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

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.

Selection and Evaluation of Reference Genes for qRT-PCR in Spodoptera frugiperda (Lepidoptera: Noctuidae)

“…The qRT-PCR is a reliable technique in gene expression analysis, with high sensitivity and specificity, and the qRT-PCR data must be normalized by suitable reference genes to avoid expression differences among samples [31]. Recently, there have many reports on reference genes selection of various insects under different abiotic and biotic conditions [32][33][34][35][36], including S. frugiperda [27]. In these studies, the expression levels of conventional reference genes in different insects are quite different, and none of them with similar expression levels under all conditions, which indicates that there is no absolute universality among homologous reference genes.…”

Evaluation of Reference Genes in Glenea cantor (Fabricius) by Using qRT-PCR

“…For example, the number of developmental stage-specific reference genes was three for T. dendrolimi Matsumura ( SOD , GAPDH , and ACTIN ) [ 53 ]. The number of reference genes was five in Apis mellifera under different viral infections (IAPV infection: ache2 , rps18 , β-actin , tbp , and tif ; CSBV treatment: rpl14 , tif , rpsa , ubc , and ache2 ; and dsRNA treatment: Rpl14 , tif , rps18 , ubc , and α-tubulin ) [ 54 ]. In conclusion, the principle for the internal reference genes is that the ratio of the expression levels of the two optimal internal reference genes is consistent, regardless of the experimental conditions, and was not affected by differences in the gene expression [ 33 ].…”

IAPV-Induced Paralytic Symptoms Associated with Tachypnea via Impaired Tracheal System Function

“…To clearly understand the cause and effect between paralytic symptoms and respiratory failure, we quantified the expression levels of target genes from bees treated with dsRNA. dsRNAs were generated as previously described [ 33 ]. Briefly, the preparation of template DNA of two respiratory chain genes (SDH and COX) and GFP was performed with gene-specific amplicons, approximately 300–500 bp in length.…”