- Email: [email protected]
Molecular cloning and characterization of GABA receptor and GluCl subunits in the western flower thrips, Frankliniella occidentalis
Pesticide Biochemistry and Physiology xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Pesticide Biochemistry and Physiology journal homepage: www.elsevier.com/locate/pest
Molecular cloning and characterization of GABA receptor and GluCl subunits in the western flower thrips, Frankliniella occidentalis Xiangkun Meng, Zhijuan Xie, Nan Zhang, Caihong Ji, Fan Dong, Kun Qian, Jianjun Wang
⁎
College of Horticulture and Plant Protection, Yangzhou University, Yangzhou 225009, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Rdl GluCl Insecticide resistance SNPs Frankliniella occidentalis Abamectin
To understand the role of target site insensitivity in abamectin resistance in the western flower thrips (WFT), Frankliniella occidentalis (Pergande), cDNAs encoding gamma-aminobutyric acid receptor subunit (FoRdl) and glutamate-gated chloride channel (FoGluCl) were cloned from WFT, and both single nucleotide polymorphisms (SNPs) and mRNA expression levels of FoRdl and FoGluCl were detected in a susceptible strain (ABA-S) and a laboratory selected strain (ABA-R) displaying 45.5-fold resistance to abamectin. Multiple cDNA sequence alignment revealed three alternative splicing variants of FoRdl and two alternative splicing variants of FoGluCl generated by alternative splicing of exon 3. While sequence comparison of FoRdl and FoGluCl in ABA-S and ABAR strains identified no resistance-associated mutations, the expression level of FoGluCl in ABA-R strain was 2.63fold higher than that in ABA-S strain. Thus, our preliminary results provide the evidence that the increased mRNA expression of FoGluCl could be an important factor in FoGluCl-mediated target site insensitivity in WFT.
1. Introduction The western flower thrips (WFT), Frankliniella occidentalis (Pergande), is one of the most important insect pests worldwide (Gao et al., 2012). The high polyphagy and fecundity, short generation time, together with haplo-diploid reproductive system enable WFT to cause serious feeding damage to fruits, vegetables and many crops (Gao et al., 2012; Kirk and Terry, 2003). In addition, as an important virus vector, WFT can transmit several plant viruses, such as Impatiens necrotic spot virus (INSV) and Tomato spotted wilt virus (TSWV) (Pappu et al., 2009; Webster et al., 2011). The use of insecticides is the primary strategy employed to control WFT, however, due to the biological attributes of WFT and the abuse of insecticides, WFT has developed resistance to a range of insecticide classes, including pyrethroids, neonicotinoids, carbamates, organophosphates, spinosad and abamectin (Gao et al., 2012; Bielza, 2008; Cloyd, 2016; Li et al., 2016; Wang et al., 2016a). Abamectin (avermectin B1) belongs to the avermectin subfamily of macrocyclic lactones, produced by fermentation of the soil-dwelling microorganism Streptomyces avermitilis (Lasota and Dybas, 1991). Abamectin exerts broad spectrum of activity against pests including WFT, and several studies have reported that WFT has developed resistance to abamectin around the world (Dagli and Tunc, 2007; Herron and James,
⁎
2007; Immaraju et al., 1992; Wang et al., 2014; Zhao et al., 2013). In a previous study, we reported that the enhanced oxidative metabolism mediated by cytochrome P450 monooxygenases (P450s) was a major mechanism for abamectin resistance in ABA-R strain of WFT (Chen et al., 2011). However, due to the fact that the piperonyl butoxide (PBO) could not completely bring the abamectin resistance to a susceptible level, additional mechanisms, such as target site insensitivity, might also be involved in abamectin resistance. In insect nervous system, both excitatory and inhibitory synaptic transmission are mediated by members of the Cys-loop ligand-gated ion channel (Cys-loop LGIC) superfamily, such as nicotinic acetylcholine receptors (nAChRs), gamma-aminobutyric acid (GABA) receptors and glutamate-gated chloride channels (GluCls) (Jones and Sattelle, 2007). Several lines of evidence have suggested that GluCls were the primary targets of macrocyclic lactones, while binding studies suggested that macrocyclic lactones also act on the insect GABA receptor subunit, Rdl (resistance to dieldrin) (Cully et al., 1994; Nakao et al., 2015; Wolstenholme, 2012; Wolstenholme and Rogers, 2005). In the present study, cDNAs encoding Rdl (FoRdl) and GluCl (FoGluCl) were cloned from WFT. Single nucleotide polymorphisms (SNPs) were detected in abamectin susceptible (ABA-S) and resistant (ABA-R) strains of WFT. The mRNA expression levels of these two target genes in ABA-S and ABA-R were also analyzed.
Corresponding author. E-mail address: [email protected] (J. Wang).
https://doi.org/10.1016/j.pestbp.2018.06.012 Received 8 May 2018; Received in revised form 8 June 2018; Accepted 16 June 2018 0048-3575/ © 2018 Elsevier Inc. All rights reserved.
Please cite this article as: Meng, X., Pesticide Biochemistry and Physiology (2018), https://doi.org/10.1016/j.pestbp.2018.06.012
Pesticide Biochemistry and Physiology xxx (xxxx) xxx–xxx
X. Meng et al.
PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, China) according to the manufacturer's instructions. The SYBR® PrimeScript™ RT-PCR Kit II (TaKaRa, Dalian, China) and gene specific primers were used for the gene expression determining (Table 1). The housekeeping gene β-actin was used as an internal control (Cifuentes et al., 2012). A 20 μL PCR reaction volume contains 10 μL SYBR Premix EX Taq™ II (2×), 2 μL diluted cDNA template with a concentration of 200 ng/μL, 0.4 μL ROX Reference Dye II (50×) and 0.4 μM of each primer. The reaction mixtures were performed on a Bio-Rad CFX 96 Real-time PCR system with a condition of 95 °C for 30 s, 40 cycles of 95 °C for 5 s and 60 °C for 30 s, and a melting curve analysis was performed at the end. The relative gene expression was calculated using 2−ΔΔCT method and normalized to β-actin in the same sample (Pfaffl, 2001). Means and standard errors were obtained from the average of three independent sample sets.
Table 1 Primers used for RT-PCR, RACE and qPCR. Primer name
Sequence (5′ to 3′)⁎
Description
192.GABAF1 193.GABAF2 474.GLURF1 476.GLURR2 212.C4.1R2 469.C4.1R6 213.C4.1F1 214.C4.1F2 506.GLUCLR1 504.GLUCLF2 471.Q2F1
TGGGTNCCNGAYACNTTYTT(WVPDTFF) TAYTTYCAYATHGCNACNAC(YFHIATT) AAYGARAARGARGGNCAYTT(NEKEGHF) CARCANGGDATRTADATYTG (QIYIPCC) TTGCTGGTGGTTGCGATGTGAA CTCGGGCTGGTGTAGCATTTCGGTTC CTGGCTGAACCGAAATGCTA GCTAATGTCCTCAACTAACGCTG GGATGAGGTAGTATGAGAACTCCCGC CTATGGGTGGACAACCAACGACTT ATGGGCAAAAGAGTAAGGCC
Rdl RT-PCR
473.W45R1 781.GuClF1
CTACTTGTCCTCCTGCAACA TACGACGCTAGAATACGACC
783.GuClR1 814.GABAF 815.GABAR 812.GuClF 813.GuClR 706.FoR2_actinF 707.FoR2_actinR
AAGTTTACTGGTCCTCGGCC CATCTACCTGGGCACCTGTT GTCGTGAACTATGAAGCGCA CAGTATTCGCATTTCGCTCA AACCGAGGAAGATGGAGGTT CGGTCAGGTCATCACCATTG TCGTCTCGTGTATTCCGCAC
GluCl RT-PCR Rdl 5′RACE Rdl 3′RACE GluCl 5′RACE GluCl 3′RACE Rdl RT-PCR for SNP analysis GluCl RT-PCR for SNP analysis
2.5. Cloning and sequence analysis
Rdl qPCR
2. Materials and methods
RT-PCR and RACE products were subcloned into the pMD18-T vector (TaKaRa, Dalian, China) and sequenced. The sequence alignment was performed using CLUSTALW with the default settings (Thompson et al., 1994). The aligned sequences were used to construct the phylogenetic tree in MEGA 5 with a bootstrapping of 1000 iterations. The molecular mass and isoelectric point were determined using ExPASy online service (http://web.expasy.org/compute_pi/). The cDNA sequences of FoRdl and FoGluCl have been deposited in the GenBank and the accession numbers were MH249047 and MH249048, respectively.
2.1. Insect strains
2.6. Data analysis
The laboratory selected ABA-R strain with 45.5-fold resistance to abamectin was derived from a susceptible strain (ABA-S) after 15 selection cycles with abamectin during 18 generations (Chen et al., 2011). About 20 2nd-instar nymphs of ABA-S or ABA-R were collected and stored at −80 °C until use for total RNA extraction. At least three repetitions were prepared for a given sample.
Statistical analysis was performed by one-way ANOVA (SPSS version 10.0, SPSS Inc., Chicago, USA) with at least three repeats, and P < 0.05 was considered to be statistically significant.
GluCl qPCR β-actin qPCR
⁎ Corresponding amino acid sequences for degenerate primers were shown in parentheses.
