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

• Published in: Scientific reports Vol. 9; no. 1; pp. 17692 - 13
• Main Authors: Freitas, Flávia C. P., Depintor, Thiago S., Agostini, Lucas T., Luna-Lucena, Danielle, Nunes, Francis M. F., Bitondi, Márcia M. G., Simões, Zilá L. P., Lourenço, Anete P.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 27-11-2019; Nature Publishing Group
• Subjects: 38/77; 631/1647/2017; 631/337/572; Animals; Bees; Bees - classification; Bees - genetics; Bees - growth & development; Bees - microbiology; Biodegradation; Endangered species; Environmental degradation; Escherichia coli; Escherichia coli Infections - genetics; Escherichia coli Infections - microbiology; Fat Body; Female; Gene expression; Gene Expression - drug effects; Genes, Essential; Glyceraldehyde-3-phosphate dehydrogenase; Head; Humanities and Social Sciences; Larva - genetics; Male; multidisciplinary; Ovary; Pesticides; Pesticides - pharmacology; Pollinators; Polymerase chain reaction; Pupa - genetics; Real-Time Polymerase Chain Reaction - methods; Reverse transcription; Science; Science (multidisciplinary); Sex
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-019-53544-0

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.