Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptomic analysis of Anopheles gambiae females subjected to physiological stress.

ABSTRACT: Overall, the study aims at obtaining a comprehensive picture of the African malaria mosquito, Anopheles gambiae, transcriptome using high-coverage RNA-seq of sexed whole-insect samples. This experiment focuses on transcriptomes of females subjected to physiological stress. The samples include adult females collected at different time intervals subject to sub-optimal relative humidity, after exposure to sublethal dose of deltamethrin, or following blood-feeding. Females kept at standard laboratory conditions without exposure to stressors, were collected at the same time intervals as control samples.

INSTRUMENT(S): Illumina HiSeq 2000

ORGANISM(S): Anopheles gambiae

SUBMITTER: Jaroslaw Krzywinski

PROVIDER: E-MTAB-2877 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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:Background: Mosquito midgut is an important target for the host parasite interaction studies as this major immune organ plays a role in parasite growth, maturation and vector competence. Most important rural malaria vector of India is Proteomic approaches coupled with bioinformatics analysis have been used to study expression of functional proteins/enzymes of An. culicifacies susceptible and refractory species midgut in order to understand the mechanism of refractoriness that may help in contributing to unravel the host pathogen interactions Methodology/Principle findings: Basic shot gun proteomics approaches along with iTRAQ based differential studies followed by LC/MS/MS were employed to study the proteome of sugar fed An. culicifacies midgut. Further bioinformatics analysis was carried out using Gene ontology, SMART analysis, CELLO algorithm etc. Here proteins identified with iTRAQ Ratio more than 1.5 was considered up regulated and less than 0.67 were considered down regulated. Ratio from 1.5 – 0.67 were considered as moderate to no change. Using these criteria we observed total 80 proteins in An. culicifacies midgut and when compared susceptible species against refractory species, total 30 proteins were found to be downregulated. 3 proteins were found to be upregulated in refractory species Conclusion/Significance: Based on the present data a significant increase in number of proteins in midgut of refractory An. culicifacies species B were found that may conclude that these proteins may be responsible for the inhibiting parasite growth and linked to the melanization of oocysts or parasite lysis mechanisms in natural populations of refractory mosquito. Hence the progress of our studies at protein level suggests that these identified annotated putative proteins/enzymes may help to explore natural vector-parasite systems and reveal valuable insights into the mechanism of refractoriness which in turn further useful for bringing of novel strategies for control of malaria.

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Transcriptomic analysis of Anopheles gambiae females subjected to physiological stress.

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