Abstract
As adult female mosquito’s salivary gland facilitate blood meal uptake and pathogen transmission e.g. Plasmodium, virus etc., a plethora of research has been focused to understand the mosquito-vertebrate-pathogen interactions. Despite the fact that mosquito spends longer time over nectar sugar source, the fundamental question ‘how adult female salivary gland’ manages molecular and functional relationship during sugar vs. blood meal uptake remains unanswered. Currently, we are trying to understand these molecular relationships under dual feeding conditions in the salivary glands of the mosquito Anopheles culicifacies. During functional annotation of salivary transcriptome database, unexpectedly we discovered a cluster of salivary transcripts encoding plant like proteins. Our multiple experimental validations confirmed that Plant like transcripts (PLTs) are of mosquito origin and may encode functional proteins. A comprehensive molecular analysis of the PLTs and ongoing metagenomic analysis of salivary microbiome provide first evidence that how mosquito may have been benefited from its association with plant host and microbes. Future understanding of the underlying mechanism of the feeding associated molecular responses may provide new opportunity to control vector borne diseases.
Introduction
Sugar feeding by adult mosquitoes is essential for regular metabolic energy production to maintain a wealth of behavioral, structural, and physiological demands. While blood feeding by adult female mosquitoes is essential to meet the extra nutrient requirement for egg production and life cycle maintenance. Thus blood and sugar feeding are mutually exclusive and antagonistic behavioral-cum-physiological properties of the conflicting demands(Foster, 1995). It is worth of interest to understand the biological consequences of the mosquito tissues e.g. salivary glands, midgut etc. involved in feeding and digestion.
Adult female mosquito salivary glands initiate first biochemical communication to facilitate sugar as well as blood feeding. Additionally, it also potentiates pathogen transmissions and therefore research has largely been focused to understand the role of salivary glands in relation to blood feeding(Das et al., 2010; Dixit et al., 2009; James, 2003; Ribeiro et al., 2010; Rodriguez and Hernandez-Hernandez Fde, 2004). Despite the mosquitoes spend longer time over plant (floral nectar) sugar source, several fundamental questions in relation to the evolution of the dual feeding behavior ‘in general’ and functional relationship of salivary glands during sugar vs. blood meal uptake ‘in specific’, remain unanswered. It is believed that dual feeding behavior evolution might have occurred from herbivores feeding behavior to blood acquisition (Lehane, 2005)
Currently we are investigating the salivary associated molecular factors that affect the mosquito feeding behavior and Plasmodium transmission(Dixit et al., 2011; Dixit et al., 2009). During ongoing annotation of our recent salivary transcriptome database (unpublished) of the Indian malarial vector Anopheles culicifacies, we unexpectedly observed a cluster of Plant like transcripts (PLTs) in the sugar fed library. A comprehensive molecular analysis of the PLTs and ongoing metagenomic analysis of salivary microbiome provide initial evidence that how mosquito evolved and adapted for feeding over plant host.
Results & Discussion
In recent years, next-generation sequencing has not only opened the door for functional genomics analysis, but also emerging as an important tool to understand the evolutionary relationship of the molecular codes identified from non-model organisms(Bao et al., 2012; Gibbons et al., 2009; Hittinger et al., 2010; Su et al., 2012; Wang et al., 2010). Accordingly, we adopted Illumina based deep sequencing approach as a proof of concept for gene discovery tool. We sequenced two cDNA libraries prepared from the salivary glands, collected from 3-4 days old either sugar or blood fed (within 1hr of blood feeding) adult female mosquitoes. This protocol in fact generated a total of ∼58.5 million raw reads, which were quality filtered and denovo assembled, yielding a set of 11,498 (5808 for sugar fed (SF) & 5690 for blood fed (BF) library) contigs. Initially, the quality of the assembly was carefully examined by multiple homology search analysis of the whole transcriptome dataset against draft genome/transcript databases for the mosquito A. culicifacies, available at www.vectorbase.org.
As expected ninety two percent transcripts yielded significant match (10−5 e-value) to the draft genome of the mosquito A. culicifacies, at nucleotide level. Later, we selected few full length cDNA transcripts (>1000bp) and compared them with previously well annotated genes identified from other mosquitoes (S1). Subsequent validation of the selected transcripts by RT-PCR based expression analysis not only confirmed the quality of the assembly, but also allowed us to find out those rare Plants like transcripts which remained previously un-noticed, as mentioned below. Detail stats of the salivary transcriptome assembly kinetics have been summarized in the S1.
