Dataset Information

Quantitative real-time PCR analysis of Anopheles dirus TEP1 and NOS during Plasmodium berghei infection, using three reference genes.

ABSTRACT: Quantitative reverse transcription PCR (qRT-PCR) has been an integral part of characterizing the immunity of Anopheles mosquitoes towards Plasmodium invasion. Two anti-Plasmodium factors of Anopheles, thioester-containing protein 1 (TEP1) and nitric oxide synthase (NOS), play a role in the refractoriness of Anopheles towards Plasmodium infection and are generally expressed during infection. However, these are less studied in Anopheles dirus, a dominant malaria vector in Southeast Asia. Furthermore, most studies used a single reference gene for normalization during gene expression analysis without proper validation. This may lead to erroneous quantification of expression levels. Therefore, the present study characterized and investigated the expression profiles of TEP1 and NOS of Anopheles dirus during P. berghei infection. Prior to that, the elongation factor 1-alpha (EF1), actin 1 (Act) and ribosomal protein S7 (S7) genes were validated for their suitability as a set of reference genes. TEP1 and NOS expressions in An. dirus were found to be significantly induced after P. berghei infection.

SUBMITTER: Liew JWK

PROVIDER: S-EPMC5533154 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Quantitative real-time PCR analysis of Anopheles dirus TEP1 and NOS during Plasmodium berghei infection, using three reference genes.

Liew Jonathan W K JWK Fong Mun Yik MY Lau Yee Ling YL

PeerJ 20170726

Quantitative reverse transcription PCR (qRT-PCR) has been an integral part of characterizing the immunity of Anopheles mosquitoes towards Plasmodium invasion. Two anti-Plasmodium factors of Anopheles, thioester-containing protein 1 (TEP1) and nitric oxide synthase (NOS), play a role in the refractoriness of Anopheles towards Plasmodium infection and are generally expressed during infection. However, these are less studied in Anopheles dirus, a dom ...[more]

PMID: 28761783

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Project description:BackgroundThe native gut microbiota of Anopheles mosquitoes is known to play a key role in the physiological function of its host. Interestingly, this microbiota can also influence the development of Plasmodium in its host mosquitoes. In recent years, much interest has been shown in the employment of gut symbionts derived from vectors in the control of vector-borne disease transmission. In this study, the midgut microbial diversity has been characterized among laboratory-reared adult Anopheles stephensi mosquitoes, from the colony created by rearing progeny of wild-caught mosquitoes (obtained from three different locations in southern India) for multiple generations, using 16S ribosomal RNA (rRNA) gene sequencing approach. Further, the influence of native midgut microbiota of mosquitoes on the development of rodent malaria parasite Plasmodium berghei in its host has been studied.MethodsThe microbial diversity associated with the midgut of An. stephensi mosquitoes was studied by sequencing V3 region of 16S ribosomal RNA (rRNA) gene. The influence of native midgut microbiota of An. stephensi mosquitoes on the susceptibility of the mosquitoes to rodent malaria parasite P. berghei was studied by comparing the intensity and prevalence of P. berghei infection among the antibiotic treated and untreated cohorts of mosquitoes.ResultsThe analysis of bacterial diversity from the midguts of An. stephensi showed Proteobacteria as the most dominant population among the three laboratory-reared strains of An. stephensi studied. Major genera identified among these mosquito strains were Acinetobacter, Pseudomonas, Prevotella, Corynebacterium, Veillonella, and Bacillus. The mosquito infectivity studies carried out to determine the implication of total midgut microbiota on P. berghei infection showed that mosquitoes whose native microbiota cleared with antibiotics had increased susceptibility to P. berghei infection compared to the antibiotic untreated mosquitoes with its natural native microbiota.ConclusionsThe use of microbial symbiont to reduce the competence of vectors involved in disease transmission has gained much importance in recent years as an emerging alternative approach towards disease control. In this context, the present study was aimed to identify the midgut microbiota composition of An. stephensi, and its effect on the development of P. berghei. Interestingly, the analysis of midgut microbiota from An. stephensi revealed the presence of genus Veillonella in Anopheles species for the first time. Importantly, the study also revealed the negative influence of total midgut microbiota on the development of P. berghei in three laboratory strains of An. stephensi, emphasizing the importance of understanding the gut microbiota in malaria vectors, and its relationship with parasite development in designing strategies to control malaria transmission.

Dataset Information

Quantitative real-time PCR analysis of Anopheles dirus TEP1 and NOS during Plasmodium berghei infection, using three reference genes.

Publications

Quantitative real-time PCR analysis of <i>Anopheles dirus TEP1</i> and <i>NOS</i> during <i>Plasmodium berghei</i> infection, using three reference genes.

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