Introduction
Aphis spiraecola Patch (Hemiptera: Aphididae), which was mistakenly synonymized with Aphis citricola (van der Goot) (Eastop and Blackman, Reference Eastop and Blackman1988), is a polyphagous aphid species that is distributed among temperate and tropical regions of the globe. In the last few decades, A. spiraecola has become a widely distributed pest of apple and citrus orchards (CABI, 2022). This invasive aphid causes the curling of young leaves, reduces the growth of infested shoots and excretes honeydew on leaves and fruit thus favouring the development of the fungal disease sooty mould, leading to economic losses (Mu et al., Reference Mu, Liu, Kong, Gao, Shi and Qiu2002).
Currently, insecticides are commonly used for managing A. spiraecola. However, the extensive use of insecticides has led to the resistance of insects to multiple insecticide classes, including organophosphates, carbamates, and neonicotinoids (Bass et al., Reference Bass, Puinean, Zimmer, Denholm, Field, Foster, Gutbrod, Nauen, Slater and Williamson2014; Chen et al., Reference Chen, Li, Chen, Ma, Liang, Liu, Song and Gao2016; Margaritopoulos et al., Reference Margaritopoulos, Kati, Voudouris, Skouras and Tsitsipis2021). Lambda-cyhalothrin is a broad-spectrum, fast-acting type-II pyrethroid insecticide, which is widely used due to its relatively low toxicity to humans and high effectiveness in controlling insects (Soderlund and Bloomquist, Reference Soderlund and Bloomquist1989; Nasuti et al., Reference Nasuti, Cantalamessa, Falcioni and Gabbianelli2003). Resistance to pyrethroid insecticides has been found in some aphid species. For instance, Myzus persicae (Sulzer) and Aphis gossypii Glover have been reported to have developed resistance to β-cypermethrin (Tang et al., Reference Tang, Ma, Hou and Gao2017; Wang et al., Reference Wang, Liang, Shang, Yu and Xue2021). On the other hand, Rhopalosiphum padi (L.) has been reported to possess resistance to lambda-cyhalothrin (Zuo et al., Reference Zuo, Wang, Zhang, Peng, Piñero and Chen2016b; Wang et al., Reference Wang, Huang, Li and Chen2018, Reference Wang, Bai, Zhao, Su, Liu, Han and Chen2020). The resistance levels of A. spiraecola to lambda-cyhalothrin have not been documented.
There are several mechanisms underlying the development of insect resistance to insecticides, and some species may exhibit more than one of these mechanisms simultaneously. Insect resistance to pesticides can be attributed to two major mechanisms: metabolic resistance and target site insensitivity (Liu, Reference Liu2015; Yang and Zhang, Reference Yang and Zhang2015; Barres et al., Reference Barres, Micoud, Corio-Costet, Debieu, Fillinger-David, Walker, Délye, Grosman and Siegwart2016). In many insect populations that have developed resistance to insecticides, the constitutive expression levels of detoxification enzyme (e.g. Glutathione S-transferases, GSTs; carboxylesterases, CarEs; and Cytochrome P450 monooxygenases, P450s) genes were significantly increased. Hu et al. (Reference Hu, Lu, Li, Su, Dong, Zhang, Liu, Shi, Wang and Chen2022b) found that the overexpression of the CYP4CJ6 gene conferred resistance to imidacloprid and thiamethoxam in the Indian grain aphid, Sitobion miscanthi (Takahashi). Some P450 gene expression were significantly increased and associated with lambda-cyhalothrin resistance in R. padi (Wang et al., Reference Wang, Bai, Zhao, Su, Liu, Han and Chen2020, Reference Wang, Zhao, Han and Chen2022). In the chlorpyrifos-resistant strain of Laodelphax striatellus (Fallen), the expression of LsCarE1 was found to be up-regulated by 32.06-fold (Zhang et al., Reference Zhang, Wang, Guo, Li, Zhang, Xie and Fang2012). GSTs can mediate insecticide resistance through various mechanisms (Pavlidi et al., Reference Pavlidi, Vontas and Van Leeuwen2018). In the codling moth, Cydia pomonella (L.), it was found that GSTs were overexpressed in the lambda-cyhalothrin resistant strain, and it was confirmed that the GSTs contribute to the resistance through the sequestration mechanism (Hu et al., Reference Hu, Liu, Wang, Mota-Sanchez, He, Shi and Yang2022a).
