Host adaptability and insecticide resistance of insects are closely related to detoxification metabolism-related proteins. In this study, the distribution and expression of glutathione s-transferase (GST) in Agrilus zanthoxylumi Hou (Coleoptera: Buprestidae) were studied. Based on the transcriptome data of A. zanthoxylumi, five GST genes were screened and cloned. The transcription levels of the five GST genes in male and female adult head, thorax, abdomen, legs and wings were determined by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) in order to provide a theoretical basis for the functional study of the gene. The results showed that all five GST genes had highly conserved N-terminal domain or C-terminal domain, belonging to two subfamilies of Delta or Sigma. The phylogenetic tree results showed that the evolutionary relationship of GST genes between A. zanthoxylumi, Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), and Tenebrio molitor Linnaeus (Coleoptera: Tenebrionidae) was the closest; RT-qPCR results showed that the five GST genes were differentially expressed in different tissues and sexes, and its expression level in each tissue of the male was higher than that of the female as a whole, especially in the head. The results of this study can provide basic data for analyzing the mechanism of detoxification resistance of A. zanthoxylumi and provide reference for its biological control and resistance research.
Agrilus zanthoxylumi Hou (Coleoptera: Buprestidae) is the main stem borer of Chinese prickly ash, Zanthoxylum bungeanum Maxim, in northern China (Yuan 2017) and can cause serious economic losses when occurring in large numbers. At present, A. zanthoxylumi is mainly based on chemical control. The mainstream chemical pesticide control technology is efficient, but it has many disadvantages, such as environmental pollution and development of resistance, which are not conducive to sustainable development. Under long-term selective pressure of pesticides, pests adapted through a variety of strategies leading to microevolution and resistance (Chen et al. 2016, Wang and Wu 2009, Zhou and Zeng 2014). Insect resistance mechanisms are diverse and complex, including behavioral resistance, decreased epidermal penetration, metabolic resistance, and target resistance. Overexpression of detoxification enzyme genes and mutation of target sites are the main causes of development of insect resistance to pesticides (Denholm et al. 2002).
In long-term evolution, phytophagous insects also formed a series of antidefense mechanisms to ensure the population reproduction, such as changing the activity levels of related physiological enzymes in the body to ensure that the population grows and reproduces successfully on the host plants (Li et al. 2018, Zhi et al. 2016). Studies have found that when insects feed on and adapt to different host plants or are challenged by a variety of pesticides, they usually rely on the detoxification enzyme system in vivo to activate and regulate their resistance mechanisms, thus showing a certain degree of detoxification ability. Moreover, this ability is regulated by its gene and is inheritable (Cai 2017).
Glutathione S-transferase (GST) is a multifunctional superfamily of enzymes involved in detoxification of a variety of exogenous substances, including pesticides (Salinas and Wong 1999). They are widely present in aerobic organisms and play a central role in detoxification of endogenous and exogenous compounds. In recent years, research of insect GSTs in resistance has mainly focused on their detoxification of exogenous compounds, especially pesticides and phytochemicals, and their role in regulating oxidative stress response. GSTs can metabolize pesticides by promoting the reductive dehydrogenation of pesticides or the coupling reaction with reduced glutathione, thereby producing water-soluble metabolites that are more easily excreted (Enayati et al. 2005). In addition, GSTs also can be used as ligand-binding proteins to capture toxic substances, with the insects exhibiting toxin resistance and tolerance (You et al. 2013). Gu et al. (2018) used real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) for quantitative analysis and found that the expression level of GSTs was reduced after the Myzus persicae Sulzer (Hemiptera: Aphididae) was transferred from the original host cabbage to the tobacco host, indicating that GSTs might contribute to the feeding of M. persicae on tobacco. The above results indicate that GSTs in insects are involved in the detoxification metabolism of insects when they fed on different hosts or responded to secondary substances stress, thus enhancing their resistance and adaptability.
Compared with other coleopteran stem borers, the transcriptome-related studies of A. zanthoxylumi mainly involve the analysis of olfactory expression profile, as well as the identification and expression of chemosensory protein genes, and there are few reports on the molecular mechanism of resistance. In this study, five GST genes with detoxification metabolism were screened, cloned, and identified based on the existing transcriptome data of A. zanthoxylumi. The similarities of detoxification enzyme-related genes with related species of Coleoptera were explored, and the expression of GST genes in different tissues and sexes of A. zanthoxylumi also was analyzed. The results could provide a basis for research on the adaptability, resistance mechanism, and inheritance of resistance to pesticides of A. zanthoxylumi and provide new ideas for formulating sustainable green prevention and control strategies of A. zanthoxylumi.
