Editorial Type:
Article Category: Research Article
 | 
Online Publication Date: 13 Jun 2024

An Epsilon Class Glutathione S-Transferase Gene Contributes to the Phytochemical Susceptibility of Tribolium castaneum (Coleoptera: Tenebrionidae)1

,
,
,
,
,
, and
Page Range: 344 – 354
DOI: 10.18474/JES23-71
Save
Download PDF

Abstract

Evidence is accumulating that insect epsilon class glutathione S-transferases (GSTs) play an important role in the resistance to xenobiotics such as insecticides. A Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) epsilon class GST gene, TcGSTe17, was previously found to be significantly induced following exposure to the plant derivative eucalyptol. However, whether TcGSTe17 is involved in resistance to eucalyptol remains unclear. In this study, TcGSTe17 was cloned from the T. castaneum. Development stage expression profiling revealed that TcGSTe17 was expressed at all development stages of T. castaneum but was highly expressed in late-stage larvae. Subsequent expression profiling of tissues showed that TcGSTe17 was highly expressed in the gut of larvae and the fat body of adults, and also revealed gender-specific expression patterns. In addition, the transcripts of TcGSTe17 were significantly increased following exposure to eucalyptol, and RNAi increased the susceptibility of the beetles to eucalyptol. Collectively, these results suggest that TcGSTe17 contributes to the susceptibility of T. castaneum to eucalyptol. These findings provide new data for the prevention of pest resistance.

Keywords: epsilon class glutathione S-transferases; Tribolium castaneum ; RNAi; phytochemical susceptibility

Glutathione-S-transferases (GSTs; EC 2.5.1.18) are a kind of superfamily of enzymes that are widely distributed in animals, plants, and microorganisms (Sheehan et al. 2001). GSTs are related to many physiological processes of organisms, including synthesis of endogenous hormones, metabolism of exogenous substances, and anti-oxidative stress (Enayati et al. 2005). In the metabolism of exogenous substances, GSTs are a class of phase II detoxification enzymes that can metabolize secondary products from phase I (Pavlidi et al. 2018). According to their subcellular location, GSTs can be classified into mitochondrial, microsomal, and cytosolic GSTs, of which only two classes (microsomal and cytosolic GSTs) have been reported in insects (Ketterman et al. 2011). Cytosolic GSTs of insects can be further classified into six groups: delta, epsilon, sigma, omega, zeta, and theta, of which only delta and epsilon are unique to insects (Ranson et al. 2002).

The epsilon class of GSTs, which is one of the largest subfamilies of insect GSTs, are mainly implicated in the resistance to xenobiotics such as insecticides (Hu et al. 2022). With the development and accessibility of sequencing, numerous GST genes, including those from the epsilon class, have been identified and characterized from insect genomes (Han et al. 2016, Hu et al. 2022, You et al. 2015), thereby facilitating the study of insect GSTs. For instance, evidence is accumulating that the epsilon class GSTs are associated with insecticide resistance in insects including Aedes aegypti (L.) (Lumjuan et al. 2005, 2011; Morou et al. 2010), Bombyx mori (Yamamoto et al. 2013, Zhou et al. 2015), Anopheles gambiae Giles (Ding et al. 2005, Muleya et al. 2008, Wang et al. 2008), Spodoptera litura F. (Deng et al. 2009, Hirowatari et al. 2018, Li et al. 2021, Xu et al. 2015), Bactrocera dorsalis (Hendel) (Lu et al. 2016, 2020), Locusta migratoria (L.) (Zhang et al. 2022), Cydia pomonella (L.) (Hu et al. 2020a), Musca domestica L. (Nakamura et al. 2013), and Leptinotarsa decemlineata (Say) (Han et al. 2016). Additionally, in the above insects, all epsilon class GSTs could be induced or overexpressed after exposure to insecticides, and reduction of the relevant epsilon class GSTs led to the enhanced susceptibility of insects to insecticides. In contrast, there is less research on insect-specific epsilon class GSTs involved in resistance to plant derivatives.

Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) is a worldwide grain storage pest, causing billions of dollars of damage annually. It has been reported that T. castaneum has developed resistance to 33 active ingredients, ranking in the top 20 arthropod resistance lists (Kalsi and Palli 2017). Therefore, it is vital to find plant-derived natural products with insecticidal activity. Eucalyptol is an effective component of secondary metabolites in many plants, including Senecio cannabifolius Less. (Wu et al. 2006, Yang et al. 2021), Cinnamum cassia Ness ex Blume, and Laurus nobilis L. (Wang et al. 2019). In addition, various studies have reported that eucalyptol has insecticidal and repellent effects on insects including A. aegypti, M. domestica, and Chrysomya megacephala (F.) (Klocke et al. 1987, Sukontason et al. 2004). In our previous study, eucalyptol could cause a time-dependent increase in T. castaneum larval mortality, and investigation of the molecular mechanism of action of eucalyptol on T. castaneum indicated that an epsilon class GST gene (TcGSTe17) was significantly upregulated after exposure to eucalyptol (Gao et al. 2023). However, whether TcGSTe17 is involved in susceptibility to eucalyptol has yet to be elucidated. Therefore, the purpose of this study was to further investigate the causal role of TcGSTe17 in susceptibility to eucalyptol. Findings from the study will provide new data for the generation and prevention of insect resistance.

Materials and Methods

Insect rearing

The Georgia-1 (GA-1) strain of T. castaneum used in this study was maintained at Nanjing Normal University for 13 years and originated from Kansas State University (Manhattan, KS). The insects were raised in jars containing wheat flour and Brewer’s yeast (19:1) maintained at 30°C and a relative humidity (RH) of 40% with a 14:10 h light:dark cycle (Gao et al. 2020, Xiong et al. 2019a).

Gene cloning and bioinformatics analysis

The open reading frame (ORF) cDNA sequence of TcGSTe17 was augmented by RT-PCR (primers listed in Table 1). The recovery-purification of the PCR product was done by using a FastPure Gel DNA Extraction Mini Kit (Vazyme, Nanjing, China), and the purified product was inserted into the PET28a (+) vector (Novagen, Darmstadt, Germany). The recombinant plasmid was transformed into Escherichia coli Trans1T1 (TransGene, Beijing, China) and sequenced by Traditional Sanger sequencing (General Biol, Nanjing, China). In addition, the sequence of TcGSTe17 (accession no. TC003347) was downloaded from the iBeetle-Base (https://ibeetle-base.uni-goettingen.de/). Prediction in the ORF sequence, deduced proteins, molecular weight, and pI were performed using the ExPASy online server (https://www.expasy.org). Signalp-5.0 (http://www.cbs.dtu.dk/services/SignalP/) was used for predicting the location of a signal peptide.

Table 1 Primers used for this research.
Table 1

Sample collection, RNA extraction, and cDNA synthesis

The different stages and tissues of T. castaneum were sampled as previously described (Xie et al. 2020). Briefly, approximately 50 mg of 1-d-old and 3-d-old embryos were sampled, whereas, three individuals were sampled for each of 1-d-old larvae, 18-d-old larvae, 1-d-old pupae, 5-d-old pupae, 1-d-old adults, and 10-d-old adults. The gut, fat body, integument, and hemolymph of the larvae were sampled from approximately 100 18-d-old larvae, and the gut, integument, ovary, fat body, testis, and head were sampled from approximately 100 10-d-old adults. Three biological replicates were conducted for each sample. The total RNA of each sample was extracted according to the manufacturer’s instructions of the RNA Extraction Reagent kit (Vazyme). Subsequently, 1 µg of purified total RNA was used for cDNA synthesis according to the instructions of HiScript® III RT SuperMix (Vazyme).

Detection of gene expression

The expression of each gene (Table 1) was determined by quantitative real-time polymerase chain reaction (qRT-PCR). According to the instructions of ChamQ SYBR qPCR Master Mix (Vazyme), qRT-PCR was conducted on an ABI Q6 instrument (Applied Biosystems, Foster City, CA) using the following conditions: 95°C for 10 min; 40 cycles of 95°C for 15 s, and 60°C for 60 s; followed by 95°C for 15 s, 60°C for 60 s, and 95°C for 15 s. The qRT-PCR system included 3.5 µl of RNase-free water, 0.25 µl of each primer, 5 µl of ChamQ SYBR qPCR Master Mix, and 1 µl of cDNA. The 2−ΔΔCT method was used to calculate gene expression from the data (Begum et al. 2009, Livak and Schmittgen 2001).

