Effects of Insecticide Stress on Expression of NlABCG Transporter Gene in the Brown Planthopper, Nilaparvata lugens

The brown planthopper (BPH), Nilaparvata lugens, is an important pest of rice that severely affects production. Insecticides are an important means of controlling BPH, but their long-term use has led to resistance. To provide insight into BPH responses to insecticide stress, we determined the expression levels of BPH ABCG transporter genes under treatment with thiamethoxam, abamectin, and cyantraniliprole at LC10, LC25, LC50, and LC90. We cloned 13 BPH ABCG transporters, named NlABCG1 to NlABCG13. Conservative domain analysis showed that all 13 transporters have one nucleotide binding domain and one transmembrane domain, typical of semi-molecular transporters. Real-time quantitative PCR showed that thiamethoxam, abamectin, and cyantraniliprole stress increased the expression of some NlABCG transporters gene in BPH. However, after treatment with thiamethoxam at LC25 and abamectin at LC10, there was no significant upregulation of NlABCG. These results indicate that the expression of NlABCG varies in response to stress from different insecticides. These findings provide baseline information for further understanding of the molecular mechanisms of insecticide resistance in BPH.


Introduction
The brown planthopper (BPH), Nilaparvata lugens, an important migratory pest of rice in Asia that consumes the phloem of the rice plant and lays eggs on the leaf sheath, causing a decline in yield. It also spreads viral diseases such as grass stunt and ragged stunt [1][2][3]. BPH is a typical r-strategic pest with a very high intrinsic growth rate and strong adaptability to the environment. Conventionally, chemical control has been used to control BPH [4,5], but resistance to organophosphates, carbamates, pyrethroids, neonicotinoids, insect growth regulators, and phenylpyrazoles [3,[6][7][8] has been observed, thereby hindering its control [9].
The ATP-binding cassette (ABC) transporter family includes various transporters present across organisms from bacteria to humans [10,11] and transport inorganic ions, sugars, amino acids, lipids, lipopolysaccharides, polypeptides, metals, xenobiotics, and chemotherapeutic drugs [12]. This group can be subdivided into eight major subfamilies (A-H) in insects [13]. Studies on ABC transporters in eukaryotes have revealed that some such transporters are capable of transporting structurally unrelated compounds [14,15], while studies on ABC transporters in insects have focused on their transport of exogenous substances and their involvement in insecticide resistance. For example, in vitro experiments with Helicoverpa armigera showed that the presence of P-glycoprotein might be one of the reasons for insecticide resistance in this insect [16]. Studies on Drosophila melanogaster using the CRISPR-Cas9 system indicate that Mdr65 knockouts were more susceptible to all neuroactive insecticides tested [17]. In addition, the ABC transporter G subfamily gene (ABCG) in Sogatella furcifera is involved in the adaptation to insecticide stress [18,19]. Eight ABC transporters in the ABCB/C/D/G subfamily may be involved in the resistance of Laodelphax striatellus to chlorpyrifos, deltamethrin, and imidacloprid [20], and the ABCB, ABCC, while the ABCG subfamily genes are highly expressed in a pyrethroid-resistant strain of Aedes aegypti [21]. These studies suggest that the presence or upregulated expression of ABC transporters is directly related to insect resistance.
ABC transporters have been systematically studied in many insects [20][21][22][23][24][25][26][27][28][29][30]. However, the effects of insecticides on ABC transporters in BPH are not fully understood. In this study, we aimed to demonstrate the role of ABCG transporter genes in the response to insecticide stress, and to lay a foundation for understanding the molecular mechanism of resistance of BPH.

