Selection and validation of reference genes for qRT-PCR expression analysis of candidate genes involved in olfactory communication in the butterfly Bicyclus anynana.

Alok Arun,Gaël Amelot,Véronique Baumlé,Caroline M Nieberding

doi:10.1371/journal.pone.0120401

Abstract

Real-time quantitative reverse transcription PCR (qRT-PCR) is a technique widely used to quantify the transcriptional expression level of candidate genes. qRT-PCR requires the selection of one or several suitable reference genes, whose expression profiles remain stable across conditions, to normalize the qRT-PCR expression profiles of candidate genes. Although several butterfly species (Lepidoptera) have become important models in molecular evolutionary ecology, so far no study aimed at identifying reference genes for accurate data normalization for any butterfly is available. The African bush brown butterfly Bicyclus anynana has drawn considerable attention owing to its suitability as a model for evolutionary ecology, and we here provide a maiden extensive study to identify suitable reference gene in this species. We monitored the expression profile of twelve reference genes: eEF-1α, FK506, UBQL40, RpS8, RpS18, HSP, GAPDH, VATPase, ACT3, TBP, eIF2 and G6PD. We tested the stability of their expression profiles in three different tissues (wings, brains, antennae), two developmental stages (pupal and adult) and two sexes (male and female), all of which were subjected to two food treatments (food stress and control feeding ad libitum). The expression stability and ranking of twelve reference genes was assessed using two algorithm-based methods, NormFinder and geNorm. Both methods identified RpS8 as the best suitable reference gene for expression data normalization. We also showed that the use of two reference genes is sufficient to effectively normalize the qRT-PCR data under varying tissues and experimental conditions that we used in B. anynana. Finally, we tested the effect of choosing reference genes with different stability on the normalization of the transcript abundance of a candidate gene involved in olfactory communication in B. anynana, the Fatty Acyl Reductase 2, and we confirmed that using an unstable reference gene can drastically alter the expression profile of the target candidate genes.

Highlights

Butterflies have fascinated mankind throughout the ages with their wing coloration, association with angiosperms and life cycle involving metamorphosis
It has been demonstrated that B. anynana males use a male sex pheromone (MSP hereafter), produced by wing structures named androconia, which plays a key role in mate choice and sexual selection [13,14,15]
The protein sequences of the reference genes from various insects were used as a query to perform a TBLASTN search in B. anynana transcriptome in order to obtain the sequences of the closest B. anynana orthologs

Summary

Introduction

Butterflies have fascinated mankind throughout the ages with their wing coloration, association with angiosperms and life cycle involving metamorphosis. B. anynana has become a model in ecology and evolution, thanks to the coupling of in-depth field studies and experimental work conducted on labadapted population This strategy has unraveled the ecological relevance of a large range of phenotypes associated, for example, with seasonal polyphenism producing adaptive phenotypes to the alternating wet and dry seasons in East Africa, life-history traits relevant to ageing research and to sexual selection [10,12]. In this regard, it has been demonstrated that B. anynana males use a male sex pheromone (MSP hereafter), produced by wing structures named androconia, which plays a key role in mate choice and sexual selection [13,14,15]. We validated the selection of our reference genes by evaluating across different tissues, the expression profile of a fatty-acyl reductase gene (FAR 2 hereafter) involved in sex pheromone biosynthesis in the Lepidoptera species B. anynana [36]

Materials and Methods

Results and Discussion

Conclusion