Qingli Shang

Over-expression of CYP6A2 is associated with spirotetramat resistance and cross-resistance in the resistant strain of Aphis gossypii Glover

A laboratory-selected spirotetramat-resistant strain (SR) of cotton aphid developed 579-fold and 15-fold resistance to spirotetramat in adult aphids and 3rd instar nymphs, respectively, compared with a susceptible strain (SS) [26]. The SR strain developed high-level cross-resistance to alpha-cypermethrin and bifenthrin and very low or no cross-resistance to the other tested insecticides. Synergist piperonyl butoxide (PBO) dramatically increased the toxicity of spirotetramat and alpha-cypermethrin in the resistant strain. RT-qPCR results demonstrated that the transcriptional levels of CYP6A2 increased significantly in the SR strain compared with the SS strain, which was consistent with the transcriptome results [30]. The depletion of CYP6A2 transcripts by RNAi also significantly increased the sensitivity of the resistant aphid to spirotetramat and alpha-cypermethrin. These results indicate the possible involvement of CYP6A2 in spirotetramat resistance and alpha-cypermethrin cross-resistance in the cotton aphid. These together with other cross-resistance results have implications for the successful implementation of resistance management strategies for Aphis gossypii.

Transcriptomic comparison of thiamethoxam-resistance adaptation in resistant and susceptible strains of Aphis gossypii Glover

A thiamethoxam-resistant strain of cotton aphid (ThR) strain displayed a 19.35-fold greater resistance to thiamethoxam compared to a susceptible cotton aphid (SS) strain. Solexa sequencing technology was used to investigate differentially expressed genes (DEGs) in cotton aphids in the context of thiamethoxam resistance. A total of 22,569,311 and 21,317,732 clean reads were obtained from the ThR and SS transcriptomes, respectively, and assembled into 35,222 non-redundant (Nr) consensus sequences. The expression of 620 unigenes changed significantly in the ThR libraries compared to the SS strain; 349 genes were up-regulated, and 271 genes were down-regulated (P≤0.001). Expression levels of ribosomal proteins, ATP synthase, cytochrome c oxidase, ecdysteroid UDP-glucosyltransferase and esterase were up-regulated significantly in the ThR strain compared to the SS strain. The genes of cuticle proteins, salivary proteins, and fibroin heavy chain decreased dramatically. One nicotinic acetylcholine receptor (nAChR) α subunit was down-regulated in the ThR strain. The expression levels of 10 differentially expressed unigenes were confirmed using real-time RT-PCR, and the observed trends in gene expression matched the Solexa expression profiles. Specific single-nucleotide polymorphisms (SNPs) in nAChRs that cause amino acid substitution were found from the ThR and SS stains respectively. These data illustrate that genetic changes in nAChR genes and up-regulated ribosomal proteins, ecdysteroid UDP-glucosyltransferase, cytochrome c oxidase, esterase and peroxidase may confer the tolerance of resistant cotton aphids to thiamethoxam.

Spirotetramat resistance adaption analysis of Aphis gossypii Glover by transcriptomic survey.

