Comparative proteomics reveals mechanisms that underlie insecticide resistance in Culex pipiens pallens Coquillett

Abstract

Mosquito control based on chemical insecticides is considered as an important element of the current global strategies for the control of mosquito-borne diseases. Unfortunately, the development of insecticide resistance of important vector mosquito species jeopardizes the effectiveness of insecticide-based mosquito control. In contrast to target site resistance, other mechanisms are far from being fully understood. Global protein profiles among cypermethrin-resistant, propoxur-resistant, dimethyl-dichloro-vinyl-phosphate-resistant and susceptible strain of Culex pipiens pallens were obtained and proteomic differences were evaluated by using isobaric tags for relative and absolute quantification labeling coupled with liquid chromatography/tandem mass spectrometric analysis. A susceptible strain of Culex pipiens pallens showed elevated resistance levels after 25 generations of insecticide selection, through iTRAQ data analysis detected 2,502 proteins, of which 1,513 were differentially expressed in insecticide-selected strains compared to the susceptible strain. Finally, midgut differential protein expression profiles were analyzed, and 62 proteins were selected for verification of differential expression using iTRAQ and parallel reaction monitoring strategy, respectively. iTRAQ profiles of adaptation selection to three insecticide strains combined with midgut profiles revealed that multiple insecticide resistance mechanisms operate simultaneously in resistant insects of Culex pipiens pallens. Significant molecular resources were developed for Culex pipiens pallens, potential candidates were involved in metabolic resistance and reducing penetration or sequestering insecticide. Future research that is targeted towards RNA interference of the identified metabolic targets, such as cuticular proteins, cytochrome P450s, glutathione S-transferases and ribosomal proteins proteins and biological pathways (drug metabolism-cytochrome P450, metabolism of xenobiotics by cytochrome P450, oxidative phosphorylation, ribosome) could lay the foundation for a better understanding of the genetic basis of insecticide resistance in Culex pipiens pallens.

Fig 1. Repeatability analysis between two replicates of LC-MS/MS experiments of Cx. pipiens pallens in response to three insecticides.

The figure shows correlation between two replicates of LC-MS/MS samples, number in the square frame means that repeatability of differentially expressed proteins between the samples, the closer correlation value to 1 between the LC-MS/MS samples, the same samples tend to be, dark blue in the figure.The smaller the value, the more statistical significance it is.

Fig 2. Significantly upregulated GO molecular function cluster in Cx. pipiens pallens in response to three insecticides.

Upregulated proteins in the molecular function category according to the Fisher’s extract test, chitin binding was upregulated by comparing Cx_pro vs.Cx_s; structural constituent of cuticle and structural molecule activity were upregulated by comparing Cx_ddv vs.Cx_s; structural constituent of cuticle, lipid transporter activity and structural molecule activity were upregulated by comparing Cx_ddv vs. Cx_pro; lipid transporter activity and chitin binding were upregulated by comparing Cx_cym vs.Cx_pro. Scale bar is the value calculated by—log 10 (Fisher exact p value), distinguished by color. The larger the value, the more red it is. The smaller the value, the more blue it is, Fisher exact p value is used to measure statistical significance.

Fig 3. Significantly downregulated GO molecular function cluster in Cx. pipiens pallens after three insecticides selection.

Downregulated proteins in the molecular function category according to the Fisher’s extract test, structural constituent of cuticle and structural molecule activity were downregulated by comparing Cx_cym, Cx_pro, Cx_ddv vs.Cx_s and Cx_cym vs. Cx_ddv, respectively; odorant binding and transferase activity were downregulated by comparing Cx_ddv vs. Cx_pro; structural constituent of cuticle was downregulated by comparing Cx_cym vs. Cx_pro. Scale bar is the value calculated by—log 10 (Fisher exact p value), distinguished by color. The larger the value, the more red it is. The smaller the value, the more blue it is, Fisher exact p value is used to measure statistical significance.

Fig 4. Cluster of orthologous groups (COG) classification of putative proteins in the midgut of Cx. pipiens pallens exposed with bti.

559 putativeproteins were classified functionally into 15 molecular familiesin theCOG database, the “J” and“Z” clusterrepresent the largest two groups. B, Chromatin structure and dynamics; C, Energy production and conversion; D, Cell cycle control, cell division, chromosome partitioning; E, Amino acid transport and metabolism; F, Nucleotide transport and metabolism; G, Carbohydrate transport and metabolism; H, Coenzyme transportand metabolism; I, Lipid transport and metabolism; J, Translation, ribosomal structure and biogenesis; O, Posttranslational modification, protein turnover, chaperones; P, Inorganic ion transport and metabolism; Q, Secondary metabolites biosynthesis, transport and catabolism; R, General function prediction only; T, Signal transduction mechanisms; Z, Cytoskeleton.

Fig 5. A treemap overview of significant GO biological processes in the midgut proteome of Cx. pipiens pallen exposed with bti.

The diagram shows that exposed with bti condition has a significant effect on proteins biosynthetic process, microtubule-based process, organic substance biosynthetic process, organonitrogen compound metabolic process, oxidoreduction coenzyme metabolic process. The relative sizes of the treemap boxes are based on the |log10(p-value)| of the respective GO term, related terms are visualized with the same color, the color represents the significant p value of this kind of term after -log10, the deeper the color was, the greater the p value was, that is, the more significant enrichment the GO terms were.