3. Results 3.1. cDNA cloning and characterization of FoRdl and FoGluCl
2.2. Reverse transcriptase-polymerase chain reaction (RT-PCR) The 4070 bp full length cDNA of FoRdl contains a 48-bp 5′-untranslated region (UTR), 1401 bp of ORF encoding a 466-amino acid residue protein with a calculated molecular mass of 52.34 kDa and an isoelectric point (pI) of 8.79, and a 2621-bp 3′-UTR (Fig. 1). Despite repeated attempts to amplify the 5′ end using conventional RACE strategy, we only obtained 1931 bp partial cDNA of FoGluCl, which encodes 437 amino acid residues and contains a 620-bp 3′-UTR. The common features of insect Cys-loop LGIC subunits, such as six ligand binding loops (Loop A-F) and four conserved transmembrane regions (TM1–4), were conserved in both FoRdl and FoGluCl (Fig. 1). An amino acid sequence alignment shows that FoRdl was 64.7% and 84.0% identical in pairwise comparisons with the Rdl amino acid sequences in Drosophila melanogaster and Tribolium castaneum, respectively. The deduced FoGluCl amino acid sequence showed 78.0% and 78.3% identities to GluCl from D. melanogaster and T. castaneum, respectively. Phylogenetic analysis reveals that Rdl proteins form a sister group relationship with Lcch3 subunits, and FoRdl and FoGluCl were closely related to blattaria and hymenoptera homologues, respectively (Fig. 2).
Total RNAs were extracted from the collected samples using an SV total RNA isolation system (Promega, Madison, WI), according to the manufacturer's instructions. First-strand cDNA was synthesized from 1 μg of total RNA using the Primescript™ First-Strand cDNA Synthesis kit (TaKaRa, Dalian, China) according to the manufacturer's instructions. Degenerate primer pairs were designed against the amino acid residues that were conserved between insects (Table 1). PCR was performed in a 25 μL reaction volume containing 20–50 ng cDNA, 0.8 μM of each degenerate primer, 0.2 mM of each dNTP, 2 mM of MgCl2, 1.25 U Ex Taq™ polymerase and 2.5 μL Ex Taq™ buffer (Takara, Dalian, China). A touchdown PCR program was used, which consisted of 1 cycle at 94 °C for 5 min, 12 cycles at 94 °C for 30 s, 52–41 °C (decreasing by −1 °C/cycle) for 30 s, and 72 °C for 2 min, followed by 25 cycles of 94 °C for 30 s, 40 °C for 30 s, and 72 °C for 2 min, and a final extension at 72 °C for 10 min. 2.3. Rapid amplification of cDNA ends (RACE) To complete the cDNA sequence of FoRdl and FoGluCl, 5′ -RACE and 3′ -RACE reactions were performed using 5′-full RACE core set and 3′full RACE core set (Takara, Dalian, China) respectively, following the manufacturer's instructions. Gene specific primers (GSP) used for the 5′ - and 3′ -RACE are listed in Table 1.
3.2. Alternative splicing The alignment of multiple cDNA clone sequences revealed three alternative splicing variants of FoRdl (FoRdl-A, FoRdl-B and FoRdl-C) and two alternative splicing variants of FoGluCl (FoGluCl-A and FoGluCl-B) in WFT (Fig. 3). Comparison of the cDNA sequences with corresponding genomic sequences derived from the whole genome shotgun database (WGS) assembly of WFT deposited at GenBank revealed that both FoRdl and FoGluCl variants were generated by
2.4. Reverse transcription quantitative PCR (RT-qPCR) Total RNA was employed for cDNA template synthesis using 2
Pesticide Biochemistry and Physiology xxx (xxxx) xxx–xxx
X. Meng et al.
Fig. 1. Amino acid sequence alignment of Rdl and GluCl from WFT, D. melanogaster and T. castaneum. The dotted line amino acid sequences represent the alternative splicing site in Rdl and GluCl of WFT. The signal peptides at the N-terminus were boxed. Lines above amino acid sequences represent the six ligand binding loops (Loop A-F) and four conserved transmembrane regions (TM1–4) of receptor protein. The ‘▲’ indicated the reported resistance mutations in insect GluCl, and the ‘▼’indicated the reported resistance mutations in insect Rdl.
alternative splicing of exon 3, designated as A, B, and C in FoRdl and A, B in FoGluCl (data not shown). Specifically, the alternative splicing site forming mutually exclusive exons lie upstream to loop D encoding region within proximity of the agonist binding site (Fig. 1). All these alternative exons were composed of 68 nucleotides encoding 23 amino acids (55–77 in FoRdl, and 62–84 in FoGluCl). The three alternative exons in FoRdl share nucleotide and amino acid identities of 70.6%–80.9% and 82.6%–95.7%, respectively, whereas the two alternative exons in FoGluCl were divergent, differing at 7 of 23 amino acid residues (Fig. 3). The splice sites are highly conserved in insect Rdl and GluCl. Exon 3 variants of FoRdl and FoGluCl showed > 91.3% amino acid identities to the corresponding variants in D. melanogaster and T. castaneum (Fig. 3).
Sequences analysis revealed 33 nucleotide polymorphisms of FoRdl in two strains, which resulted in 23 amino acids replacement (Table 2). The extraordinarily high number of SNPs in FoRdl is not unusual, as high nucleotide diversity has been found in the para-like voltage-sensitive sodium channel gene in this insect, which is probably a direct consequence of pest migration (Forcioli et al., 2002). However, most of these replacements occurred in both ABA-S and ABA-R strains, and no putative resistance-associated mutations were detected in ABA-R strain (Table 2). A total of 23 nucleotide polymorphisms of FoGluCl were found in ABA-R and ABA-S strains, which caused 8 amino acid substitutions (Table 3). However, two abamectin resistance-associated mutations (A309V and G315E) conserved in several insects and mites (Wang et al., 2017) were not detected in FoGluCl of ABA-R strain.
3.3. SNPs of FoRdl and FoGluCl
3.4. mRNA expression levels of FoRdl and FoGluCl in ABA-R and ABA-S starins
To preliminarily explore the resistance-associated mutations in ABA-R strain, cDNA fragments of both FoRdl (1401 bp) and FoGluCl (1308 bp) in ABA-R and ABA-S strains were amplified, and a total of 7 positive clones for each strain and target gene were sequenced.
The mRNA expression levels of both FoRdl and FoGluCl were compared between ABA-R and ABA-S strains. While no difference of FoRdl expression was found between ABA-R and ABA-S strains, the mRNA 3
Pesticide Biochemistry and Physiology xxx (xxxx) xxx–xxx
X. Meng et al.
Fig. 2. Phylogenetic analysis of GABA receptor subunits and GluCl from WFT and other insects.
compared with that of the unselected strain (Wang and Wu, 2007). In Liriomyza sativae, GST activities in two abamectin-resistant strains were significantly higher than that in susceptible strain, indicating the involvement of GST in conferring abamectin resistance (Wei et al., 2015). In Plutella xylostella, elevated levels of P450 monooxygenase activity was shown to be associated with resistance to abamectin (Qian et al., 2008), and a recent study reported that the overexpression of CYP340W1 plays an important role in abamectin resistance (Gao et al., 2016). Consisting with these results, our previous study with in vivo synergism assessments and in vitro metabolic enzyme assays showed that increased cytochrome P450s enzyme activity was a major factor conferring abamectin resistance in the WFT (Chen et al., 2011). In addition to enhanced detoxification, target site insensitivity was
expression level of FoGluCl in ABA-R strain was 2.63-fold higher than that in ABA-S strain (Fig. 4). 4. Discussion Generally, the mechanisms of insecticide resistance include behavioral resistance, reduced penetration of toxicants, enhanced insecticide metabolism, and alterations of insecticide targets (Brattsten et al., 1986). It has been reported that abamectin resistance in several insects were driven by metabolic resistance mechanisms. In a laboratory selected Bemisia tabaci strain with 14.5-fold resistance to abamectin, P450 monooxygenase activity and glutathione S-transferase (GST) activity were elevated to 2.1- and 2.0-fold, respectively,
Fig. 3. Sequence alignments of Rdl and GluCl alternative splicing variants. (A) Nucleotide sequences of three FoRdl and two FoGluCl splicing variants. (B) Amino acid sequence alignments of Rdl and GluCl variants from WFT, D. melanogaster and T. castaneum. 4
Pesticide Biochemistry and Physiology xxx (xxxx) xxx–xxx
X. Meng et al.
Table 2 Polymorphisms of FoRdl in the susceptible and abamectin resistant strains of WFT. Nucleotide positiona
Nucleotideb
Nucleotide substitutionc ABA-S Strain
20, 21 22 102 155, 156 259 269 385 430 464 480 490 544 547, 548 557 618 669 672 684 778 892 909 959 965 1089 1100 1117 1201 1221 1289 1299 a b c d
ACC AGC ATT AAC CAA ACG TAC CAC CTA TGT CTT GGG TAT AGG TCT AGT CTG AAC TCC AAG AAA CTT GAG GCG ACG GTT GCC CTG CGA TTC
6
1
ACC , AAA AGC6, TGC1 ATT7 AAC6, AGA1 CAA6, TAA1 ACG7 TAC7 CAC6, AAC1 CTA7 TGT7 CTT6, TGG1 GGG6, TGG1 TAT7 AGG6, ACG1 TCT6, TCC1 AGT7 CTG7 AAC6, AAA1 TCC6, CCC1 AAG6, GAG1 AAA7 CTT6, CCT1 GAG7 GCG6, GCA1 ACG6, AGG1 GTT7 GCC6, CCC1 CTG7 CGA6, CAA1 TTC6, TTT1
Amino acid ABA-R Strain 3
2
Amino acid substitutiond ABA-S Strain
2
ACC , AAA , AAC AGC7 ATT6, ATA1 AAC7 CAA7 ACG6, ATG1 TAC6, CAC1 CAC7 CTA6, ATA1 TGT6, TGC1 CTT7 GGG7 TAT5, CAT1, TTT1 AGG7 TCT7 AGT6, AGG1 CTG6, CTA1 AAC7 TCC7 AAG7 AAA6, AAT1 CTT7 GAG6, GGG1 GCG7 ACG7 GTT6, TTT1 GCC7 CTG6, CTT1 CGA7 TTC7
T7 S8 I34 N52 Q87 T90 Y129 H144 T155 C160 L164 G182 Y183 R186 S206 S223 L224 N228 S260 K298 K303 L320 E322 A363 T367 V373 A401 L407 R430 F433
6
1
ABA-R Strain
T ,K S6, C1
T3, K2, N2 S7
N6, R1 Q6, *1 T7 Y7 H6, N1 T7
N7 Q7 T6, M1 Y6, H1 H7 T6, N1
L6, F1 G6, W1 Y7 R6, T1
L7 G7 Y5, H1, F1 R7
S7
S6, R1
N6, K1 S6, P1 K6, E1 K7 L6, P1 E7
N7 S7 K7 K6, N1 L7 E6, G1
T6, R1 V7 A6, P1
T7 V6 , F 1 A7
R6, Q1
R7
The position of A in the initial methionine codon is numbered 1. The substituted nucleotides were italicized and bolded. The numbers indicated the amount of nucleotide substitutions in 7 clones. Only sense substitutions were shown, and ‘*’represent the termination codon.