Bioassay to Determine Insecticide Concentrations for Treatment
A bioassay to identify the LC 10 -LC 90 (resulting in 10% -25% mortality) for each insecticide was carried out using third instar nymphs according to a previously reported rice stem dipping method [31,32]. Thiamethoxam, abamectin, and cyantraniliprole were dissolved in acetone and diluted to the required concentrations with 0.1% Triton X-100, respectively, and 0.1% Triton X-100 was also used as a blank control. Rice plants at the tillering stage were selected, washed, cut into stems of approximately 25 cm in length, and dried in the shade. These were soaked in the experimental insecticide solutions for 30 s, then dried and placed in glass tubes (300 mm height × 30 mm diameter) placed horizontally and open at both ends. Twenty healthy nymphs of similar size were selected and placed in the glass tubes, which were then placed upright after all nymphs climbed onto the rice stems. The experimental conditions were as follows: temperature 25 ± 1 • C, relative humidity 70 ± 10%, and illumination 16:8 in an artificial climate chamber for 48 h. At this point, the number of dead insects was counted. A toxicological regression equation was fitted using the Probit model in SPSS 22.0, and LC 10 , LC 25 , LC 50 , and LC 90 were calculated.

Insecticide Stress at Known Concentrations
Following quantitation of LC 10 , LC 25 , LC 50 , and LC 90 for thiamethoxam, abamectin, and cyantraniliprole, these concentrations were used in a similar format to induce insecticide stress in a further 300 nymphs using the same rice stem dipping method. As before, 0.1% Triton X-100 aqueous solution was used as a blank control. Rearing conditions were as described in the previous section. After 48 h, 15 nymphs were taken from each sample and stored at −80 • C. Each treatment was repeated three times.

Total RNA Extraction and cDNA Synthesis
Reserved nymphs were placed in a grinding tube and disrupted using Precellys ® 24 lysis/homogenizer (Bertin Technologies, Montigny le Bretonneux, France). Total RNA was extracted using E.Z.N.A. ® HP Total RNA Kit (Omega Bio-Tek Inc., Norcross, GA, USA) according to the manufacturer's recommended procedure. Total RNA quality was detected using agarose gel electrophoresis, and RNA concentration was determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The first strand of cDNA was synthesized according to the PrimeScriptTM RT reagent Kit with gDNA Eraser (Takara, Dalian, China) kit instructions, and amplified as a PCR template.

Cloning of NlABCG
Transcriptome data of BPH were downloaded from the NCBI SRA database (SRR8840386-SRR8840388) and assembled using Genomis R9 [33] with reference to the SfABCG genes. Using the BLAST tool on the NCBI website for alignment, we confirmed that the assembled genes were NlABCG. Gene-specific primers (Table S1) were designed using Primer Premier 6.0 software, and the resulting sequences were verified by RT-PCR under the following amplification conditions: pre-denaturation at 95 • C for 3 min, denaturation at 95 • C for 30 s, annealing at 55 • C for 30 s, extension at 72 • C for 1-3 min for 30 cycles, and final extension at 72 • C for 10 min. The PCR product was tested using electrophoresis and then purified and ligated to the vector for cloning. The cloned products were further expanded and cultured, and sent to a sequencing company (Sangon Biotech, Shanghai, China) for sequencing. The sequencing results were aligned using Blastx.

Sequence Analysis of NlABCG
Nucleotide and amino acid sequence similarity were searched in the BLAST database based on the full-length sequence of ABCG genes. DNA MAN software version 6.0 (Lynnon Biosoft, Quebec, Canada) was used to analyze and predict putative amino acid sequences. ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) was used to identify the open reading frames of NlABCG, and the "Compute pI/Mw" (http://au.expasy.org/tools/pi_tool.html) in SWISS-PROT (ExPASy Server) was used to calculate molecular weight and theoretical isoelectric points. Pfam (http://pfam.xfam.org/) and SMART (http://smart.embl-heidelberg.de/) were used to identify conserved nucleotide binding and transmembrane domains of all putative NlABCG genes. A phylogenetic tree was constructed using the neighbor-joining method in MEGA 6.0 software [34] with 1000 runs.

