCHITsandTuCHITs are evolutionarily related and possibly share similar physiological functions.Figure 2
Phylogenic analysis of TcCHITs. Maximum likelihood tree constructed by MEGA 5.0. Phylogeny testing was conducted via the bootstrap method with 1000 replications. Sequences used for constructing the tree are listed in Supplementary Table S4.


## 3.4. Expression Patterns ofTcCHITs in Different Developmental Stages and upon Acaricide Treatment

qPCR was performed to evaluateTcCHITgene expression levels during different developmental stages (egg, larva, nymph, and adult) and upon acaricidal treatment. Results showed that the 12 CHIT genes(TcCHIT1 to -12) were expressed throughout all life stages, suggesting the involvement ofTcCHITsin biological processes in all developmental and growth stages. Specifically,TcCHITswere highly expressed during the larval and nymphal stages compared to in other developmental stages;TcCHITexpression levels were the lowest during the egg stage (Figure 3). Statistical analysis suggests that relative expression levels ofTcCHITstotaled 0.012, 0.056, 0.038, 0.027, 0.514, 0.254, 0.029, 0.004, 0.019, 0.120, 0.126, and 0.004 during the egg stage; 1.009, 0.845, 0.677, 1.009, 1.982, 1.006, 6.434, 1.318, 2.648, 5.192, 6.243, and 0.650 during the larval stage; 0.745, 2.130, 1.583, 1.024, 0.834, 0.672, 1.730, 0.078, 14.873, 2.902, 0.981, and 0.181 during the nymphal stage; and 0.029, 0.006, 0.175, 0.002, 0.275, 0.416, 1.504, 0.057, 0.051, 1.000, 1.000, and 0.129 during the adult stage (Figure 3).Figure 3
RT-qPCR evaluation of the developmentally specific expression patterns of the 12 CHIT genes in T. cinnabarinus. The following life stages were analyzed: egg, larvae, nymph, and adult. Error bars represent the standard error of the calculated mean based on three biological replicates. Different letters on the error bars indicate significant difference according to Duncan’s multiple tests (alpha = 0.05), that is, no statistical difference between “A” and “A”; significant difference among “A,” “B,” “C,” and “D.”RPS18 was used as the reference gene. (a) to (l) wereTcCHIT1~12, respectively.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
(k)
(l)Results of scopoletin treatment experiment showed that, compared with the genes in the control group, all 12 CHIT genes(TcCHIT1 to -12) were downregulated after 24 h of exposure to scopoletin (Figure 4). Statistical analysis suggests that compared with expression levels of the control (CK), relative expression levels ofTcCHITswere 1.5-, 1.1-, 1.3-, 3.5-, 1.7-, 2.9-, 1.3-, 0.9-, 1.3-, 2.3-, 1.6-, and 1.7-fold lower at LC10 doses of scopoletin; 1.8-, 4.8-, 2.3-, 6.0-, 1.3-, 12.4-, 2.3-, 7.7-, 5.2-, 6.7-, 3.7-, and 5.2-fold lower at LC30 doses of scopoletin; and 1.8-, 1.4-, 1.1-, 1.7-, 1.2-, 3.2-, 1.1-, 1.0-, 1.7-, 2.9-, 0.9-, and 2.2-fold lower at LC50 doses of scopoletin. However, relative expression levels of all the 12 CHIT genes were upregulated and were 1.1-, 1.2-, 1.1-, 1.3-, 1.1-, 1.2-, 1.3-, 1.7-, 1.6- 1.3-, 1.5, and 1.1-fold higher than those in the control (CK) after treatment with DFB at LC50.Figure 4
Expression profiles of TcCHITs transcripts after scopoletin treatment for 24 h at three different concentrations. Error bars represent the standard error of the calculated mean based on three biological replicates. Water containing 0.1% Tween-80 was used as the control treatment (CK). LC50 of DFB was used as the positive control. Asterisk (∗) on the error bar indicates a significant difference between the treatment and group (CK) according tot-tests, (p∗<0.05) or (p∗∗<0.01).RPS18 was used as the reference gene. (a) to (l) wereTcCHIT1~12, respectively.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
(k)
(l)


