Project description:Background: Anopheles culicifacies is a rural vector of malaria in tropical and sub tropical South East Asian region. The salivary gland of the mosquito is the target for sporozoite interaction, blood feeding behavior, haemostasis and vector-parasite interactions. Malaria parasite matures inside the salivary gland, gain competence and transmitted to the host along with the saliva during biting. The importance of the proteins expressed in salivary gland is the first step in understanding the physiology of blood feeding and may provide insights into vector- parasite interactions. Since, no genomic or transcriptomics information is available of Anopheles culicifacies, therefore locally expressed functional proteins in salivary glands are of much importance. . Method: In this study, 1DE protein and in solution digestion was combined with tandem mass spectrometry (nano LC-MS/MS) and computational bioinformatics for data mining was employed to study the proteome profile of salivary glands of sugar fed An. culicifacies mosquito species. Functional annotation of all the identified proteins was carried out using gene ontology tools, CELLO and SMART analysis software. Results: Total 102 proteins were identified and analysed by SEQUEST algorithm against mosquito protein database from Uniprot/NCBI. Out of which 81 proteins were identified using gel free approach and 21 proteins using in-gel approach and 15 were common among these two approaches. All the identified proteins were categorized in to 23 groups of biological processes using GO tool. 7 proteins were depicted to be secretary in nature by investigating the signal peptide present. Potential proteins with unknown function were predicted by analyzing their functional association with other characterized proteins by STRING algorithm and were categorized in cell adhesion, cytoskeleton and membrane trafficking networks. Conclusion: Our study elucidates the first proteomic dataset of An. culicifacies salivary gland proteins. Functional annotation of salivary proteins and complementary gene ontology assignments in An. culicifacies species may contribute towards understanding the complex physiology of the tissues in this species. This proteome baseline data may facilitate the discernment of salivary glands and parasite correlation during blood feeding. Furthermore, this mass spectrometry based proteomic data may also provide insights into the elucidation of role of differential functional proteins present in refractory An. culicifacies mosquito and may be useful for development of effective malaria control strategies.

Project description:In addressing R. microplus - A. marginale interactions, we propose and test three linked hypotheses. The first is that the tick gene response is organ specific: the midgut gene regulation is unique during feeding and during acquisition of A. marginale as compared to the salivary gland. This distinction is relevant as the two organs serve very different roles in the transmission biology of A. marginale with early survival and replication within the midgut epithelium, composed of highly phagocytic cells, required for initial colonization while a second round of replication in the salivary gland acini, composed of highly secretory cells, is required for transmission of an infectious dose in the saliva. Importantly, both the midgut epithelium and salivary glands have been identified as separate and distinct barriers for transmission of A. marginale and thus represent two potential sites where transmission could be blocked. The second hypothesis to be tested is that the salivary gland transcriptome is temporally dynamic. Initiation of tick attachment and feeding involves secretion of a virtual pharmacopeia including lytic enzymes, anticoagulants, and inhibitors of the mammalian innate immune and nocioceptive systems. Concomitantly, the acini provide an environment where A. marginale replicates >100 fold and are secreted into the saliva. Prior studies show that duration of feeding is a critical component of transmission efficiency, with increased efficiency positively correlated with time of tick feeding. The third hypothesis to be tested is that A. marginale colonization does not significantly modulate the tick midgut and salivary gland transcriptome. This hypothesis is based on observations by ourselves and others that tick infection does not impart a significant fitness cost on the vector. This is in contrast to other bacterial and protozoal pathogens that have dramatic effects on success of tick attachment, engorgement, and survival. A. marginale, similar to other tick-borne pathogens in the Family Anaplasmataceeae, is believed to have evolved from an arthropod-specific bacterium with relatively late adaptation to specific niches in mammalian hosts. Consequently, we predict that A. marginale is well adapted to its tick vector and utilizes the normal signaling pathways of the feeding tick with few, if any, effects on the midgut and salivary gland transcriptome. In this manuscript, we report the testing of these three hypotheses and present the results in context of the vector-pathogen-mammalian host interaction at the time of transmission. A Roche NimbleGen high-density gene expression microarray was custom designed based on the expressed sequence tag (EST) database, B. microplus Gene Index Version 2 (BmiGI V2) for R. microplus. The expression level of 14,447 R. microplus genes was analyzed from total RNA extracted from 10 different tick tissue samples; 30 arrays were included since triplicates of each different sample were analyzed as follow: unfed (midgut and salivary glands), blood feeding (2 days midgut and 2, 6 and 9 days salivary glands), A. marginale-infected blood feeding (2 days midgut and 2, 6 and 9 days salivary glands).

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Mosquito metagenome

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