Expression Analysis of NlABCG under Insecticide Stress
Real-time quantitative PCR (RT-qPCR) primers (Table S1) were designed based on the full-length cDNA sequence NlABCG, and cDNA obtained by reverse transcription was used as a template. RT-qPCR was used to detect NlABCG expression in each experimental treatment. Reference gene primers were designed based on the cDNA sequence of the BPH 18S gene [35]. Each RT-qPCR was conducted in a 20 µL mixture containing 1 µL sample cDNA, 1 µL of each primer (10 µM), 7 µL diethyl pyrocarbonate-treated H 2 O, and 10 µL FastStart Essential DNA Green Master Mix. The qPCR cycling parameters were as follows: 95 • C for 10 min, followed by 40 cycles of 95 • C for 30 s and 60 • C for 30 s. Melting curve generation was performed from 65 to 95 • C. Three biological replicates and three technical replicates were performed for each treatment.

Data Analysis
The relative expression of 13 NlABCG genes after treatment with different insecticides was calculated using the 2 −∆∆Ct method. RT-qPCR expression data were analyzed using SPSS 22.0 statistical software, and multiple comparisons were made using one-way ANOVA and LSD methods.  (Table 1). In addition, the order of toxicity of the three insecticides to the third instar BPH nymphs, based on the LC 50 value, was abamectin > thiamethoxam > cyantraniliprole.

Identification and Characterization of NlABCG Transporter Genes
We obtained 13 NlABCG transporters, which we named NlABCG1 to NlABCG13 (Table 2). ORFs (open reading frames) of all gene sequences ranged from 603 to 910 amino acids. SMART online software analysis revealed that the NlABCG genes contained six or seven transmembrane regions. With the exception of NlABCG5, the remaining 12 NlABCG genes contained AAA domains (nucleotide binding domains (NBDs); Figure 1A). In order to verify whether there was an error in the NlABCG5 protein sequence, we analyzed similar sequences from S. furcifera, L. striatellus, Bemisia tabaci, and Tribolium castaneum. The results showed that this gene in other species had the same AAA domain deletion ( Figure S1) according to SMART software. In contrast, analysis using Pfam online software showed that NlABCG5 contained an AAA domain and an ABC2 transmembrane domain (TMD), but NlABCG10 lacked ABC2 TMD ( Figure 1B). In addition, sequence analysis indicated that the NlABCG transporters genes contained signature C, Walker A/P-loop, Walker B, D-loop, Q-loop/Lid, and H-loop/switch domains ( Figure 2).  (Table 1). In addition, the order of toxicity of the three insecticides to the third instar BPH nymphs, based on the LC50 value, was abamectin > thiamethoxam > cyantraniliprole.

Identification and Characterization of NlABCG Transporter Genes
We obtained 13 NlABCG transporters, which we named NlABCG1 to NlABCG13 (Table 2). ORFs (open reading frames) of all gene sequences ranged from 603 to 910 amino acids. SMART online software analysis revealed that the NlABCG genes contained six or seven transmembrane regions. With the exception of NlABCG5, the remaining 12 NlABCG genes contained AAA domains (nucleotide binding domains (NBDs); Figure 1A). In order to verify whether there was an error in the NlABCG5 protein sequence, we analyzed similar sequences from S. furcifera, L. striatellus, Bemisia tabaci, and Tribolium castaneum. The results showed that this gene in other species had the same AAA domain deletion ( Figure S1) according to SMART software. In contrast, analysis using Pfam online software showed that NlABCG5 contained an AAA domain and an ABC2 transmembrane domain (TMD), but NlABCG10 lacked ABC2 TMD ( Figure 1B). In addition, sequence analysis indicated that the NlABCG transporters genes contained signature C, Walker A/P-loop, Walker B, D-loop, Qloop/Lid, and H-loop/switch domains ( Figure 2).  A phylogenetic tree based on BPH NlABCG transporters aligned with other insect ABC transporter amino acid sequences was divided into eight major branches (A-H) (Table 2, Figure 3). NlABCG from BPH are clustered on the branches of the G subfamily, further demonstrating that the ABC transporters of BPH belong to the G subfamily of proteins. Three species of planthoppers were close to one another, particularly, L. striatellus and BPH. A phylogenetic tree based on BPH NlABCG transporters aligned with other insect ABC transporter amino acid sequences was divided into eight major branches (A-H) (Table 2, Figure 3). NlABCG from BPH are clustered on the branches of the G subfamily, further demonstrating that the ABC transporters of BPH belong to the G subfamily of proteins. Three species of planthoppers were close to one another, particularly, L. striatellus and BPH.

