Ajeet Kumar Mohanty

A proteogenomic analysis of Anopheles gambiae using high-resolution Fourier transform mass spectrometry

Anopheles gambiae is a major mosquito vector responsible for malaria transmission, whose genome sequence was reported in 2002. Genome annotation is a continuing effort, and many of the approximately 13,000 genes listed in VectorBase for Anopheles gambiae are predictions that have still not been validated by any other method. To identify protein-coding genes of An. gambiae based on its genomic sequence, we carried out a deep proteomic analysis using high-resolution Fourier transform mass spectrometry for both precursor and fragment ions. Based on peptide evidence, we were able to support or correct more than 6000 gene annotations including 80 novel gene structures and about 500 translational start sites. An additional validation by RT-PCR and cDNA sequencing was successfully performed for 105 selected genes. Our proteogenomic analysis led to the identification of 2682 genome search-specific peptides. Numerous cases of encoded proteins were documented in regions annotated as intergenic, introns, or untranslated regions. Using a database created to contain potential splice sites, we also identified 35 novel splice junctions. This is a first report to annotate the An. gambiae genome using high-accuracy mass spectrometry data as a complementary technology for genome annotation.

A compendium of molecules involved in vector-pathogen interactions pertaining to malaria

Malaria is a vector-borne disease causing extensive morbidity, debility and mortality. Development of resistance to drugs among parasites and to conventional insecticides among vector-mosquitoes necessitates innovative measures to combat this disease. Identification of molecules involved in the maintenance of complex developmental cycles of the parasites within the vector and the host can provide attractive targets to intervene in the disease transmission. In the last decade, several efforts have been made in identifying such molecules involved in mosquito-parasite interactions and, subsequently, validating their role in the development of parasites within the vector. In this study, a list of mosquito proteins, which facilitate or inhibit the development of malaria parasites in the midgut, haemolymph and salivary glands of mosquitoes, is compiled. A total of 94 molecules have been reported and validated for their role in the development of malaria parasites inside the vector. This compendium of molecules will serve as a centralized resource to biomedical researchers investigating vector-pathogen interactions and malaria transmission.

Rapid Identification of Polyhydroxyalkanoate Accumulating Members of Bacillales Using Internal Primers for phaC Gene of Bacillus megaterium

Bacillus megaterium is gaining recognition as an experimental model and biotechnologically important microorganism. Recently, descriptions of new strains of B. megaterium and closely related species isolated from diverse habitats have increased. Therefore, its identification requires several tests in combination which is usually time consuming and difficult to do. We propose using the uniqueness of the polyhydroxyalkanoate synthase C gene of B. megaterium in designing primers that amplify the 0.9 kb region of the phaC for its identification. The PCR method was optimized to amplify 0.9 kb region of phaC gene. After optimization of the PCR reaction, two methods were investigated in detail. Method I gave an amplification of a single band of 0.9 kb only in B. megaterium and was demonstrated by several strains of B. megaterium isolated from different habitats. The use of Method I did not result in the amplification of the phaC gene with other members of Bacillales. The specificity for identification of B. megaterium was confirmed using sequencing of amplicon and RT-PCR. Method II showed multiple banding patterns of nonspecific amplicons among polyhydroxyalkanoate accumulating members of Bacillales unique to the respective species. These methods are rapid and specific for the identification of polyhydroxyalkanoate accumulating B. megaterium and members of Bacillales.

Mosquito-Borne Diseases and Omics: Tissue-Restricted Expression and Alternative Splicing Revealed by Transcriptome Profiling of Anopheles stephensi

Malaria is one of the most debilitating mosquito-borne diseases with high global health burdens. While much of the research on malaria and mosquito-borne diseases is focused on Africa, Southeast Asia accounts for a sizable portion of the global burden of malaria. Moreover, about 50% of the Asian malaria incidence and deaths have been from India. A promising development in this context is that the completion of genome sequence of Anopheles stephensi, a major malaria vector in Asia, offers new opportunities for global health innovation, including the progress in deciphering the vectorial ability of this mosquito species at a molecular level. Moving forward, tissue-based expression profiling would be the next obvious step in understanding gene functions of An. stephensi. We report in this article, to the best of our knowledge, the first in-depth study on tissue-based transcriptomic profile of four important organs (midgut, Malpighian tubules, fat body, and ovary) of adult female An. stephensi mosquitoes. In all, we identified over 20,000 transcripts corresponding to more than 12,000 gene loci from these four tissues. We present and discuss the tissue-based expression profiles of majority of annotated transcripts in An. stephensi genome, and the dynamics of their alternative splicing in these tissues, in this study. The domain-based Gene Ontology analysis of the differentially expressed transcripts in each of the mosquito tissue indicated enrichment of transcripts with proteolytic activity in midgut; transporter activity in Malpighian tubules; cell cycle, DNA replication, and repair activities in ovaries; and oxidoreductase activities in fat body. Tissue-based study of transcript expression and gene functions markedly enhances our understanding of this important malaria vector, and in turn, offers rationales for further studies on vectorial ability and identification of novel molecular targets to intercept malaria transmission.