Pilot discovery of Plant like transcripts
BLAST2GO analysis of both the libraries independently showed distinct annotation kinetics (S2). Unexpectedly, species distribution analysis revealed several ‘Plant Like Transcripts’ (472 PLTs/∼8%), with highest similarities (>95% identity) to the ‘plants species’ in the sugar fed, but absent in the blood fed transcriptome database (Fig.1a/ST-1). To find out the possible evolutionary relationship of putative PLTs, we performed extensive BLASTX analysis against either NR database or Insect specific database at NCBI. From this analysis we further characterized three categories of transcript(s) (i) one transcript: encoding highly conserved alpha-tubulin (cytoskeleton associated protein), showing highest identity (>95%) to plant and (85-90%) identity to insect; (ii) two transcripts: encoding aquaporin (water channel membrane protein) (Maurel et al., 2008) and active site of the cysteine protease (protein chewing enzyme)(Grudkowska and Zagdanska, 2004) showing highest (>90%) identity to plant and 40-52% identity to insect (iii) two transcripts: encoding dehydrin (cold stress response protein) (Hanin et al., 2011) and expansin (plant cell wall loosening protein)(McQueen-Mason and Cosgrove, 1995) only matched to plants, but remained unmatched to any insect database (S3a,b,c).
The above fascinating observations prompted to know whether such PLTs have previously been predicted or identified from any other mosquito species. To clarify, we performed extensive homology search and surprisingly find out one ESTs dataset of Aedes aegypti larval cDNA library, where several PLTs have been recorded and submitted in the unigene database at NCBI (http://www.ncbi.nlm.nih.gov/unigene?term=Aae.16606&cmd=DetailsSearch/S4a/b). We believe such putative PLTs remain undescribed, probably due to tissue contamination suspect (Venancio et al., 2009). Nevertheless, in our case we collected the salivary tissues from the same cohort of the mosquitoes offered sugar or blood meal, and processed both the samples for library sequencing in identical conditions. Thus, we believe the absence of PLTs in blood fed transcriptome; nullify the chances of any suspect of contamination. However, above observations still rose several puzzling, but important arguable questions: (i) do these PLTs really express in mosquito tissue (ii) if express, do these transcripts have any evolutionary significance in relation to feeding preference and adaptation?
PLTs are of mosquito origin
First, we did a deep enquiry with technical staff and confirmed that under standard rearing facilities, mosquitoes are never exposed to any plant material. However to rule out the possibilities of any contamination, for each experimental analysis, we separately maintained the experimental mosquitoes as detailed in the methodology section. For technical validation of the PLTs origin, we conducted a series of experiments: (i) in two independent experiments, first we examined and verified the RT-PCR based expression of at least 10 selected PLTs (Fig1b/S5a), in the sugar fed salivary glands of adult male and female mosquitoes; (ii) interestingly, we also observed that PLTs expression is not only restricted to the mosquito tissues, but also express during the aquatic developmental stages viz. egg, larva, and pupa of the laboratory reared mosquitoes (Fig.1b). Here, it is also important to mention that mosquito egg and pupa stages are metabolically active, but never feed; (iii) we also observed similar amplification of selected PLTs through genomic DNA PCR (Fig.1c); (iv) we further carried out the functional validation of one of the plant homolog PLT encoding dehyrin protein, by Real-Time PCR as well as Immunoblot analysis (see fig. 4a/b); (v) lastly, from ongoing annotation of another independent transcriptome sequence database originated from non-salivary tissue of adult female mosquito Anopheles culicifacies (unpublished), we again observed similar PLTs (see S5b). Taken, together our experimental data strongly validate that mosquito genome may code plant like proteins. The poor match of PLTs to the available A. culicifacies mosquito genome sequences, could be due to following possible reasons: (i) strain specificity i.e. we worked on Indian strain, while available sequence data are originated from wild caught Iranian strain of A. culicifacies (www.vectorbase.org; Neafsey et al., 2013); (ii) incomplete annotation i.e. the quality and annotation of draft genome of Iranian strain may be incomplete (iii) genome assembly pipelines i.e. it is usual practice to filter out poorly matched, non-linage specific orphan genes etc. as ‘junk DNA’, during genome assembly from raw data; (iv) we believe that observed PLTs in our transcriptome screen may be an added benefit of deep sequencing approach which allowed to recover the rare transcripts, but may not be easy to recover in case of genome sequence.