The α subunit of the insect sodium channel is composed of four structurally similar domains (I–IV), each containing six hydrophobic transmembrane helices (S1–S6) and a P-loop. The voltage sensing module is formed by segments S1–S4, while segments S5 and S6, along with the P-loop, form the sodium ion passage pore module (Dong et al., Reference Dong, Du, Rinkevich, Nomura, Xu, Wang, Silver and Zhorov2014). Mutations associated with knockdown resistance (kdr) mostly occur in the S5 and S6 transmembrane helices of domain II in the sodium channel. For instance, one mutation involves the substitution of phenylalanine with leucine at position 1014 in the transmembrane segment IIS6 (kdr, L1014F, Martinez-Torres et al., Reference Martinez-Torres, Foster, Field, Devonshire and Williamson1999). Another mutation involves the substitution of leucine with methionine at position 918 in the transmembrane segment IIS4-S5 (super-kdr, M918L, Eleftherianos et al., Reference Eleftherianos, Foster, Williamson and Denholm2008). There were multiple leucine substitutions at position 918 and other site-specific mutations may also be present in the sodium channel (Dong et al., Reference Dong, Du, Rinkevich, Nomura, Xu, Wang, Silver and Zhorov2014). Mutations in the target site of the voltage-gated sodium channel (VGSC) gene are involved in aphid resistance to pyrethroids (Guillemaud et al., Reference Guillemaud, Brun, Anthony, Sauge, Boll, Delorme, Fournier, Lapchin and Vanlerberghe-Masutti2003; Bass et al., Reference Bass, Puinean, Zimmer, Denholm, Field, Foster, Gutbrod, Nauen, Slater and Williamson2014; Scott, Reference Scott2019). For instance, the M918 T mutation in the sodium channel of the English grain aphid, Sitobion avenae (Fabricius), played a significant role in conferring resistance to pyrethroids (Foster et al., Reference Foster, Paul, Slater, Warren, Denholm, Field and Williamson2014). Similarly, the M918 T/L/V and L1014F mutations contributed to pyrethroid resistance in various insects such as M. persicae, A. gossypii, and R. padi. (Eleftherianos et al., Reference Eleftherianos, Foster, Williamson and Denholm2008; Chen et al., Reference Chen, Tie, Chen, Ma, Li, Liang, Liu, Song and Gao2017; Wang et al., Reference Wang, Bai, Zhao, Su, Liu, Han and Chen2020; Munkhbayar et al., Reference Munkhbayar, Liu, Li and Qiu2021). However, the underlying mechanisms involved in pesticide resistance of A. spiraecola are still unclear.
In this study, we quantified the toxicity of lambda-cyhalothrin to field populations of A. spiraecola from different regions of the Shaanxi province, China. The changes in detoxification enzyme activities and gene transcription levels of A. spiraecola in response to sublethal doses of lambda-cyhalothrin were quantified. Furthermore, we characterized the mutation target sites of VSGC and examined the mutation rates of L1014F. Our main objective was to investigate the initial resistance mechanisms of A. spiraecola to lambda-cyhalothrin and to establish a theoretical foundation for effective pest resistance management programmes of the aphid.
