Evaluation of reference genes for gene expression analysis by real-time quantitative PCR (qPCR) in three stingless bee species (Hymenoptera: Apidae: Meliponini)

Flávia C P Freitas,Thiago S Depintor,Danielle Luna-Lucena,Francis M F Nunes,Márcia M G Bitondi,Lucas T Agostini,Zilá L P Simões,Anete P Lourenço

doi:10.1038/s41598-019-53544-0

Abstract

Stingless bees are generalist pollinators distributed through the pantropical region. There is growing evidence that their wild populations are experiencing substantial decline in response to habitat degradation and pesticides. Policies for conservation of endangered species will benefit from studies focusing on genetic and molecular aspects of their development and behavior. The most common method for looking at gene expression is real-time quantitative polymerase chain reaction preceded by reverse transcription (RT-qPCR) of the mRNA of interest. This method requires the identification of reliable reference genes to correctly estimate fluctuations in transcript levels. To contribute to molecular studies on stingless bees, we used Frieseomelitta varia, Melipona quadrifasciata, and Scaptotrigona bipunctata species to test the expression stability of eight reference genes (act, ef1-α, gapdh, rpl32, rps5, rps18, tbp, and tbp-af) in RT-qPCR procedures in five physiological and experimental conditions (development, sex, tissues, bacteria injection, and pesticide exposure). In general, the rpl32, rps5 and rps18 ribosomal protein genes and tpb-af gene showed the highest stability, thus being identified as suitable reference genes for the three stingless bee species and defined conditions. Our results also emphasized the need to evaluate the stability of candidate genes for any designed experimental condition and stingless bee species.

Highlights

A global effort to sequence genomes of all living species on Earth is in progress[1] with the aims of mitigating the impact of climate changes on biodiversity and preserving endangered species and ecosystems
The specificity of amplification of the eight candidate reference genes was initially tested for each species by conventional PCR and quantitative PCR (qPCR) using a pool of complementary DNA (cDNA) samples and genomic DNA
The expression of reference genes is used to correct the fluctuations in the target gene expression levels caused by technical variations in the quantity of total RNA or in the cDNA synthesis

Summary

Introduction

A global effort to sequence genomes of all living species on Earth is in progress[1] with the aims of mitigating the impact of climate changes on biodiversity and preserving endangered species and ecosystems. The expanded availability of genomic data during the last decade created a favorable condition for researchers to investigate a myriad of biological processes and their molecular bases, and explore the application of this knowledge to the areas of health, agriculture, industry, and ecology In this context, more than thirteen bee species have had their genomes sequenced, among which most are from the main tribes of corbiculate Apidae: Apini (Apis cerana, A. dorsata, A. florea, A. mellifera), Euglossini (Eufriesea mexicana, Euglossa dilemma), Bombini (Bombus terrestris, B. impatiens) and Meliponini (Melipona quadrifasciata)[3,4,5,6,7,8]. Our work contributes to studies that aim to investigate the molecular bases of events related to development, sex, tissues, immunity, and pesticide exposure in the three different models of stingless bee species here addressed

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Results

Conclusion