It has long been accepted and proved that a significant variation exists in the chromosomal DNA as well as genome size within Anopheline and other mosquito species(Rai, 1999; Neafsey et al., 2013), but how these variations differentially affect the mosquito biology viz. behavior, physiology, immunity and vectorial capacity etc., are poorly understood at molecular level. Thus, we believe that unexpected finding of PLTs may be one of the valuable source databases to improve our knowledge and understanding the biological meaning of complex genomic variations within mosquito species.
Phylogenomic analysis of Plant like transcripts
Excitingly, the above data confirmation, prompted to follow up the associated evolutionary consensus, favoring plant-mosquito relationship: a parallelism setting where different species from unrelated taxa faces the common selective pressure (Zhen et al., 2012). Initial multiple sequence alignment analysis revealed significant heterogeneity (substitution/deletion) of amino acid residues, but also indicated unique conservation of insect or plant specific residues within the mosquito A. culicifacies, result a clade formation with plant species (Fig.2a/b/S6). Subsequently, we also tested whether evolution of common traits from unrelated taxa owing to similar selection pressure favors adaptive significance.
A maximum likelihood (ML) estimation was applied to calculate and compare the sitewise likelihood (ΔSSLS) values between two hypothesis i.e. mosquito-mosquito species evolution (H0) and mosquito-plant convergent adaptive evolution (H1), for the selected PLTs. The site wise log likelihood plot indicator i.e. divergence towards negative (ΔSSLS) was compared with LRT (likelihood ratio test), using parametric bootstrap at 1000 replicate analysis (cut off p-value 5%). Final data analysis and comparison stats favored the convergent hypothesis, (Arendt and Reznick, 2008) demonstrating that mosquito A. culicifacies PLTs followed convergent model favoring (H1), an adaptive evolution for sugar feeding associated functional relationship with plants (Fig.2c/S6). Our analysis also support the previous observations noted for the evolution of echolocating gene clusters among bats and bottlenose dolphins(Parker et al., 2013). Additionally, the predicted 3D structural analysis revealed fine conservation of the active functional domains in the mosquito and plants proteins e.g. cysteine protease (Fig.2d/S7). From these studies, we concluded that mosquito feeding associated genes are not only evolving actively, but also acquiring new genes (e.g. dehydrin, expansin), to adapt successfully over plant host.
Feeding associated molecular complexity of ‘salivary-sugar-microbe’: A tripartite interaction
Evolution of herbivores insect-plant association represents one of the dominant interactions over millions of years(Agrawal et al., 2012; Ehrlich, 1964; Fraenkel, 1959). These interactions are thought to play an important role in the co-evolution of molecular effectors arms, enabling effective adaptation over each other (Hogenhout and Bos, 2011). Uncovering of the molecular mechanisms of the herbivores insect-plant interaction has greatly facilitated the design of molecular strategies to save valuable crops from insect pests (Baldwin et al., 2001; Felton and Tumlinson, 2008; Ferry et al., 2004; Maffei et al., 2007). However, such studies have not been given special attention to mosquitoes. From the unexpected findings of the mosquito PLTs, we interpreted that either studies in relation to the sugar feeding associated salivary biology has largely been ignored (Dixit et al., 2011; Juhn et al., 2011) or the mosquito A. culicifacies may have evolved with more complex genetic architecture favoring evolution of environmentally-guided several traits viz. carbon metabolism; light mediated photo conditions for mating, feeding, survival etc. Therefore, to predict sugar metabolism associated molecular and functional relationship of salivary PLTs, initially we analyzed all the putative plant like transcripts against three databases (Reactome; KEGG and Biocycles) annotated for Arabidopsis thaliana, using KOBAS online software (http://kobas.cbi.pku.edu.cn/home.do).