Materials and Methods
Sample preparation and total RNA extraction. The insects used in this study were collected from May to June 2020 in Puhua Town, Lantian County, Shaanxi Province (109°31′E; 34°13′N) where the average annual temperature is 12°C. The head, thorax, abdomen, legs, and wings of male and female adults were removed and placed individually into centrifuge tubes without RNA enzyme and held at –80°C until assayed.
Total RNA was extracted using the column type insect RNA extraction kit (BTN81220, Biolab Company, Beijing). Following the kit instructions, total RNA was extracted from the five tissues of male and female insects. During the entire extraction process, masks and gloves were worn to prevent the degradation of RNA quality caused by contamination.
Primer design and gene cloning. Five GST gene sequences were screened from the transcriptome data of A. zanthoxylumi, named as AzanGST1, AzanGST3, AzanGST4, AzanGST5, and AzanGST11. The primers were designed for quantitative analysis by Primer-BLAST with the annealing temperature controlled at 50°C–55°C. The designed length of each primer was 16–22 bp, which were sent to Shanghai Biotech Co., Ltd. for synthesis (Table 1).
The first cDNA strand was synthesized using the Prime Script Reverse Transcription System Reverse Transcription Kit (R133-01, Vazyme, Beijing) with the total RNA of each tissue of A. zanthoxylumi as template. The nucleotide sequence encoding the gene was amplified in PCR. The PCR reaction conditions were as follows: 95°C for 3 min, 35 cycles of 94°C for 30 s, 60°C for 30 s, 72°C 30 s, and 72°C for 10 min. The amplified products were detected by 1% agarose gel electrophoresis, and the length of DNA amplified fragment was determined according to the detection results.
Bioinformatics analysis. The nucleic acid sequence of GST gene of other insects were downloaded from National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/guide/) using the online Blast tool (BLAST http://www.ncbi.nlm.gov/BLAST/) to compare the similarity between GST gene of A. zanthoxylumi and other insects. The phylogenetic tree of A. zanthoxylumi and other insects was constructed by using Neighbor-Joining (NJ) method in MEGA software. The Bootstrap value was set to 1,000. ExPASy (http://www.expasy.org/tools/protparam.html) was used to analyze the relative molecular weight, isoelectric point, and amino acid sequence composition of the GST gene encoded protein of A. zanthoxylumi.
Real-time fluorescence quantitative PCR detection. The synthesized cDNA first strand from each tissue was used as a template to detect the expression amount of the GST gene by RT-qPCR, using the following protocol: predenatured at 95°C for 30 s, 40 cycles of 95°C for 0.05 s, 60°C for 30 s, 72°C for 30 s. The relative expression was calculated according to the Ct value obtained by 2–ΔΔCt. Statistical Product and Service Solutions 19.0 software was used to analyze the differences between different organizations (analysis of variance, Tukey Honestly Significant Difference (HSD), P < 0.05).
Preliminary identification of five GST genes from A. zanthoxylumi. Based on the transcriptome data of A. zanthoxylumi and comparison validation in NR database, five GST genes were identified, which were named as AzanGST1, AzanGST3, AzanGST4, AzanGST5, and AzanGST11. BLAST results showed five candidate GST genes that exhibited had high matching degree with the GST genes of Agrilus planipennis Fairmaire (Coleoptera: Buprestidae) and Anoplophora glabripennis Motschulsky (Coleoptera: Cerambycidae) (80–90%). These results indicated that the five candidate GST genes of A. zanthoxylumi are GST genes (Table 2).
Phylogenetic analysis of AzanGSTs gene. The phylogenetic tree of five GST genes from A. zanthoxylumi and other coleopterans was constructed by the NJ method. As can be seen from the Fig. 1, AzanGSTs and other coleopteran GST genes are closely related, basically with the related species clustered into a branch. Among them, AzanGST4, AzanGST11, and A. planipennis GST gene are clustered into a branch; AzanGST3, AzanGST5, and Tenebrio molitor GST gene are clustered into a branch; and AzanGST1 and Lissorhoptrus oryzophilus Kuschel (Coleoptera: Elephantidae) GST gene are clustered into a branch. These results indicate that A. zanthoxylumi has the closest evolutionary relationship with GST genes of A. planipennis and T. molitor.
Cloning and analysis of AzanGSTs gene. All the five genes obtained by PCR gene cloning have complete open reading frames. The obtained five amplification products were basically consistent with the expected values. The corresponding amino acid sequence characteristics were analyzed and compared showing that all the five genes have GST-N or GST-C domains (Table 3). AzanGST3 and AzanGST5 only have GST-C domains and belong to Delta family. AzanGST1, AzanGST4, and AzanGST11 have both GST-C and GST-N domains. After comparison and identification, AzanGST1 and AzanGST4 were determined to belong to the Delta family and AzanGST11 to the Sigma family. The amino acid number of AzanGST1 was more than 900 amino acids and the molecular weight was above 100 ku. The numbers of amino acids of the other four GST genes were 140–570 and the molecular weights were 16–65 ku. The isoelectric points of the five GST genes ranged from 5.02 to 8.97.