## 3.5. RNAi via dsRNA Knockdown

To validate existence of offsite effects, expressions of all 12 CHIT genes and 2 CHS genes were detected when a mixture of 12TcCHIT dsRNAs was applied (Figure 5). mRNA expressions ofTcCHITs significantly decreased but not those ofTcCHSs whenTcCHIT dsRNAs were applied at the adult stage in mites (Figure 5). Results showed that transcript levels ofTcCHITs significantly decreased to 57.30%, 53.87%, 32.23%, 60.37%, 72.32%, 61.78%, 55.49%, 70.14%, 81.16%, 51.02%, 77.57%, and 35.50% compared with transcript levels ofTcCHITs after DEPC-water treatment (Figure 5). No significant difference in transcript efficiency existed between the two controls (water and dsGFP) (Figure 5). These results indicate the absence of offsite effect of RNAi experiments in this study. At 48 h after feeding ofTcCHITdsRNAs, 53.3% of mites died because of integument shrinkage or damage (Figures 6(b) and 7). By contrast, the mites fed with dsGFP showed 4.7% mortality. These results reveal successful knockdown ofTcCHITtranscripts by RNAi inTetranychus cinnabarinus.Figure 5
Quantitative PCR detection of target gene expression after dsRNA-TcCHITs feeding relative to expression levels after DEPC-water treatment. dsGFP was adopted as negative control.RPS18 was used as the reference gene. Asterisk (∗) on the error bar indicates a significant difference between the treatment and group (DEPC-water) according tot-tests, p<0.05.Figure 6
The phenotypes of T. cinnabarinus after DEPC-water exposure (a), dsRNA-TcCHITs feeding (b), scopoletin (c), and DFB (d) exposure for 48 h at the adult stage. Adult mites were treated with the LC50 of scopoletin and DFB. Note that dsRNA-TcCHITsfeeding (b), scopoletin (c), and DFB (d) exposure exhibited similar death symptoms: the adult individuals integument shrinkage or damage.
(a)
(b)
(c)
(d)Figure 7
Mortality rate of T. cinnabarinus after being artificially fed with dsGFP or dsRNA-TcCHITs at the adult stage after 48 h. Means were compared byt-tests, p<0.05 (n=3, including 180 mites).To explore the biological functions ofTcCHITs, RNAi method was applied to knock downTcCHITsexpression at the larval stage. Consequently, at 48 h after dsRNA feeding, 92.7% of mites died because of failure to molt or to undergo dysecdysis (Figures 8 and 9, resp.). By contrast, the mites fed with dsGFP exhibited only 1.4% mortality. At 72 h after dsRNA feeding, the remaining mites still failed to molt after treatment withTcCHITdsRNAs, whereas all the mites in the control group successfully turned into nymphs. At 72 h after dsRNA feeding, molting rate reached 7.3% during treatment ofTcCHITs dsRNAs and totaled 98.6% in dsGFP treatment (Figure 9).Figure 8
The phenotypes of T. cinnabarinus after being artificially fed with dsGFP or dsRNA-TcCHITs at the larval stage after 72 h. The artificial feeding of dsTcCHITs resulted in a lethal phenotype: the larva individuals failed molting or undergoing dysecdysis, whereas mites fed with dsGFP developed normally.Figure 9
Molting rate of T. cinnabarinus after being artificially fed with dsGFP or dsRNA-TcCHITs at the larval stage after 72 h. Means were compared byt-tests, p<0.05.