A resistant strain of the cotton aphid (SR) developed 441.26-fold and 11.97-fold resistance to spirotetramat for adult aphids and nymphs, respectively, compared with the susceptible (SS) strain. Solexa sequencing technology was employed to identify differentially expressed genes (DEGs) in the spirotetramat-resistant cotton aphid. Respective totals of 22,430,522 and 21,317,732 clean reads were obtained from SR and SS cDNA libraries and assembled into 35,222 non-redundant (Nr) consensus sequences. A total of 14,913, 9,220, 7,922, 4,314 and 4,686 sequences were annotated using Nr, Swiss-Prot, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Clusters of Orthologous Groups (COG), respectively. Compared with the SS strain, the SR strain had 1287 significantly changed unigenes, of which 130 genes were up-regulated and 1157 genes were down-regulated (P ≤ 0.001). Among these genes, 440 unigenes were annotated, consisting of 114 up-regulated and 326 down-regulated genes. The expression levels of heat shock protein 70 (Hsp70) and UDP-glucuronosyltransferase were significantly up-regulated in the SR strain compared to the SS strain. The genes encoding cuticle proteins, salivary glue protein, fibroin heavy chain, energy ATP synthase, and cytochrome c oxidase were dramatically decreased. Among the DEGs, cytochrome P450 6A2 (c20965.graph_c0) was the only P450 gene up-regulated in the SR strain. The expression levels of 10 DEGs were confirmed by real-time qPCR, and the trends in gene expression observed by qPCR matched those of the Solexa expression profiles. The acetyl-CoA carboxylase (ACC) genes in the SR and SS libraries both contain four single nucleotide polymorphisms (SNPs), with three common SNPs: 1227 (C/T), 1811 (A/T: F/Y) and 3759 (C/T); however, 7540 (A/T) and 108 (G/A) occurred solely in the SS and SR strains, respectively.

Elevated expression of esterase and cytochrome P450 are related with lambda–cyhalothrin resistance and lead to cross resistance in Aphis glycines Matsumura

A resistant strain of the Aphis glycines Matsumura (CRR) has developed 76.67-fold resistance to lambda-cyhalothrin compared with the susceptible (CSS) strain. Synergists piperonyl butoxide (PBO), S,S,S-Tributyltrithiophosphate (DEF) and triphenyl phosphate (TPP) dramatically increased the toxicity of lambda-cyhalothrin to the resistant strain. Bioassay results indicated that the CRR strain had developed high levels of cross-resistance to chlorpyrifos (11.66-fold), acephate (8.20-fold), cypermethrin (53.24-fold), esfenvalerate (13.83-fold), cyfluthrin (9.64-fold), carbofuran (14.60-fold), methomyl (9.32-fold) and bifenthrin (4.81-fold), but did not have cross-resistance to chlorfenapyr, imidacloprid, diafenthiuron, abamectin. The transcriptional levels of CYP6A2-like, CYP6A14-like and cytochrome b-c1 complex subunit 9-like increased significantly in the resistant strain than that in the susceptible. Similar trend were observed in the transcripts and DNA copy number of CarE and E4 esterase. Overall, these results demonstrate that increased esterase hydrolysis activity, combined with elevated cytochrome P450 monooxygenase detoxicatication, plays an important role in the high levels of lambda-cyhalothrin resistance and can cause cross-resistance to other insecticides in the CRR strain.

A transferrin gene associated with development and 2‐tridecanone tolerance in Helicoverpa armigera

The full‐length cDNA (2320 bp) encoding a putative iron‐binding transferrin protein from Helicoverpa armigera was cloned and named HaTrf. The putative HaTrf sequence included 670 amino acids with a molecular mass of approximately 76 kDa. Quantitative PCR results demonstrated that the transcriptional level of HaTrf was significantly higher in the sixth instar and pupa stages as compared with other developmental stages. HaTrf transcripts were more abundant in fat bodies and in the epidermis than in malpighian tubules. Compared with the control, the expression of HaTrf increased dramatically 24 h after treatment with 2‐tridecanone. Apparent growth inhibition with a dramatic body weight decrease was observed in larvae fed with HaTrf double‐stranded RNA (dsRNA), as compared with those fed with green fluorescent protein dsRNA. RNA interference of HaTrf also significantly increased the susceptibility of larvae to 2‐tridecanone. These results indicate the possible involvement of HaTrf in tolerance to plant secondary chemicals.