Table 3 Polymorphisms of FoGluCl in the susceptible and abamectin resistant strains of WFT. Nucleotide positiona
Nucleotideb
Nucleotide substitution ABA-S Strain
269 435 450 511 588 618 705 759 855 903 954 1080 1104 1107 1115 1139 1164 1173 1205 1213 1236 1269 1270 a b c d
CTT TAC CCG AAG TTT GTT AGT CAG ACT CTG CTC CCC AAC ACC CCG CAC TGC ACT CGC GTC CCG TGG TCC
6
1
CTT , CCT TAC6, TAT1 CCG6, CCA1 AAG6, GAG1 TTT5, TTC2 GTT5, GTC2 AGT6, AGA1 CAG6, CAA1 ACT6, ACC1 CTG6, CTA1 CTC6, CTG1 CCC6, CCG1 AAC7 ACC6, ACT1 CCG6, CCC1 CAC7 TGC6, TGT1 ACT6, ACG1 CGC7 GTC6, ATC1 CCG7 TGG5, TGA2 TCC6, CCC1
c
Amino acid ABA-R Strain 7
CTT TAC7 CCG7 AAG7 TTT5, TTC2 GTT5, GTC2 AGT7 CAG7 ACT5, ACC2 CTG7 CTC7 CCC7 AAC6, AAT1 ACC6, ACT1 CCG6, CCC1 CAC6, CTC1 TGC7 ACT7 CGC6, CTC1 GTC7 CCG5, CCA2 TGG7 TCC7
The position of A in the initial threonine codon is numbered 1. The substituted nucleotides were italicized and bolded. The numbers indicated the amount of nucleotide substitutions in 7 clones. Only sense substitutions were shown, and ‘*’represent the termination codon. 5
Amino acid substitutiond ABA-S Strain
L90 Y145 P150 K171 F196 V206 S235 Q253 T285 L301 L318 P360 N368 T369 P372 H380 C388 T391 R402 V405 P412 W423 S424
6
ABA-R Strain
1
L ,P
L7
K6, E1
K7
S 6 , R1
S7
H7
H6, L1
R7 V6, I1
R6, L1 V7
W5, *2 S6, P 1
W7 S7
Pesticide Biochemistry and Physiology xxx (xxxx) xxx–xxx
X. Meng et al.
abamectin were decreased when the expression of GluCl was knocked down using RNA interference (Shi et al., 2012; Wei et al., 2018). This discrepancy can be explained by the fact that GluCl plays important role in the development of insects (Boumghar et al., 2012; Chiang et al., 2002; El Hassani et al., 2012; Kita et al., 2013), and RNAi-mediated down-regulation of GluCl definitely have negative effect on the insect viability as well as tolerance against insecticides. Alternative splicing is a key posttranscriptional processing mechanism for generating protein diversity, and it appears to be of central importance for ion channels as insecticide targets, such as Rdl and GluCl. Alternative splicing of exon 3 in Rdl and GluCl have been found in many insects. Two Rdl variants derived from the splicing of exon 3 in D. melanogaster showed different pharmacological properties (Buckingham et al., 2005). Three GluCl variants generated by the alternative splicing of exon 3 were present in T. castaneum, Apis mellifera, Laodelphax striatellus and Musca domestica (Jones and Sattelle, 2007; El Hassani et al., 2012; Eguchi et al., 2006; Wu et al., 2017), and showed various physiological functions in A. mellifera (Boumghar et al., 2012). In addition to the alternative splicing of exon 3, insect Rdl variants could also derive from the alternative splicing of exon 6 (Jones and Sattelle, 2007). In this study, we found three Rdl variants derived from the splicing of exon 3 in WFT, however, alternative splicing of exon 6 was not detected. In contrast, among three insect GluCl variants, only two variants were found in WFT. The pharmacological and physiological functions of FoRdl and FoGluCl splicing variants need further study.
Fig. 4. mRNA expression of FoRdl and FoGluCl genes in ABA-R and ABA-S strains of WFT. *Significant difference by one-way ANOVA test (P < 0.05).
also reported to contribute to abamectin resistance in several insects and mites. A G323D mutation in GluCl1 and G326E in GluCl3 of Tetranychus urticae conferred 18-fold and 2000-fold resistance to abamectin in this mite, respectively (Dermauw et al., 2012; Kwon et al., 2010). Recently, both electrophysiology and homology modelling studies demonstrated that two mutations (A309V, corresponding to A310 in WFT FoGluCl, and G315E, corresponding to G323 in TuGluCl1, G326 in TuGluCl3 and G316 in WFT FoGluCl) and a 36-bp deletion in P. xylostella GluCl contribute to target-site resistance to abamectin (Wang et al., 2017; Liu et al., 2014; Wang et al., 2016b). In this study, while sequence analysis revealed multiple nucleotide polymorphisms of FoGluCl in ABA-S and ABA-R strains of WFT, none of the above-mentioned mutations were detected in ABA-R strain. We also did not find any putative resistance-associated mutations in the secondary target of abamectin, FoRdl. However, it is interesting to note that two amino acid replacements of Rdl including A302S and V327I, which were associated with cylcodiene insecticide resistance in several insects (FfrenchConstant et al., 1993; Wondji et al., 2011), were found in FoRdl of both ABA-S and ABA-R strains (S276 and I307). The A302S substitution was also detected in one of the duplicated Rdl genes, CsRDL2S, in Chilo suppressalis (Sheng et al., 2018), however, a tBLASTn search of the WGS assembly of WFT deposited at GenBank revealed the presence of single Rdl gene in WFT. These preliminary data suggest that the target site mutation did not contribute to abamectin resistance in ABA-R strain, and the ancestor population of ABA-S strain might have been under strong selection of cylcodiene insecticides with little negative fitness of these two resistance-associated mutations. Theoretically, the change of mRNA expression of target gene should have effects on the sensitivity of insects against insecticides. This theory has been confirmed by increasing number of studies. For example, in the greenbug Schizaphis graminum, northern blot analysis showed that the amount of acetylcholinesterase mRNA in the organophosphate resistant clones was approximately 1.5-fold higher than that in the susceptible clone (Gao and Zhu, 2002). In a laboratory flubendiamide-selected P. xylostella strain, the relative ryanodine receptor mRNA transcript level was 2.93-fold as compared with the susceptible strain (Yan et al., 2014). In this study, the mRNA expression of FoGluCl in ABA-R was 2.63-fold higher than that in ABA-S, suggesting the role of up-regulation of target gene in abamectin resistance in WFT. This result was consistent with the studies reporting association of up-regulation of GluCl with abamectin resistance in P. xylostella (Liang, 2009; Liu, 2011). However, in both B. tabaci and P. xylostella, susceptibilities to
Acknowledgements This work was supported by National Natural Science Foundation of China (31701807, 31572000) and Jiangsu Province Science Foundation for Youths (BK20170491). References Bielza, P., 2008. Insecticide resistance management strategies against the western flower thrips, Frankliniella occidentalis. Pest Manag. Sci. 64, 1131–1138. Boumghar, K., Couret-Fauvel, T., Garcia, M., Armengaud, C., 2012. Evidence for a role of GABA- and glutamate-gated chloride channels in olfactory memory. Pharmacol. Biochem. Be. 103, 69–75. Brattsten, L.B., Holyoke Jr., C.W., Leeper, J.R., Raffa, K.F., 1986. Insecticide resistance: challenge to pest management and basic research. Science 231, 1255–1260. Buckingham, S.D., Biggin, P.C., Sattelle, B.M., Brown, L.A., Sattelle, D.B., 2005. Insect GABA receptors: splicing, editing, and targeting by antiparasitics and insecticides. Mol. Pharmacol. 68, 942–951. Chen, X., Yuan, L., Du, Y., Zhang, Y., Wang, J., 2011. Cross-resistance and biochemical mechanisms of abamectin resistance in the western flower thrips, Frankliniella occidentalis. Pestic. Biochem. Physiol. 101, 34–38. Chiang, A.S., Pszczolkowski, M.A., Liu, H.P., Lin, S.C., 2002. Ionotropic glutamate receptors mediate juvenile hormone synthesis in the cockroach, Diploptera punctata. Insect Biochem. Mol. Biol. 32, 669–678. Cifuentes, D., Chynoweth, R., Guillen, J., De La Rua, P., Bielza, P., 2012. Novel cytochrome P450 genes, CYP6EB1 and CYP6EC1, are over-expressed in acrinathrin-resistant Frankliniella occidentalis (Thysanoptera: Thripidae). J. Econ. Entomol. 105, 1006–1018. Cloyd, R.A., 2016. Western flower thrips (Thysanoptera: Thripidae) and insecticide resistance: an overview and strategies to mitigate insecticide resistance development. J. Entomol. Sci. 51, 257–273. Cully, D.F., Vassilatis, D.K., Liu, K.K., Paress, P.S., Van der Ploeg, L.H., Schaeffer, J.M., Arena, J.P., 1994. Cloning of an avermectin-sensitive glutamate-gated chloride channel from Caenorhabditis elegans. Nature 371, 707–711. Dagli, F., Tunc, I., 2007. Insecticide resistance in Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) collected from horticulture and cotton in Turkey. Aust. J. Entomol. 46, 320–324. Dermauw, W., Ilias, A., Riga, M., Tsagkarakou, A., Grbic, M., Tirry, L., Van Leeuwen, T., Vontas, J., 2012. The cys-loop ligand-gated ion channel gene family of Tetranychus urticae: implications for acaricide toxicology and a novel mutation associated with abamectin resistance. Insect Biochem. Mol. Biol. 42, 455–465. Eguchi, Y., Ihara, M., Ochi, E., Shibata, Y., Matsuda, K., Fushiki, S., Sugama, H., Hamasaki, Y., Niwa, H., Wada, M., Ozoe, F., Ozoe, Y., 2006. Functional characterization of Musca glutamate- and GABA-gated chloride channels expressed independently and coexpressed in Xenopus oocytes. Insect Mol. Biol. 15, 773–783. El Hassani, A.K., Schuster, S., Dyck, Y., Demares, F., Leboulle, G., Armengaud, C., 2012. Identification, localization and function of glutamate-gated chloride channel receptors in the honeybee brain. Eur. J. Neurosci. 36, 2409–2420.
