Validation of quantitative real-time PCR reference genes for the determination of seasonal and labor-specific gene expression profiles in the head of Western honey bee, Apis mellifera

Kyunghwan Moon,Si Hyeock Lee,Young Ho Kim,Wolfgang Blenau

doi:10.1371/journal.pone.0200369

Kyunghwan Moon, Si Hyeock Lee + Show 2 more

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https://doi.org/10.1371/journal.pone.0200369

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Abstract

Honey bee is not only considered an important pollinator in agriculture, but is also widely used as a model insect in biological sciences, thanks to its highly evolved sociality, specialization of labor division, and flexibility of colony management. For an intensive investigation of the seasonal and labor-dependent expression patterns of its genes, accurate quantification of the target gene transcription level is a fundamental step. To date, quantitative real-time PCR (qRT-PCR) has been widely used for rapid quantification of gene transcripts, with reliable reference gene(s) for normalization. To this end, in an attempt to search for reliable reference genes, the amplification efficiencies of six candidate reference genes (rp49, rpL32, rpS18, tbp, tub, and gapdh) were determined. Subsequently, four genes (rpL32, rpS18, tbp, and gapdh) with PCR efficiencies of 90% to 110% were evaluated for their expression stabilities with three programs (geNorm, NormFinder, and BestKeeper) and used for normalization of seasonal expression patterns of target genes in the forager and nurse heads. Although the three programs revealed slightly different results, two genes, rpS18 and gapdh, were suggested to be the optimal reference genes for qRT-PCR-based determination of seasonal and labor-specific gene expression profiles. Furthermore, the combined use of these two genes yielded a more accurate normalization, compared with the use of a single gene in the head of honey bee. The validated reference genes can be widely used for quantification of target gene expression in honey bee head although it is still remained to be elucidated the expression levels of the selected reference genes in specific tissues in head.

Highlights

The Western honey bee, Apis mellifera L., is a keystone pollinator in both natural and agricultural ecosystems
Six potential reference genes were initially amplified by reverse transcription PCR with designed primer sets (S1 Table), with total RNA extracted from the head of honey bees as a template, and the amplicons were visualized on the 2% agarose gel
We investigate the transcript levels of four candidate reference genes and evaluate their expression stability using three different algorithms, in the head of foragers and nurses collected over a yearlong cycle on a monthly basis

Summary

Introduction

The Western honey bee, Apis mellifera L., is a keystone pollinator in both natural and agricultural ecosystems. In the United States, the monetary value of honey bees as commercial pollinators is estimated at more than $15 billion annually: they pollinate almost 80% of the agricultural fruits and vegetables. The many tasks associated with colony maintenance and growth, such as hive clearing, brood rearing, and foraging, are divided among the worker bees in an age-based fashion, a phenomenon known as polyethism [3, 4]. From days 13 to 20, the middle-age bees engage in hive maintenance and food storage in the peripheral region. At day 20, the bees defend the colony at the entrance of the hive as a guard, and after a few days, they forage for nectar and pollen outside the nest until they die [6, 7]

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