Dip bioassays of eucalyptol

Eucalyptol (99%, CAS: 470-82-6) was obtained from the Aladdin Company (Shanghai, China). The median lethal concentration (LC50) (43.294 mg/ml) of eucalyptol to T. castaneum was prepared by dissolving eucalyptol in acetone (Gao et al. 2023). Subsequently, dip bioassays of eucalyptol were used to treat beetles (Lu et al. 2012). Briefly, 60 12-d-old larvae were divided into two groups, and one group was treated with 100 µL of LC50 eucalyptol for 1 min, and the other group was treated with the same volume of acetone for 1 min. Each treatment was performed in three independent biological replicates. Subsequently, the treated insect life stages were dried on filter paper for 2 min and transferred to glass vials containing wheat flour. Mortality was measured at 12, 24, 36, 48, 60, and 72 h after exposure. The larvae were considered dead if they do not respond when touched with a brush. Meanwhile, the surviving larvae were sampled at 12, 24, 36, 48, 60, and 72 h after exposure. The total RNA of each sample was extracted and reverse transcribed into cDNA for qRT-PCR.

dsRNA synthesis, injection, and eucalyptol bioassay after RNAi

According to the instructions of the TranscriptAid T7 High Yield Transcription Kit (Fermentas, Vilnius, Lithuania), the dsRNA of genes (Table 1) was synthesized and 200 nl was then injected into the body of each larva by microinjector. Simultaneously, each larva was injected with an equal volume of IB (Injection Buffer) or dsVer (T. castaneum vermillion, accession no. AY052390) as a control. The interference efficiency of the dsRNA was detected on the third day. For the eucalyptol bioassay following RNAi of TcGSTe17, the dsRNA-treated larvae of T. castaneum were treated with eucalyptol by drip method (Zhang et al. 2022). Briefly, 90 12-d-old larvae were divided into three groups, and these groups were injected with IB, dsVer, or dsGSTe17, respectively. Three biological replicates were administered for each injection treatment. The surviving larvae were treated with eucalyptol on the third day after injection, and mortality was recorded at 72 h after exposure.

Results and Discussion

cDNA cloning, sequence analysis, and molecular docking analysis

The cDNA of TcGSTe17 was further cloned and analyzed to characterize TcGSTe17 (accession no. TC003347). Sequence analysis revealed that full-length TcGSTe17 was 1263 bp, which contained an ORF of 597 nucleotides encoding 198 amino acids (Fig. 1). The predicted molecular mass and isoelectric point of TcGSTe17 were 22.5 kDa and 6.61, respectively. In most organisms, the molecular mass of cytosolic GSTs ranges from 21 to 29 kDa (Blanchette et al. 2007, Mannervik et al. 1988), indicating that TcGSTe17 is in accordance with the characteristic of GSTs. In addition, the predicted TcGSTe17 protein contained GST_N_3 and GST_C, and no signal peptide was predicted at the N-terminus of TcGSTe17 (Fig. 1). This finding may be related to the fact that TcGSTe17 is a cytosolic GST (Hu et al. 2022).

Fig. 1.Fig. 1.Fig. 1.
Fig. 1. Nucleotide and deduced amino acid sequences of TcGSTe17 from T. castaneum. The red frame represents the N-terminus of TcGSTe17, and the blue frame represents the C-terminus.

Citation: Journal of Entomological Science 59, 3; 10.18474/JES23-71

Expression profile of TcGSTe17

The spatio-temporal expression of TcGSTe17 was detected to further analyze the function of TcGSTe17. Development stage expression profiling indicated that the transcripts of TcGSTe17 were expressed at all development stages of T. castaneum and were highly expressed in late-stage larvae (Fig. 2), which may be related to the resistance to xenobiotics during insect development. The period when insects consume most is the larval stage, and some studies have shown that continued development and increased food intake of larvae determine the increase of GST expression (You et al. 2015). This could explain why TcGSTe17 was highly expressed in the larvae stage. Similar results have been reported in several epsilon class GST genes from other insects, including C. pomonella (Hu et al. 2020a, 2022), Plutella xylostella (L.) (You et al. 2015), Bombyx mori L. (Yu et al. 2008), Nilaparvata lugens (Stal) (Sun et al. 2013), Chironomus tentans F. (Li et al. 2009), and Pieris rapae (L.) (Liu et al. 2017).

Fig. 2.Fig. 2.Fig. 2.
Fig. 2. The expression of TcGSTe17 in different developmental stages of T. castaneum. The gene rps3 was used as a housekeeping gene for the normalization of data. The error bars indicate the standard errors for three independent biological replicates. One-way analysis of variance (ANOVA) in combination with a least significant difference (LSD) test was performed for the significance analysis of all developmental stages. Different letters at the top of the bars indicate significant differences under P < 0.05.