6
Pesticide Biochemistry and Physiology xxx (xxxx) xxx–xxx
X. Meng et al.
Sheng, C., Jia, Z., Ozoe, Yoshihisa, Huang, Q., Han, Z., Zhao, C., 2018. Molecular cloning, spatiotemporal and functional expression of GABA receptor subunits RDL1 and RDL2 of the rice stem borer Chilo suppressalis. Insect Biochem. Mol. Biol. 94, 18–27. Shi, X., Guo, Z., Zhu, X., Wang, S., Xu, B., Xie, W., Zhang, Y., Wu, Q., 2012. RNA interference of the inhibitory glutamate receptor in Plutella xylostella (Lepidoptera Plutellidae). Acta Entomol. Sin. 55, 1331–1336. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positionspecific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. Wang, L., Wu, Y., 2007. Cross-resistance and biochemical mechanisms of abamectin resistance in the B-type Bemisia tabaci. J. Appl. Entomol. 131, 98–103. Wang, S., Zhang, A., Li, L., Men, X., Zhou, X., Zhai, Y., Liu, Y., Wei, S., Yu, Y., 2014. Insecticide resistance status of field populations of Frankliniella occidentalis (Thysanoptera: Thripidae) in China and its control strategies. Acta Entomol. Sin. 57, 621–630. Wang, Z., Gong, Y., Jin, G., Li, B., Chen, J., Kang, Z., Zhu, L., Gao, Y., Reitz, S., Wei, S., 2016a. Field-evolved resistance to insecticides in the invasive western flower thrips Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) in China. Pest Manag. Sci. 72, 1440–1444. Wang, X., Wang, R., Yang, Y., Wu, S., O'Reilly, A.O., Wu, Y., 2016b. A point mutation in the glutamate-gated chloride channel of Plutella xylostella is associated with resistance to abamectin. Insect Mol. Biol. 25, 116–125. Wang, X., Puinean, A.M., O'Reilly, A.O., Williamson, M.S., Smelt, C.L.C., Millar, N.S., Wu, Y., 2017. Mutations on M3 helix of Plutella xylostella glutamate-gated chloride channel confer unequal resistance to abamectin by two different mechanisms. Insect Biochem. Mol. Biol. 86, 50–57. Webster, C.G., Reitz, S.R., Perry, K.L., Adkins, S., 2011. A natural M RNA reassortant arising from two species of plant- and insect-infecting bunyaviruses and comparison of its sequence and biological properties to parental species. Virology 413, 216–225. Wei, Q., Lei, Z., Nauen, R., Cai, D., Gao, Y., 2015. Abamectin resistance in strains of vegetable leafminer, Liriomyza sativae (Diptera: Agromyzidae) is linked to elevated glutathione S-transferase activity. Insect Sci. 22, 243–250. Wei, P., Che, W., Wang, J., Xiao, D., Wang, R., Luo, C., 2018. RNA interference of glutamate-gated chloride channel decreases abamectin susceptibility in Bemisia tabaci. Pestic. Biochem. Physiol. 145, 1–7. Wolstenholme, A.J., 2012. Glutamate-gated chloride channels. J. Biol. Chem. 287, 40232–40238. Wolstenholme, A.J., Rogers, A.T., 2005. Glutamate-gated chloride channels and the mode of action of the avermectin/milbemycin anthelmintics. Parasitology 131, S85–S95. Wondji, C.S., Dabire, R.K., Tukur, Z., Irving, H., Djouaka, R., Morgan, J.C., 2011. Identification and distribution of a GABA receptor mutation conferring dieldrin resistance in the malaria vector Anopheles funestus in Africa. Insect Biochem. Mol. Biol. 41, 484–491. Wu, S., Mu, X., Dong, Y., Wang, L., Wei, Q., Gao, C., 2017. Expression pattern and pharmacological characterisation of two novel alternative splice variants of the glutamate-gated chloride channel in the small brown planthopper Laodelphax striatellus. Pest Manag. Sci. 73, 590–597. Yan, H., Xue, C., Li, G., Zhao, X., Che, X., Wang, L., 2014. Flubendiamide resistance and Bi-PASA detection of ryanodine receptor G4946E mutation in the diamondback moth (Plutella xylostella L.). Pestic. Biochem. Physiol. 115, 73–77. Zhao, H., Yi, X., Deng, Y., Hu, M., Zhong, G., Wang, P., 2013. Resistance to fenpropathrin, chlorpyriphos and abamectin in different populations of Amblyseius longispinosus (Acari: Phytoseiidae) from vegetable crops in South China. Biol. Control 67, 61–65.
Ffrench-Constant, R.H., Rocheleau, T.A., Steichen, J.C., Chalmers, A.E., 1993. A point mutation in a Drosophila GABA receptor confers insecticide resistance. Nature 363, 449–451. Forcioli, D., Frey, B., Frey, J.E., 2002. High nucleotide diversity in the Para-like voltagesensitive sodium channel gene sequence in the western flower thrips (Thysanoptera: Thripidae). J. Econ. Entomol. 95, 838–848. Gao, J., Zhu, K., 2002. Increased expression of an acetylcholinesterase gene may confer organophosphate resistance in the greenbug, Schizaphis graminum (Homoptera: Aphididae). Pestic. Biochem. Physiol. 73, 164–173. Gao, Y., Lei, Z., Reitz, S.R., 2012. Western flower thrips resistance to insecticides: detection, mechanisms and management strategies. Pest Manag. Sci. 68, 1111–1121. Gao, X., Yang, J., Xu, B., Xie, W., Wang, S., Zhang, Y., Yang, F., Wu, Q., 2016. Identification and characterization of the gene CYP340W1 from Plutella xylostella and its possible involvement in resistance to abamectin. Int. J. Mol. Sci. 17, 274. Herron, G.A., James, T.M., 2007. Insecticide resistance in Australian populations of western flower thrips, Frankliniella occidentalis (pergande) (thysanoptera: thripidae). Gen. Appl. Entomol. 36, 1–5. Immaraju, J.A., Paine, T.D., Bethke, J.A., Robb, K.L., Newman, J.P., 1992. Western flower thrips (Thysanoptera: Thripidae) resistance to insecticides in coastal California greenhouses. J. Econ. Entomol. 85, 9–14. Jones, A.K., Sattelle, D.B., 2007. The cys-loop ligand-gated ion channel gene superfamily of the red flour beetle, Tribolium castaneum. BMC Genomics 8. Kirk, W.D.J., Terry, L.I., 2003. The spread of the western flower thrips Frankliniella occidentalis (Pergande). Agr. For. Entomol. 5, 301–310. Kita, T., Ozoe, F., Azuma, M., Ozoe, Y., 2013. Differential distribution of glutamate- and GABA-gated chloride channels in the housefly Musca domestica. J. Insect Physiol. 59, 887–893. Kwon, D.H., Yoon, K.S., Clark, J.M., Lee, S.H., 2010. A point mutation in a glutamategated chloride channel confers abamectin resistance in the two-spotted spider mite, Tetranychus urticae Koch. Insect Mol. Biol. 19, 583–591. Lasota, J.A., Dybas, R.A., 1991. Avermectins, a novel class of compounds: implications for use in arthropod pest control. Annu. Rev. Entomol. 36, 91–117. Li, D., Shang, X., Reitz, S., Nauen, R., Lei, Z., Si Hyeock, L., Gao, Y., 2016. Field resistance to spinosad in western flower thrips Frankliniella occidentalis (Thysanoptera: Thripidae). J. Integr. Agric. 15, 2803–2808. Liang, Y., 2009. Studies on Molecular Mechanism of Resistance to Avermectin in Plutella xylostella. Gansu Agricultural University. Liu, F., 2011. The Role of GluCl Receptor in the Resistance of Plutella xylostella (Lepidoptera: Plutellidae) to Abamectin. Chinese Academy of Agricultural Sciences. . Liu, F., Shi, X., Liang, Y., Wu, Q., Xu, B., Xie, W., Wang, S., Zhang, Y., Liu, N., 2014. A 36bp deletion in the alpha subunit of glutamate-gated chloride channel contributes to abamectin resistance in Plutella xylostella. Entomol. Exp. Appl. 153, 85–92. Nakao, T., Banba, S., Hirase, K., 2015. Comparison between the modes of action of novel meta-diamide and macrocyclic lactone insecticides on the RDL GABA receptor. Pestic. Biochem. Physiol. 120, 101–108. Pappu, H.R., Jones, R.A.C., Jain, R.K., 2009. Global status of tospovirus epidemics in diverse cropping systems: successes achieved and challenges ahead. Virus Res. 141, 219–236. Pfaffl, M.W., 2001. A new mathematical model for relative quantification in real-time RTPCR. Nucleic Acids Res. 29, 2002–2007. Qian, L., Cao, G., Song, J., Yin, Q., Han, Z., 2008. Biochemical mechanisms conferring cross-resistance between tebufenozide and abamectin in Plutella xylostella. Pestic. Biochem. Physiol. 91, 175–179.
