Over the last decade, RNAi has been successfully adopted as
the major genetic tool for gene function analysis in honey bees [3–5]. However, there can be
considerable variability in the levels and duration of transcript knockdowns between tissues,
individuals and experiments. In order to take full advantage of this powerful technology to study gene
function in honey bees and other insects, it is necessary to develop a complete understanding of the
mechanisms by which RNAi decreases transcript abundance and, in particular, to examine and
characterize any off-target effects.

Off-target effects are non-specific and caused by undesired base-pairing of non-target genes with
small interfering RNA (siRNA) derived from double-stranded RNA (dsRNA) [6]. Off-target effects
can be widespread and can alter expression of large numbers of genes, as previously reported in RNAi
experiments involving plants, invertebrates, vertebrates [7–9], as well as honey bees [10].
A green fluorescent protein (GFP)-derived dsRNA (dsRNA-GFP) has been used as an exogenous
control for RNAi assays in several arthropod species. Its gene sequence is not found in the honey bee genome. 

Although dsRNA-GFP is not expected to trigger an RNAi response in treated bees, undesirable effects on gene
expression, pupal pigmentation or developmental timing have been routinely observed. To better
understand the molecular and phenotypic effects of dsRNA-GFP in honey bees and to evaluate its use
as a control for RNAi studies, we examined the impact of dsRNA-GFP on global gene expression
patterns in developing workers. The dsRNA-GFP was introduced using a non-invasive feeding
protocol [23]. We found that dsRNA-GFP causes large-scale changes in gene expression associated
with multiple biological processes. 

Insects 2013, 4, 90-103; doi:10.3390/insects4010090

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