Expression analysis of AzanGSTs gene in different tissues. The AzanGSTs gene was differentially expressed in different tissues and genders (Fig. 2). The expression level in male tissues was higher than that in female tissues as a whole. The expression levels in the male head, abdomen, and wings were relatively high. Five genes in the male head are higher than that in the female head. In particular, the expression level of AzanGST3 in the male head was about 27× greater than that in the female head. In AzanGST5, the expression level in male head was about 13× more than that in the female head, and the expression level in the male abdomen was about 19× more than that in the female abdomen. It is noteworthy that the expression of AzanGST11 in other tissues is generally lower than that of the other four genes except for the high expression in the abdomen. Overall, the expression of AzanGST genes in males was higher than that in females, especially in the head. The highest expression level in female A. zanthoxylumi was in the wings followed by legs and abdomen. The highest expression level in male A. zanthoxylumi was in the head followed by abdomen and wings.
Insect GST is an important metabolic enzyme in insects that is involved in the metabolism of exogenous and endogenous toxic substances (Li 2014). It is divided into six families designated as Delta, Omega, Epsilon, Theta, Sigma, and Zeta and an “unclassified” subfamily (Ketterman et al. 2011, Liu et al. 2017, You et al. 2013). In this study, we obtained five high-expression GST genes (AzanGST1, AzanGST3, AzanGST4, AzanGST5, and AzanGST11) from transcriptome data of A. zanthoxylumi. By analyzing the gene sequences obtained from full-length cloning and constructing the phylogenetic tree in comparison with other related species, we confirmed that these five genes belonged to the Delta and Sigma families. The matching degree of GST gene between A. zanthoxylumi and other coleopterans was high, and the evolutionary relationship of A. zanthoxylumi was closest to that of A. planipennis and T. molitor. Sigma family GSTs widely exist in organisms, while Delta is an insect-specific family (Enayati et al. 2005). Ranson et al. (2002) found through functional verification that the Delta family GSTs play an important role in the detoxification and metabolism of exogenous substances, which can enhance the adaptability of insects to the environment. However, whether it is an insect-specific GST gene or not, it plays an important role in metabolism of endogenous and exogenous compounds (Oakeshott et al. 2010).
In this study, five GST genes were expressed in different tissues of A. zanthoxylumi, and the expression levels in different tissues of male were generally higher than those of the female, in the head and abdomen were relatively high. The four Delta family genes AzanGST1, AzanGST3, AzanGST4, and AzanGST5 in the head of the male A. zanthoxylumi were significantly higher than those in other tissues. This is similar to previous studies. DaGSTe1 gene of Dendroctonus armandii Tsai et Li (Coleoptera: Scolytidae) is highly expressed in males, and CpomGSTd2 gene of codling moth Cydia pomonella Linnaeus (Lepidoptera: Tortricidae) is expressed in antennae higher than that of females. It is speculated that the GST gene may be involved in the degradation of toxic substances and regulation of sex pheromones (Huang et al. 2017, Ma et al. 2015). The specific detoxification effects of AzanGSTs on exogenous toxic substances need to be further explored and verified.
The results of this study can provide a basis for further research on the adaptability of A. zanthoxylumi to insecticides and the mechanism and inheritance of toxin resistance. However, it should be noted that in this research, the distribution of GST gene was detected only in the adult tissue of A. zanthoxylumi, and the expression in the detoxification metabolic organs of larvae, such as midgut, Malpighian tubules, and fat body, was not detected. Thus, the function of these genes in the physiological metabolism of larvae remains unclear. In addition to GST, the specific enzymes involved in the detoxification metabolism of endogenous and exogenous secondary substances by A. zanthoxylumi require further study.
This study was supported by the “Green Pollution Control Technology Based on Chemical Pheromone in Agrilus zanthoxylum”, The National Public welfare forestry projects in China (Grant No. 201504324); “Forestry Science and Technology Promotion and Demonstration Project in Yunnan Province: Promotion and Application Demonstration of Green Control Technology for Main Pests and Diseases of Zanthoxylum bungeanum Maxim (yun TG01)”; “Shaanxi Province's Second Batch of Special Support Program Leading Talent Project”. Mention of a commercial or proprietary product does not constitute an endorsement of the product by the Northwest Agriculture & Forestry University.