7
Contents lists available at ScienceDirect
Pesticide Biochemistry and Physiology journal homepage: www.elsevier.com/locate/pest
Molecular cloning and characterization of GABA receptor and GluCl subunits in the western flower thrips, Frankliniella occidentalis Xiangkun Meng, Zhijuan Xie, Nan Zhang, Caihong Ji, Fan Dong, Kun Qian, Jianjun Wang
⁎
College of Horticulture and Plant Protection, Yangzhou University, Yangzhou 225009, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Rdl GluCl Insecticide resistance SNPs Frankliniella occidentalis Abamectin
To understand the role of target site insensitivity in abamectin resistance in the western flower thrips (WFT), Frankliniella occidentalis (Pergande), cDNAs encoding gamma-aminobutyric acid receptor subunit (FoRdl) and glutamate-gated chloride channel (FoGluCl) were cloned from WFT, and both single nucleotide polymorphisms (SNPs) and mRNA expression levels of FoRdl and FoGluCl were detected in a susceptible strain (ABA-S) and a laboratory selected strain (ABA-R) displaying 45.5-fold resistance to abamectin. Multiple cDNA sequence alignment revealed three alternative splicing variants of FoRdl and two alternative splicing variants of FoGluCl generated by alternative splicing of exon 3. While sequence comparison of FoRdl and FoGluCl in ABA-S and ABAR strains identified no resistance-associated mutations, the expression level of FoGluCl in ABA-R strain was 2.63fold higher than that in ABA-S strain. Thus, our preliminary results provide the evidence that the increased mRNA expression of FoGluCl could be an important factor in FoGluCl-mediated target site insensitivity in WFT.
1. Introduction The western flower thrips (WFT), Frankliniella occidentalis (Pergande), is one of the most important insect pests worldwide (Gao et al., 2012). The high polyphagy and fecundity, short generation time, together with haplo-diploid reproductive system enable WFT to cause serious feeding damage to fruits, vegetables and many crops (Gao et al., 2012; Kirk and Terry, 2003). In addition, as an important virus vector, WFT can transmit several plant viruses, such as Impatiens necrotic spot virus (INSV) and Tomato spotted wilt virus (TSWV) (Pappu et al., 2009; Webster et al., 2011). The use of insecticides is the primary strategy employed to control WFT, however, due to the biological attributes of WFT and the abuse of insecticides, WFT has developed resistance to a range of insecticide classes, including pyrethroids, neonicotinoids, carbamates, organophosphates, spinosad and abamectin (Gao et al., 2012; Bielza, 2008; Cloyd, 2016; Li et al., 2016; Wang et al., 2016a). Abamectin (avermectin B1) belongs to the avermectin subfamily of macrocyclic lactones, produced by fermentation of the soil-dwelling microorganism Streptomyces avermitilis (Lasota and Dybas, 1991). Abamectin exerts broad spectrum of activity against pests including WFT, and several studies have reported that WFT has developed resistance to abamectin around the world (Dagli and Tunc, 2007; Herron and James,
⁎
2007; Immaraju et al., 1992; Wang et al., 2014; Zhao et al., 2013). In a previous study, we reported that the enhanced oxidative metabolism mediated by cytochrome P450 monooxygenases (P450s) was a major mechanism for abamectin resistance in ABA-R strain of WFT (Chen et al., 2011). However, due to the fact that the piperonyl butoxide (PBO) could not completely bring the abamectin resistance to a susceptible level, additional mechanisms, such as target site insensitivity, might also be involved in abamectin resistance. In insect nervous system, both excitatory and inhibitory synaptic transmission are mediated by members of the Cys-loop ligand-gated ion channel (Cys-loop LGIC) superfamily, such as nicotinic acetylcholine receptors (nAChRs), gamma-aminobutyric acid (GABA) receptors and glutamate-gated chloride channels (GluCls) (Jones and Sattelle, 2007). Several lines of evidence have suggested that GluCls were the primary targets of macrocyclic lactones, while binding studies suggested that macrocyclic lactones also act on the insect GABA receptor subunit, Rdl (resistance to dieldrin) (Cully et al., 1994; Nakao et al., 2015; Wolstenholme, 2012; Wolstenholme and Rogers, 2005). In the present study, cDNAs encoding Rdl (FoRdl) and GluCl (FoGluCl) were cloned from WFT. Single nucleotide polymorphisms (SNPs) were detected in abamectin susceptible (ABA-S) and resistant (ABA-R) strains of WFT. The mRNA expression levels of these two target genes in ABA-S and ABA-R were also analyzed.
Corresponding author. E-mail address: [email protected] (J. Wang).
https://doi.org/10.1016/j.pestbp.2018.06.012 Received 8 May 2018; Received in revised form 8 June 2018; Accepted 16 June 2018 0048-3575/ © 2018 Elsevier Inc. All rights reserved.
Please cite this article as: Meng, X., Pesticide Biochemistry and Physiology (2018), https://doi.org/10.1016/j.pestbp.2018.06.012
Pesticide Biochemistry and Physiology xxx (xxxx) xxx–xxx
X. Meng et al.
PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, China) according to the manufacturer's instructions. The SYBR® PrimeScript™ RT-PCR Kit II (TaKaRa, Dalian, China) and gene specific primers were used for the gene expression determining (Table 1). The housekeeping gene β-actin was used as an internal control (Cifuentes et al., 2012). A 20 μL PCR reaction volume contains 10 μL SYBR Premix EX Taq™ II (2×), 2 μL diluted cDNA template with a concentration of 200 ng/μL, 0.4 μL ROX Reference Dye II (50×) and 0.4 μM of each primer. The reaction mixtures were performed on a Bio-Rad CFX 96 Real-time PCR system with a condition of 95 °C for 30 s, 40 cycles of 95 °C for 5 s and 60 °C for 30 s, and a melting curve analysis was performed at the end. The relative gene expression was calculated using 2−ΔΔCT method and normalized to β-actin in the same sample (Pfaffl, 2001). Means and standard errors were obtained from the average of three independent sample sets.
Table 1 Primers used for RT-PCR, RACE and qPCR. Primer name
Sequence (5′ to 3′)⁎
Description
192.GABAF1 193.GABAF2 474.GLURF1 476.GLURR2 212.C4.1R2 469.C4.1R6 213.C4.1F1 214.C4.1F2 506.GLUCLR1 504.GLUCLF2 471.Q2F1
TGGGTNCCNGAYACNTTYTT(WVPDTFF) TAYTTYCAYATHGCNACNAC(YFHIATT) AAYGARAARGARGGNCAYTT(NEKEGHF) CARCANGGDATRTADATYTG (QIYIPCC) TTGCTGGTGGTTGCGATGTGAA CTCGGGCTGGTGTAGCATTTCGGTTC CTGGCTGAACCGAAATGCTA GCTAATGTCCTCAACTAACGCTG GGATGAGGTAGTATGAGAACTCCCGC CTATGGGTGGACAACCAACGACTT ATGGGCAAAAGAGTAAGGCC
Rdl RT-PCR
473.W45R1 781.GuClF1
CTACTTGTCCTCCTGCAACA TACGACGCTAGAATACGACC
783.GuClR1 814.GABAF 815.GABAR 812.GuClF 813.GuClR 706.FoR2_actinF 707.FoR2_actinR
AAGTTTACTGGTCCTCGGCC CATCTACCTGGGCACCTGTT GTCGTGAACTATGAAGCGCA CAGTATTCGCATTTCGCTCA AACCGAGGAAGATGGAGGTT CGGTCAGGTCATCACCATTG TCGTCTCGTGTATTCCGCAC
GluCl RT-PCR Rdl 5′RACE Rdl 3′RACE GluCl 5′RACE GluCl 3′RACE Rdl RT-PCR for SNP analysis GluCl RT-PCR for SNP analysis
2.5. Cloning and sequence analysis
Rdl qPCR
2. Materials and methods
RT-PCR and RACE products were subcloned into the pMD18-T vector (TaKaRa, Dalian, China) and sequenced. The sequence alignment was performed using CLUSTALW with the default settings (Thompson et al., 1994). The aligned sequences were used to construct the phylogenetic tree in MEGA 5 with a bootstrapping of 1000 iterations. The molecular mass and isoelectric point were determined using ExPASy online service (http://web.expasy.org/compute_pi/). The cDNA sequences of FoRdl and FoGluCl have been deposited in the GenBank and the accession numbers were MH249047 and MH249048, respectively.
2.1. Insect strains
2.6. Data analysis
The laboratory selected ABA-R strain with 45.5-fold resistance to abamectin was derived from a susceptible strain (ABA-S) after 15 selection cycles with abamectin during 18 generations (Chen et al., 2011). About 20 2nd-instar nymphs of ABA-S or ABA-R were collected and stored at −80 °C until use for total RNA extraction. At least three repetitions were prepared for a given sample.
Statistical analysis was performed by one-way ANOVA (SPSS version 10.0, SPSS Inc., Chicago, USA) with at least three repeats, and P < 0.05 was considered to be statistically significant.
GluCl qPCR β-actin qPCR
⁎ Corresponding amino acid sequences for degenerate primers were shown in parentheses.
3. Results 3.1. cDNA cloning and characterization of FoRdl and FoGluCl
2.2. Reverse transcriptase-polymerase chain reaction (RT-PCR) The 4070 bp full length cDNA of FoRdl contains a 48-bp 5′-untranslated region (UTR), 1401 bp of ORF encoding a 466-amino acid residue protein with a calculated molecular mass of 52.34 kDa and an isoelectric point (pI) of 8.79, and a 2621-bp 3′-UTR (Fig. 1). Despite repeated attempts to amplify the 5′ end using conventional RACE strategy, we only obtained 1931 bp partial cDNA of FoGluCl, which encodes 437 amino acid residues and contains a 620-bp 3′-UTR. The common features of insect Cys-loop LGIC subunits, such as six ligand binding loops (Loop A-F) and four conserved transmembrane regions (TM1–4), were conserved in both FoRdl and FoGluCl (Fig. 1). An amino acid sequence alignment shows that FoRdl was 64.7% and 84.0% identical in pairwise comparisons with the Rdl amino acid sequences in Drosophila melanogaster and Tribolium castaneum, respectively. The deduced FoGluCl amino acid sequence showed 78.0% and 78.3% identities to GluCl from D. melanogaster and T. castaneum, respectively. Phylogenetic analysis reveals that Rdl proteins form a sister group relationship with Lcch3 subunits, and FoRdl and FoGluCl were closely related to blattaria and hymenoptera homologues, respectively (Fig. 2).
