Assessment of Aedes albopictus reference genes for quantitative PCR at different stages of development.

Najat Dzaki,Ghows Azzam,Luciano Andrade Moreira

doi:10.1371/journal.pone.0194664

Abstract

Members of the Aedes genus of mosquitoes are widely recognized as vectors of viral diseases. Ae.albopictus is its most invasive species, and are known to carry viruses such as Dengue, Chikugunya and Zika. Its emerging importance puts Ae.albopictus on the forefront of genetic interaction and evolution studies. However, a panel of suitable reference genes specific for this insect is as of now undescribed. Nine reference genes, namely ACT, eEF1-γ, eIF2α, PP2A, RPL32, RPS17, PGK1, ILK and STK were evaluated. Expression patterns of the candidate reference genes were observed in a total of seventeen sample types, separated by stage of development and age. Gene stability was inferred from obtained quantification data through three widely cited evaluation algorithms i.e. BestKeeper, geNorm, and NormFinder. No single gene showed a satisfactory degree of stability throughout all developmental stages. Therefore, we propose combinations of PGK and ILK for early embryos; RPL32 and RPS17 for late embryos, all four larval instars, and pupae samples; eEF1-γ with STK for adult males; eEF1-γ with RPS17 for non-blood fed females; and eEF1-γ with eIF2α for both blood-fed females and cell culture. The results from this study should be able to provide a more informed selection of normalizing genes during qPCR in Ae.albopictus.

Highlights

Aedes albopictus (Ae. albopictus) is the most widely-travelled member of the Aedes genus [1]
Nine shortlisted candidates progressed through to the stages of analysis. They can be divided into four functional classes: (i) ribosomal and protein-production genes: RPS17, RPL32, eEF1-γ, and eIF2α; (ii) metabolism-related gene: PP2A; (iii) signal-transduction genes: PGK1, ILK and STK; and (iv) structural integrity gene: ACT
A standard curve for each primer pair was generated with pooled cDNA serially diluted by a factor of 5

Summary

Introduction

Aedes albopictus (Ae. albopictus) is the most widely-travelled member of the Aedes genus [1]. Due to increased trade and ease of transcontinental travel [2,3], they are found abundantly in the tropical Asian countries from which they originate, and temperate Asia, Europe, the Americas, Australia, and Africa [4]. Though less notorious than Aedes aegypti (Ae. Aegypti) as a vector of arboviruses, Ae. albopictus is acknowledged as an efficient vector of at least 22 viral strains [5]. Aegypti) as a vector of arboviruses, Ae. albopictus is acknowledged as an efficient vector of at least 22 viral strains [5] It carries a multitude of cross-species infecting bacteria [6,7].

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