## 3.6. Susceptibility Test ofTetranychus cinnabarinus to Acaricides after RNAi ofTcCHITs

Susceptibilities to the two acaricides at 48 h afterTcCHITsdsRNA feeding at the adult stage were detected by the slip-dip method.TcCHITstranscripts in the LC50 and LC30 assays of scopoletin were knocked down by RNAi inTetranychus cinnabarinus. Mortality significantly increased to 16.92% and 20.12% in the mites fed withTcCHITsdsRNAs compared with mites treated with DEPC-water (Figure 10). Contradictory results were generated whenTcCHITstranscripts in the LC50 and LC30 assays of DFB were knocked down by RNAi inTetranychus cinnabarinus. Mortality significantly decreased to 18.43% and 14.88% in the mites fed withTcCHITsdsRNAs compared with those treated with DEPC-water (Figure 10). No significant difference in mortality existed between DEPC-water and dsRNA-GFPtreatments (Figure 10). These results demonstrate that RNAi of CHIT genes increased susceptibility ofTetranychus cinnabarinusto scopoletin but reduced that to DFB. These results indicate that CHIT genes may play a crucial role in the acaricidal effects of scopoletin and DFB.Figure 10
Knockdown of TcCHITs expressions increased susceptibility to scopoletin and reduced susceptibility to DFB in mites. (a, b) Mortality ofTcCHITs-silencedT. cinnabarinusto scopoletin at LC50 and LC30, respectively. (c, d) Mortality ofTcCHITs-silencedT. cinnabarinusto DFB at LC50 and LC30, respectively. dsGFP was adopted as negative control. Error bars represent the standard error of the calculated mean based on three biological replicates. Different letters on the error bars indicate significant difference according to Duncan’s multiple tests (alpha = 0.05) that is, no statistical difference between “A” and “A”; significant difference between “A” and “B”.
(a)
(b)
(c)
(d)