Cross-resistance pattern and basis of resistance in a thiamethoxam-resistant strain of Aphis gossypii Glover

A thiamethoxam-resistant strain of cotton aphid (ThR) displayed a 13.79-fold greater resistance to thiamethoxam than a susceptible cotton aphid (SS) strain. Piperonyl butoxide (PBO) and triphenyl phosphate (TPP) synergistically increased the toxicity of thiamethoxam in the resistant strain, whereas diethyl maleate (DEM) did not exhibit significant synergistic effects. Bioassay results indicated that the ThR strain developed increased levels of cross-resistance to bifenthrin (11.71 fold), cyfluthrin (17.90 fold), esfenvalerate (6.85 fold), clothianidin (6.56 fold), methidathion (5.34 fold) and alpha-cypermethrin (4.53 fold) but did not show cross-resistance to malathion, omethoate, acephate, chlorpyrifos, methomyl, sulfoxaflor or imidacloprid. PBO and TPP increased bifenthrin toxicity in the resistant strain by 2.38 and 4.55 fold, respectively. Quantitative real-time PCR results indicated that the mRNA expression levels of the α1, α4-1, α4-2, α5 and α7 subunits decreased significantly by 3.32, 1.60, 2.05, 5.41 and 1.48 fold, respectively, in the resistant strain compared with those in the susceptible strain. However, significant differences were not observed in the expression of the α2, α3 and β1 subunits. No target-site mutations within the α1, α2 and β1 subunits of nicotinic acetylcholine receptors (nAChRs) were detectable in the ThR strain. In conclusion, the levels of thiamethoxam resistance and cross-resistance to other insecticides observed in the ThR strain are likely regulated by two mechanisms, which include the overexpression of detoxification-related P450s and esterase. These results should be useful for the understanding thiamethoxam resistance mechanism and the management of insecticide-resistant cotton aphids in China.

The retardant effect of 2-Tridecanone, mediated by Cytochrome P450, on the Development of Cotton bollworm, Helicoverpa armigera

BackgroundPlant allelochemicals act as toxins, inhibitors of digestion, and deterrents that affect the fecundity of insects. These compounds have attracted significant research attention in recent decades, and much is known about the effects of these xenobiotic plant secondary metabolites on insect development. To date, although ecological interactions between xenobiotic plant secondary chemicals that retard insect growth have been observed in many species, it remains unclear how particular allelochemicals influence insect development in a life stage-dependent manner.ResultsWe found that 2-tridecanone can affect insect development; this effect appears similar to the symptoms induced by the physiological imbalance between juvenile and molting hormones in cotton bollworm. We later detected that a decrease in the concentration of 20-hydroxyecdysone occurred alongside the observed symptoms. We next identified the transcriptome of Helicoverpa armigera and eightdigital gene expression libraries for shading light on how 2-tridecanone retarded the development of cotton bollworm. The expression of CYP314A1, CYP315A1, CYP18A1, CYP307A1, and CYP306A1 (unigenes 16487, 15409, 40026, 41217, 35643, 16953, 8199, 13311, and 13036) were found to be induced by 2-tridecanone; these are known to be related to the biosynthesis or metabolism of 20-hydroxyecdysone. Expression analysis and RNA interference studies established that the retardant effect of 2-tridecanone on the development of cotton bollworm is mediated by P450 genes.ConclusionsThe candidate P450 gene approach described and exploited here is useful for identifying potential causal genes for the influence of plant allelochemicals on insect development.

Comparative proteomic analysis in Aphis glycines Mutsumura under lambda-cyhalothrin insecticide stress

Lambda-cyhalothrin is now widely used in China to control the soybean aphid Aphis glycines. To dissect the resistance mechanism, a laboratory-selected resistant soybean aphid strain (CRR) was established with a 43.42-fold resistance ratio to λ-cyhalothrin than the susceptible strain (CSS) in adult aphids. In this study, a comparative proteomic analysis between the CRR and CSS strains revealed important differences between the susceptible and resistant strains of soybean aphids for λ-cyhalothrin. Approximately 493 protein spots were detected in two-dimensional polyacrylamide gel electrophoresis (2-DE). Thirty-six protein spots displayed differential expression of >2-fold in the CRR strain compared to the CSS strain. Out of these 36 protein spots, 21 had elevated and 15 had decreased expression. Twenty-four differentially expressed proteins were identified by MALDI TOF MS/MS and categorized into the functional groups cytoskeleton-related protein, carbohydrate and energy metabolism, protein folding, antioxidant system, and nucleotide and amino acid metabolism. Function analysis showed that cytoskeleton-related proteins and energy metabolism proteins have been associated with the λ-cyhalothrin resistance of A. glycines. The differential expression of λ-cyhalothrin responsive proteins reflected the overall change in cellular structure and metabolism after insecticide treatment in aphids. In summary, our studies improve understanding of the molecular mechanism resistance of soybean aphid to lambda-cyhalothrin, which will facilitate the development of rational approaches to improve the management of this pest and to improve the yield of soybean.