Dynamics of Plasmodium vivax sporogony in wild Anopheles stephensi in a malaria-endemic region of Western India

BackgroundIn global efforts to track mosquito infectivity and parasite elimination, controlled mosquito-feeding experiments can help in understanding the dynamics of parasite development in vectors. Anopheles stephensi is often accepted as the major urban malaria vector that transmits Plasmodium in Goa and elsewhere in South Asia. However, much needs to be learned about the interactions of Plasmodium vivax with An. stephensi. As a component of the US NIH International Center of Excellence for Malaria Research (ICEMR) for Malaria Evolution in South Asia (MESA), a series of membrane-feeding experiments with wild An. stephensi and P. vivax were carried out to better understand this vector-parasite interaction.MethodsWild An. stephensi larvae and pupae were collected from curing water in construction sites in the city of Ponda, Goa, India. The larvae and pupae were reared at the MESA ICEMR insectary within the National Institute of Malaria Research (NIMR) field unit in Goa until they emerged into adult mosquitoes. Blood for membrane-feeding experiments was obtained from malaria patients at the local Goa Medical College and Hospital who volunteered for the study. Parasites were counted by Miller reticule technique and correlation between gametocytaemia/parasitaemia and successful mosquito infection was studied.ResultsA weak but significant correlation was found between patient blood gametocytaemia/parasitaemia and mosquito oocyst load. No correlation was observed between gametocytaemia/parasitaemia and oocyst infection rates, and between gametocyte sex ratio and oocyst load. When it came to development of the parasite in the mosquito, a strong positive correlation was observed between oocyst midgut levels and sporozoite infection rates, and between oocyst levels and salivary gland sporozoite loads. Kinetic studies showed that sporozoites appeared in the salivary gland as early as day 7, post-infection.ConclusionsThis is the first study in India to carry out membrane-feeding experiments with wild An. stephensi and P. vivax. A wide range of mosquito infection loads and infection rates were observed, pointing to a strong interplay between parasite, vector and human factors. Most of the present observations are in agreement with feeding experiments conducted with P. vivax elsewhere in the world.

Mapping Anopheles stephensi midgut proteome using high-resolution mass spectrometry

Anopheles stephensi Liston is one of the major vectors of malaria in urban areas of India. Midgut plays a central role in the vector life cycle and transmission of malaria. Because gene expression of An. stephensi midgut has not been investigated at protein level, an unbiased mass spectrometry-based proteomic analysis of midgut tissue was carried out. Midgut tissue proteins from female An. stephensi mosquitoes were extracted using 0.5% SDS and digested with trypsin using two complementary approaches, in-gel and in-solution digestion. Fractions were analysed on high-resolution mass spectrometer, which resulted in acquisition of 494,960 MS/MS spectra. The MS/MS spectra were searched against protein database comprising of known and predicted proteins reported in An. stephensi using Sequest and Mascot software. In all, 47,438 peptides were identified corresponding to 5,709 An. stephensi proteins. The identified proteins were functionally categorized based on their cellular localization, biological processes and molecular functions using Gene Ontology (GO) annotation. Several proteins identified in this data are known to mediate the interaction of the Plasmodium with vector midgut and also regulate parasite maturation inside the vector host. This study provides information about the protein composition in midgut tissue of female An. stephensi, which would be useful in understanding vector parasite interaction at molecular level and besides being useful in devising malaria transmission blocking strategies. The data of this study is related to the research article “Integrating transcriptomics and proteomics data for accurate assembly and annotation of genomes”.