Citation: Journal of Entomological Science 59, 3; 10.18474/JES23-71

The expression profiling of T. castaneum tissues indicated that TcGSTe17 was highly expressed in the gut of larvae (Fig. 3a) and the fat body of adults (Fig. 3b). The gut and fat body are important metabolic tissues of insects and are usually associated with the detoxification of xenobiotics (Hu et al. 2020a, b). The high expression of TcGSTe17 in these tissues implies that this gene also might be associated with the detoxification of xenobiotics. SoGSTe12, involved in the tolerance of phosphine, is highly expressed in the gut of Sitophilus oryzae (L.) (Hu et al. 2018), which is consistent with our results. In addition, TcGSTe17 is highly expressed in the gonads including ovary and testis (Fig. 3b). Similarly, some epsilon class GST genes showed gender-specific expression patterns in C. pomonella (Hu et al. 2020a, 2022) and B. dorsalis (Lu et al. 2016), indicating that this is a common phenomenon, and the detoxification capacity between males and females probably differs.

Fig. 3.Fig. 3.Fig. 3.
Fig. 3. The expression of TcGSTe17 in different tissues of T. castaneum. The gene rps3 was used as a housekeeping gene for the normalization of data. The error bars indicate the standard errors for three independent biological replicates. One-way analysis of variance (ANOVA) in combination with a least significant difference (LSD) test was performed for the significance analysis of all tissues. Different letters at the top of the bars indicate significant differences under P < 0.05.

Citation: Journal of Entomological Science 59, 3; 10.18474/JES23-71

The effects of eucalyptol on the expression profile of TcGSTe17

qRT-PCR was performed to investigate the effect of eucalyptol on the transcripts of TcGSTe17. In comparison to the control, the transcripts of TcGSTe17 were significantly upregulated following exposure to eucalyptol (Fig. 4), suggesting that TcGSTe17 could be significantly induced by eucalyptol. Insect GSTs are instrumental in the tolerance of xenobiotics such as insecticides and phytochemicals. For example, LdGSTe2a and LdGSTe2b were significantly overexpressed in L. decemlineata under the stress of three different insecticides (Han et al. 2016). Similarly, the mRNA levels of PrGSTe1, PrGSTe2, and PrGSTe3 in P. rapae were upregulated following exposure to different insecticides (Liu et al. 2017). The mRNA levels of TcGSTe17 were increased in the samples at 12 and 24 h after eucalyptol exposure and then decreased at 36, 48, 60, and 72 h (Fig. 4). This is a normal phenomenon of the stress response of insects. In general, when insects are attacked by xenobiotics, the expression level of detoxification enzyme genes will increase in response to the stress of foreign substances, but over time, these detoxification enzyme genes slowly return to normal levels of expression (Xiong et al. 2019b). This could explain the tendency of relative mRNA expression of TcGSTe17 after eucalyptol exposure. Consistent with the trend in our results, the transcripts of CpGSTe1 in C. pomonella first increased and then decreased with extended exposure time (Hu et al. 2022).

Fig. 4.Fig. 4.Fig. 4.
Fig. 4. The expression of TcGSTe17 in T. castaneum after exposure to eucalyptol. The 12-day-old T. castaneum larvae were treated with acetone (control) or eucalyptol. Then, the expression of TcGSTe17 was detected at 12, 24, 36, 48, 60, and 72 h by qRT-PCR. The gene rps3 was used as a housekeeping gene for the normalization of data. The error bars indicate the standard errors for three independent biological replicates. Student’s t tests were performed for the significance analysis. The asterisks at the top of bars indicate significant differences between control and treatment (*P < 0.05, **P < 0.01, ***P < 0.001).

Citation: Journal of Entomological Science 59, 3; 10.18474/JES23-71

Functional analysis of TcGSTe17 by RNAi

To further investigate whether TcGSTe17 is involved in resistance to eucalyptol in T. castaneum, 12-d-old larvae were injected with dsGSTe17 and then exposed to eucalyptol. The interference efficiency of the dsRNA was detected on the third day. Compared with the control, the transcripts of TcGSTe17 were significantly decreased (Fig. 5a). Subsequently, the surviving larvae were treated with eucalyptol to assess the tolerance of dsGSTe17-treated larvae to eucalyptol. In comparison to the control (IB- or dsVer-treated larvae), the mortality of dsGSTe17-treated larvae was markedly increased after exposure to eucalyptol (Fig. 5b), indicating that TcGSTe17 contributes to the susceptibility of T. castaneum to eucalyptol. Similarly, RNAi of epsilon class SlGSTe12 in S. litura (Li et al. 2021), BdGSTe2, BdGSTe3, BdGSTe4, and BdGSTe8 in B. dorsalis (Lu et al. 2016, 2020), LmGSTe4 in L. migratoria (Zhang et al. 2022), and AaGSTe2 and AaGSTe7 in A. aegypti (Lumjuan et al. 2011) also significantly increased the mortality from insecticides, indicating that epsilon class GSTs are involved in insecticide susceptibility. As a plant-derived compound, eucalyptol is not an insecticide but has significant activity against insects (Klocke et al. 1987, Sukontason et al. 2004). Similar results were found in our previous study (Gao et al. 2023), which suggests that eucalyptol is a potential active ingredient for controlling pests. Our study also demonstrates that an epsilon class GST (TcGSTe17) is associated with phytochemical tolerance, which could further validate the contribution of epsilon class GSTs in phytochemical resistance and provide strong support in the development of new strategies to control pests.

