Dataset Information

Molecular and Functional Characterization of Trehalase in the Mosquito Anopheles stephensi.

ABSTRACT: Like other insects, in blood-feeding mosquitoes, trehalase (TRE; EC 3.2.1.28), an enzyme that metabolizes trehalose, may influence a wide array of functions including flight, survival, reproduction, and vectorial capacity, but its role has not been investigated in detail. Here, we characterized a 1,839-bp-long transcript, encoding a 555-aa-long trehalase-2 homolog protein from the mosquito Anopheles stephensi. With a conserved insect homology, and in silico predicted membrane-bound protein, we tested whether trehalase (As-TreH) also plays a role in mosquito physiologies. Constitutive expression during aquatic development or adult mosquito tissues, and a consistent upregulation until 42 h of starvation, which was restored to basal levels after sugar supply, together indicated that As-TreH may have a key role in stress tolerance. A multifold enrichment in the midgut (p < 0.001819) and salivary glands (p < 4.37E-05) of the Plasmodium vivax-infected mosquitoes indicated that As-TreH may favor parasite development and survival in the mosquito host. However, surprisingly, after the blood meal, a consistent upregulation until 24 h in the fat body, and 48 h in the ovary, prompted to test its possible functional correlation in the reproductive physiology of the adult female mosquitoes. A functional knockdown by dsRNA-mediated silencing confers As-TreH ability to alter reproductive potential, causing a significant loss in the egg numbers (p < 0.001), possibly by impairing energy metabolism in the developing oocytes. Conclusively, our data provide initial evidence that As-TreH regulates multiple physiologies and may serve as a suitable target for designing novel strategies for vector control.

SUBMITTER: Tevatiya S

PROVIDER: S-EPMC7710876 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Molecular and Functional Characterization of Trehalase in the Mosquito Anopheles stephensi.

Tevatiya Sanjay S Kumari Seena S Sharma Punita P Rani Jyoti J Chauhan Charu C Das De Tanwee T Pandey Kailash C KC Pande Veena V Dixit Rajnikant R

Frontiers in physiology 20201119

Like other insects, in blood-feeding mosquitoes, trehalase (TRE; EC 3.2.1.28), an enzyme that metabolizes trehalose, may influence a wide array of functions including flight, survival, reproduction, and vectorial capacity, but its role has not been investigated in detail. Here, we characterized a 1,839-bp-long transcript, encoding a 555-aa-long trehalase-2 homolog protein from the mosquito Anopheles stephensi. With a conserved insect homology, and in silico predicted membrane-bound ...[more]

PMID: 33329025

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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.

Project description:Strains of Serratia marcescens, originally isolated from the gut lumen of adult female Anopheles stephensi mosquitoes, established persistent infection at high rates in adult A. stephensi whether fed to larvae or in the sugar meal to adults. By contrast, the congener S. fonticola originating from Aedes triseriatus had lower infection in A. stephensi, suggesting co-adaptation of Serratia strains in different species of host mosquitoes. Coinfection at high infection rate in adult A. stephensi resulted after feeding S. marcescens and Elizabethkingia anophelis in the sugar meal, but when fed together to larvae, infection rates with E. anophelis were much higher than were S. marcescens in adult A. stephensi, suggesting a suppression effect of coinfection across life stages. A primary isolate of S. marcescens was resistant to all tested antibiotics, showed high survival in the mosquito gut, and produced alpha-hemolysins which contributed to lysis of erythrocytes ingested with the blood meal. Genomes of two primary isolates from A. stephensi, designated S. marcescens ano1 and ano2, were sequenced and compared to other Serratia symbionts associated with insects, nematodes and plants. Serratia marcescens ano1 and ano2 had predicted virulence factors possibly involved in attacking parasites and/or causing opportunistic infection in mosquito hosts. S. marcescens ano1 and ano2 possessed multiple mechanisms for antagonism against other microorganisms, including production of bacteriocins and multi-antibiotic resistance determinants. These genes contributing to potential anti-malaria activity including serralysins, hemolysins and chitinases are only found in some Serratia species. It is interesting that genome sequences in S. marcescens ano1 and ano2 are distinctly different from those in Serratia sp. Ag1 and Ag2 which were isolated from Anopheles gambiae. Compared to Serratia sp. Ag1 and Ag2, S. marcescens ano1 and ano2 have more rRNAs and many important genes involved in commensal and anti-parasite traits.

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.

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.

Dataset Information

Molecular and Functional Characterization of Trehalase in the Mosquito Anopheles stephensi.

Publications

Molecular and Functional Characterization of Trehalase in the Mosquito <i>Anopheles stephensi</i>.

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