Materials and methods
Insects
The A. spiraecola laboratory susceptible strain (SS) was collected from apple orchards in Yangling (Shaanxi Province) in 2020. The SS strain was reared on apple seedlings without exposure to any insecticides. The A. spiraecola field populations were collected from apple trees in eight regions of Shaanxi province, one of the most important apple-growing areas in China (table 1). All aphids were reared on apple seedlings at 25 ± 1℃, 60 ± 10% relative humidity, and a 16:8 h (L:D) photoperiod in the laboratory.
Table 1. Sampling locations for A. spiraecola in apple orchards of Shaanxi
Susceptibility of A. spiraecola to lambda-cyhalothrin
Lambda-cyhalothrin, [cyano- (3-phenoxyphenyl) methyl] 3-(2-chloro-33,3-trifluoroprop-1-enyl)-2,2-dimethylcyclopropane-1-carboxylate (95% purity, Ningbo Sanjiang Yinong Chemical Co., Ltd., China) was used in this study. A leaf dip method modified by Zuo et al. (Reference Zuo, Wang, Zhang, Peng, Piñero and Chen2016b) was used for the bioassays. Five to six serial concentrations of lambda-cyhalothrin were prepared using 0.1% tween-100 as solvent. Leaves with 30 apterous adult aphids were dipped in the insecticide dilutions for 10 s. Then, the leaves were removed from the solution, and residual droplets on the leaves were adsorbed with clean, dry filter paper. One replicate consisted of 30 aphids exposed to serial concentrations of insecticide. There were three replicates of each concentration. The treated aphids were kept at 25 ± 1℃, 60 ± 10% RH, and 16 h / 8 h dark-light cycle. After 24 h, mortality was assessed with a microscope. Aphids were considered dead if they did not move after gentle prodding with a fine brush.
Assay of detoxification enzyme activity in A. spiraecola
Protein content determination
For this assay, we used ten adults of A. spiraecola from each region. Three detoxification enzymes (P450, CarE, and GST) were extracted with 1 mL of PBS (phosphate-buffered saline) buffer. The detoxification enzymes were homogenized with PBS of different pH (pH8.0 for GST, pH7.8 for P450, and pH7.0 for CarE). The homogenate was centrifuged under 12,000 RPM at 4 °C for 30 min. The method of Bradford (Reference Bradford1976) was used to measure the protein content and detected by a BCA protein assay kit (Beijing Solar Science and Technology Co., Ltd., Beijing, China).
Detoxification enzyme activity
The activity of the three detoxifying enzymes (P450, GST, and CarE) was measured following the method of Su et al. (Reference Su, Jian, Zhang, Fang, Peng, Piñero and Chen2021). For GST activity, CDNB (1-chloro-2,4-dinitrobenzene) and GSH (reduced glutathione) were used as the substrate for the reaction with enzyme solution, and the changed absorbance was measured at 340 nm for 5 min. In contrast, P450 was measured at 400 nm after the p-nitroanisole and NADPH reacted with the enzyme solution at 30 °C for 2 h and was calculated with PBS buffer as a control. Chromogenic agents and α-naphthyl acetate were used for CarE. Each of them was mixed with the enzyme solution and reacted at 30 °C for 10 min, and the absorbance at 600 nm was measured, which also used PBS buffer as a control. Chromogenic agents consisted of 5% sodium dodecyl sulphonate and 1% fast blue B salt solution with a volume ratio of 5:2. All works were replicated three times.
RNA extraction and cDNA synthesis
Ten A. spiraecola apterous adult aphids were placed in 1.5 mL RNase-free centrifuge tubes (30 adults per treatment) and stored in a refrigerator at −80℃ for RNA extraction. Total RNA was extracted by TRIGeneⓇ Reagent (TRIGeneⓇ Biotech (Beijing) Co., Ltd., Beijing, China). DNase I (Takara, Kyoto, Japan) for DNA decontamination in total RNA was performed. HiScript® II Q RT SuperMix Kit (Vazyme Biotech Co., Ltd, Nanjing, China) was used to reverse transcribe first-strand cDNA with RNA as a template. The system was 1 μl total RNA (1 μg), 4 μl 4 × gDNA Wiper Mix, and 11 μl RNase-free water, reacted at 42℃ for 2 min. 4 μl 5 × HiScript II qRT SuperMix II was added to the previous system at 50 °C for 15 min and 85 °C for 15 s. The first-strand cDNA was used as a template for quantitative real-time PCR (hereafter referred to as qPCR).