Fig. 5.Fig. 5.Fig. 5.
Fig. 5. The effect of RNAi treatment on the expression of TcGSTe17 (a) and the susceptibility of T. castaneum larvae to eucalyptol (b). The gene rps3 was employed as a housekeeping gene for the normalization of data. The error bars indicate the standard errors for three independent biological replicates. One-way analysis of variance (ANOVA) in combination with a least significant difference (LSD) test was performed for the significance analysis. Different letters at the top of the bars indicate significant differences under P < 0.05.

Citation: Journal of Entomological Science 59, 3; 10.18474/JES23-71

Acknowledgments

This study was sponsored by the Staring Foundation for the Doctor, Anyang Institute of Technology (grant number BSJ2019009, BSJ2021040 and BSJ2021041); the Staring Foundation of Innovation and Practice Base for Postdoctors, Anyang Institute of Technology (grant numbers BHJ2020008); major scientific and technological program of Anyang (grant numbers 2021A01NY004); the Scientific and Technological Project of Henan Province (grant number 212102110444); Anyang Science and Technology Research Project (grant number 2022C01NY015). The authors declare that they have no conflicts of interest.

References Cited

  • Begum, K., Li B., Beemanand R. W. and ParkY. 2009. Functions of ion transport peptide and ion transport peptide-like in the red flour beetle Tribolium castaneum. Insect Biochem. Mol. Biol. 39: 717–725.
  • Blanchette, B., Fengand X. and SinghB. R. 2007. Marine glutathione S-transferases. Marine Biotech. 9: 513–542.
  • Deng, H., Huang Y., Fengand Q. and ZhengS. 2009. Two epsilon glutathione S-transferase cDNAs from the common cutworm, Spodoptera litura: characterization and developmental and induced expression by insecticides. J. Insect Physiol. 55: 1174–1183.
  • Ding, Y., Hawkes N., Meredith J., Eggleston P., Hemingway J. and RansonH. 2005. Characterization of the promoters of epsilon glutathione transferases in the mosquito Anopheles gambiae and their response to oxidative stress. Biochem. J. 387: 879–888.
  • Enayati, A. A., Ransonand H. and HemingwayJ. 2005. Insect glutathione transferases and insecticide resistance. Insect Mol. Biol. 14: 3–8.
  • Gao, S., Huo Z., Guo M., Zhang K., Zhang Y., Wang X. and LiR. 2023. Contact toxicity of eucalyptol and RNA sequencing of Tribolium castaneum after exposure to eucalyptol. Entomol. Res. 53: 226–237.
  • Gao, S., Zhang K., Wei L., Wei G., Xiong W., Lu Y., Zhang Y., Gao A. and LiB. 2020. Insecticidal activity of Artemisia vulgaris essential oil and transcriptome analysis of Tribolium castaneum in response to oil exposure. Front. Genet. 11: 589.
  • Han, J. B., Li G. Q., Wan P. J., Zhu T. T. and MengQ. W. 2016. Identification of glutathione S-transferase genes in Leptinotarsa decemlineata and their expression patterns under stress of three insecticides. Pestic. Biochem. Physiol. 133: 26–34.
  • Hirowatari, A., Chen Z., Mita K. and YamamotoK. 2018. Enzymatic characterization of two epsilon-class glutathione S-transferases of Spodoptera litura. Arch. Insect Biochem. Physiol. 97: e21443.
  • Hu, C., Liu J. Y., Wang W., Mota-Sanchez D., He S., Shi Y. and YangX. Q. 2022. Glutathione S-transferase genes are involved in lambda-cyhalothrin resistance in Cydia pomonella via sequestration. J. Agric. Food Chem. 70: 2265–2279.
  • Hu, C., Wang W., Ju D., Chen G. M., Tan X. L., Mota-Sanchez D. and YangX. Q. 2020a. Functional characterization of a novel λ-cyhalothrin metabolizing glutathione S-transferase, CpGSTe3, from the codling moth Cydia pomonella. Pest Manag. Sci. 76: 1039–1047.
  • Hu, C., Wei Z. H., Li P. R., Harwood J. D., Li X. Y. and YangX. Q. 2020b. Identification and functional characterization of a sigma glutathione S-transferase CpGSTs2 involved in λ-cyhalothrin resistance in the codling moth Cydia pomonella. J. Agric. Food Chem. 68: 12585–12594.
  • Hu, F., Ye K., Tu X.-F., Lu Y.-J., Thakur K., Jiang L. and WeiZ.-J. 2018. Identification and expression profiles of twenty-six glutathione S-transferase genes from rice weevil, Sitophilus oryzae (Coleoptera: Curculionidae). Intern. J. Biol. Macromol. 120: 1063–1071.
