A physical map for an Asian malaria mosquito, Anopheles stephensi.
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ABSTRACT:
SUBMITTER: Sharakhova MV
PROVIDER: S-EPMC2963963 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Anopheles stephensi is one of the major vectors of malaria in the Middle East and Indo-Pakistan subcontinent. Understanding the population genetic structure of malaria mosquitoes is important for developing adequate and successful vector control strategies. Commonly used markers for inferring anopheline taxonomic and population status include microsatellites and chromosomal inversions. Knowledge about chromosomal locations of microsatellite markers with respect to polymorphic inversions could be useful for better understanding a genetic structure of natural populations. However, fragments with microsatellites used in population genetic studies are usually too short for successful labeling and hybridization with chromosomes. We designed new primers for amplification of microsatellite loci identified in the A. stephensi genome sequenced with next-generation technologies. Twelve microsatellites were mapped to polytene chromosomes from ovarian nurse cells of A. stephensi using fluorescent in situ hybridization. All microsatellites hybridized to unique locations on autosomes, and 7 of them localized to the largest arm 2R. Ten microsatellites were mapped inside the previously described polymorphic chromosomal inversions, including 4 loci located inside the widespread inversion 2Rb. We analyzed microsatellite-based population genetic data available for A. stephensi in light of our mapping results. This study demonstrates that the chromosomal position of microsatellites may affect estimates of population genetic parameters and highlights the importance of developing physical maps for nonmodel organisms.
| S-EPMC3198509 | biostudies-literature
Project description:BACKGROUND:The Asian malaria mosquito, Anopheles stephensi, is a major urban malaria vector in the Middle East and on the Indian subcontinent. Early zygotic transcription, which marks the maternal-to-zygotic transition, has not been systematically studied in An. stephensi or any other Anopheles mosquitoes. Improved understanding of early embryonic gene expression in An. stephensi will facilitate genetic and evolutionary studies and help with the development of novel control strategies for this important disease vector. RESULTS:We obtained RNA-seq data in biological triplicates from four early An. stephensi embryonic time points. Using these data, we identified 70 and 153 pure early zygotic genes (pEZGs) under stringent and relaxed conditions, respectively. We show that these pEZGs are enriched in functional groups related to DNA-binding transcription regulators, cell cycle modulators, proteases, transport, and cellular metabolism. On average these pEZGs are shorter and have less introns than other An. stephensi genes. Some of the pEZGs may arise de novo while others have clear non-pEZG paralogs. There is no or very limited overlap between An. stephensi pEZGs and Drosophila melanogaster or Aedes aegypti pEZGs. Interestingly, the upstream region of An. stephensi pEZGs lack significant enrichment of a previously reported TAGteam/VBRGGTA motif found in the regulatory region of pEZGs in D. melanogaster and Ae. aegypti. However, a GT-rich motif was found in An. stephensi pEZGs instead. CONCLUSIONS:We have identified a number of pEZGs whose predicted functions and structures are consistent with their collective roles in the degradation of maternally deposited components, activation of the zygotic genome, cell division, and metabolism. The pEZGs appear to rapidly turn over within the Dipteran order and even within the Culicidae family. These pEZGs, and the shared regulatory motif, could provide the promoter or regulatory sequences to drive gene expression in the syncytial or early cellular blastoderm, a period when the developing embryo is accessible to genetic manipulation. In addition, these molecular resources may be used to achieve sex separation of mosquitoes for sterile insect technique.