Quantitative real-time PCR (qPCR)
For analyses of P450 gene expression in aphids from field populations and SS strain, 10 aphids were randomly taken for RNA extraction and cDNA synthesis for each of the three replicates. The baseline toxicity of SS was determined by the above bioassay method, and P450 gene expression was examined by treating SS of A. spiraecola with the λ-cyhalothrin LC50 concentration. Twenty-five apterous adult aphids were treated with LC50 concentrations of λ-cyhalothrin. The carrier (ddH2O with 0.01% (v/v) Triton X-100) was used as a control. Ten surviving aphids were collected from each of the three replicates 24 h after treatment for RNA extraction and cDNA synthesis.
Twenty-five P450 genes of A. spiraecola were identified from the transcriptome sequence. Primer Premier 5.0 (Tsingke Biological Technology Co., Ltd., Beijing, China) was used to design primers, as shown in table S1. The qPCR analysis was performed with ChamQ SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd, Nanjing, China) in Rotor Q thermocycler (Qiagen, Hilden, Germany). For the system and conditions of the reaction referred to Su et al. (Reference Su, Jian, Zhang, Fang, Peng, Piñero and Chen2021). Lambda-actin gene and elongation factor 1-alpha (EF1-α) were regarded as internal reference genes (Wang et al., Reference Wang, Huang, Li and Chen2018; Fan et al., Reference Fan, Han, Gao, Liu, Zhang, Yang and Fan2019; Li et al., Reference Li, Li, Wang, Li, Zhu, Zhang, Li, Yang and Zhu2021). Primers for qPCR amplification efficiency were determined by a 5-fold serial dilution with a cDNA template. Each sample was subjected to three biological replicates and three mechanical replicates. The LightCycler 480 system (Roche) was adapted for qPCR detection and then the relative gene expression level was analysed by the 2−ΔΔCT method (Livak and Schmittgen, Reference Livak and Schmittgen2001).
Mutation detection
Mutation sites in the IIS4-IIS6 region of VGSC had been reported to be linked with pyrethroid resistance in many pests, in particular M918L and L1014F (Foster et al., Reference Foster, Paul, Slater, Warren, Denholm, Field and Williamson2014; Chen et al., Reference Chen, Tie, Chen, Ma, Li, Liang, Liu, Song and Gao2017; Wang et al., Reference Wang, Bai, Zhao, Su, Liu, Han and Chen2020). Based on the sequence of the sodium channel α subunit, we genotyped for the mutation of the VGSC gene from individuals of A. spiraecola field and laboratory SSs. The EZNA® Tissue DNA Kit (Omega Bio-Tek Inc., Norcross, GA, USA) was used to extract genomic DNA (gDNA) from each individual aphid according to the recommended protocol. The gDNA was used as a template and the primer pairs of AsVGSC are shown in table S1. The PCR reaction system was 25 μl, including 2.5 μl 2 × SuperStar HiFi PCR Mix (GenStarⓇ Biotech (Beijing) Co., Ltd., Beijing, China), 1 μl 10 μM upstream/downstream primers, and 1 μl genomic DNA, and the rest was supplemented with RNase-free water to 25 μl. The reaction conditions are as follows: 94℃ for 3 min, then 35 cycles of 94℃ for 30 s, 60–65℃ for 30 s, 72℃ for 1 min, and finally 72℃ for 5 min. PCR products were analysed on 1% agarose gel (40 ml 1 × TAE buffer with 0.4 g agarose) and coloured under a DL2000 DNA marker (Dining Biotech Co., Ltd., Beijing, China). Finally, it was sent to Shanghai Sangon Biotech Co., Ltd., (Shanghai, China) for sequencing.