  • Kalsi, M. and PalliS. R. 2017. Cap and collar transcription factor regulates multiple genes coding for proteins involved in insecticide detoxification in the red flour beetle, Tribolium castaneum. Insect Biochem. Mol. Biol. 90: 43–52.
  • Ketterman, A. J., Saisawang C. and WongsantichonJ. 2011. Insect glutathione transferases. Drug Metab. Rev. 43: 253–265.
  • Klocke, J. A., Darlington M. V. and BalandrinM. F. 1987. 1, 8-cineole (eucalyptol), a mosquito feeding and ovipositional repellent from volatile oil of Hemizonia fitchii (Asteraceae). J. Chem. Ecol. 13: 2131–2141.
  • Li, D., He C., Xie L., Kong F., Wu Y., Shi M., Liu R. and XuL. 2021. Functional analysis of SlGSTE12 in pyrethroid and organophosphate resistance in Spodoptera litura. J. Agric. Food Chem. 69: 5840–5848.
  • Li, X., Zhang X., Zhang J., Zhang X., Starkey S. R. and ZhuK. Y. 2009. Identification and characterization of eleven glutathione S-transferase genes from the aquatic midge Chironomus tentans (Diptera: Chironomidae). Insect Biochem. Mol. Biol. 39: 745–754.
  • Liu, S., Zhang Y.-X., Wang W.-L., Zhang B.-X. and LiS.-G. 2017. Identification and characterisation of seventeen glutathione S-transferase genes from the cabbage white butterfly Pieris rapae. Pestic. Biochem. Physiol. 143: 102–110.
  • Livak, K. J. and SchmittgenT. D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods25: 402–408.
  • Lu, X. P., Wang L. L., Huang Y., Dou W., Chen C. T., Wei D. and WangJ. J. 2016. The epsilon glutathione S-transferases contribute to the malathion resistance in the oriental fruit fly, Bactrocera dorsalis (Hendel). Comp. Biochem. Physiol. C Toxicol. Pharmacol. 180: 40–48.
  • Lu, X. P., Xu L., Meng L. W., Wang L. L., Niu J. and WangJ. J. 2020. Divergent molecular evolution in glutathione S-transferase conferring malathion resistance in the oriental fruit fly, Bactrocera dorsalis (Hendel). Chemosphere242: 125203.
  • Lu, Y., Park Y., Gao X., Zhang X., Yao J., Pang Y. P., Jiang H. and ZhuK. Y. 2012. Cholinergic and non-cholinergic functions of two acetylcholinesterase genes revealed by gene-silencing in Tribolium castaneum. Sci. Rep. 2: 288.
  • Lumjuan, N., McCarroll L., Prapanthadara L. A., Hemingway J. and RansonH. 2005. Elevated activity of an epsilon class glutathione transferase confers DDT resistance in the dengue vector, Aedes aegypti. Insect Biochem. Mol. Biol. 35: 861–871.
  • Lumjuan, N., Rajatileka S., Changsom D., Wicheer J., Leelapat P., Prapanthadara L. A., Somboon P., Lycett G. and RansonH. 2011. The role of the Aedes aegypti epsilon glutathione transferases in conferring resistance to DDT and pyrethroid insecticides. Insect Biochem. Mol. Biol. 41: 203–209.
  • Mannervik, B., Helena Danielson U. and KettererB. 1988. Glutathione transferases—structure and catalytic activity. Crit. Rev. Biochem. 23: 283–337.
  • Morou, E., Dowd A. J., Rajatileka S., Steven A., Hemingway J., Ranson H., Paine M. and VontasJ. 2010. A simple colorimetric assay for specific detection of glutathione-S transferase activity associated with DDT resistance in mosquitoes. PLoS Negl. Trop. Dis. 4: e808.
  • Muleya, V., Hayeshi R., Ranson H., Abegaz B., Bezabih M. T., Robert M., Ngadjui B. T., Ngandeu F. and MukanganyamaS. 2008. Modulation of Anopheles gambiae epsilon glutathione transferase activity by plant natural products in vitro. J. Enzy. Inhib. Med. Chem. 23: 391–399.
  • Nakamura, C., Yajima S., Miyamoto T. and SueM. 2013. Structural analysis of an epsilon-class glutathione transferase from housefly, Musca domestica. Biochem. Biophys. Res. Commun. 430: 1206–1211.
  • Pavlidi, N., Vontas J. and Van LeeuwenT. 2018. The role of glutathione S-transferases (GSTs) in insecticide resistance in crop pests and disease vectors. Curr. Opin. Insect Sci. 27: 97–102.
  • Ranson, H., Claudianos C., Ortelli F., Abgrall C., Hemingway J., Sharakhova M. V., Unger M. F., Collins F. H. and FeyereisenR. 2002. Evolution of supergene families associated with insecticide resistance. Science298: 179–181.