Effect of Thiamethoxam on NlABCG Expression
Under treatment with thiamethoxam LC 10 , expression of NlABCG1, NlABCG9, and NlABCG11 were significantly upregulated, while expression of NlABCG2, NlABCG3, NlABCG4, NlABCG5, and NlABCG8 were significantly inhibited ( Figure 4). However, expression levels of none of the NlABCG genes were significantly upregulated at LC 25 . In addition, NlABCG1 and NlABCG9 were significantly upregulated under thiamethoxam LC 50 , and NlABCG1 was significantly upregulated under LC 90 . With the exception of the inhibition observed at LC 25 , the expression of NlABCG1 was significantly upregulated under thiamethoxam. genes were significantly upregulated at LC25. In addition, NlABCG1 and NlABCG9 were significantly upregulated under thiamethoxam LC50, and NlABCG1 was significantly upregulated under LC90. With the exception of the inhibition observed at LC25, the expression of NlABCG1 was significantly upregulated under thiamethoxam.

Effect of Abamectin on NlABCG Expression
After abamectin LC10 treatment, the expression of only NlABCG4 and NlABCG12 was significantly inhibited (P <0.05), while the expression levels of other NlABCG family genes changed compared with the control, but the difference was not significant ( Figure 5). The expression levels of other genes changed, but not significantly. At LC25, NlABCG1, NlABCG3, NlABCG8, and NlABCG9 were significantly upregulated, while NlABCG4 was significantly downregulated. The expression of other genes did not change significantly. At LC50, NlABCG3 and NlABCG9 were significantly upregulated and NlABCG4 and NlABCG5 were significantly downregulated. Interestingly, after treatment at LC90, eight NlABCG genes were significantly upregulated, while the expression of the other five was unchanged. At LC25, LC50, and LC90, NlABCG3 and NlABCG9 were significantly upregulated, but these genes were not significantly affected at LC10.

Effect of Abamectin on NlABCG Expression
After abamectin LC 10 treatment, the expression of only NlABCG4 and NlABCG12 was significantly inhibited (P < 0.05), while the expression levels of other NlABCG family genes changed compared with the control, but the difference was not significant ( Figure 5). The expression levels of other genes changed, but not significantly. At LC 25 , NlABCG1, NlABCG3, NlABCG8, and NlABCG9 were significantly upregulated, while NlABCG4 was significantly downregulated. The expression of other genes did not change significantly. At LC 50 , NlABCG3 and NlABCG9 were significantly upregulated and NlABCG4 and NlABCG5 were significantly downregulated. Interestingly, after treatment at LC 90 , eight NlABCG genes were significantly upregulated, while the expression of the other five was unchanged. At LC 25 , LC 50 , and LC 90 , NlABCG3 and NlABCG9 were significantly upregulated, but these genes were not significantly affected at LC 10 .