Statistical analysis
LC50 values were calculated and compared against the control. The two compared values were considered significantly different if their respective 95% CIs did not overlap (Litchfield and Wilcoxon, Reference Litchfield and Wilcoxon1949; Wolfe and Hanley, Reference Wolfe and Hanley2002). The activities of three detoxification enzymes (P450, GST, and CarE) and the relative expression levels of P450 genes were compared statistically using SPSS 25.0 (SPSS, Chicago, IL) with α = 0.05. Resistance ratios (RR) were used to determine levels of resistance of A. spiraecola to lambda-cyhalothrin. The resistance levels used were based on Shen and Wu (Reference Shen and Wu1995): susceptible (RR < 3-fold), decreased susceptibility (3- < RR ≤ 5-fold), low resistance (RR = 5- to 10-fold), moderate resistance (10- < RR ≤ 40-fold), high resistance (RR = 40-160-fold), and extremely high resistance (RR > 160-fold). Linear regression analyses were used to establish the relationship between the median lethal concentration of lambda-cyhalothrin and the mutation rates of sodium channel L1014F. All bioassay analyses were conducted using DPS software (Zhejiang University, Hangzhou, China).
Results
Susceptibility of field populations of A. spiraecola to lambda-cyhalothrin
The susceptibility of A. spiraecola to lambda-cyhalothrin was found to differ from one region to another and the LC50 values varied between years in the same area (table 2). The LC50 values recorded in four regions - BS, QX, FF, and LC - showed an increasing trend from 2021 to 2022, while such an increase of LC50 values was not found in the other regions. The LC50 of the SS in the laboratory was 2.07 (1.686–2.742) mg l–1. The RR values of A. spiraecola to lambda-cyhalothrin were highest in the XY and XP areas in 2021 (174.230 and 123.240, respectively). However, for both locations, the RR values decreased in 2022 to 111.082 in XY and to 103.197 in XP. The lowest RR values were recorded in the BS and FF locations.
Table 2. Susceptibility of eight A. spiraecola field populations to lambda-cyhalothrin
SEM, Standard error of the mean; χ2, Chi-square value; CL, 95% confidence limits; SS, Susceptible strain.
a RR: Resistance ratios = resistant strain LC50 of lambda-cyhalothrin/ LC50 of susceptible strain.
Metabolic enzymes activity of A. spiraecola
In 2021, Cytochrome P450 activity in field populations was significantly higher (t-test; P < 0.01) in five locations (XY, LQ, FX, LC, and XP) compared to the control (SS, fig. 1A). Compared with the SS, the field population did not show a significant increase in the GST enzyme activity, except in the case of the LC and FF populations in 2021 (fig. 1B). Similarly, the CarE enzyme activity was not significantly increased (fig. 1C). The activity levels of P450 differed significantly between field strains and SS in 2022 (fig. 1a). GST and CarE activity were significantly lower in the samples from different regions in 2022 compared with SS, except the CarE enzyme in LQ population (fig. 1b and 1c).
Figure 1. Specific activities of detoxifying enzymes of A. spiraecola in field populations relative to the susceptible laboratory strain (SS). (A)-(C) The specific activity of three detoxification enzymes in samples collected in 2021. (A) The specific activity of P450; (B) The specific activity of GST; (C) The specific activity of CarE. (a)–(c) The specific activity of three detoxification enzymes in samples collected in 2022. (a) The specific activity of P450; (b) The specific activity of GST; (c) The specific activity of CarE. Data are shown as mean ± SEM (standard error of mean). The asterisks above bars denote significant differences between treatments and SS according to the t-test. ns = not significant; *, P < 0.05; **, P < 0.01.