Susceptibility of wild and colonized Anopheles stephensi to Plasmodium vivax infection

BackgroundAs much as 80% of global Plasmodium vivax infections occur in South Asia and there is a shortage of direct studies on infectivity of P. vivax in Anopheles stephensi, the most common urban mosquito carrying human malaria. In this quest, the possible effects of laboratory colonization of mosquitoes on infectivity and development of P. vivax is of interest given that colonized mosquitoes can be genetically less divergent than the field population from which they originated.MethodsPatient-derived P. vivax infected blood was fed to age-matched wild and colonized An. stephensi. Such a comparison requires coordinated availability of same-age wild and colonized mosquito populations. Here, P. vivax infection are studied in colonized An. stephensi in their 66th–86th generation and fresh field-caught An. stephensi. Wild mosquitoes were caught as larvae and pupae and allowed to develop into adult mosquitoes in the insectary. Parasite development to oocyst and sporozoite stages were assessed on days 7/8 and 12/13, respectively.ResultsWhile there were batch to batch variations in infectivity of individual patient-derived P. vivax samples, both wild and colonized An. stephensi were roughly equally susceptible to oocyst stage Plasmodium infection. At the level of sporozoite development, significantly more mosquitoes with sporozoite load of 4+ were seen in wild than in colonized populations.ConclusionsOverall at the level of oocyst development, significant difference was found between the colonized and wild Anopheles stephensi in their susceptibility to P. vivax. For initial understanding of infections with local strains of P. vivax, colonized Anopheles stephensi will serve as a good model. For experiments, where high number of sporozoites are necessary, wild mosquitoes provide distinct advantage over the colonized vector populations. Understanding the molecular mechanism modulating this variability between these two populations will be prime area of focus in future studies.

Quantitative proteome of midgut, Malpighian tubules, ovaries and fat body from sugar-fed adult An. stephensi mosquitoes

The data presented in this article is associated with the quantitative proteomic analysis of four mosquito tissues – midgut, Malpighian tubules, ovaries and fat body from female Anopheles stephensi mosquitoes. To identify the proteins that were expressed in a tissue-specific manner, the four mosquito tissues were labelled with iTRAQ labels and analyzed using a high-resolution mass spectrometer. Database searches of the 1,10,616 raw spectra from 23 peptide fractions resulted in the identification of 84,733 peptide spectrum matches corresponding to 16,278 peptides and 3372 proteins. Of these, 959 proteins were found to be differentially expressed across the tissues. Gene ontology-based bioinformatic analysis of the differentially expressed proteins are also provided in the article. The data in this article has been deposited in the (ProteomeXchange) Consortium via the PRIDE repository and can be accessed through the accession ID, PXD001128.

Proteome data of Anopheles stephensi hemolymph using high resolution mass spectrometry

The article provides insights into the protein expression in Anopheles stephensi hemolymph. We carried out data acquisition using a high-resolution LTQ-Orbitrap Velos mass spectrometer to identify the hemolymph proteins of An. stephensi. Experimentally derived mass spectrometry data was analyzed using Proteome Discoverer 2.1 software using two different search algorithms SEQUEST and MASCOT. A total of 1091 proteins were identified from the hemolymph. The identified proteins were categorized for their role in biological processes and molecular functions. The interactions between these proteins were predicted using STRING online tool. Relation can be drawn between the data provided in this study to the already published article “Integrating transcriptomics and proteomics data for accurate assembly and annotation of genomes” (Prasad et al., 2017) [1].

Data from salivary gland proteome analysis of female Aedes aegypti Linn

Salivary gland proteins from female Aedes aegypti mosquito were extracted and analyzed on high-resolution mass spectrometry. Proteomic data was analysed using two search algorithms SEQUEST and Mascot, which results in acquisition of 83,836 spectra which were assigned to 5417 peptides belonging to 1208 proteins.These proteins were then assigned molecular functions and further analysis revealed biological processes they are involved in using Gene Ontology annotations. Several immunity related pathways were found to be enriched in salivary gland.The data of this study are also related to the research article “Mosquito-Borne Diseases and Omics: Salivary gland proteome of the female Aedes aegypti mosquito” (Dhawan et al., 2017) [1]. These data are deposited in ProteomeXchange in the public dataset PXD002468. In addition,a scientific interpretation of this dataset by Dhawan et al. [1] is available at http://dx.doi.org/10.1089/journal.omi.2016.0160.