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

Author(s)

Freitas, Flavia C. P. ^{[1, 2]} ; Depintor, Thiago S. ^[1] ; Agostini, Lucas T. ^[3] ; Luna-Lucena, Danielle ^[1] ; Nunes, Francis M. F. ^{[1, 4]} ; Bitondi, Marcia M. G. ^[3] ; Simoes, Zila L. P. ^{[3, 1]} ; Lourenco, Anete P. ^{[1, 5]}

Affiliation

^[1] Univ Sao Paulo, Fac Med Ribeirao Preto, Dept Genet, Ribeirao Preto, SP - Brazil

^[2] Univ Fed Alfenas, Inst Ciencias Biomed, Alfenas, MG - Brazil

^[3] Univ Sao Paulo, Fac Filosofia Ciencias & Letras Ribeirao Preto, Dept Biol, Ribeirao Preto, SP - Brazil

^[4] Univ Fed Sao Carlos, Ctr Ciencias Biol & Saude, Dept Genet & Evolucao, Sao Carlos, SP - Brazil

^[5] Univ Fed Vales Jequitinhonha & Mucuri, Dep Ciencias Biol, Diamantina, MG - Brazil

• Document type: Journal article
• Source: SCIENTIFIC REPORTS; v. 9, NOV 27 2019.
• Web of Science Citations: 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-alpha, 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. (AU)

• FAPESP's process: 16/06657-0 - Analysis of the endocrine system and circadian cycle as modulators of Apis mellifera development
• Grantee: Zilá Luz Paulino Simões
• Support Opportunities: Regular Research Grants