Stable reference genes for the measurement of transcript abundance during larval caste development in the honeybee

Abstract

-Many genes are differentially regulated by caste development in the honeybee. Identifying and understanding these differences is key to discovering the mechanisms underlying this process. To identify these gene expression differences requires robust methods to measure transcript abundance. RT-qPCR is currently the gold standard to measure gene expression, but requires stable reference genes to compare gene expression changes. Such reference genes have not been established for honeybee caste development. Here, we identify and test potential reference genes that have stable expression throughout larval development between the two female castes. In this study, 15 candidate reference genes were examined to identify the most stable reference genes. Three algorithms (GeNorm, Bestkeeper and NormFinder) were used to rank the candidate reference genes based on their stability between the castes throughout larval development. Of these genes Ndufa8 (the orthologue of a component of complex one of the mitochondrial electron transport chain) and Pros54 (orthologous to a component of the 26S proteasome) were identified as being the most stable. When these two genes were used to normalise expression of two target genes (previously found to be differentially expressed between queen and worker larvae by microarray analysis) they were able to more accurately detect differential expression than two previously used reference genes (awd and RpL12). The identification of these novel reference genes will be of benefit to future studies of caste development in the honeybee.caste development / larval development / gene expression

Immunogene and viral transcript dynamics during parasitic Varroa destructor mite infection of developing honey bee (Apis mellifera) pupae

“…One solution is to search for suitable reference genes to normalize quantitative PCR results until specific reference genes for an experimental paradigm have been identified (Cameron et al, 2013;Reim et al, 2013). However, large sets of candidate genes may have to be studied without a priori information, particularly in two-factorial experimental designs that cross developmental stages (Cameron et al, 2013), such as ours. To reduce normalization bias, multiple reference genes can be combined (Lourenco et al, 2008).…”

“…However, raw C t values of all honey bee transcripts were more correlated with each other than ∆C t values. Therefore, we preferentially present and discuss the relative differences of immune genes but recommend a wider set of reference genes (but see Boncristiani et al, 2013) or specific sets of reference genes (Cameron et al, 2013;Reim et al, 2013) in the future. Regardless of the method of quantification, the experimental treatments unambiguously and consistently affected the titer of DWV in the developing bees.…”

Starvation stress during larval development facilitates an adaptive response in adult worker honey bees (Apis mellifera L.)

“…Each biological sample was run in technical triplicate on an ABI Prism 7500 Real-Time PCR system (Applied Biosystems) for measuring candidate gene transcript levels, in comparison with those of the reference gene actin by means of the ΔΔCt method (Livak and Schmittgen, 2001). Studies have shown that actin is stably expressed during honey bee development (Lourenco et al, 2008;Reim et al, 2012) as well as in adults (Cameron et al, 2013). Therefore, actin is commonly used as a reference gene in studies of honey bee gene expression (Scharlaken et al, 2008;de Azevedo and Hartfelder, 2008;Lourenco et al, 2008;Wang et al, 2009).…”

Insulin-like peptides (AmILP1 and AmILP2) differentially affect female caste development in the honey bee (Apis mellifera)

“…Each biological sample was run in technical triplicate on an ABI Prism 7500 Real-Time PCR system (Applied Biosystems) for measuring AmILP1 and AmILP2 transcript levels in comparison with those of the reference gene Amrp49 by means of the ΔΔCt method (Livak and Schmittgen, 2001). Studies have shown that Amrp49, which has been renamed as rpl23 (AF441189) in the honey bee genome version Amel 4.5, is stably expressed during larval development (Lourenço et al, 2008;Reim et al, 2013) and in adults (Cameron et al, 2013). Therefore Amrp49 is commonly used as a reference gene during the larval stage (de Azevedo and Hartfelder, 2008;Martins et al, 2010) and also the adult stage of honey bees (Ben-Shahar et al, 2003;Navajas et al, 2008).…”