Insecticide resistance levels
After 25 generations of larval selection with the insecticides cypermethrin, propoxur and DDV, bioassays revealed a constitutive increased resistance of each selected strain to its respective insecticide as compared to the parental susceptible strain (Table 1). Resistance levels of the Cx_pro and Cx_ddv strains were moderate but significant (11.34 fold and 8.17 fold respectively). Although significant, the resistance level of the Cx_cym strain to cypermethrin susceptible was considerably higher (243.00 fold).
Protein identification and differentially expressed proteins (DEP) screening
A total of 2,502 protein were identified in two replicates of the LC-MS/MS experiments with a high confidence of peptide selection (FDR=0.01). Among them, 1513 protein obtained quantitative message, all the proteins were completed annotated with the accession number for each identified protein arranged by the Gene Ontology, KEGG pathway, Domain, Cluster of Orthologous Groups (COG) function catalogues and Subcellular Location analyses (Additional file 1: Table S1). Our analysis revealed significant changes in proteins in these Cx_cym, Cx_pro, Cx_ddv, and Cx_s strains, there were 164 proteins with significant differential expression (fold change > 1.2 and p value < 0.05) between the Cx_cym and Cx_s control groups, including 54 up-regulated and 110 down-regulated proteins. 156 proteins changed dynamically between the Cx_pro and Cx_s control groups, including 59 up-regulated and 97 down-regulated proteins, whereas 81 proteins changed between the Cx_ddv and Cx_s control groups, with 31 up and 50 down-regulated proteins, respectively.
Bioinformatics analyses of the altered proteins
To better understand the characteristics of the altered proteins in response to insecticide selection, GO ontology was performed to analyze the upregulated and downregulated proteins among Cx_cym, Cx_pro, Cx_ddv, and Cx_s strains of Cx. pipiens pallens (Figure 1). As shown, the most enriched GO terms regarding molecular function of the upregulated proteins were related to the structural constituent of cuticle, structural molecule activity, lipid transporter activity, chitin binding, transferase activity, transferring acyl groups and odorant binding, which had a higher fold enrichment (log2) and Fisher's exact test p-value (-log10). Whereas with the downregulated proteins, structural constituent of cuticle were the most enriched GO terms.
Hierarchical clustering was employed to characterize changes in the Cx_cym, Cx_pro, Cx_ddv, and Cx_s strains, all quantified proteins were classified into 7 clusters based on their expression level, revealed clearly distinguishable increasing or decreasing expression (Figure 2). In KEGG enrichment analysis, drug metabolism - cytochrome P450 (map00982) and metabolism of xenobiotics by cytochrome P450 (map00980) pathways exist in response to insecticide selection conditions, implicating P450s are the important regulatory enzyme associated with metabolic resistance in Cx. pipiens pallens under insecticide selection. Furthermore, the differential changes in glutathione S-transferase (B0W6D0), microsomal glutathione s-transferase (B0X075) and glutathione-s-transferase theta (B0XGK3) proteins in both pathways suggest a critical regulatory role associated with P450, these findings indicate that glutathione S-transferase may play an important role in the regulation of metabolic resistance pathway in Cx. pipiens pallens (Figure 3A and Figure 3B).
The KEGG pathways annotation of comparison shows that oxidative phosphophrylation (map00190) (Additional file 2: Figure S1) is activated associated with electron-transport chain subunits (Ndufa2, Ndufa4, Ndufa5, Ndufa7, Ndufa8, Ndufa9, Ndufab1, COX5), ribosome (03010) (Additional file 2: Figure S2) is activated associated with S15e, L34e, S6e, and L18Ae, respectively. Interestingly, those subunits quantitation deviated simultaneously towards different direction, such as Ndufs4, Ndufs decrease while Ndufa4 increase, in agreement, oxidation phosphorylation and membrane located proteins are critical nodes in glycolytic flux and transit intracellular signal avoiding malfunction.
Comparing the pattern of expression of different proteins among Cx_cym, Cx_pro, Cx_ddv, and Cx_s strains of Cx. pipiens pallens, all of these proteins are significantly differentially expressed in resistant strains when compared to their expression in the susceptible strain, almost all protein enrichment changes at the peak and the lowest valley were in Cx_cym, Cx_pro and Cx_ddv, indicating that most of these protein showed an increase or decrease in expression following the insecticides selection from susceptible to resistance strains in Cx. pipiens pallens, the insecticide selection alters the normal developmental pathway into an alternative one (Figure 4).
In Cx_cym vs. Cx_s, upregulated proteins, TGS-like, OBG-type guanine nucleotide-binding (G) domain, GTP binding domain, MD-2-related lipid-recognition domain, Beta-grasp domain, Chaperonin Cpn60/TCP-1 family, whereas downregulated proteins, Chitin-binding type R&R consensus, Insect cuticle protein, Actin, conserved site, actin/actin-like conserved site, actin family, vitellinogen, open beta-sheet. In Cx_pro vs. Cx_s, upregulated proteins, FAD dependent oxidoreductase, chitin binding domain, serine proteases, trypsin family, serine active site, Serine proteases, trypsin family, histidine active site, Peptidase S1A, chymotrypsin family, whereas downregulated proteins, Insect cuticle protein, Chitin-binding type R&R consensus, Actin, conserved site, Actin/actin-like conserved site, Actin family, Vitellinogen, open beta-sheet. In Cx_ddv vs. Cx_s, upregulated proteins, Immunoglobulin E-set, Ribosomal protein L30, ferredoxin-like fold domain, ribosomal protein L30, conserved site, chitin-binding type R&R consensus, hemocyanin, N-terminal, whereas downregulated proteins, insect cuticle protein, chitin-binding type R&R consensus, adult cuticle protein 1, insect odorant-binding protein A10/ejaculatory bulb-specific protein 3 (Figure 5).
Prediction of the PPI network under insecticide selection
Because the protein–protein interaction plays important roles in most cellular biological processes, the PPI network were predicted under insecticides selection conditions using the STRING (www.string-db.org) online PPI prediction software, to further analyze the functional correlation of differentially expressed proteins between each group (Figure 6, Additional file 2: Figure S3-S4).
Validation of proteomics data by parallel reaction monitoring
To validate the accuracy and reproducibility of the proteomics analysis results, parallel reaction monitoring (PRM)-quantifiable strategy analyses were performed, 65 proteins selected as those that may play important roles in pyrethroid resistance, the differentially expressed levels of these proteins were quantified in Cx_cym, Cx_pro, Cx_ddv and Cx_s strains of Cx. pipiens pallens were verification and validation, all proteins, peptides, and measured peptide peak areas are listed (Additional file 3: Table S2).
Differential protein expression in the midgut of Cx. pipiens pallen induced by Bti
A total of 564 protein were identified with 559 protein obtained quantitative message, there were 66 proteins with significant differential expression (fold change > 1.2 and p value < 0.05) between the Cx_bti and Cx_nbti control groups, including 33 up-regulated and 33 down-regulated proteins (Additional file 4). Most of the proteins coding for cuticular, cytoskeleton related proteins, metabolic enzymes are enrichment differential changes in the digestive tract linings dissected from Cx_bti strain of Cx. pipiens pallens (Additional file 2: Figures S5-S8). Given our interest in the role of P450s in metabolic resistance, we further profiled the differential protein expression of Cytochrome P450 3A19 (B0X758) in the midgut of third instar larvae of Cx. pipiens pallen, P450 protein showed higher expression levels in Cx_bti strains.