Bacterial diversity analysis of larvae and adult midgut microflora using culture-dependent and culture-independent methods in lab-reared and field-collected Anopheles stephensi-an Asian malarial vector

<p>Abstract</p> <p>Background</p> <p>Mosquitoes are intermediate hosts for numerous disease causing organisms. Vector control is one of the most investigated strategy for the suppression of mosquito-borne diseases. <it>Anopheles stephensi </it>is one of the vectors of malaria parasite <it>Plasmodium vivax</it>. The parasite undergoes major developmental and maturation steps within the mosquito midgut and little is known about <it>Anopheles</it>-associated midgut microbiota. Identification and characterization of the mosquito midgut flora is likely to contribute towards better understanding of mosquito biology including longevity, reproduction and mosquito-pathogen interactions that are important to evolve strategies for vector control mechanisms.</p> <p>Results</p> <p>Lab-reared and field-collected <it>A. stephensi </it>male, female and larvae were screened by "culture-dependent and culture-independent" methods. Five 16S rRNA gene library were constructed form lab and field-caught <it>A. stephensi </it>mosquitoes and a total of 115 culturable isolates from both samples were analyzed further. Altogether, 68 genera were identified from midgut of adult and larval <it>A. stephensi</it>, 53 from field-caught and 15 from lab-reared mosquitoes. A total of 171 and 44 distinct phylotypes having 85 to 99% similarity with the closest database matches were detected among field and lab-reared <it>A. stephensi </it>midgut, respectively. These OTUs had a Shannon diversity index value of 1.74–2.14 for lab-reared and in the range of 2.75–3.49 for field-caught <it>A. stephensi </it>mosquitoes. The high species evenness values of 0.93 to 0.99 in field-collected adult and larvae midgut flora indicated the vastness of microbial diversity retrieved by these approaches. The dominant bacteria in field-caught adult male <it>A. stephensi </it>were uncultured <it>Paenibacillaceae </it>while in female and in larvae it was <it>Serratia marcescens</it>, on the other hand in lab-reared mosquitoes, <it>Serratia marcescens </it>and <it>Cryseobacterium meninqosepticum </it>bacteria were found to be abundant.</p> <p>Conclusion</p> <p>More than fifty percent of the phylotypes were related to uncultured class of bacteria. Interestingly, several of the bacteria identified are related to the known symbionts in other insects. Few of the isolates identified in our study are found to be novel species within the gammaproteobacteria which could not be phylogenetically placed within known classes. To the best of our knowledge, this is the first attempt to study the midgut microbiota of <it>A. stephensi </it>from lab-reared and field-collected adult and larvae using "culture-dependent and independent methods".</p

Determination of nitric oxide metabolites, nitrate and nitrite, in Anopheles culicifacies mosquito midgut and haemolymph by anion exchange high-performance liquid chromatography: plausible mechanism of refractoriness

<p>Abstract</p> <p>Background</p> <p>The diverse physiological and pathological role of nitric oxide in innate immune defenses against many intra and extracellular pathogens, have led to the development of various methods for determining nitric oxide (NO) synthesis. NO metabolites, nitrite (NO₂^-) and nitrate (NO₃^-) are produced by the action of an inducible <it>Anopheles culicifacies </it>NO synthase (AcNOS) in mosquito mid-guts and may be central to anti-parasitic arsenal of these mosquitoes.</p> <p>Method</p> <p>While exploring a plausible mechanism of refractoriness based on nitric oxide synthase physiology among the sibling species of <it>An. culicifacies</it>, a sensitive, specific and cost effective high performance liquid chromatography (HPLC) method was developed, which is not influenced by the presence of biogenic amines, for the determination of NO₂^-and NO₃^-from mosquito mid-guts and haemolymph.</p> <p>Results</p> <p>This method is based on extraction, efficiency, assay reproducibility and contaminant minimization. It entails de-proteinization by centrifugal ultra filtration through ultracel 3 K filter and analysis by high performance anion exchange liquid chromatography (Sphereclone, 5 μ SAX column) with UV detection at 214 nm. The lower detection limit of the assay procedure is 50 pmoles in all midgut and haemolymph samples. Retention times for NO₂^-and NO₃^-in standards and in mid-gut samples were 3.42 and 4.53 min. respectively. Assay linearity for standards ranged between 50 n<it>M </it>and 1 m<it>M</it>. Recoveries of NO₂^-and NO₃^-from spiked samples (1–100 μ<it>M</it>) and from the extracted standards (1–100 μ<it>M</it>) were calculated to be 100%. Intra-assay and inter assay variations and relative standard deviations (RSDs) for NO₂^-and NO₃^-in spiked and un-spiked midgut samples were 5.7% or less. Increased levels NO₂^-and NO₃^-in midguts and haemolymph of <it>An. culicifacies </it>sibling species B in comparison to species A reflect towards a mechanism of refractoriness based on AcNOS physiology.</p> <p>Conclusion</p> <p>HPLC is a sensitive and accurate technique for identification and quantifying pmole levels of NO metabolites in mosquito midguts and haemolymph samples that can be useful for clinical investigations of NO biochemistry, physiology and pharmacology in various biological samples.</p