Notably, we observed that 18 salivary transcripts encoding proteins related to at least five Biocyclic pathways linked to photosynthetic organelles viz. plastid in plants (S8a/T-1; see also S2). To verify above predicted ‘plastid’ related salivary transcripts, Fisher’s exact test analysis identified a pool of 11 transcripts differentially expressed in the sugar fed mosquitoes (Fisher test p<0.001; S8b.); encoding important enzymes/proteins, associated with one of the key pathway “Carbon fixation in Photosynthetic Organisms” (Fig.3a). Further, we also identified four unique salivary transcripts encoding different enzymes linked to three other secondary metabolite synthesis pathways:-namely ‘Trepenoid Backbone Biosynthesis’ (4-hydroxy-3-methylbut-2-enyl diphosphate reductase/E.C.1.17.1.2, LYTB); ‘Carotenoid Biosynthesis’ (Phytoene Synthase/E.C.2.5.1.32, PS); and ‘Flavonoid Biosynthesis’ (3-dioxigenase/E.C.1.14.11.9 & 3’ beta-hydroxylase/E.C.1.14.13.88) pathways restricted to the plants (S9). A comprehensive molecular and phylogenetic analysis of few selected transcript, encoding an enzyme 4-hydroxy-3-methylbut-2-enyl diphosphate reductase/E.C.1.17.1.2 (LYTB) and phytoene synthase/E.C.2.5.1.32 (PS); exclusively revealed unique evolutionary relationship to the cyanobacteria, algae, plants and aphid Acyrithosiphon pisum (Fig. 3b,c S9).
In fact during its early development mosquito larvae start to feed diverse micronutrients e.g. bacteria, algae, fungi etc., and switch to feed on nectar sugars in adult mosquito stage. Thus, it could be possible that a longer association and regular microbes-mosquito-plant interactions(Bennett, 2013; Pieterse and Dicke, 2007), might have favored insect/mosquito to adapt, feed, digest sugar and selective synthesis of secondary metabolites/pigments, essential for specific phenotype e.g. visual pigmentation/dark body coloration(Benedict and Seawright, 1987). Our recent metagenomic analysis of salivary microbiome identified several diverse unique bacterium phyla including Chlorobium, Cyanobacteria, Nitrospira and other phototrophic bacteria associated with salivary glands (Fig.3d), but absent in the gut of the laboratory reared 3-4 days old adult female mosquito Anopheles culicifacies (Sharma et al., 2014).
These findings further support the hypothesis that mosquito may have feeding associated distinct plant like molecular machinery components, partly shared by residing symbiotic bacterial community for diverse carbon/nitrogen rich plant sugar source metabolism. For example, finding of prominent salivary associated Acidobacteria (2.4%), may facilitate the utilization of plant polymer viz. cellulose/xylan sugars of diverse origin(Eichorst et al., 2011), as reported in the gut of the wood feeding larvae of Huhu Beetle (Prionoplus reticulari)(Reid et al., 2011). Furthermore, recent study on light-induced ATP synthesis from the chloroplastidic-like carotene pigments in ’Acyrithosiphon pisum’, a plant sap sucking aphid, provides first molecular evidence that aphid genome may carry plant like photosynthesis machinery components(Valmalette et al., 2012). A fungal mediated lateral horizontal gene transfer mechanism has been proposed for the evolution of carotenoid biosynthesis gene in this aphid(Moran and Jarvik, 2010).
Although, accumulating evidences of genetic material transfer within metazoan are still at premature stage but strongly suggest that acquisition of new beneficial traits, may favor improved survival and adaptation values in changing ecologies (Boto, 2014). Thus we believe our finding may begin to unlock previously unexplored biology, to rebuild new hypothesis “how insects are most successful” to feed, adapt and survive in the diverse environments. In support of these observation, next we attempted to validate whether PLTs encodes a functional protein for specific function (see below).
Mosquito encoded Plant-homolog Dehydrin: a functional validation
Dehydrins are low-temperature (LT) acclimation and evolutionarily conserved proteins that allows to develop efficient tolerance to drought and cold stress among photosynthetic as well as in some non-photosynthetic organisms such as yeast (Campbell and Close, 1997; Close and Lammers, 1993; Li et al., 1998; Mtwisha et al., 1998). Dehydrins are characterized by conserved K-segment comprising consensus KIKEKLPG amino acid sequence towards the C-terminus and may be repeated one to many times to encode 9 -200 kDa protein (Close, 1997; Close et al., 1993; Ouellet et al., 1993; Takahashi et al., 1994).
Unlike plants, insect dehyrin have not been reported so far, though a putative transcript AGAP000328 has been predicted from mosquito A. gambiae genome, carrying (PF00257 domain) a signature of dehyrin like proteins (S10). In our RT-PCR analysis, we observed a constitutive expression of AcDehydrin, throughout the aquatic developmental stages of the mosquito, indicating that identified PLT AcDehydrin transcript may encode a putative functional protein. Thus we characterized mosquito encoded plant homolog protein dehydrin in detail (S10). Our real-time PCR analysis repeatedly confirmed that dehydrins highly express in the egg than larva or pupae (Fig. 4a/S10), suggesting an abundant accumulation in the egg.