The expression levels of P450 genes in A. spiraecola
The relative expression levels of 25 P450 genes (table S1) identified from the transcriptome are shown in figs 2 and 3. The results indicated that compared with the control (SS strain), the expression levels of 14 P450 genes in XY were significantly up-regulated, while 18 P450 genes in XP were significantly higher than the SS (fig. 2). The qPCR analyses showed that the expression levels of one P450 gene were not significantly affected, while nine P450 genes were significantly down-regulated (fig. 3A) and fifteen P450 genes were significantly up-regulated after treatment with a sublethal concentration of lambda-cyhalothrin (fig. 3B). There were eight P450 genes were expressed with up-regulated levels in both sublethal effects and field populations.
Figure 2. Relative expression levels of P450 genes of A. spiraecola and the susceptible laboratory strain (SS) that were up-regulated in two field populations: XY (A) and XP (B). Data are shown as mean ± SEM values. The asterisks above bars denote significant differences between treatments according to the t-test. ns = not significant; *, P < 0.05; **, P < 0.01.
Figure 3. Relative expression levels of P450 genes of A. spiraecola and the susceptible laboratory strain (SS) treated with the LC50 concentrations of lambda-cyhalothrin. (A) P450 genes were not up-regulated; and (B) P450 genes were up-regulated. Data are shown as mean ± SEM values. Asterisks above bars denote significant differences between treatments according to Student's t-test. ns = not significant; *, P < 0.05; **, P < 0.01.
Mutations of sodium channel site in A. spiraecola
A 486 bp with M918 site and a 605 bp with L1014 site of AsVGSC were amplified. The L1014F mutation in the VGSC gene was found to be present in A. spiraecola, while the M918L was not found (fig. 4). The CTT to TTT appeared in all the sequence chromatogram with a mixed peak, indicating that the L1014F was heterozygous (fig. 4B). The results showed that the mutation frequency of L1014F in the SS strain was 3.3%, while the mutation frequency was 80.0%, 70.6%, and 76.7% in the population from XY, LQ, and XP, respectively (table 3). There was a significant positive linear correlation between the L1014F mutation frequency and LC50 of lambda-cyhalothrin in A. spiraecola (r = 0.767, P < 0.05) (fig. 5).
Figure 4. Detection of voltage-gated sodium channel mutation in A. spiraecola. (A) and (B) Partial nucleotide sequence chromatograms of the M918 site in A. spiraecola voltage-gated sodium channel between Susceptible Strain (A) and field populations (B). No mutation was found in the field population. (C) and (D) Partial sequence of the nucleotide at the L1014F mutation site in the A. spiraecola voltage-gated sodium channel in Susceptible Strain (C) and field population (D). The field strains of A. spiraecola showed the heterozygous mutation.
Table 3. Mutation frequencies of L1014F in the sodium channel of A. spiraecola
Figure 5. Linear regression analysis of the LC50 of lambda-cyhalothrin and the mutation frequencies of L1014F in the sodium channel of A. spiraecola.
Discussion
While pyrethroid insecticides are being increasingly applied in pest control (Bass et al., Reference Bass, Puinean, Zimmer, Denholm, Field, Foster, Gutbrod, Nauen, Slater and Williamson2014; Scott et al., Reference Scott, Yoshimizu and Kasai2015; Wang et al., Reference Wang, Bai, Zhao, Su, Liu, Han and Chen2020), long-term pyrethroid use and overuse have produced varying levels of resistance in aphid species such as R. padi. For instance, this insect pest has been found to possess resistance levels of 18.2, 13.1, and 24.14 to bifenthrin, deltamethrin, and lambda-cyhalothrin, respectively (Zuo et al., Reference Zuo, Wang, Zhang, Peng, Piñero and Chen2016b; Wang et al., Reference Wang, Huang, Li and Chen2018, Reference Wang, Bai, Zhao, Su, Liu, Han and Chen2020). A. gossypii had more than 5000-fold resistance to beta-cypermethrin (Chen et al., Reference Chen, Li, Chen, Ma, Liang, Liu, Song and Gao2016, Reference Chen, Tie, Chen, Ma, Li, Liang, Liu, Song and Gao2017). Tang et al. (Reference Tang, Ma, Hou and Gao2017) found that the resistant strain of M. persicae had 260.80- and 123.15-fold resistance levels to β-cypermethrin and cypermethrin, respectively, relative to the SS. In this study, we found that the resistance of A. spiraecola to lambda-cyhalothrin ranged from moderate to extremely high levels.
