Screening and Validation of Reference Genes for RT-qPCR Under Different Honey Bee Viral Infections and dsRNA Treatment.

Yanchun Deng,Chunsheng Hou,Sa Yang,Hongxia Zhao,Li Zhang,Lina Zhang

doi:10.3389/fmicb.2020.01715

Abstract

Honey bee viruses are one of the most important pathogens that have contributed to the decrease in honey bee colony health. To analyze the infection dynamics of honey bee viruses, quantification of viral gene expression by RT-qPCR is necessary. However, suitable reference genes have not been reported from viral and RNAi studies of honey bee. Here, we evaluated the expression of 11 common reference genes (ache2, rps18, β-actin, tbp, tif, rpl32, gadph, ubc, α-tubulin, rpl14, and rpsa) from Apis mellifera (Am) and Apis cerana (Ac) under Israeli acute paralysis virus (IAPV), chronic bee paralysis virus (CBPV), and Chinese sacbrood virus (CSBV) infection as well as dsRNA-PGRP-SA treatment, and we confirmed their validation by evaluating the levels of the defensin 1 and prophenoloxidase (ppo) genes during viral infection. Our results showed that the expression of selected genes varied under different viral infections. ache2, rps18, β-actin, tbp, and tif can be used to normalize expression levels in Apis mellifera under IAPV infection, while the combination of actin and tif is suitable for CBPV-infected experiments. The combination of rpl14, tif, rpsa, ubc, and ache2 as well as more reference genes is suitable for CSBV treatment in Apis cerana. Rpl14, tif, rps18, ubc, and α-tubulin were the most stable reference genes under dsRNA treatment in Apis mellifera. Furthermore, the geNorm and NormFinder algorithms showed that tif was the best suitable reference gene for these four treatments. This study screened and validated suitable reference genes for the quantification of viral levels in honey bee, as well as for RNAi experiments.

Highlights

Honey bees provide a significant pollination service for many agricultural crops and wild plants
The first, second, and third groups consisted of bees treated with Israeli acute bee paralysis virus (IAPV), chronic bee paralysis virus (CBPV), and Chinese sacbrood virus (CSBV), respectively; the fourth group consisted of bees treated with double stranded RNA (dsRNA)-pgrpsa; and the fifth group was used to test the reliability of the reference genes
RT-qPCR is an important, simple and practical technique for assessing gene expression when compared to other quantitative methods such as Northern blotting, in situ hybridization and RNA-seq technology (Peng et al, 2018; Adeyinka et al, 2019; He et al, 2019)

Summary

INTRODUCTION

Honey bees provide a significant pollination service for many agricultural crops and wild plants. Reference Genes for Viral Infections pathogens play an important role in colony loss (Brutscher et al, 2015) Among these pathogens, RNA viruses are the major impacting factors, including two Apis mellifera viruses, Israeli acute bee paralysis virus (IAPV) (Maori et al, 2007) and chronic bee paralysis virus (CBPV) (Genersch and Aubert, 2010), as well as one Apis cerana virus, namely, Chinese sacbrood virus (CSBV) (Li et al, 2018). Suitable candidate reference genes have not been validated during honey bee viral infection Most of these genes displayed variable expression levels under different experimental conditions (Thellin et al, 2009). We tested the reliability of the reference genes by investigating the expression levels of two immune genes, defensin 1 and prophenoloxidase (ppo), under different viral infections. We hope that this report will provide a basis for future studies on gene expression in honey bee under viral infection

MATERIALS AND METHODS

RESULTS

DISCUSSION

Findings

DATA AVAILABILITY STATEMENT