Total RNAs were extracted from the collected samples using an SV total RNA isolation system (Promega, Madison, WI), according to the manufacturer's instructions. First-strand cDNA was synthesized from 1 μg of total RNA using the Primescript™ First-Strand cDNA Synthesis kit (TaKaRa, Dalian, China) according to the manufacturer's instructions. Degenerate primer pairs were designed against the amino acid residues that were conserved between insects (Table 1). PCR was performed in a 25 μL reaction volume containing 20–50 ng cDNA, 0.8 μM of each degenerate primer, 0.2 mM of each dNTP, 2 mM of MgCl2, 1.25 U Ex Taq™ polymerase and 2.5 μL Ex Taq™ buffer (Takara, Dalian, China). A touchdown PCR program was used, which consisted of 1 cycle at 94 °C for 5 min, 12 cycles at 94 °C for 30 s, 52–41 °C (decreasing by −1 °C/cycle) for 30 s, and 72 °C for 2 min, followed by 25 cycles of 94 °C for 30 s, 40 °C for 30 s, and 72 °C for 2 min, and a final extension at 72 °C for 10 min. 2.3. Rapid amplification of cDNA ends (RACE) To complete the cDNA sequence of FoRdl and FoGluCl, 5′ -RACE and 3′ -RACE reactions were performed using 5′-full RACE core set and 3′full RACE core set (Takara, Dalian, China) respectively, following the manufacturer's instructions. Gene specific primers (GSP) used for the 5′ - and 3′ -RACE are listed in Table 1.
3.2. Alternative splicing The alignment of multiple cDNA clone sequences revealed three alternative splicing variants of FoRdl (FoRdl-A, FoRdl-B and FoRdl-C) and two alternative splicing variants of FoGluCl (FoGluCl-A and FoGluCl-B) in WFT (Fig. 3). Comparison of the cDNA sequences with corresponding genomic sequences derived from the whole genome shotgun database (WGS) assembly of WFT deposited at GenBank revealed that both FoRdl and FoGluCl variants were generated by
2.4. Reverse transcription quantitative PCR (RT-qPCR) Total RNA was employed for cDNA template synthesis using 2
Pesticide Biochemistry and Physiology xxx (xxxx) xxx–xxx
X. Meng et al.
Fig. 1. Amino acid sequence alignment of Rdl and GluCl from WFT, D. melanogaster and T. castaneum. The dotted line amino acid sequences represent the alternative splicing site in Rdl and GluCl of WFT. The signal peptides at the N-terminus were boxed. Lines above amino acid sequences represent the six ligand binding loops (Loop A-F) and four conserved transmembrane regions (TM1–4) of receptor protein. The ‘▲’ indicated the reported resistance mutations in insect GluCl, and the ‘▼’indicated the reported resistance mutations in insect Rdl.
alternative splicing of exon 3, designated as A, B, and C in FoRdl and A, B in FoGluCl (data not shown). Specifically, the alternative splicing site forming mutually exclusive exons lie upstream to loop D encoding region within proximity of the agonist binding site (Fig. 1). All these alternative exons were composed of 68 nucleotides encoding 23 amino acids (55–77 in FoRdl, and 62–84 in FoGluCl). The three alternative exons in FoRdl share nucleotide and amino acid identities of 70.6%–80.9% and 82.6%–95.7%, respectively, whereas the two alternative exons in FoGluCl were divergent, differing at 7 of 23 amino acid residues (Fig. 3). The splice sites are highly conserved in insect Rdl and GluCl. Exon 3 variants of FoRdl and FoGluCl showed > 91.3% amino acid identities to the corresponding variants in D. melanogaster and T. castaneum (Fig. 3).
Sequences analysis revealed 33 nucleotide polymorphisms of FoRdl in two strains, which resulted in 23 amino acids replacement (Table 2). The extraordinarily high number of SNPs in FoRdl is not unusual, as high nucleotide diversity has been found in the para-like voltage-sensitive sodium channel gene in this insect, which is probably a direct consequence of pest migration (Forcioli et al., 2002). However, most of these replacements occurred in both ABA-S and ABA-R strains, and no putative resistance-associated mutations were detected in ABA-R strain (Table 2). A total of 23 nucleotide polymorphisms of FoGluCl were found in ABA-R and ABA-S strains, which caused 8 amino acid substitutions (Table 3). However, two abamectin resistance-associated mutations (A309V and G315E) conserved in several insects and mites (Wang et al., 2017) were not detected in FoGluCl of ABA-R strain.
3.3. SNPs of FoRdl and FoGluCl
3.4. mRNA expression levels of FoRdl and FoGluCl in ABA-R and ABA-S starins
To preliminarily explore the resistance-associated mutations in ABA-R strain, cDNA fragments of both FoRdl (1401 bp) and FoGluCl (1308 bp) in ABA-R and ABA-S strains were amplified, and a total of 7 positive clones for each strain and target gene were sequenced.
The mRNA expression levels of both FoRdl and FoGluCl were compared between ABA-R and ABA-S strains. While no difference of FoRdl expression was found between ABA-R and ABA-S strains, the mRNA 3
Pesticide Biochemistry and Physiology xxx (xxxx) xxx–xxx
X. Meng et al.
Fig. 2. Phylogenetic analysis of GABA receptor subunits and GluCl from WFT and other insects.
compared with that of the unselected strain (Wang and Wu, 2007). In Liriomyza sativae, GST activities in two abamectin-resistant strains were significantly higher than that in susceptible strain, indicating the involvement of GST in conferring abamectin resistance (Wei et al., 2015). In Plutella xylostella, elevated levels of P450 monooxygenase activity was shown to be associated with resistance to abamectin (Qian et al., 2008), and a recent study reported that the overexpression of CYP340W1 plays an important role in abamectin resistance (Gao et al., 2016). Consisting with these results, our previous study with in vivo synergism assessments and in vitro metabolic enzyme assays showed that increased cytochrome P450s enzyme activity was a major factor conferring abamectin resistance in the WFT (Chen et al., 2011). In addition to enhanced detoxification, target site insensitivity was
expression level of FoGluCl in ABA-R strain was 2.63-fold higher than that in ABA-S strain (Fig. 4). 4. Discussion Generally, the mechanisms of insecticide resistance include behavioral resistance, reduced penetration of toxicants, enhanced insecticide metabolism, and alterations of insecticide targets (Brattsten et al., 1986). It has been reported that abamectin resistance in several insects were driven by metabolic resistance mechanisms. In a laboratory selected Bemisia tabaci strain with 14.5-fold resistance to abamectin, P450 monooxygenase activity and glutathione S-transferase (GST) activity were elevated to 2.1- and 2.0-fold, respectively,
Fig. 3. Sequence alignments of Rdl and GluCl alternative splicing variants. (A) Nucleotide sequences of three FoRdl and two FoGluCl splicing variants. (B) Amino acid sequence alignments of Rdl and GluCl variants from WFT, D. melanogaster and T. castaneum. 4
Pesticide Biochemistry and Physiology xxx (xxxx) xxx–xxx
X. Meng et al.
Table 2 Polymorphisms of FoRdl in the susceptible and abamectin resistant strains of WFT. Nucleotide positiona
Nucleotideb
Nucleotide substitutionc ABA-S Strain
20, 21 22 102 155, 156 259 269 385 430 464 480 490 544 547, 548 557 618 669 672 684 778 892 909 959 965 1089 1100 1117 1201 1221 1289 1299 a b c d
ACC AGC ATT AAC CAA ACG TAC CAC CTA TGT CTT GGG TAT AGG TCT AGT CTG AAC TCC AAG AAA CTT GAG GCG ACG GTT GCC CTG CGA TTC
6
1
ACC , AAA AGC6, TGC1 ATT7 AAC6, AGA1 CAA6, TAA1 ACG7 TAC7 CAC6, AAC1 CTA7 TGT7 CTT6, TGG1 GGG6, TGG1 TAT7 AGG6, ACG1 TCT6, TCC1 AGT7 CTG7 AAC6, AAA1 TCC6, CCC1 AAG6, GAG1 AAA7 CTT6, CCT1 GAG7 GCG6, GCA1 ACG6, AGG1 GTT7 GCC6, CCC1 CTG7 CGA6, CAA1 TTC6, TTT1
Amino acid ABA-R Strain 3
2
Amino acid substitutiond ABA-S Strain
2
ACC , AAA , AAC AGC7 ATT6, ATA1 AAC7 CAA7 ACG6, ATG1 TAC6, CAC1 CAC7 CTA6, ATA1 TGT6, TGC1 CTT7 GGG7 TAT5, CAT1, TTT1 AGG7 TCT7 AGT6, AGG1 CTG6, CTA1 AAC7 TCC7 AAG7 AAA6, AAT1 CTT7 GAG6, GGG1 GCG7 ACG7 GTT6, TTT1 GCC7 CTG6, CTT1 CGA7 TTC7
T7 S8 I34 N52 Q87 T90 Y129 H144 T155 C160 L164 G182 Y183 R186 S206 S223 L224 N228 S260 K298 K303 L320 E322 A363 T367 V373 A401 L407 R430 F433
6
1
ABA-R Strain
T ,K S6, C1
T3, K2, N2 S7
N6, R1 Q6, *1 T7 Y7 H6, N1 T7
N7 Q7 T6, M1 Y6, H1 H7 T6, N1
L6, F1 G6, W1 Y7 R6, T1
L7 G7 Y5, H1, F1 R7
S7
S6, R1
N6, K1 S6, P1 K6, E1 K7 L6, P1 E7
N7 S7 K7 K6, N1 L7 E6, G1
T6, R1 V7 A6, P1
T7 V6 , F 1 A7
R6, Q1
R7
The position of A in the initial methionine codon is numbered 1. The substituted nucleotides were italicized and bolded. The numbers indicated the amount of nucleotide substitutions in 7 clones. Only sense substitutions were shown, and ‘*’represent the termination codon.
