Project description:With their genome sequenced, Anopheles gambiae mosquitoes now serve as a powerful tool for basic research in comparative, evolutionary and developmental biology. The knowledge generated by these studies is expected to reveal molecular targets for novel vector control and pathogen transmission blocking strategies. Comparisons of gene-expression profiles between adult male and nonblood-fed female Anopheles gambiae mosquitoes revealed that roughly 22% of the genes showed sex-dependent regulation. Blood-fed females switch the majority of their metabolism to blood digestion and egg formation within 3 h after the meal is ingested, in detriment to other activities such as flight and response to environment stimuli. Changes in gene expression are most evident during the first, second and third days after a blood meal, when as many as 50% of all genes showed significant variation in transcript accumulation. After laying the first cluster of eggs (between 72 and 96 h after the blood meal), mosquitoes return to a nongonotrophic stage, similar but not identical to that of 3-dayold nonblood-fed females. Ageing and/or the nutritional state of mosquitoes at 15 days after a blood meal is reflected by the down-regulation of 5% of all genes. A full description of the large number of genes regulated at each analysed time point and each biochemical pathway or biological processes in which they are involved is not possible within the scope of this contribution. Therefore, we present descriptions of groups of genes displaying major differences in transcript accumulation during the adult mosquito life. However, a publicly available searchable database (Anopheles gambiae Gene Expression Database at UC Irvine) has been made available so that detailed analyses of specific groups of genes based on their descriptions, functions or levels of gene expression variation can be performed by interested investigators according to their needs. Keywords: response to bloodmeal
Project description:The Anopheles gambiae midgut harbors bacteria that proliferate upon a blood feed. We used microarrays to examine the midgut gene expression response at early stages (3hours) after an artifitial meal containing heat killed bacteria.
Project description:With their genome sequenced, Anopheles gambiae mosquitoes now serve as a powerful tool for basic research in comparative, evolutionary and developmental biology. The knowledge generated by these studies is expected to reveal molecular targets for novel vector control and pathogen transmission blocking strategies. Comparisons of gene-expression profiles between adult male and nonblood-fed female Anopheles gambiae mosquitoes revealed that roughly 22% of the genes showed sex-dependent regulation. Blood-fed females switch the majority of their metabolism to blood digestion and egg formation within 3 h after the meal is ingested, in detriment to other activities such as flight and response to environment stimuli. Changes in gene expression are most evident during the first, second and third days after a blood meal, when as many as 50% of all genes showed significant variation in transcript accumulation. After laying the first cluster of eggs (between 72 and 96 h after the blood meal), mosquitoes return to a nongonotrophic stage, similar but not identical to that of 3-dayold nonblood-fed females. Ageing and/or the nutritional state of mosquitoes at 15 days after a blood meal is reflected by the down-regulation of 5% of all genes. A full description of the large number of genes regulated at each analysed time point and each biochemical pathway or biological processes in which they are involved is not possible within the scope of this contribution. Therefore, we present descriptions of groups of genes displaying major differences in transcript accumulation during the adult mosquito life. However, a publicly available searchable database (Anopheles gambiae Gene Expression Database at UC Irvine) has been made available so that detailed analyses of specific groups of genes based on their descriptions, functions or levels of gene expression variation can be performed by interested investigators according to their needs. Experiment Overall Design: 12 Samples analyzed, 3 replicates per sample, Affymetrix internal controls
Project description:The effect of chloroquine on mosquitoes transcript abundance was assayed by comparing gene expression between mosquitoes fed on a blood meal containing 50 mg/Kg of chloroquine and those that had fed on a normal blood meal. Pools of 50 midguts were dissected and were hybridize with MMC1 (or 20K) microarrays. <br><br>Anopheles gambiae female mosquitoes were blood fed on BALB/c mice infected with P. berghei and intraperitonally pre-treated with 50 mg/kg of chloroquine. As controls, mosquitoes were blood fed on untreated P. berghei infected mice.<br><br>Mosquitoes were collected 24 hours post-blood feeding and pools of 50 midguts were dissected and processed for hybridization with MMC1 (or 20K) microarrays.Two different biological experiments were performed for each treatment.
Project description:Proteomic analysis of Anopheles gambiae brain tissue after in-gel trypsin digestion. To gain insights into neurobiology of the Anopheles gambiae mosquito, we carried out a proteomic analysis of its brain using a comprehensive proteomic approach.
Project description:Anopheles gambiae mosquitoes transmit the human malaria parasite Plasmodium falciparum, which causes the majority of fatal malaria cases worldwide. The hematophagous life style defines the mosquito reproductive biology and is exploited by P. falciparum for its own sexual reproduction and transmission. The two main phases of the mosquito reproductive cycle, pre-vitellogenic (PV) and post-blood meal (PBM) shape its capacity to transmit malaria. Transition between these phases is tightly coordinated to ensure homeostasis between mosquito tissues and successful reproduction. One layer of control is provided by microRNAs, well-known regulators of blood meal digestion and egg development in mosquitoes. Here, we report a global overview of tissue-specific miRNA expression during the PV and PBM phases and identify miRNAs regulated during PV to PBM transition. The observed coordinated changes in the expression levels of a set of miRNAs in the energy-storing tissues suggest a role in the regulation of blood meal-induced metabolic changes.