Comparative proteomics of salivary glands of Anopheles culicifacies mosquitoes using tandem mass tag (TMT) mass spectrometry

Background & objectives: Salivary gland proteins play a pivotal role in blood feeding, epithelial interactions, and parasite transmission in mosquito vectors. Anopheles culicifacies is a complex of five sibling species, viz. A, B, C, D, and E, with diverse geographical distribution patterns. Among these, sibling species B has been identified as poor vector. Exploring the differentially expressed salivary proteins in An. culicifacies may potentially identify refractoriness factors during malaria parasite maturation and may help to elucidate the mechanism of refractoriness. Methods: A comparative proteomic analysis was carried out using tandem mass tag (TMT) technology combined with LC-MS/MS mass spectrometry and bioinformatics analysis, to identify the differentially expressed salivary gland proteins among An. culicifacies species A (susceptible) and An. culicifacies species B (refractory) mosquitoes. Results: A total of 82 proteins were found to be differentially expressed. Out of these, seven proteins including TRIO, translation initiation factor 5C, glutathione S-transferase, and 5’ nucleotidase were up-regulated, and 75 proteins including calreticulin, elongation factors, fructose biphosphatase, isocitrate dehydrogenase, histone proteins and anti-platelet proteins, etc. were down-regulated in refractory species. Analysis of KEGG pathways showed that the up-regulated proteins were related to fatty acid metabolism and RNA transport pathways. Interpretation & conclusion: This comparative proteomic analysis of susceptible and refractory An. culicifacies salivary gland proteins identifies the plausible role of the differential proteome in immune responses, digestion, energy, and carbon metabolic pathways. This information may serve as a basis for future work concerning the possible role of these proteins in refractoriness dependent metabolic function of mosquitoes

Proteome-wide analysis of Anopheles culicifacies mosquito midgut: new insights into the mechanism of refractoriness

Abstract Background Midgut invasion, a major bottleneck for malaria parasites transmission is considered as a potential target for vector-parasite interaction studies. New intervention strategies are required to explore the midgut proteins and their potential role in refractoriness for malaria control in Anopheles mosquitoes. To better understand the midgut functional proteins of An. culicifacies susceptible and refractory species, proteomic approaches coupled with bioinformatics analysis is an effective means in order to understand the mechanism of refractoriness. In the present study, an integrated in solution- in gel trypsin digestion approach, along with Isobaric tag for relative and absolute quantitation (iTRAQ)–Liquid chromatography/Mass spectrometry (LC/MS/MS) and data mining were performed to identify the proteomic profile and differentially expressed proteins in Anopheles culicifacies susceptible species A and refractory species B. Results Shot gun proteomics approaches led to the identification of 80 proteins in An. culicifacies susceptible species A and 92 in refractory species B and catalogue was prepared. iTRAQ based proteomic analysis identified 48 differentially expressed proteins from total 130 proteins. Of these, 41 were downregulated and 7 were upregulated in refractory species B in comparison to susceptible species A. We report that the altered midgut proteins identified in naturally refractory mosquitoes are involved in oxidative phosphorylation, antioxidant and proteolysis process that may suggest their role in parasite growth inhibition. Furthermore, real time polymerase chain reaction (PCR) analysis of few proteins indicated higher expression of iTRAQ upregulated protein in refractory species than susceptible species. Conclusion This study elucidates the first proteome of the midguts of An. culicifacies sibling species that attempts to analyze unique proteogenomic interactions to provide insights for better understanding of the mechanism of refractoriness. Functional implications of these upregulated proteins in refractory species may reflect the phenotypic characteristics of the mosquitoes and will improve our understandings of blood meal digestion process, parasite vector interactions and proteomes of other vectors of human diseases for development of novel vector control strategies

Additional file 2: of Proteome-wide analysis of Anopheles culicifacies mosquito midgut: new insights into the mechanism of refractoriness

Table S2. A catalogue of midgut proteins identified using in-solution digestion strategy and LC/MS/MS in in refractory An. culicifacies species B. (DOCX 27 kb

Additional file 5: of Proteome-wide analysis of Anopheles culicifacies mosquito midgut: new insights into the mechanism of refractoriness

Table S5. A catalogue of identified putative proteins found in both species A and species B of An. culicifacies using iTRAQ labeling method. (DOCX 31 kb

Additional file 4: of Proteome-wide analysis of Anopheles culicifacies mosquito midgut: new insights into the mechanism of refractoriness

Table S4. A catalogue of midgut proteins identified using in-gel digestion strategy coupled with LC/MS/MS in An. culicifacies species B. (DOC 97 kb

Additional file 1: of Proteome-wide analysis of Anopheles culicifacies mosquito midgut: new insights into the mechanism of refractoriness

Table S1. A catalogue of midgut proteins identified using in-solution digestion strategy and LC/MS/MS in susceptible An. culicifacies species A. (DOCX 27 kb

Additional file 3: of Proteome-wide analysis of Anopheles culicifacies mosquito midgut: new insights into the mechanism of refractoriness

Table S3. A catalogue of midgut proteins identified using in-gel digestion strategy coupled with LC/MS/MS in An. culicifacies species A. (DOC 95 kb