Table 3 Polymorphisms of FoGluCl in the susceptible and abamectin resistant strains of WFT. Nucleotide positiona
Nucleotideb
Nucleotide substitution ABA-S Strain
269 435 450 511 588 618 705 759 855 903 954 1080 1104 1107 1115 1139 1164 1173 1205 1213 1236 1269 1270 a b c d
CTT TAC CCG AAG TTT GTT AGT CAG ACT CTG CTC CCC AAC ACC CCG CAC TGC ACT CGC GTC CCG TGG TCC
6
1
CTT , CCT TAC6, TAT1 CCG6, CCA1 AAG6, GAG1 TTT5, TTC2 GTT5, GTC2 AGT6, AGA1 CAG6, CAA1 ACT6, ACC1 CTG6, CTA1 CTC6, CTG1 CCC6, CCG1 AAC7 ACC6, ACT1 CCG6, CCC1 CAC7 TGC6, TGT1 ACT6, ACG1 CGC7 GTC6, ATC1 CCG7 TGG5, TGA2 TCC6, CCC1
c
Amino acid ABA-R Strain 7
CTT TAC7 CCG7 AAG7 TTT5, TTC2 GTT5, GTC2 AGT7 CAG7 ACT5, ACC2 CTG7 CTC7 CCC7 AAC6, AAT1 ACC6, ACT1 CCG6, CCC1 CAC6, CTC1 TGC7 ACT7 CGC6, CTC1 GTC7 CCG5, CCA2 TGG7 TCC7
The position of A in the initial threonine codon is numbered 1. The substituted nucleotides were italicized and bolded. The numbers indicated the amount of nucleotide substitutions in 7 clones. Only sense substitutions were shown, and ‘*’represent the termination codon. 5
Amino acid substitutiond ABA-S Strain
L90 Y145 P150 K171 F196 V206 S235 Q253 T285 L301 L318 P360 N368 T369 P372 H380 C388 T391 R402 V405 P412 W423 S424
6
ABA-R Strain
1
L ,P
L7
K6, E1
K7
S 6 , R1
S7
H7
H6, L1
R7 V6, I1
R6, L1 V7
W5, *2 S6, P 1
W7 S7
Pesticide Biochemistry and Physiology xxx (xxxx) xxx–xxx
X. Meng et al.
abamectin were decreased when the expression of GluCl was knocked down using RNA interference (Shi et al., 2012; Wei et al., 2018). This discrepancy can be explained by the fact that GluCl plays important role in the development of insects (Boumghar et al., 2012; Chiang et al., 2002; El Hassani et al., 2012; Kita et al., 2013), and RNAi-mediated down-regulation of GluCl definitely have negative effect on the insect viability as well as tolerance against insecticides. Alternative splicing is a key posttranscriptional processing mechanism for generating protein diversity, and it appears to be of central importance for ion channels as insecticide targets, such as Rdl and GluCl. Alternative splicing of exon 3 in Rdl and GluCl have been found in many insects. Two Rdl variants derived from the splicing of exon 3 in D. melanogaster showed different pharmacological properties (Buckingham et al., 2005). Three GluCl variants generated by the alternative splicing of exon 3 were present in T. castaneum, Apis mellifera, Laodelphax striatellus and Musca domestica (Jones and Sattelle, 2007; El Hassani et al., 2012; Eguchi et al., 2006; Wu et al., 2017), and showed various physiological functions in A. mellifera (Boumghar et al., 2012). In addition to the alternative splicing of exon 3, insect Rdl variants could also derive from the alternative splicing of exon 6 (Jones and Sattelle, 2007). In this study, we found three Rdl variants derived from the splicing of exon 3 in WFT, however, alternative splicing of exon 6 was not detected. In contrast, among three insect GluCl variants, only two variants were found in WFT. The pharmacological and physiological functions of FoRdl and FoGluCl splicing variants need further study.
Fig. 4. mRNA expression of FoRdl and FoGluCl genes in ABA-R and ABA-S strains of WFT. *Significant difference by one-way ANOVA test (P < 0.05).
also reported to contribute to abamectin resistance in several insects and mites. A G323D mutation in GluCl1 and G326E in GluCl3 of Tetranychus urticae conferred 18-fold and 2000-fold resistance to abamectin in this mite, respectively (Dermauw et al., 2012; Kwon et al., 2010). Recently, both electrophysiology and homology modelling studies demonstrated that two mutations (A309V, corresponding to A310 in WFT FoGluCl, and G315E, corresponding to G323 in TuGluCl1, G326 in TuGluCl3 and G316 in WFT FoGluCl) and a 36-bp deletion in P. xylostella GluCl contribute to target-site resistance to abamectin (Wang et al., 2017; Liu et al., 2014; Wang et al., 2016b). In this study, while sequence analysis revealed multiple nucleotide polymorphisms of FoGluCl in ABA-S and ABA-R strains of WFT, none of the above-mentioned mutations were detected in ABA-R strain. We also did not find any putative resistance-associated mutations in the secondary target of abamectin, FoRdl. However, it is interesting to note that two amino acid replacements of Rdl including A302S and V327I, which were associated with cylcodiene insecticide resistance in several insects (FfrenchConstant et al., 1993; Wondji et al., 2011), were found in FoRdl of both ABA-S and ABA-R strains (S276 and I307). The A302S substitution was also detected in one of the duplicated Rdl genes, CsRDL2S, in Chilo suppressalis (Sheng et al., 2018), however, a tBLASTn search of the WGS assembly of WFT deposited at GenBank revealed the presence of single Rdl gene in WFT. These preliminary data suggest that the target site mutation did not contribute to abamectin resistance in ABA-R strain, and the ancestor population of ABA-S strain might have been under strong selection of cylcodiene insecticides with little negative fitness of these two resistance-associated mutations. Theoretically, the change of mRNA expression of target gene should have effects on the sensitivity of insects against insecticides. This theory has been confirmed by increasing number of studies. For example, in the greenbug Schizaphis graminum, northern blot analysis showed that the amount of acetylcholinesterase mRNA in the organophosphate resistant clones was approximately 1.5-fold higher than that in the susceptible clone (Gao and Zhu, 2002). In a laboratory flubendiamide-selected P. xylostella strain, the relative ryanodine receptor mRNA transcript level was 2.93-fold as compared with the susceptible strain (Yan et al., 2014). In this study, the mRNA expression of FoGluCl in ABA-R was 2.63-fold higher than that in ABA-S, suggesting the role of up-regulation of target gene in abamectin resistance in WFT. This result was consistent with the studies reporting association of up-regulation of GluCl with abamectin resistance in P. xylostella (Liang, 2009; Liu, 2011). However, in both B. tabaci and P. xylostella, susceptibilities to
Acknowledgements This work was supported by National Natural Science Foundation of China (31701807, 31572000) and Jiangsu Province Science Foundation for Youths (BK20170491). References Bielza, P., 2008. Insecticide resistance management strategies against the western flower thrips, Frankliniella occidentalis. Pest Manag. Sci. 64, 1131–1138. Boumghar, K., Couret-Fauvel, T., Garcia, M., Armengaud, C., 2012. Evidence for a role of GABA- and glutamate-gated chloride channels in olfactory memory. Pharmacol. Biochem. Be. 103, 69–75. Brattsten, L.B., Holyoke Jr., C.W., Leeper, J.R., Raffa, K.F., 1986. Insecticide resistance: challenge to pest management and basic research. Science 231, 1255–1260. Buckingham, S.D., Biggin, P.C., Sattelle, B.M., Brown, L.A., Sattelle, D.B., 2005. Insect GABA receptors: splicing, editing, and targeting by antiparasitics and insecticides. Mol. Pharmacol. 68, 942–951. Chen, X., Yuan, L., Du, Y., Zhang, Y., Wang, J., 2011. Cross-resistance and biochemical mechanisms of abamectin resistance in the western flower thrips, Frankliniella occidentalis. Pestic. Biochem. Physiol. 101, 34–38. Chiang, A.S., Pszczolkowski, M.A., Liu, H.P., Lin, S.C., 2002. Ionotropic glutamate receptors mediate juvenile hormone synthesis in the cockroach, Diploptera punctata. Insect Biochem. Mol. Biol. 32, 669–678. Cifuentes, D., Chynoweth, R., Guillen, J., De La Rua, P., Bielza, P., 2012. Novel cytochrome P450 genes, CYP6EB1 and CYP6EC1, are over-expressed in acrinathrin-resistant Frankliniella occidentalis (Thysanoptera: Thripidae). J. Econ. Entomol. 105, 1006–1018. Cloyd, R.A., 2016. Western flower thrips (Thysanoptera: Thripidae) and insecticide resistance: an overview and strategies to mitigate insecticide resistance development. J. Entomol. Sci. 51, 257–273. Cully, D.F., Vassilatis, D.K., Liu, K.K., Paress, P.S., Van der Ploeg, L.H., Schaeffer, J.M., Arena, J.P., 1994. Cloning of an avermectin-sensitive glutamate-gated chloride channel from Caenorhabditis elegans. Nature 371, 707–711. Dagli, F., Tunc, I., 2007. Insecticide resistance in Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) collected from horticulture and cotton in Turkey. Aust. J. Entomol. 46, 320–324. Dermauw, W., Ilias, A., Riga, M., Tsagkarakou, A., Grbic, M., Tirry, L., Van Leeuwen, T., Vontas, J., 2012. The cys-loop ligand-gated ion channel gene family of Tetranychus urticae: implications for acaricide toxicology and a novel mutation associated with abamectin resistance. Insect Biochem. Mol. Biol. 42, 455–465. Eguchi, Y., Ihara, M., Ochi, E., Shibata, Y., Matsuda, K., Fushiki, S., Sugama, H., Hamasaki, Y., Niwa, H., Wada, M., Ozoe, F., Ozoe, Y., 2006. Functional characterization of Musca glutamate- and GABA-gated chloride channels expressed independently and coexpressed in Xenopus oocytes. Insect Mol. Biol. 15, 773–783. El Hassani, A.K., Schuster, S., Dyck, Y., Demares, F., Leboulle, G., Armengaud, C., 2012. Identification, localization and function of glutamate-gated chloride channel receptors in the honeybee brain. Eur. J. Neurosci. 36, 2409–2420.
