Tianfei Peng

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.

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.

Transcription factor aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator is involved in regulation of the xenobiotic tolerance‐related cytochrome P450 CYP6DA2 in Aphis gossypii Glover

The cotton aphid, Aphis gossypii, is one of the most economically important agricultural pests worldwide as it is polyphagous and resistant to many classes of insecticides. Overexpression of the cytochrome P450 monooxygenase (P450) CYP6DA2 has previously been found to be associated with gossypol and spirotetramat tolerance in the cotton aphid. In the present study, the elements located in the promoter region (‐357:‐343; ‐250:‐241; ‐113:‐104) of CYP6DA2 were shown to control promoter activity, and gossypol induction was observed. We hypothesized that the expression of CYP6DA2 is subject to transcriptional regulation. To investigate the underlying mechanism, we assessed two transcription factors, aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator (ARNT), and found that the abundance of AhR was highly correlated with CYP6DA2 abundance. RNA interference of AhR or ARNT significantly decreased the levels of the target gene as well as those of its counterpart, and both dramatically repressed CYP6DA2 expression. Cotransfection of the ARNT, AhR, or AhR plus ARNT and CYP6DA2 promoter constructs elevated CYP6DA2 promoter activity, with the AhR plus ARNT cotransfection being the most effective. Thus, these elements located in the promoter were responsible for CYP6DA2 transcription, and CYP6DA2 expression was regulated by the transcription factors AhR and ARNT.

Novel mutations and expression changes of acetyl‐coenzyme A carboxylase are associated with spirotetramat resistance in Aphis gossypii Glover

Acetyl‐coenzyme A carboxylase (ACC) catalyses the carboxylation of acetyl‐coenzyme A (acetyl‐CoA) to produce malonyl‐CoA during the de novo synthesis of fatty acids. Spirotetramat, an inhibitor of ACC, is widely used to control a range of sucking insects, including the Aphis gossypii. In the present study, Reverse transcription quantitative real‐time PCR (RT‐qPCR) results demonstrated that ACC was significantly overexpressed in a laboratory‐selected spirotetramat‐resistant strain compared with the susceptible strain. ACC RNA interference significantly suppressed fecundity and led to cuticle formation deficiencies in resistant adults and nymphs compared with the control. The full‐length ACC gene was sequenced from both resistant and susceptible cotton aphids, and a strong association was found between spirotetramat resistance and 14 amino acid substitutions in the biotin carboxylase domain and carboxyl transferase domain of the ACC gene. Furthermore, ACC activity was higher in resistant aphids than in the susceptible strain, and ACC in the resistant aphids exhibited significant insensitivity to spirotetramat and spirotetramat‐enol. The results indicate that the overexpressed insensitive (mutated) ACC target played an important role in the high levels of spirotetramat resistance observed here. This association of amino acid substitution with resistance is the first report of a potential target site mechanism affecting spirotetramat in the cotton aphid.

PROTEOMICS ANALYSIS OF OVEREXPRESSED PLASMA PROTEINS IN RESPONSE TO COLD ACCLIMATION IN Ostrinia furnacalis.

Many insects in temperate regions overwinter in diapause. In these insects, one of the metabolic adaptations to cold stress is the synthesis of responsive proteins. Using proteomic analysis, an investigation aimed to a better understanding of the molecular adaptation mechanisms to cold stress was carried out in Ostrinia furnacalis larva. Proteins were extracted from the larval hemolymph collected from both control and overwintering larva. By polyethylene glycol precipitation, approximately 560 protein spots were separated and visualized on two-dimensional (2D) gels after silver staining. Eighteen protein spots were found to be upregulated in overwinter larval plasma in different patterns. As an initial work, 13 of these proteins were identified using MALDI TOF/TOF MS. The differentially overexpressed proteins include heat shock 70 kDa cognate protein, small heat shock protein (sHSP), putative aliphatic nitrilase, arginine kinase, phosphoglyceromutase, triosephosphateisomerase, and glutathione transferase. Alterations in the levels of these proteins were further confirmed by qPCR. This study is the first analysis of differentially expressed plasma proteins in O. furnacalis diapause larvae under extremely low temperature conditions and gives new insights into the acclimation mechanisms responsive to cold stress. Our results also support the idea that energy metabolism, alanine and proline metabolism, and antioxidative reaction act in the cold acclimation of O. furnacalis diapause larvae.

Rapid evolution of symbiotic bacteria populations in spirotetramat-resistant Aphis gossypii glover revealed by pyrosequencing

Aphis gossypii is one of the most economically important insect pests for agriculture worldwide. Aphids have developed symbiotic associations with bacterial species, which has led to morphological and molecular differences, such as body color and insecticide resistance. Adults and 3rd instar nymphs of a laboratory-selected spirotetramat-resistant strain of cotton aphid presented 579-fold and 15-fold higher resistance to spirotetramat, respectively, than a susceptible strain (Pan et al., 2015; Peng et al., 2016). In this study, we found that antibiotics, especially ampicillin and tetracycline, increased spirotetramat toxicity in resistant aphids. We also characterized all of the bacterial endosymbionts in these two clones by sequencing the 16S rRNA genes of the endosymbiont. The total reads could be clustered into 3534 operational taxonomic units (OTUs) that showed 97% similarity and belonged to six abundant phyla. Proteobacteria and Firmicutes dominated in the two strains, and the most abundant families were Enterobacteriaceae, Lactobacillaceae and Rhodobiaceae. The genera Arsenophonus, Anderseniella, Buchnera and Lactobacillus were most abundant in the susceptible strain, whereas a significant decrease in abundance of Anderseniella and a great increase in abundance of Arsenophonus and Lactobacillus were observed in the resistant strain. Certain identified species had low sequence similarity to the reported species, which indicates the possibility of novel taxa. The type and abundance of different bacterial groups varied significantly between the two strains. The insecticide selection pressure could be the reason for the observed shift in the bacteria groups. These results increase our understanding of the symbiotic relationships between bacteria and their hosts under insecticide stress and provide clues for the development of potential control techniques against this cotton aphid.