For functional validation of AcDehydrin protein, we examined the developmental expression of the dehydrin protein through immuno-blotting assay using anti-dehyrin antibody (kind gift from Dr. Timothy Close). In these experiments we used wheat seedling protein sample as positive reference control, while bacterial protein sample as negative reference. The anti-dehydrin antibody not only recognized the expected (28, 53 and 62 kDa) protein band in the wheat samples(Borovskii et al., 2002), but also identified two proteins (28 and 62kDa) abundantly expressing in the egg as compared to other developmental stages of the mosquito (Fig.4b). Taken together, our functional validation suggests that mosquito encoded AcDehydrin protein may have functional role similar to plant.
Like other late embryogenesis abundant proteins (LEA), dehydrins accumulate to high amounts in plant embryos, but remains undetectable in other vegetative tissues until their exposure to cellular dehydration stress. The stress exposure results in their rapid induction and binding to multiple proteins, probably through intramolecular hydrogen bonding to protect tissue damage from dehydration/cold stress (Hanin et al., 2011).We hypothesize that mosquito A. culicifacies, may have survival benefits of cold stress tolerance. Future studies involving dsRNA mediated gene silencing approach may unravel molecular and functional relationship of the PLTs, controlling feeding and adaptation phenotypes in the mosquito (Lu et al., 2012; Scott et al., 2013).
Material & Methods
Mosquito Rearing: Cyclic colony of the mosquito Anopheles culicifacies sibling species A, were reared and maintained at 28 ±2°C /RH 80% in the insectary fitted with a simulated dawn and dusk machine, essentially required for proper mating and feeding at NIMR (Adak et al., 1999). All protocols for rearing, maintenance of the mosquito culture were approved by ethical committee of the institute. For our specific research work, we harvested 80-100 pupa and allowed them to emerge in standard mosquito cages. For regular sugar supply the mosquitoes were offered sterile 5% sugar solution (crystal sugar dissolved 5g/100ml water) using cotton swab, while blood meal was offered to adult mosquitoes through rabbit. For aquatic development, gravid female were allowed to lay egg on moistened filter paper mounted inside small plastic cup (e.g. ice cream cup), semi-filled with water. Hatched larvae were feed on mixed dried powder of fish food and yeast.
cDNA library preparation & sequencing: Total RNA isolation and double stranded cDNA library preparation was done by PCR-based protocol as described previously(Dixit et al., 2009). Briefly, DNase-treated total RNA was reverse-transcribed and amplified to make double-stranded cDNA (ds-cDNA) through a PCR-based protocol using SMART cDNA synthesis kit (catalogue No. 634902; BD Clontech, Palo Alto, CA, USA), following the manufacturer’s instructions. For deep sequencing, Single-End RNA-seq protocol was used for each sugar fed and blood fed salivary gland tissue libraries, by commercial service providers (NxGenBio Life Sciences, New Delhi, India). The tagged Single-End RNA-Seq libraries were diluted and pooled in equimolar concentrations and sequenced using TruSeqTM SBS Kit V2 on Illumina GAIIx (Illumina, San Diego, CA) for generating 1x36bp single end sequencing reads.
Transcriptome assembly: Following sequencing, the low quality bases were filtered or trimmed using in-house Perl scripts. All the bases, above Q20 phred score were used for further downstream analysis. De-novo transcriptome assembly was performed using Trinity assembler (Grabherr, 2013) with the default settings k-mer size of 25, minimum contig length of 200, paired fragment length of 500, 16 CPUs, with butterfly Heap space of 100G (allocated Memory). Prior to submission of the data to the Transcriptome Shotgun Assembly Sequence Database (TSA), assembled transcripts were blasted to NCBI’s UniVec database (http://www.ncbi.nlm.nih.gov/VecScreen/UniVec.html) to identify segments with adapter contamination and trimmed when significant hits were found. This adapter contamination may result from sequencing into the 3' ligated adapter of small fragments (<100 bp).