| S-EPMC6304767 | biostudies-literature
Project description:BackgroundmicroRNAs (miRNAs) are non-coding RNAs that are now recognized as a major class of gene-regulating molecules widely distributed in metozoans and plants. miRNAs have been found to play important roles in apoptosis, cancer, development, differentiation, inflammation, longevity, and viral infection. There are a few reports describing miRNAs in the African malaria mosquito, Anopheles gambiae, on the basis of similarity to known miRNAs from other species. An. stephensi is the most important malaria vector in Asia and it is becoming a model Anopheline species for physiological and genetics studies.ResultsWe report the cloning and characterization of 27 distinct miRNAs from 17-day old An. stephensi female mosquitoes. Seventeen of the 27 miRNAs matched previously predicted An. gambiae miRNAs, offering the first experimental verification of miRNAs from mosquito species. Ten of the 27 are miRNAs previously unknown to mosquitoes, four of which did not match any known miRNAs in any organism. Twenty-five of the 27 Anopheles miRNAs had conserved sequences in the genome of a divergent relative, the yellow fever mosquito Aedes aegypti. Two clusters of miRNAs were found within introns of orthologous genes in An. gambiae, Ae. aegypti, and Drosophila melanogaster. Mature miRNAs were detected in An. stephensi for all of the nine selected miRNAs, including the four novel miRNAs (miR-x1- miR-x4), either by northern blot or by Ribonuclease Protection Assay. Expression profile analysis of eight of these miRNAs revealed distinct expression patterns from early embryo to adult stages in An. stephensi. In both An. stephensi and Ae. aegypti, the expression of miR-x2 was restricted to adult females and predominantly in the ovaries. A significant reduction of miR-x2 level was observed 72 hrs after a blood meal. Thus miR-x2 is likely involved in female reproduction and its function may be conserved among divergent mosquitoes. A mosquito homolog of miR-14, a regulator of longevity and apoptosis in D. melanogaster, represented 25% of all sequenced miRNA clones from 17-day old An. stephensi female mosquitoes. An. stephensi miR-14 displayed a relatively strong signal from late embryonic to adult stages. miR-14 expression is consistent during the adult lifespan regardless of age, sex, and blood feeding status. Thus miR-14 is likely important across all mosquito life stages.ConclusionThis study provides experimental evidence for 23 conserved and four new microRNAs in An. stephensi mosquitoes. Comparisons between miRNA gene clusters in Anopheles and Aedes mosquitoes, and in D. melanogaster suggest the loss or significant change of two miRNA genes in Ae. aegypti. Expression profile analysis of eight miRNAs, including the four new miRNAs, revealed distinct patterns from early embryo to adult stages in An. stephensi. Further analysis showed that miR-x2 is likely involved in female reproduction and its function may be conserved among divergent mosquitoes. Consistent expression of miR-14 suggests that it is likely important across all mosquito life stages from embryos to aged adults. Understanding the functions of mosquito miRNAs will undoubtedly contribute to a better understanding of mosquito biology including longevity, reproduction, and mosquito-pathogen interactions, which are important to disease transmission.
| S-EPMC2430712 | biostudies-literature
Project description:A piggyBac transposon-based gene trap element was transformed into the Asian malaria vector, Anopheles stephensi, and remobilized using the jumpstarter approach using genetic crosses. Individuals that displayed a gene trap remobilization event were then photodocumented and their RNA and DNA complements were extracted. The DNA compelement was used to determine the genomic insertion site, while the RNA was used to determine the transcript coverage of the genes into which the transposons inserted. In nearly half of the cases, insertion was identified to fall within introns present in the 5'-UTR of transcripts- which are not indicated in the current ab initio models for Anopheles stephensi. The ability to utilize next generation RNA-Seq elucidated the functionality of the gene trap elements that inserted outside of the ab initio gene models, providing clear evidence that not only was the gene trap element working properly, but that it also had a bias towards 5'-end insertion, in particular, 5'-UTR intronic insertion.
2016-03-01 | GSE78771 | GEO
Project description:A piggyBac transposon-based gene trap element was transformed into the Asian malaria vector, Anopheles stephensi, and remobilized using the jumpstarter approach using genetic crosses. Individuals that displayed a gene trap remobilization event were then photodocumented and their RNA and DNA complements were extracted. The DNA compelement was used to determine the genomic insertion site, while the RNA was used to determine the transcript coverage of the genes into which the transposons inserted. In nearly half of the cases, insertion was identified to fall within introns present in the 5'-UTR of transcripts- which are not indicated in the current ab initio models for Anopheles stephensi. The ability to utilize next generation RNA-Seq elucidated the functionality of the gene trap elements that inserted outside of the ab initio gene models, providing clear evidence that not only was the gene trap element working properly, but that it also had a bias towards 5'-end insertion, in particular, 5'-UTR intronic insertion. RNA from 200 pooled individually-extracted An. stephensi that demonstrated a gene trap remobilizable event.
2016-03-01 | E-GEOD-78771 | biostudies-arrayexpress
Project description:Insect odorant-binding proteins (OBPs) are small, water-soluble molecules that are thought to transport the hydrophobic odorants to their receptors in the chemosensory neurones. Here we report the identification and molecular characterization of the Anopheles stephensi odorant-binding protein 1 gene (AsteObp1), an Obp1 gene in An. stephensi, a major malaria vector in Asia. We show that AsteObp1 and Anopheles gambiae Obp1 (AgamObp1) are orthologues. These two genes share similar coding sequences and conserved noncoding sequences (CNSs) that may be involved in their regulation. Transcript of AsteObp1 was observed in larvae and reached a relatively high level in late pupae. Quantitative real-time PCR on female adult chemosensory tissues showed approximately 900-fold higher expression of AsteObp1 in antennae than in maxillary palp and proboscis. The amount of AsteObp1 in female legs was approximately 15-fold lower than that of maxillary palp and proboscis. The level of AsteObp1 transcript was seven and 85-fold higher in females than in males in the antennae, and maxillary palp and proboscis, respectively. Moreover, the AsteObp1 level was reduced by approximately 20-fold in maxillary palp and proboscis 24 h after a bloodmeal. Our results indicate that AsteObp1 is likely to function in the female olfactory response and may also be involved in blood-feeding behaviour.