In recent years, apple trees have been severely damaged by A. spiraecola, leading to extensive use of lambda-cyhalothrin spray in the regions. The frequent applications of insecticides had caused the rapid increasing in insecticide resistance among A. spiraecola populations. One mechanism underlying insect resistance is metabolic resistance, manifested by the variation of activity of detoxifying enzymes and the related gene expression. In the present study, the activity level of P450 detoxification enzyme was significantly increased in XY, LQ, FX, LC, and XP populations of A. spiraecola when compared to the SS in 2021, but was not up-regulated in the three population (BS, QX, and FF) with lower resistant to lambda-cyhalothrin. However, the resistant level of BS, QX, and FF populations increased in 2022, and the activity of P450 enzymes was significantly higher than SS in all regions in this year, indicating the P450 may contribute to the resistance. It has been widely reported that the upregulation of P450 gene can lead to the insect resistance to pyrethroids (Xi et al., Reference Xi, Pan, Bi, Gao, Chen, Peng, Zhang, Zhang, Hu and Shang2015; Wang et al., Reference Wang, Jiang, Mota-Sanchez, Wang, Li, Gao, Lu and Yang2019, Reference Wang, Bai, Zhao, Su, Liu, Han and Chen2020). The elevated P450 activity was the primary mechanism of lambda-cyhalothrin resistance in Helicoverpa armigera (Wang et al. (Reference Wang, Jiang, Mota-Sanchez, Wang, Li, Gao, Lu and Yang2019). The increased activity of the P450s leads to the development of lambda-cyhalothrin resistance in Aphis glycines (Xi et al., Reference Xi, Pan, Bi, Gao, Chen, Peng, Zhang, Zhang, Hu and Shang2015). Notably, the P450 enzyme activity in the XY population of A. spiraecola was lower than that in XP population. However, the LC50 of XY population for lambda-cyhalothrin was the highest, which could potentially be attributed to the L1014F mutation of the sodium channel gene in XY population. The mutation rates of the sodium channel were closely associated with the RRs of A. spiraecola population to lambda-cyhalothrin. The mutation rate of the L1014F in the sodium channel in the XY population was highest among all regions.
Eight P450 genes (CYP6CY7, CYP6CY14, CYP6CY22, P4504C1-like, P4506a13, CYP4CZ1, CYP380C47, and CYP4CJ2) were up-regulated in both A. spiraecola field populations and under exposure to LC50 concentration of lambda-cyhalothrin in SS. Five P450 genes (P4504C1-like, P4506a13, CYP6CY14, CYP4CJ1, and CYP4CJ2) were highly expressed in A. spiraecola under exposure to LC50 concentration of lambda-cyhalothrin. Overexpression of detoxification enzyme genes can contribute to insecticide resistance, as found in A. glycines (Xi et al., Reference Xi, Pan, Bi, Gao, Chen, Peng, Zhang, Zhang, Hu and Shang2015). Similarly, overexpression of CYP6A2 was related to pyrethroid resistance in A. gossypii (Peng et al., Reference Peng, Pan, Yang, Gao, Xi, Wu, Huang, Zhu, Xin, Zhan and Shang2016). Wang et al. (Reference Wang, Bai, Zhao, Su, Liu, Han and Chen2020) showed that the overexpression CYP6DC1 and CYP380C47 played important roles in the pyrethroid resistance of R. padi. Our results revealed that the P450 genes were related to lambda-cyhalothrin resistance in A. spiraecola. The documented high levels of resistance in the FF and LC populations may also be associated with GST enzyme activities. The effect of GST enzyme activities on pyrethroid resistance in insects has been documented in various species of insects (Pavlidi et al., Reference Pavlidi, Vontas and Van Leeuwen2018). GSTs contributed to resistance of C. pomonella to pyrethroid (Wei et al., Reference Wei, Liu, Hu and Yang2020; Hu et al., Reference Hu, Liu, Wang, Mota-Sanchez, He, Shi and Yang2022a). The enhanced GST enzyme activity had been found to provide protection to tissues from oxidative damage and increase resistance to permethrin and lambda-cyhalothrin in Nilaparvata lugens (Vontas et al., Reference Vontas, Small and Hemingway2001). Further research is needed to investigate the role of GSTs in the lambda-cyhalothrin resistance of A. spiraecola.