Functional annotation: Following DENOVO clustering, CAP3 assembly using desktop cDNA annotation system (Guo et al., 2009) was used to build final conting/transcripts dataset for functional annotation. The assembled transcripts were subjected to similarity search against NCBI’s NR database using the BLASTx algorithm (Altschul et al., 1990), with a cut-off E-value of ≤10-3 using BLOSUM62 matrix as well as GO annotation/Interproscan analysis using BLAS2GO(Conesa et al., 2005). Biocyclic pathway analysis for PLTs KOBAS online (http://kobas.cbi.pku.edu.cn/home.do) software (Xie et al., 2011).
PCR based gene expression analysis:The desired tissues viz. salivary glands, midgut and hemocyte (Rodrigues et al., 2010) or the whole body, were directly in the Trizol. Total RNA was isolated using standard Trizol method, followed by first-strand cDNA synthesis using Oligo-dT or Random Hexamer primers (Verso kit). For differential expression analysis, routine RT-PCR and agarose gel electrophoresis protocols were used. Relative gene expression was assessed by QuantiMix SYBR green dye (Biotool Biolabs, Madriad, Spain) in Eco-Real-Time PCR Machine (Illumina). PCR cycle parameters involved an initial denaturation at 95°C for 15 min, 40 cycles of 10 s at 94°C, 20 s at 55°C, and 30 s at 72°C. Fluorescence readings were taken at 72°C after each cycle. A final extension at 72°C for 5 min was completed before deriving a melting curve, to confirm the identity of the PCR product. Actin gene was used as an internal control in all qPCR measurements, where minimum two technical replicates were used in each Real-Time experiment. To better evaluate the relative expression, each experiment was performed in three independent biological replicates. The relative quantification results were normalized with internal control Actin gene and analyzed by 2−ΔΔCt method (Livak and Schmittgen, 2001).
Phylogenomics analysis: Following primary BLASTX analysis, the reference sequences from the selected top hits were retrieved and edited for subsequent analysis in the FASTA format. Multiple sequence alignment was performed using ClustaX2 (Larkin et al., 2007). The CLC Sequence viewer (http://www.clcbio.com) software was used for better quality graphics. The phylogenetic relationship was inferred through MEGA 5.1 (http://www.megasoftware.net/) software. The evolutionary history was inferred using the Neighbor-Joining method, with and percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates). The evolutionary distances were computed using the p-distance method, presented in the units of the number of amino acid differences per site. A work flow for the Phylogenomic analysis has been presented in the supplemental document S6. Following major steps were followed:
Alignment of orthologous sequences for the selected genes Cysteine Protease, Aquaporin and Alphatubulin using MAFFT v6.864 at default parameters (Auto (FFT-NS-1, FFT-NS-2, FFT-NS-i or L-INS-i) with Amino Acid substitution matrix (BLOSUM62), Gap Penalty (1.53), offset penalty (0.123) and saved in Phylip Interleaved alignment format.
Alignment was used to generate RAxML tree, using T-REX online (Boc et al., 2012), at following parameters for generating de-novo phylogeny at following parameters: PROTCATDAYHOFF substitution model, Hill Climbing Alogrithm, Number of alternative runs on distinct starting trees =100, Rapid bootstrap random seed =12345, Bootstrap random seed=12345. This alternate phylogeny was called H1, as compared to commonly accepted Species phylogeny which was called H0 (the null hypothesis).
For Delta SSLS estimation, site wise log likelihood values were calculated using (Jobb et al., 2004) for both H0 and H1 phylogeny. Difference in Sitewise Log likelihood was calculated (Delta SSLS= H0-H1), where negative value supports convergent evolution and positive value supports species phylogeny.
For LRT test (Tree Finder), Phylogenetic reconstruction for H0 and H1 was done under WAG substitution model & Likelihood method for identifying best fit protein model with optimized frequencies with Heterogeneity models (G, GI and I). Parametric bootstrapping analysis was done to compare the two evolutionary hypotheses ‘H0’ and ‘H1’. The resulting p-value is the probability that the likelihood ratio simulated under the null hypothesis is less or equal than the observed. Given a level of significance of 5%, a p-value greater than 95% indicates that H1 is better than H0, and a p-value less than 5% indicates that H1 is worse.
Modeling Procedure & 3D structural prediction analysis: All structures of representative protein were retrieved from the Protein Data Bank (www.rcsb.org) and aligned using the structure alignment program STAMP (Russell and Barton, 1992). Models using all four structures as template were generated using Modeller9v10 (Sali and Blundell, 1993). 3D representation of the model was prepared in VMD (Visual Molecular dynamics tool) (Humphrey et al., 1996).