Effect of Abamectin on NlABCG Expression
After abamectin LC10 treatment, the expression of only NlABCG4 and NlABCG12 was significantly inhibited (P <0.05), while the expression levels of other NlABCG family genes changed compared with the control, but the difference was not significant ( Figure 5). The expression levels of other genes changed, but not significantly. At LC25, NlABCG1, NlABCG3, NlABCG8, and NlABCG9 were significantly upregulated, while NlABCG4 was significantly downregulated. The expression of other genes did not change significantly. At LC50, NlABCG3 and NlABCG9 were significantly upregulated and NlABCG4 and NlABCG5 were significantly downregulated. Interestingly, after treatment at LC90, eight NlABCG genes were significantly upregulated, while the expression of the other five was unchanged. At LC25, LC50, and LC90, NlABCG3 and NlABCG9 were significantly upregulated, but these genes were not significantly affected at LC10.   90 . Mean values ± SE were used to analyze relative expression levels under different concentrations of insecticides using the 2 −∆∆Ct method; the 18S gene of BPH was used as the internal reference gene, and non-insecticide treatment as a control. Three biological replicates and three technical replicates were performed for each treatment. Different letters indicate significant differences at P < 0.05 among different treatment concentrations. LC, Lethal concentration; SE, Standard error.

Effect of Cyantraniliprole on NlABCG Expression
At cyantraniliprole LC 10 , NlABCG7 and NlABCG13 were significantly upregulated, while NlABCG8 was significantly downregulated, and other genes were not significantly affected (Figure 6). At LC 25

Effect of Cyantraniliprole on NlABCG Expression
At cyantraniliprole LC10, NlABCG7 and NlABCG13 were significantly upregulated, while NlABCG8 was significantly downregulated, and other genes were not significantly affected ( Figure  6). At LC25, NlABCG1, NlABCG2, NlABCG9, and NlABCG11 were significantly upregulated. At LC50, NlABCG1, NlABCG2, and NlABCG6 were significantly upregulated. At LC90, NlABCG1, NlABCG2, and NlABCG9 were significantly upregulated. At cyantraniliprole LC25, LC50, and LC90, NlABCG1 and NlABCG2 genes showed a significant increase in expression. . Mean values ± SE were used to analyze relative expression levels under different concentrations of insecticides using the 2 −ΔΔCt method; the 18S gene of BPH was used as the internal reference gene, and non-insecticide treatment as a control. Three biological replicates and three technical replicates were performed for each treatment. Different letters indicate significant differences at P <0.05 among different treatment concentrations. LC, Lethal concentration; SE, Standard error.

Co-Induced Expression of NlABCG Genes by Three Insecticide Treatments
To better illustrate the upregulated expression of the NlABCG genes under different insecticide treatments, we generated a Venn diagram for the upregulated genes at each concentration (Figure 7). NlABCG was not upregulated at LC10 of any pesticide ( Figure 7A). NlABCG1 and NlABCG9 were both significantly upregulated after treatment with cyantraniliprole and abamectin at LC25 (Figure 7B). At LC50, both cyantraniliprole and thiamethoxam caused upregulation of NlABCG1, and thiamethoxam and abamectin caused significant upregulation of NlABCG9. However, at LC50, no NlABCG gene was significantly affected by any of the three insecticides ( Figure 7C). Three insecticidal LC90 treatments caused significant upregulation of NlABCG1; in addition, abamectin and cyantraniliprole also caused significant upregulation of NlABCG2 and NlABCG9 ( Figure 7D). . Mean values ± SE were used to analyze relative expression levels under different concentrations of insecticides using the 2 −∆∆Ct method; the 18S gene of BPH was used as the internal reference gene, and non-insecticide treatment as a control. Three biological replicates and three technical replicates were performed for each treatment. Different letters indicate significant differences at P < 0.05 among different treatment concentrations. LC, Lethal concentration; SE, Standard error.

Co-Induced Expression of NlABCG Genes by Three Insecticide Treatments
To better illustrate the upregulated expression of the NlABCG genes under different insecticide treatments, we generated a Venn diagram for the upregulated genes at each concentration (Figure 7). NlABCG was not upregulated at LC 10 of any pesticide ( Figure 7A). NlABCG1 and NlABCG9 were both significantly upregulated after treatment with cyantraniliprole and abamectin at LC 25 ( Figure 7B). At LC 50 , both cyantraniliprole and thiamethoxam caused upregulation of NlABCG1, and thiamethoxam and abamectin caused significant upregulation of NlABCG9. However, at LC 50 , no NlABCG gene was significantly affected by any of the three insecticides ( Figure 7C). Three insecticidal LC 90 treatments caused significant upregulation of NlABCG1; in addition, abamectin and cyantraniliprole also caused significant upregulation of NlABCG2 and NlABCG9 ( Figure 7D).