| S-EPMC2823854 | biostudies-literature
Project description:Anopheles stephensi is a principal vector of urban malaria on the Indian subcontinent and an emerging model for molecular and genetic studies of mosquito biology. To enhance our understanding of female mosquito reproduction, and to develop new tools for basic research and for genetic strategies to control mosquito-borne infectious diseases, we identified 79 genes that displayed previtellogenic germline-specific expression based on RNA-Seq data generated from 11 life stage-specific and sex-specific samples. Analysis of this gene set provided insights into the biology and evolution of female reproduction. Promoters from two of these candidates, vitellogenin receptor and nanos, were used in independent transgenic cassettes for the expression of artificial microRNAs against suspected mosquito maternal-effect genes, discontinuous actin hexagon and myd88. We show these promoters have early germline-specific expression and demonstrate 73% and 42% knockdown of myd88 and discontinuous actin hexagon mRNA in ovaries 48 hr after blood meal, respectively. Additionally, we demonstrate maternal-specific delivery of mRNA and protein to progeny embryos. We discuss the application of this system of maternal delivery of mRNA/miRNA/protein in research on mosquito reproduction and embryonic development, and for the development of a gene drive system based on maternal-effect dominant embryonic arrest.
| S-EPMC4321024 | biostudies-literature
Project description:Small laboratory cage trials of non-drive and gene-drive strains of the Asian malaria vector mosquito, Anopheles stephensi, were used to investigate release ratios and other strain properties for their impact on transgene spread during simulated population modification. We evaluated the effects of transgenes on survival, male contributions to next-generation populations, female reproductive success and the impact of accumulation of gene drive-resistant genomic target sites resulting from nonhomologous end-joining (NHEJ) mutagenesis during Cas9, guide RNA-mediated cleavage. Experiments with a non-drive, autosomally-linked malaria-resistance gene cassette showed 'full introduction' (100% of the insects have at least one copy of the transgene) within 8 weeks (? 3 generations) following weekly releases of 10:1 transgenic:wild-type males in an overlapping generation trial design. Male release ratios of 1:1 resulted in cages where mosquitoes with at least one copy of the transgene fluctuated around 50%. In comparison, two of three cages in which the malaria-resistance genes were linked to a gene-drive system in an overlapping generation, single 1:1 release reached full introduction in 6-8 generations with a third cage at ~80% within the same time. Release ratios of 0.1:1 failed to establish the transgenes. A non-overlapping generation, single-release trial of the same gene-drive strain resulted in two of three cages reaching 100% introduction within 6-12 generations following a 1:1 transgenic:wild-type male release. Two of three cages with 0.33:1 transgenic:wild-type male single releases achieved full introduction in 13-16 generations. All populations exhibiting full introduction went extinct within three generations due to a significant load on females having disruptions of both copies of the target gene, kynurenine hydroxylase. While repeated releases of high-ratio (10:1) non-drive constructs could achieve full introduction, results from the 1:1 release ratios across all experimental designs favor the use of gene drive, both for efficiency and anticipated cost of the control programs.
| S-EPMC6922335 | biostudies-literature
Project description:Transposon-based forward and reverse genetic technologies will contribute greatly to ongoing efforts to study mosquito functional genomics. A piggyBac transposon-based enhancer-trap system was developed that functions efficiently in the human malaria vector, Anopheles stephensi. The system consists of six transgenic lines of Anopheles stephensi, each with a single piggyBac-Gal4 element in a unique genomic location; six lines with a single piggyBac-UAStdTomato element; and two lines, each with a single Minos element containing the piggyBac-transposase gene under the regulatory control of the hsp70 promoter from Drosophila melanogaster. Enhancer detection depended upon the efficient remobilization of piggyBac-Gal4 transposons, which contain the yeast transcription factor gene Gal4 under the regulatory control of a basal promoter. Gal4 expression was detected through the expression of the fluorescent protein gene tdTomato under the regulatory control of a promoter with Gal4-binding UAS elements. From five genetic screens for larval- and adult-specific enhancers, 314 progeny were recovered from 24,250 total progeny (1.3%) with unique patterns of tdTomato expression arising from the influence of an enhancer. The frequency of piggyBac remobilization and enhancer detection was 2.5- to 3-fold higher in female germ lines compared with male germ lines. A small collection of enhancer-trap lines are described in which Gal4 expression occurred in adult female salivary glands, midgut, and fat body, either singly or in combination. These three tissues play critical roles during the infection of Anopheles stephensi by malaria-causing Plasmodium parasites. This system and the lines generated using it will be valuable resources to ongoing mosquito functional genomics efforts.
| S-EPMC3484661 | biostudies-literature
Project description: Not available
| S-EPMC10953274 | biostudies-literature