  • Sheehan, D., Meade G., Foley V. M. and DowdC. A. 2001. Structure, function and evolution of glutathione transferases: implications for classification of non-mammalian members of an ancient enzyme superfamily. Biochem. J. 360: 1–16.
  • Sukontason, K. L., Boonchu N., Sukontason K. and ChoochoteW. 2004. Efeitos do eucaliptol sobre a Musca domestica (Diptera: Muscidae) e Mosca varejeira (Diptera: Calliphoridae). Rev. Instit. Med. Trop. São Paulo46: 97–101.
  • Sun, X. Q., Zhang M. X., Yu J. Y., Jin Y., Ling B., Du J. P., Li G. H., Qin Q. M. and CaiQ. N. 2013. Glutathione S-transferase of brown planthoppers (Nilaparvata lugens) is essential for their adaptation to gramine-containing host plants. PLoS One8: e64026.
  • Wang, Y., Qiu L., Ranson H., Lumjuan N., Hemingway J., Setzer W. N., Meehan E. J. and ChenL. 2008. Structure of an insect epsilon class glutathione S-transferase from the malaria vector Anopheles gambiae provides an explanation for the high DDT-detoxifying activity. J. Struct. Biol. 164: 228–235.
  • Wang, Y., Zhang L.-T., Feng Y.-X., Zhang D., Guo S.-S., Pang X., Geng Z.-F., Xi C. and DuS.-S. 2019. Comparative evaluation of the chemical composition and bioactivities of essential oils from four spice plants (Lauraceae) against stored-product insects. Indust. Crops Prod. 140: 111640.
  • Wu, B., Lin W. H., Gao H. Y., Zheng L., Wu L. J. and KimC. S. 2006. Four new antibacterial constituents from Senecio cannabifolius. Pharm. Biol. 44: 440–444.
  • Xie, J., Sang M., Song X., Zhang S., Kim D., Veenstra J. A., Park Y. and LiB. 2020. A new neuropeptide insect parathyroid hormone iPTH in the red flour beetle Tribolium castaneum. PLoS Genet. 16: e1008772.
  • Xiong, W., Gao S., Lu Y., Wei L., Mao J., Xie J., Cao Q., Liu J., Bi J., Song X. and LiB. 2019a. Latrophilin participates in insecticide susceptibility through positively regulating CSP10 and partially compensated by OBPC01 in Tribolium castaneum. Pestic. Biochem. Physiol. 159: 107–117.
  • Xiong, W., Gao S., Mao J., Wei L., Xie J., Liu J., Bi J., Song X. and LiB. 2019b. CYP4BN6 and CYP6BQ11 mediate insecticide susceptibility and their expression is regulated by latrophilin in Tribolium castaneum. Pest Manag. Sci. 75: 2744–2755.
  • Xu, Z. B., Zou X. P., Zhang N., Feng Q. L. and ZhengS. C. 2015. Detoxification of insecticides, allechemicals and heavy metals by glutathione S-transferase SlGSTE1 in the gut of Spodoptera litura. Insect Sci. 22: 503–511.
  • Yamamoto, K., Aso Y. and YamadaN. 2013. Catalytic function of an ε-class glutathione S-transferase of the silkworm. Insect Mol. Biol. 22: 523–531.
  • Yang, P., Jia M. and ZhuL. 2021. Acaricidal activity of the essential oil from Senecio cannabifolius and its constituents eucalyptol and camphor on engorged females and larvae of Rhipicephalus microplus (Acari: Ixodidae). Exp. Appl. Acarol. 83: 411–426.
  • You, Y., Xie M., Ren N., Cheng X., Li J., Ma X., Zou M., Vasseur L., Gurr G. M. and YouM. 2015. Characterization and expression profiling of glutathione S-transferases in the diamondback moth, Plutella xylostella (L.). BMC Genomics16: 152.
  • Yu, Q., Lu C., Li B., Fang S., Zuo W., Dai F., Zhang Z. and XiangZ. 2008. Identification, genomic organization and expression pattern of glutathione S-transferase in the silkworm, Bombyx mori. Insect Biochem. Mol. Biol. 38: 1158–1164.
  • Zhang, J., Ma W., Yin F., Park Y., Zhu K. Y., Zhang X., Qin X. and LiD. 2022. Evaluations of two glutathione S-transferase epsilon genes for their contributions to metabolism of three selected insecticides in Locusta migratoria. Pestic. Biochem. Physiol. 183: 105084.
  • Zhang, Y., Gao S., Zhang P., Sun H., Lu R., Yu R., Li Y., Zhang K. and LiB. 2022. Response of xenobiotic biodegradation and metabolic genes in Tribolium castaneum following eugenol exposure. Mol. Gen. Genomics297: 801–815.
  • Zhou, L., Fang S. M., Huang K., Yu Q. Y. and ZhangZ. 2015. Characterization of an epsilon-class glutathione S-transferase involved in tolerance in the silkworm larvae after long term exposure to insecticides. Ecotoxicol. Environ. Saf. 120: 20–26.
Fig. 1.
Fig. 1.