Discussion
ATP-binding cassette (ABC) transporters are an important class of transmembrane transporters. The ABC transporter exists in the form of a full-molecule or a semi-molecular transporter in the organism. The full-molecule transporter contains two NBDs and two TMDs, while the semimolecular transporter contains one NBD and one TMD [36]. According to sequence similarity of NBD, ABC transporters are divided into eight subfamilies, ABCA to ABCH. Full-molecule transporters are always present in the ABCA, ABCB, and ABCC subfamilies, while semi-molecular transporters are commonly found in the ABCB, ABCD, ABCG, and ABCH subfamilies [20]. In the present study, 13 ABCG transporters were identified and their conserved domains were analyzed and found to have one NBD and one TMD (Figure 1). These transporters constitute semi-transporter proteins, consistent with the findings from L. striatellus and S. furcifera [19,20]. However, we used Pfam and SMART to analyze the conservative domain of NlABCG genes. We found that the results of NlABCG5 and NlABCG10 using the two online software were inconsistent. This difference could be attributed to the defects in different prediction software. Therefore, we suggest using at least two online prediction software to analyze gene domains. These studies have shown a conserved sequence of approximately 200 amino acids in the NBD, which contains Walker A (GXXGXGK(S/T), Walker B (XXXLDEP), ABC signature (LSGGQ), D-loop, H-loop, and Q-loop conserved motifs [15]. The 13 NlABCG genes obtained in this study had corresponding motifs (Figure 2).
The detoxification metabolic process of insects to insecticides mainly involves detoxifying enzymes and transporters, while ABC transporters are important transporters involved in the transport of insecticide metabolites [37,38]. Additionally, some ABC transporters of members of the G subfamily have been demonstrated to confer resistance to xenobiotics, including insecticides [39]. For example, in L. striatellus, numerous ABCG genes are significantly upregulated in resistant populations to chlorpyrifos, deltamethrin, and imidacloprid [20]. Meanwhile, LC10 and LC25 concentrations of thiamethoxam, buprofezin, and abamectin can induce the expression of some ABCG genes in S. furcifera [19]. Permethrin LD50 significantly induces the expression of ABCG in Anopheles gambiae [40]. Indoxacarb, chlorpyrifos, abamectin, and lambda-cyhalothrin LC50 treatment significantly increase the expression of ABCG gene in Plutella xylostella larvae after 48 h [30]. The results of the present study indicate that the ABCG transporter in BPH shows varying responses to different levels of thiamethoxam, abamectin, and cyantraniliprole stress. Similar results have been observed in Anopheles sinensis [29], Bactrocera dorsalis [22], and Cnaphalocrocis medinalis [41]. Although these findings do not directly suggest that the ABCG transporter is involved in the development of

