Project description:Background: The Anopheles gambiae salivary glands play a major role in malaria transmission and express a variety of bioactive components that facilitate blood-feeding by preventing platelet aggregation, blood clotting, vasodilatation, and inflammatory and other reactions at the probing site on the vertebrate host. Results: We have performed a global transcriptome analysis of the A. gambiae salivary gland response to blood-feeding, to identify candidate genes that are involved in hematophagy. A total of 4,978 genes were found to be transcribed in this tissue. A comparison of salivary gland transcriptomes prior to and after blood-feeding identified 52 and 41 transcripts that were significantly up-regulated and down-regulated, respectively. Ten genes were further selected to assess their role in the blood-feeding process using RNAi-mediated gene silencing methodology. Depletion of the salivary gland genes encoding D7L2, anophelin, peroxidase, the SG2 precursor, and a 5'nucleotidase gene significantly increased probing time of A. gambiae mosquitoes and thereby their capacity to blood-feed. Conclusions: The salivary gland transcriptome comprises approximately 38% of the total mosquito transcriptome and a small proportion of it is dynamically changing already at two hours in response to blood feeding. A better understanding of the salivary gland transcriptome and its function can contribute to the development of pathogen transmission control strategies and the identification of medically relevant bioactive compounds.
Project description:Background: The Anopheles gambiae salivary glands play a major role in malaria transmission and express a variety of bioactive components that facilitate blood-feeding by preventing platelet aggregation, blood clotting, vasodilatation, and inflammatory and other reactions at the probing site on the vertebrate host. Results: We have performed a global transcriptome analysis of the A. gambiae salivary gland response to blood-feeding, to identify candidate genes that are involved in hematophagy. A total of 4,978 genes were found to be transcribed in this tissue. A comparison of salivary gland transcriptomes prior to and after blood-feeding identified 52 and 41 transcripts that were significantly up-regulated and down-regulated, respectively. Ten genes were further selected to assess their role in the blood-feeding process using RNAi-mediated gene silencing methodology. Depletion of the salivary gland genes encoding D7L2, anophelin, peroxidase, the SG2 precursor, and a 5'nucleotidase gene significantly increased probing time of A. gambiae mosquitoes and thereby their capacity to blood-feed. Conclusions: The salivary gland transcriptome comprises approximately 38% of the total mosquito transcriptome and a small proportion of it is dynamically changing already at two hours in response to blood feeding. A better understanding of the salivary gland transcriptome and its function can contribute to the development of pathogen transmission control strategies and the identification of medically relevant bioactive compounds. Salivary glands from blood-fed vs. unfed A. gambiae. 3 replicates.
Project description:We have developed an oligoGEArray with 434 genes from Ae. aegypti salivary gland. OligoGEArray was customized with the salivary gland genes chosen from the transcriptome source published by Ribeiro et al (2007). Oligonucleotides (60bp) were designed and array were manufactured by SABiosciences. Using this OligoGEArray, we analyzed the differential expression of salivary trancritptome upon blood feeding.
Project description:We have developed an oligoGEArray with 434 genes from Ae. aegypti salivary gland. OligoGEArray was customized with the salivary gland genes chosen from the transcriptome source published by Ribeiro et al (2007). Oligonucleotides (60bp) were designed and array were manufactured by SABiosciences. Using this OligoGEArray, we analyzed the differential expression of salivary trancritptome upon blood feeding. Salivary glands were dissected from 1, 3, 24 and 48 hours post fed (hpf) and unfed Ae. aegypti. Total RNA were extracted and the differential expression of transcriptome were analysed. Quantitative Real-Time PCR was performed on selected genes to validate the OligoGEArray data.
Project description:Salivary gland proteins of Anopheles mosquitoes offer attractive targets to understand interactions with sporozoites, blood feeding behavior, homeostasis and immunological evaluation of malaria vectors and parasite interactions. To date limited studies have been carried out to elucidate salivary proteins of An. stephensi salivary glands. The aim of the present study was to provide detailed analytical attributives of functional salivary gland proteins of urban malaria vector An. stephensi. A proteomic approach combining one-dimensional electrophoresis (1DE), ion trap liquid chromatography mass spectrometry (LC/MS/MS) and computational bioinformatic analysis was adopted to provide the first direct insight into identification and functional characterization of known salivary proteins and novel salivary proteins of An. stephensi. Computational studies by online servers namely, Mascot and OMSSA algorithms identified a total of 36 known salivary proteins and 123 novel proteins analysed by LC/MS/MS. This first report describes a baseline proteomic catalogue of 159 salivary proteins belonging to various categories of signal transduction, regulation of blood coagulation cascade, and various immune and energy pathways of An. stephensi sialo-transcriptome by mass spectrometry. Our results may serve as basis to provide a putative functional role of proteins into concept of blood feeding, biting behavior and other aspects of vector-parasite host interactions for parasite development in anopheline mosquitoes.
Project description:Aedes aegypti is a major vector for dengue, chikungunya and yellow fever. Though salivary gland plays a crucial role in transmission of virus and vector life cycle, there is no global and unbiased published report on salivary gland proteome of Ae. aegypti. In this study, we have carried out mass spectrometry based proteomic analysis of salivary gland from adult female Ae. aegypti mosquitoes. By fractionating the proteins isolated from salivary gland on SDS-PAGE and analyzing the in-gel digested bands on high resolution mass spectrometry, we identified 1,205 proteins. This is by far the largest catalogue of salivary gland proteome of Ae. aegypti. These proteins were then assigned molecular functions and biological processes. Several immunity related pathways were found to be enriched in salivary gland. In addition, subset of proteins was predicted to secretory in nature and may play an important role during blood feeding. This study provides a useful resource of proteins expressed in salivary gland of Ae. aegypti female mosquitoes and will aid in biomedical research focused on development of transmission blocking vaccine.
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).