It is known that target-site insensitivity can contribute to pesticide resistance in insects. The L1014F and M918L mutations in sodium channel reduced insect susceptibility to pyrethroid (Rinkevich et al., Reference Rinkevich, Du and Dong2013; Foster et al., Reference Foster, Paul, Slater, Warren, Denholm, Field and Williamson2014; Field et al., Reference Field, Davies, O'Reilly, Williamson and Wallace2017; Mingeot et al., Reference Mingeot, Hautier and Jansen2021). In the case of M. persicae, for example, mutations such as L1014F and M918 T/L have been associated with resistance to pyrethroids. (Martinez-Torres et al., Reference Martinez-Torres, Foster, Field, Devonshire and Williamson1999; Eleftherianos et al., Reference Eleftherianos, Foster, Williamson and Denholm2008; Fontaine et al., Reference Fontaine, Caddoux, Brazier, Bertho, Bertolla, Micoud and Roy2011). The mutation M918L in R. padi (Zuo et al., Reference Zuo, Peng, Wang, Lin, Li and Chen2016a; Wang et al., Reference Wang, Bai, Zhao, Su, Liu, Han and Chen2020) and A. gossypii (Chen et al., Reference Chen, Tie, Chen, Ma, Li, Liang, Liu, Song and Gao2017) has been observed to result in significant resistance to beta-cypermethrin and deltamethrin, respectively. In this study, the mutation rates of L1014F were above 60% in A. spiraecol from all regions. The documented levels of resistance in A. spiraecola observed in these regions were associated with L1014F mutation at the sodium channel, which is comparable to the high levels of resistance to pyrethroids contributed by sodium channel mutations in M. persicae, R. padi, and A. gossypii. Therefore, the resistance of A. spiraecola to lambda-cyhalothrin is linked to the L1014F mutation in the sodium channel.
In conclusion, A. spiraecola has developed resistance to lambda-cyhalothrin in apple orchards in the Shaanxi province. Resistance was associated with increased activity of P450 detoxification enzymes and the overexpression of P450 genes. The L1014F mutation in the sodium channel was detected and played a role in the resistance of A. spiraecola to lambda-cyhalothrin. We postulate that a variety of resistance mechanisms may be involved in the occurrence of resistance of A. spiraecola to lambda-cyhalothrin. This study provides a theoretical basis for the scientific management of this global pest and an initial investigation into the resistance mechanism of the aphid to pyrethroids.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0007485323000603.
Acknowledgements
We thank to the support of the funds from the National Key R&D Program of China (grant no. 2022YFD1400900) and the National Natural Science Foundation of China (Grant No. 31972263 and 32172407).
Authors’ contributions
MC conceived the study. HT and MC designed the experiments. HT, YZ, JZ, and YW performed the experiments. HT and XP analysed data. HT wrote the manuscript. HT, XP, JP, and MC revised the manuscript. All authors read and approved the final manuscript.
Competing interests
None.