Discussion
ATP-binding cassette (ABC) transporters are an important class of transmembrane transporters. The ABC transporter exists in the form of a full-molecule or a semi-molecular transporter in the organism. The full-molecule transporter contains two NBDs and two TMDs, while the semi-molecular transporter contains one NBD and one TMD [36]. According to sequence similarity of NBD, ABC transporters are divided into eight subfamilies, ABCA to ABCH. Full-molecule transporters are always present in the ABCA, ABCB, and ABCC subfamilies, while semi-molecular transporters are commonly found in the ABCB, ABCD, ABCG, and ABCH subfamilies [20]. In the present study, 13 ABCG transporters were identified and their conserved domains were analyzed and found to have one NBD and one TMD (Figure 1). These transporters constitute semi-transporter proteins, consistent with the findings from L. striatellus and S. furcifera [19,20]. However, we used Pfam and SMART to analyze the conservative domain of NlABCG genes. We found that the results of NlABCG5 and NlABCG10 using the two online software were inconsistent. This difference could be attributed to the defects in different prediction software. Therefore, we suggest using at least two online prediction software to analyze gene domains. These studies have shown a conserved sequence of approximately 200 amino acids in the NBD, which contains Walker A (GXXGXGK(S/T), Walker B (XXXLDEP), ABC signature (LSGGQ), D-loop, H-loop, and Q-loop conserved motifs [15]. The 13 NlABCG genes obtained in this study had corresponding motifs (Figure 2).
The detoxification metabolic process of insects to insecticides mainly involves detoxifying enzymes and transporters, while ABC transporters are important transporters involved in the transport of insecticide metabolites [37,38]. Additionally, some ABC transporters of members of the G subfamily have been demonstrated to confer resistance to xenobiotics, including insecticides [39]. For example, in L. striatellus, numerous ABCG genes are significantly upregulated in resistant populations to chlorpyrifos, deltamethrin, and imidacloprid [20]. Meanwhile, LC 10 and LC 25 concentrations of thiamethoxam, buprofezin, and abamectin can induce the expression of some ABCG genes in S. furcifera [19]. Permethrin LD 50 significantly induces the expression of ABCG in Anopheles gambiae [40]. Indoxacarb, chlorpyrifos, abamectin, and lambda-cyhalothrin LC 50 treatment significantly increase the expression of ABCG gene in Plutella xylostella larvae after 48 h [30]. The results of the present study indicate that the ABCG transporter in BPH shows varying responses to different levels of thiamethoxam, abamectin, and cyantraniliprole stress. Similar results have been observed in Anopheles sinensis [29], Bactrocera dorsalis [22], and Cnaphalocrocis medinalis [41]. Although these findings do not directly suggest that the ABCG transporter is involved in the development of insect resistance, our results provide insight into the function of the ABCG transporter. In addition, studies have shown that knocking down the expression of Drosophila melanogaster MDr50, Mdr65, and MRP1 by RNAi can significantly increase the sensitivity of D. melanogaster to DDT (Dichlorodiphenyltrichloroethane) [42]. Knocking down Mdr65 increased the susceptibility of D. melanogaster to nine different insecticides [43]. Therefore, on the basis of these studies and our findings, we believe that the overexpression of the ABCG transporter can contribute to insecticide resistance. However, its specific mechanism warrants further study.
Interestingly, after treatment with thiamethoxam LC 25 and abamectin LC 10 , no NlABCG gene was significantly affected, suggesting that NlABCG is not involved in the response of BPH to the insecticides at these concentrations. ABC transporters constitute a large family with numerous members. In L. striatellus, the ABCB/C/D subfamily was involved in the resistance to insecticides [20]. Therefore, in future studies, the genomic data of BPH should be combined to comprehensively study the role of the ABC transporter in its response to insecticide stress.

Conclusions
In this study, we cloned 13 BPH ABCG transporter genes and analyzed their conserved domains and evolutionary relationships. We also determined the expression levels of 13 ABCG genes following treatment of third instar nymphs with thiamethoxam, abamectin, and cyantraniliprole at LC 10 , LC 25 , LC 50 , and LC 90 for 48 h, and identified variable responses in upregulation. Our findings indicate that the BPH ABCG transporter is able to respond to insecticide stress and contribute to insecticide resistance in BPH.

Supplementary Materials:
The following are available online at http://www.mdpi.com/2075-4450/10/10/334/s1, Figure S1. Conserved domain analysis by SMART online software of Nilaparvata lugens ABCG5. Table S1. The primers of ABC transporter G subfamily gene and 18S gene for RT-PCR and RT-qPCR. Table S2. Species name, gene name and accession number of Phylogenetic tree constructed.