Annotated differentially expressed salivary proteins of susceptible and insecticide-resistant mosquitoes of Anopheles stephensi

Abstract

Vector control is one of the major global strategies for control of malaria. However, the major obstacle for vector control is the development of multiple resistances to organochlorine, organophosphorus insecticides and pyrethroids that are currently being used in public health for spraying and in bednets. Salivary glands of vectors are the first target organ for human-vector contact during biting and parasite-vector contact prior to parasite development in the mosquito midguts. The salivary glands secrete anti-haemostatic, anti-inflammatory biologically active molecules to facilitate blood feeding from the host and also inadvertently inject malaria parasites into the vertebrate host. The Anopheles stephensi mosquito, an urban vector of malaria to both human and rodent species has been identified as a reference laboratory model to study mosquito-parasite interactions. In this study, we adopted a conventional proteomic approach of 2D-electrophoresis coupled with MALDI-TOF mass spectrometry and bioinformatics to identify putative differentially expressed annotated functional salivary proteins between An. stephensi susceptible and multiresistant strains with same genetic background. Our results show 2D gel profile and MALDI-TOF comparisons that identified 31 differentially expressed putative modulated proteins in deltamethrin/DDT resistant strains of An. stephensi. Among these 15 proteins were found to be upregulated and 16 proteins were downregulated. Our studies interpret that An. stephensi (multiresistant) caused an upregulated expression of proteins and enzymes like cytochrome 450, short chain dehyrdogenase reductase, phosphodiesterase etc that may have an impact in insecticide resistance and xenobiotic detoxification. Our study elucidates a proteomic response of salivary glands differentially regulated proteins in response to insecticide resistance development which include structural, redox and regulatory enzymes of several pathways. These identified proteins may play a role in regulating mosquito biting behavior patterns and may have implications in the development of malaria parasites in resistant mosquitoes during parasite transmission.

Fig 1. Representative 2D gel picture of An. stephensi salivary gland proteins.

Molecular weight (kDa) is shown in the middle of gel. (A) Silver-stained gel of An. stephensi susceptible strain numbered from 1 to 43 using ImageMaster 2D Platinum 7.0 software (B) Silver-stained gel of An. stephensi multi resistant strain numbered from 1 to 43 using ImageMaster 2D Platinum 7.0 software.

Fig 2. Scatter plot showing volume of all protein spots of An. stephensi salivary gland.

Blue line shows linear regression. X axis represents the volumes of protein spots in An. stephensi susceptible species and Y axis represents volumes of protein spots in An. stephensi resistant species. Correlation coefficient was calculated and indicated at the bottom.

Fig 3. Differential spot selection of salivary gland protein in An. stephensi species.

Representation of the protein spots showing upregulated and downregulated expression in An. stephensi strains are marked by green line. Molecular weights (kDa) are shown in the middle of 2D gel. (A) Silver-stained gel of An. stephensi susceptible strain (B) Silver-stained gel of An. stephensi multi-resistant strain. Depiction of total 16 spots marked using ImageMaster 2D Platinum 7.0 software. Downregulated proteins: Match spot id (3,13,14,21,29,35,39); Upregulated proteins: Match spot id (4,8,9,10,11,19,36,41,42).

Fig 4. Peak spectrum analyzed by MALDI-TOF in An. stephensi multi-resistant species.

(A) Representative PMF spectrum of over expressed protein i.e Short-chain dehydrogenase reductase (DNIKVTLISPGYINTALSLNALTGTGASYGK) (spot no. 4) (B) Representative PMF spectrum of down regulated protein i.e 26S proteasome non-ATPase regulatory subunit (VSQTAAAAAPADPIVDVEMESAEDAEAAK) (spot no.39).

Fig 5. Illustration of identified upregulated & downregulated salivary proteins analyzed by gene ontology tool.

(A) GO function of identified upregulated proteins. (B) GO function of identified downregulated proteins. (C) GO component of identified proteins.

Fig 6. Evidence view of upregulated proteins in resistant strain showing interaction with other proteins.

(A) SDR protein (AGAP005532) (B) Tyrosyl DNA phosphodiesterase. Different line colors represent the types of evidence for the association. Green color depicts neighborhood; red color: gene fusion; pink color: experiments; light green color: text mining; blue color: co-occurrence; dark blue color: co-expression; purple color: homology. Circle nodes indicated different proteins.