6
Pesticide Biochemistry and Physiology xxx (xxxx) xxx–xxx
X. Meng et al.
Sheng, C., Jia, Z., Ozoe, Yoshihisa, Huang, Q., Han, Z., Zhao, C., 2018. Molecular cloning, spatiotemporal and functional expression of GABA receptor subunits RDL1 and RDL2 of the rice stem borer Chilo suppressalis. Insect Biochem. Mol. Biol. 94, 18–27. Shi, X., Guo, Z., Zhu, X., Wang, S., Xu, B., Xie, W., Zhang, Y., Wu, Q., 2012. RNA interference of the inhibitory glutamate receptor in Plutella xylostella (Lepidoptera Plutellidae). Acta Entomol. Sin. 55, 1331–1336. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positionspecific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. Wang, L., Wu, Y., 2007. Cross-resistance and biochemical mechanisms of abamectin resistance in the B-type Bemisia tabaci. J. Appl. Entomol. 131, 98–103. Wang, S., Zhang, A., Li, L., Men, X., Zhou, X., Zhai, Y., Liu, Y., Wei, S., Yu, Y., 2014. Insecticide resistance status of field populations of Frankliniella occidentalis (Thysanoptera: Thripidae) in China and its control strategies. Acta Entomol. Sin. 57, 621–630. Wang, Z., Gong, Y., Jin, G., Li, B., Chen, J., Kang, Z., Zhu, L., Gao, Y., Reitz, S., Wei, S., 2016a. Field-evolved resistance to insecticides in the invasive western flower thrips Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) in China. Pest Manag. Sci. 72, 1440–1444. Wang, X., Wang, R., Yang, Y., Wu, S., O'Reilly, A.O., Wu, Y., 2016b. A point mutation in the glutamate-gated chloride channel of Plutella xylostella is associated with resistance to abamectin. Insect Mol. Biol. 25, 116–125. Wang, X., Puinean, A.M., O'Reilly, A.O., Williamson, M.S., Smelt, C.L.C., Millar, N.S., Wu, Y., 2017. Mutations on M3 helix of Plutella xylostella glutamate-gated chloride channel confer unequal resistance to abamectin by two different mechanisms. Insect Biochem. Mol. Biol. 86, 50–57. Webster, C.G., Reitz, S.R., Perry, K.L., Adkins, S., 2011. A natural M RNA reassortant arising from two species of plant- and insect-infecting bunyaviruses and comparison of its sequence and biological properties to parental species. Virology 413, 216–225. Wei, Q., Lei, Z., Nauen, R., Cai, D., Gao, Y., 2015. Abamectin resistance in strains of vegetable leafminer, Liriomyza sativae (Diptera: Agromyzidae) is linked to elevated glutathione S-transferase activity. Insect Sci. 22, 243–250. Wei, P., Che, W., Wang, J., Xiao, D., Wang, R., Luo, C., 2018. RNA interference of glutamate-gated chloride channel decreases abamectin susceptibility in Bemisia tabaci. Pestic. Biochem. Physiol. 145, 1–7. Wolstenholme, A.J., 2012. Glutamate-gated chloride channels. J. Biol. Chem. 287, 40232–40238. Wolstenholme, A.J., Rogers, A.T., 2005. Glutamate-gated chloride channels and the mode of action of the avermectin/milbemycin anthelmintics. Parasitology 131, S85–S95. Wondji, C.S., Dabire, R.K., Tukur, Z., Irving, H., Djouaka, R., Morgan, J.C., 2011. Identification and distribution of a GABA receptor mutation conferring dieldrin resistance in the malaria vector Anopheles funestus in Africa. Insect Biochem. Mol. Biol. 41, 484–491. Wu, S., Mu, X., Dong, Y., Wang, L., Wei, Q., Gao, C., 2017. Expression pattern and pharmacological characterisation of two novel alternative splice variants of the glutamate-gated chloride channel in the small brown planthopper Laodelphax striatellus. Pest Manag. Sci. 73, 590–597. Yan, H., Xue, C., Li, G., Zhao, X., Che, X., Wang, L., 2014. Flubendiamide resistance and Bi-PASA detection of ryanodine receptor G4946E mutation in the diamondback moth (Plutella xylostella L.). Pestic. Biochem. Physiol. 115, 73–77. Zhao, H., Yi, X., Deng, Y., Hu, M., Zhong, G., Wang, P., 2013. Resistance to fenpropathrin, chlorpyriphos and abamectin in different populations of Amblyseius longispinosus (Acari: Phytoseiidae) from vegetable crops in South China. Biol. Control 67, 61–65.
Ffrench-Constant, R.H., Rocheleau, T.A., Steichen, J.C., Chalmers, A.E., 1993. A point mutation in a Drosophila GABA receptor confers insecticide resistance. Nature 363, 449–451. Forcioli, D., Frey, B., Frey, J.E., 2002. High nucleotide diversity in the Para-like voltagesensitive sodium channel gene sequence in the western flower thrips (Thysanoptera: Thripidae). J. Econ. Entomol. 95, 838–848. Gao, J., Zhu, K., 2002. Increased expression of an acetylcholinesterase gene may confer organophosphate resistance in the greenbug, Schizaphis graminum (Homoptera: Aphididae). Pestic. Biochem. Physiol. 73, 164–173. Gao, Y., Lei, Z., Reitz, S.R., 2012. Western flower thrips resistance to insecticides: detection, mechanisms and management strategies. Pest Manag. Sci. 68, 1111–1121. Gao, X., Yang, J., Xu, B., Xie, W., Wang, S., Zhang, Y., Yang, F., Wu, Q., 2016. Identification and characterization of the gene CYP340W1 from Plutella xylostella and its possible involvement in resistance to abamectin. Int. J. Mol. Sci. 17, 274. Herron, G.A., James, T.M., 2007. Insecticide resistance in Australian populations of western flower thrips, Frankliniella occidentalis (pergande) (thysanoptera: thripidae). Gen. Appl. Entomol. 36, 1–5. Immaraju, J.A., Paine, T.D., Bethke, J.A., Robb, K.L., Newman, J.P., 1992. Western flower thrips (Thysanoptera: Thripidae) resistance to insecticides in coastal California greenhouses. J. Econ. Entomol. 85, 9–14. Jones, A.K., Sattelle, D.B., 2007. The cys-loop ligand-gated ion channel gene superfamily of the red flour beetle, Tribolium castaneum. BMC Genomics 8. Kirk, W.D.J., Terry, L.I., 2003. The spread of the western flower thrips Frankliniella occidentalis (Pergande). Agr. For. Entomol. 5, 301–310. Kita, T., Ozoe, F., Azuma, M., Ozoe, Y., 2013. Differential distribution of glutamate- and GABA-gated chloride channels in the housefly Musca domestica. J. Insect Physiol. 59, 887–893. Kwon, D.H., Yoon, K.S., Clark, J.M., Lee, S.H., 2010. A point mutation in a glutamategated chloride channel confers abamectin resistance in the two-spotted spider mite, Tetranychus urticae Koch. Insect Mol. Biol. 19, 583–591. Lasota, J.A., Dybas, R.A., 1991. Avermectins, a novel class of compounds: implications for use in arthropod pest control. Annu. Rev. Entomol. 36, 91–117. Li, D., Shang, X., Reitz, S., Nauen, R., Lei, Z., Si Hyeock, L., Gao, Y., 2016. Field resistance to spinosad in western flower thrips Frankliniella occidentalis (Thysanoptera: Thripidae). J. Integr. Agric. 15, 2803–2808. Liang, Y., 2009. Studies on Molecular Mechanism of Resistance to Avermectin in Plutella xylostella. Gansu Agricultural University. Liu, F., 2011. The Role of GluCl Receptor in the Resistance of Plutella xylostella (Lepidoptera: Plutellidae) to Abamectin. Chinese Academy of Agricultural Sciences. . Liu, F., Shi, X., Liang, Y., Wu, Q., Xu, B., Xie, W., Wang, S., Zhang, Y., Liu, N., 2014. A 36bp deletion in the alpha subunit of glutamate-gated chloride channel contributes to abamectin resistance in Plutella xylostella. Entomol. Exp. Appl. 153, 85–92. Nakao, T., Banba, S., Hirase, K., 2015. Comparison between the modes of action of novel meta-diamide and macrocyclic lactone insecticides on the RDL GABA receptor. Pestic. Biochem. Physiol. 120, 101–108. Pappu, H.R., Jones, R.A.C., Jain, R.K., 2009. Global status of tospovirus epidemics in diverse cropping systems: successes achieved and challenges ahead. Virus Res. 141, 219–236. Pfaffl, M.W., 2001. A new mathematical model for relative quantification in real-time RTPCR. Nucleic Acids Res. 29, 2002–2007. Qian, L., Cao, G., Song, J., Yin, Q., Han, Z., 2008. Biochemical mechanisms conferring cross-resistance between tebufenozide and abamectin in Plutella xylostella. Pestic. Biochem. Physiol. 91, 175–179.
7