Immunoblot analysis:
Wheat seedling protein sample Preparation:
Wheat seeds were surface sterilized, imbibed for two consecutive days on moist filter pads placed in the glass Petridis, under deprived light, given alternate 16h/8h light/dark cycle for 3 days and then processed as described previously (Close et al., 1989). Briefly, crude protein extract was prepared by homogenization of seeds in Phosphate buffered saline (PBS) buffer with added benzamidine hydrochloride (1mM) and phenylmethylsulfonyl fluoride (PMSF) (1mM) followed by centrifugation at 15,000 rpm for 30 minutes at 4°C. Supernatant was collected to quantify and optimize the protein sample concentration for SDS PAGE with different amount of protein (viz. 20 μg, 50μg, 100μg, 200μg and 400μg). For further experiments 200μg amount was selected as an optimal concentration for Immunoblot analysis.
Mosquito developmental stage (Egg, Larva, Pupa) samples:
Different stages of mosquito viz. egg, larva, pupa were collected in PBS containing benzamidine hydrochloride (1mM) and phenylmethylsulfonyl fluoride (PMSF) (1mM) protease inhibitors. The collected mosquito whole body samples were homogenized on ice for 10 minutes, followed by centrifugation at 15,000 rpm for 15 min at 4°C. The clean supernatant was collected and quantified for subsequent analysis as described below.
Bacterial protein sample:
BL21* cells of E. coli (2ml) were grown in LB media containing Ampicilin (100 μg/ml) at 37°C till optical density (OD: 600) reached 0.4-0.6. Harvested cells were spinned down at 12000 rpm and re-suspended with 200ul re-suspension buffer containing 50mM NaH2PO4 pH 8.0, 300mM NaCl, 10mM Imidazole. Cell lysate was then centrifuged at 12000 rpm for 5 min and clear supernatant was analyzed through SDS-PAGE.
SDS-PAGE and Immunoblot analysis:
Protein samples (200μg each) were separated on SDS-polyacrylamide gel with Amersham mini vertical electrophoresis system and transferred to nitrocellulose membrane. Membranes were blocked with 1.5% (w/v) gelatin in PBST and incubated with anti dehydrin primary antibody (1:1000). The unbound antibody was washed three times for 5 min with PBST. Membranes were then incubated with Anti-rabbit HRP secondary antibody (1:60,000) for 1 hour. Unbound secondary antibody was washed for 5 minutes three times with PBST at room temperature. The blots were visualized using Amersham ECL Prime Western blotting detection reagent containing Solution A: luminol enhancer and Solution B: peroxide and developed on X-ray films by developer and bands were readily fixed in fixer solution.
Genomic DNA isolation & PCR: For the genomic DNA extraction, a total of five adult female mosquitoes, decapitated with head and wing, were collected in extraction buffer and processed as described earlier (Sharma et al., 2014). All the PCR amplifications conditions and parameters were identical as described above for RT-PCR analysis.
Conclusion:
Evolution and adaptation to dual feeding (sugar vs. blood) behavior of adult female mosquito still remains a central question, a knowledge critical to design vector borne disease management strategies. Comparative salivary transcriptomic and meta-genomics analysis provide initial evidence that mosquito Anopheles culicifacies, may have acquired and evolved with plant like machinery components partly shared by salivary associated microbes, together facilitating feeding preference and adaptation over plants grown in the plain agricultural area of rural India.
Acknowledgement:
We are thankful to Dr. S.K. Subbarao for expert comments on the manuscript. We thank Dr. Timothy close, for kind gift of Anti-dehydrin antibody. We thank DBT and ICMR for financial support to conduct the research at NIMR. We thank Kunwarjeet Singh for technical assistance and mosquito rearing.
Author’s Contribution:
Conceived and designed the experiments: RD, PS,NV, KCP. Performed the Experiments: PS, SS, TT,TDD, RD,VK,SLR Analyzed the data: PS, AKM, SS, RD,SV.
Contributed reagents/materials/analysis tools: NS, RD, NV, KCP. Wrote the paper: RD, PS, NV, KCP
Author Information:
The sequence data has been submitted to NCBI SRA database under following accession number: AC-SG-SF: SRR1017392 & AC-SG-BF: SRR1011070. There is no competing financial interest to declare. Correspondence and request for material should be addressed to RD (dixit2k{at}yahoo.com).