## 4. Discussion

As an important phenolic phytoalexin in plants, scopoletin features numerous pharmacological activities, such as antitumor activity. Scopoletin can affect and disrupt growth, proliferation, metastasis, and metabolism of tumor cells and induce apoptosis [47, 48]. However, the potential acaricidal mechanism of scopoletin as a plant-derived acaricide againstTetranychus cinnabarinus remains unknown.Identification and characterization of CHIT genes from mites will aid in determining the involvement of CHITs in responses of mites to specific acaricides. Findings of the present study will also help us to better understand the biological functions of CHITs. We cloned and characterized 12 full-length CHIT cDNAs inTetranychus cinnabarinus(TcCHIT1to-6; Wang et al. [26]). In 1993, full-length cDNA of the first insect CHIT gene was cloned and identified from the tobacco hornwormManduca sexta [49]. Since then, CHIT genes of various insects have been cloned and identified. The encoded CHITs inTribolium castaneum have been divided into eight subgroups on the basis of sequence similarity and domain architecture [50].Structural analysis ofTcCHITsdemonstrated that these genes possess a multidomain structural organization, which includes 1–3 catalytic domains (GH-18 domain), 0-1 cysteine-rich chitin-binding domains (peritrophin-A domain/CBM-14 domain), and serine/threonine-rich regions that can be heavily glycosylated (Figure 1).TcCHITs are predicted to feature a signal peptide (TcCHT1, TcCHT2, TcCHT3, TcCHT4, TcCHT7, TcCHT8, TcCHT9, TcCHT10, TcCHT11,andTcCHT12) or a transmembrane (TcCHT3, TcCHT5,andTcCHT6) span domain as they are targeted either to the extracellular space or sorted into the plasma membrane, in both cases facing carbohydrates of the extracellular matrix (Figure 1). Differences in their domain architecture indicate distinctive biological functions for specific CHITs. For example, Xia et al. [34] reported thatCHIT5,TcCHT7, andTcCHT10, which are also expressed at all developmental stages, play critical roles in digesting the old pupal cuticle, wing/elytra extension, and molting inTribolium castaneum, respectively.TcCHIT transcripts were detected in all four tested developmental stages ofTetranychus cinnabarinus, indicating that the CHIT gene family plays an important role during the entire life cycle of mites. However,TcCHIT expression levels during larval and nymphal stages were significantly higher than in other developmental stages ofTetranychus cinnabarinus, agreeing with the results of Yang et al. [51]. Our present results showed that the CHIT gene family exhibits different developmental patterns of expression inTetranychus cinnabarinus. Expression level ofTcCHIT9 during the larval and nymphal stages was approximately 250-fold higher than those during the egg and adult stages, whereasTcCHT3,TcCHT7,TcCHT10, andTcCHT11 were approximately 10-fold higher. Differences in expression levels may be related to the structure and function of CHIT genes. Different developmental patterns of expression indicate functional specialization in the CHIT gene family ofTetranychus cinnabarinus during molting. In addition, RNAi studies inTribolium castaneum have provided strong experimental evidence for different developmental patterns of expression and tissue-specific expression of different CHIT genes [34].Results of the present study further showed that 24 h after scopoletin exposure,TcCHITexpression levels were downregulated and that the challenge of inhibitingTcCHIT1,TcCHT6,TcCHT7,TcCHT9,andTcCHT10expressions was more significant than that of inhibiting the remaining CHIT genes inTetranychus cinnabarinus. Specifically, statistical analysis suggests thatTcCHITexpression levels were downregulated more significantly with LC30 dose of scopoletin than with LC10 and LC50 doses of scopoletin than those of the control. This result suggests the possible benefits of using appropriate doses of scopoletin. This study also showed thatTcCHITsexpression levels were significantly downregulated during the adult stage of mites. The preceding results indicate that the acaricidal mechanism of scopoletin may degrade chitin biodegradation inTetranychus cinnabarinus by decreasing CHIT gene expression. However, relative expression levels ofTcCHIT8 andTcCHT9were significantly upregulated after DFB treatment (positive control). Several studies have shown that the action mechanism of DFB may occur through direct inhibition of chitin synthase activity; inhibition of zymogen activation process; activation of CHITs; interference with hormonal balance; interference with nerve-secreting cells of the brain; and interference with nucleic acid, protein synthesis, and metabolism in several different insect species [52]. However, the acaricidal mechanism of DFB remains unknown. These results suggest thatTcCHITsplay an essential role in acaricidal mechanisms of scopoletin and DFB inTetranychus cinnabarinus.In this study, we employed RNAi to investigate the biological roles of mite CHIT genes inTetranychus cinnabarinus. RNAi has become an increasingly common method to knock down expression of genes of interest in insects and mites. RNAi also features potential applications in screening and identification of pharmaceutical targets. Previous studies have demonstrated that expressions of specific CHIT genes inTribolium castaneumcan be knocked down by microinjection assays with gene-specific RNAi [34]. For example, Xia et al. [34] reported that specific knockdown ofCHT10 transcripts ofTribolium castaneum,which contains multiple catalytic domains, prevented embryo hatching, larval molting, pupation, and adult metamorphosis. In this study, results of qPCR analyses showed thatTcCHITtranscript levels significantly decreased to 30%–80% in mites 48 h after feeding withTcCHITsdsRNAs. In this study,TcCHITs silencing in larvae and adult samples resulted in high mortality rate. These results not only demonstrated successful dsRNA-mediated knockdown ofTcCHITstranscripts but also showed suitability of dsRNA delivery via the leaf-disc method for RNAi inTetranychus cinnabarinus.Several studies have shown that activation or inhibition of CHITs kills insects or mites. Ding et al. [53] reported that mortality ofM. sexta larvae was higher when treated with a sublethal dose ofBacillus thuringiensis(Bt) toxin on transgenic tobacco lines that expressM. sextaCHIT (Group I). This result suggests that overexpression of CHIT increases susceptibility ofM. Sexta to Bt. In addition, Sakuda et al. [25] showed that allosamidin strongly inhibits insect CHITs and exerts insecticidal effects by preventing molting of insect larvae and pupae. These results indicate that inhibition of CHITs can lead to insect death. Our results showed thatTcCHITs play a similar role in acaricidal mechanisms of scopoletin and DFB. Expressions ofTcCHITswere downregulated 24 h after scopoletin exposure. Specifically, scopoletin susceptibility increased whenTcCHITsin LC50 and LC30 assays were suppressed via RNAi. These results suggest that the acaricidal mechanism of scopoletin may transpire by inhibition of expressions of CHIT genes. However,TcCHITexpressions were upregulated 24 h after DFB treatment. Meanwhile, DFB susceptibility decreased whenTcCHITtranscripts were knocked down by RNAi inTetranychus cinnabarinus. These results indicate that the acaricidal mechanism of DFB may transpire by activation of CHIT gene expression. The function of CHIT genes ofTetranychus cinnabarinus has not been reported at present. However, Zhang et al. [27] reported that CHITs are closely related to insect molting inTribolium castaneum, suggesting that acaricidal mechanism of scopoletin and DFB possibly prevents mites from undergoing normal growth and development by destroying the integument ofTetranychus cinnabarinus. This paper is the first to report that knockdown of CHIT gene expression inTetranychus cinnabarinus increases susceptibility to scopoletin but reduces that to DFB.



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