Nucleotide and deduced amino acid sequences of TcGSTe17 from T. castaneum. The red frame represents the N-terminus of TcGSTe17, and the blue frame represents the C-terminus.

Fig. 2.
Fig. 2.

The expression of TcGSTe17 in different developmental stages of T. castaneum. The gene rps3 was used as a housekeeping gene for the normalization of data. The error bars indicate the standard errors for three independent biological replicates. One-way analysis of variance (ANOVA) in combination with a least significant difference (LSD) test was performed for the significance analysis of all developmental stages. Different letters at the top of the bars indicate significant differences under P < 0.05.

Fig. 3.
Fig. 3.

The expression of TcGSTe17 in different tissues of T. castaneum. The gene rps3 was used as a housekeeping gene for the normalization of data. The error bars indicate the standard errors for three independent biological replicates. One-way analysis of variance (ANOVA) in combination with a least significant difference (LSD) test was performed for the significance analysis of all tissues. Different letters at the top of the bars indicate significant differences under P < 0.05.

Fig. 4.
Fig. 4.

The expression of TcGSTe17 in T. castaneum after exposure to eucalyptol. The 12-day-old T. castaneum larvae were treated with acetone (control) or eucalyptol. Then, the expression of TcGSTe17 was detected at 12, 24, 36, 48, 60, and 72 h by qRT-PCR. The gene rps3 was used as a housekeeping gene for the normalization of data. The error bars indicate the standard errors for three independent biological replicates. Student’s t tests were performed for the significance analysis. The asterisks at the top of bars indicate significant differences between control and treatment (*P < 0.05, **P < 0.01, ***P < 0.001).

Fig. 5.
Fig. 5.

The effect of RNAi treatment on the expression of TcGSTe17 (a) and the susceptibility of T. castaneum larvae to eucalyptol (b). The gene rps3 was employed as a housekeeping gene for the normalization of data. The error bars indicate the standard errors for three independent biological replicates. One-way analysis of variance (ANOVA) in combination with a least significant difference (LSD) test was performed for the significance analysis. Different letters at the top of the bars indicate significant differences under P < 0.05.

Contributor Notes

5 Corresponding author (email: zhangkunpengag@163.com).
2 Department of Food and Bioengineering, Anyang Institute of Technology, Anyang, Henan, 455000, China.
3 Taihang Mountain, Forest Pests Observation and Research Station of Henan Province, Linzhou, Henan, 456550, China.
4 College of Life Sciences, Nanjing Normal University, Nanjing, Jiangsu, 210023, China.
Received: 26 Sept 2023
Accepted: 19 Oct 2023
  • Download PDF