Project description:Numerous studies have examined changes in transcript levels after Anopheles gambiae takes a blood meal. Marinotti et al. (2006) used microarrays and reported massive changes in transcript levels 3 h after feeding (BF3h) compared to non-blood fed (NBF). We were intrigued by the number of transcripts for structural cuticular proteins (CPs) that showed such major differences in levels and employed paired-end (50 bp) RNA-seq technology to compare whole body transcriptomes from 5-day-old females NBF and BF3h. We detected transcripts for the majority of CPs (164/243) but levels of only 12 were significantly altered by the blood meal. While relative transcript levels of NBF females were somewhat similar to the microarray data, there were major differences in BF3h animals, resulting in levels of many transcripts, both for CPs and other genes changing in the opposite direction. We compared our data also to other studies done with both microarrays and RNA-seq. Findings were consistent that a small number of CP genes have transcripts that persist even in 5-day-old adults. Some of these transcripts showed diurnal rhythms (Rund et al., 2013; Rinker et al., 2013). In situ hybridization revealed that transcripts for several of these CP genes were found exclusively or predominantly in the eye. Transcripts other than for CPs that changed in response to blood-feeding were predominantly expressed in midgut and Malpighian tubules. Even in these tissues, genes responsible for proteins with similar functions, such as immunity or digestion, responded differently, with transcript levels for some rising and others falling. These data demonstrate that genes coding for some CPs are dynamic in expression even in adults and that the response to a blood meal is rapid and precisely orchestrated.
Project description:Transmission of malaria is dependent on the successful completion of the Plasmodium lifecycle in the Anopheles vector. Major obstacles are encountered in the midgut tissue, where most parasites are killed by the mosquito’s immune system. In the present study, DNA microarray analyses have been used to compare Anopheles gambiae responses to invasion of the midgut epithelium by the ookinete stage of the human pathogen Plasmodium falciparum and the rodent experimental model pathogen P. berghei. Invasion by P. berghei had a more profound impact on the mosquito transcriptome, including a variety of functional gene classes, while P. falciparum elicited a broader immune response at the gene transcript level. Ingestion of human malaria-infected blood lacking invasive ookinetes also induced a variety of immune genes, including several anti-Plasmodium factors. Keywords: Anopheles gambiae, Plasmodium falciparum, ookinete, invasion, innate immunity
Project description:Numerous studies have examined changes in transcript levels after Anopheles gambiae takes a blood meal. Marinotti et al. (2006) used microarrays and reported massive changes in transcript levels 3 h after feeding (BF3h) compared to non-blood fed (NBF). We were intrigued by the number of transcripts for structural cuticular proteins (CPs) that showed such major differences in levels and employed paired-end (50 bp) RNA-seq technology to compare whole body transcriptomes from 5-day-old females NBF and BF3h. We detected transcripts for the majority of CPs (164/243) but levels of only 12 were significantly altered by the blood meal. While relative transcript levels of NBF females were somewhat similar to the microarray data, there were major differences in BF3h animals, resulting in levels of many transcripts, both for CPs and other genes changing in the opposite direction. We compared our data also to other studies done with both microarrays and RNA-seq. Findings were consistent that a small number of CP genes have transcripts that persist even in 5-day-old adults. Some of these transcripts showed diurnal rhythms (Rund et al., 2013; Rinker et al., 2013). In situ hybridization revealed that transcripts for several of these CP genes were found exclusively or predominantly in the eye. Transcripts other than for CPs that changed in response to blood-feeding were predominantly expressed in midgut and Malpighian tubules. Even in these tissues, genes responsible for proteins with similar functions, such as immunity or digestion, responded differently, with transcript levels for some rising and others falling. These data demonstrate that genes coding for some CPs are dynamic in expression even in adults and that the response to a blood meal is rapid and precisely orchestrated. Five females 3h after the blood meal (BF3h) and non-blood fed (NBF, controls) were collected for each RNA extraction. RNA preparations were sent to HudsonAlpha where eight libraries were prepared (four replicates for each treatments) with poly-A selection and preparation of 200 bp fragments using the Illumina True-Seq RNA Sample Prep kit and analyzed with HiSEQ paired-end reads of 50 nt.
Project description:We custom-built a bioinformatics pipeline to search for 20E-modifying enzymes in the accessory glands of Anopheles gambiae males, searching for ecdysteroid kinases (EcK), ecdysone oxidases (EO), and ecdysteroid-phosphate phosphatases (EPP). To this end, we generated RNAseq datasets of different An. gambiae tissues dissected from virgin and mated females and males, and produced similar datasets for Anopheles albimanus, a South American species that does not synthetize and transfer ecdysteroids during mating. These analyses led to the identification of one candidate EPP and two potential EcKs (EcK1 and EcK2), which we demonstrated are involved in the activity of a male-specific oxidized ecdysteroid (3D20E). We further determined that 3D20E is specifically produced by the An. gambiae male accessory glands and is transferred to females during copulation, where it triggers a series of post-mating responses.