Characterization of the transcriptome of the Asian gypsy moth Lymantria dispar identifies numerous transcripts associated with insecticide resistance.

Although the Asian gypsy moth Lymantria dispar causes extensive forest damage worldwide, little is known regarding the genes involved in its development or response to insecticides. Accordingly, characterization of the transcriptome of L. dispar larvae would promote the development of toxicological methods for its control. RNA-seq analysis of L. dispar larvae messenger RNA (mRNA) generated 62,063 unigenes with N50 of 993 bp, from which 23,975 unique sequences (E-value < 10(-5)) were identified using a BLASTx search of the NCBI non-redundant (nr) database. Using functional classification in the Gene Ontology (GO) and Clusters of Orthologous Groups (COG) databases, 7,309 indentified sequences were categorized into 51 functional groups and 8,079 sequences were categorized into 25 functional groups, respectively. Moreover, we identified a large number of transcripts encoding known insecticide targets, or proteins involved in the metabolism of insecticides. Reads per kilobase of unigene length per million mapped reads (RPKM) analysis identified 39 high abundance transcripts, of which 27 exhibited significantly altered expression patterns across the egg, larvae, pupae, male and female adult stages. Our study provides the most comprehensive transcriptomic sequence resource for L. dispar, which will form the basis for future identification of candidate insecticide resistance genes in L. dispar.

Contribution of cytochrome P450 monooxygenase CYP380C6 to spirotetramat resistance in Aphis gossypii Glover

The cytochrome P450 monooxygenases play a key role in detoxification mechanism for spirotetramat resistance in Aphis gossypii Glover. However, only one P450 genes (CYP6DA2), among thirty-five P450 genes identified from Aphis gossypii transcriptome database, has been reported to play important role in spirotetramat resistance in previous resistance level until now. In this study, after the confirmation of the rise of resistance level and important roles of P450s in spirotetramat resistance by the synergism analysis, the gene expression changes were determined for P450 genes in spirotetramat susceptible and resistant strains. Compared with the susceptible strain, CYP6CY4, CYP6CY14, CYP6CY18 and CYP6DC1 in CYP3 Clade were up-regulated in resistant nymphs, with the CYP6CY14, CYP6CY4, CYP6DC1, and CYP6CY18 increased to 2.54-, 1.51-, 1.31- and 1.29-fold, respectively. Eight genes in CYP3 Clade, three genes in CYP4 Clade and one gene in Mito Clade were down-regulated. In resistant adult aphids, CYP380C6 in CYP4 Clade, CYP353B1 in CYP2 Clade, and CYP307A1 in Mito Clade were up-regulated under spirotetramat stress, with the CYP380C6, CYP353B1 and CYP307A1 increased to 2.89-, 1.91-, and 1.38-fold, respectively. In contrast, the other P450 genes were almost down-regulated, especially these P450 genes in CYP3 Clade, CYP4 Clade and Mito Clade. RNA interference of CYP380C6 significantly increased the sensitivity of the resistant adults and nymphs to spirotetramat, while suppression of CYP6CY14 could not increase the toxicity of spirotetramat. These results indicate the possible involvement of the CYP380C6 genes in spirotetramat resistance at present very high resistance levels. Screening the expression changes of P450 genes under different spirotetramat resistance levels in the genome-scale will provide an overall view on the possible metabolic factors in the resistance development. The results may facilitate further work to validate the roles of P450 in spirotetramat resistance with heterologous expression.