Comparative analysis of reference genes in honey bees, <i>Apis cerana</i> and <i>Apis mellifera</i>

Honey bees are vital pollinators in agriculture and important model insects. To understand the genetic and molecular aspects in their development, a reverse transcription quantitative polymerase chain (RT-qPCR) is used to investigate the target genes. However, it is essential to use the appropriate reference genes as endogenous controls for accurate normalization of target genes. To identify stable reference genes in two honey bee species, [<i>Apis mellifera</i> (<i>Am</i>) and <i>Apis cerana</i> (<i>Ac</i>)], we evaluated eight candidate reference genes including, <i>actin</i>, <i>atub</i>, <i>ef1α</i>, <i>gapdh</i>, <i>rpl13a</i>, <i>rpl32</i>, <i>rps18</i> and <i>tif</i>. Worker bees belonging to the two species were collected at each developmental day during the embryonic and postembryonic developmental stages. The <i>tyrosine hydroxylase</i> (<i>th</i>) gene was used as the target gene to validate the selected reference genes. Our results revealed that <i>rpl13a</i> was the most stable reference gene at all developmental stages of <i>Am</i> and <i>Ac</i>. In addition, gene combinations, including <i>Amrpl13a</i> &amp; <i>Amrps18</i> &amp; <i>Amactin</i>, <i>Amrpl13a</i> &amp; <i>Amrpl32</i>, <i>Acrpl13a</i> &amp; <i>Acrpl32</i>, <i>Acrpl13a</i> &amp; <i>Acrpl32</i> &amp; <i>Acef1α</i> followed by other combinations effectively normalized the expression of the target genes during the embryonic and postembryonic developmental stages of <i>Am</i> and <i>Ac</i>, respectively. Our findings provide a foundation for standardized RT-qPCR analysis to improve the accuracy of genes normalization during the different developmental stages of honey bees.