title: Non-Target Effects of Green Fluorescent Protein (GFP)-Derived Double-Stranded RNA (dsRNA-GFP) Used in Honey Bee RNA Interference (RNAi) Assays.
abstract: RNA interference has been frequently applied to modulate gene function in organisms where the production and maintenance of mutants is challenging, as in our model of study, the honey bee, Apis mellifera. A green fluorescent protein (GFP)-derived double-stranded RNA (dsRNA-GFP) is currently commonly used as control in honey bee RNAi experiments, since its gene does not exist in the A. mellifera genome. Although dsRNA-GFP is not expected to trigger RNAi responses in treated bees, undesirable effects on gene expression, pigmentation or developmental timing are often observed. Here, we performed three independent experiments using microarrays to examine the effect of dsRNA-GFP treatment (introduced by feeding) on global gene expression patterns in developing worker bees. Our data revealed that the expression of nearly 1,400 genes was altered in response to dsRNA-GFP, representing around 10% of known honey bee genes. Expression changes appear to be the result of both direct off-target effects and indirect downstream secondary effects; indeed, there were several instances of sequence similarity between putative siRNAs generated from the dsRNA-GFP construct and genes whose expression levels were altered. In general, the affected genes are involved in important developmental and metabolic processes associated with RNA processing and transport, hormone metabolism, immunity, response to external stimulus and to stress. These results suggest that multiple dsRNA controls should be employed in RNAi studies in honey bees. Furthermore, any RNAi studies involving these genes affected by dsRNA-GFP in our studies should use a different dsRNA control.
authors.affiliation: Departamento de Genética, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, 14049-900, Brazil. francis.nunes@usp.br.
authors.forename: Francis M F
authors.initials: FM
authors.lastname: Nunes
authors.affiliation: Departamento de Genética, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, 14049-900, Brazil. alinea@usp.br.
authors.forename: Aline C
authors.initials: AC
authors.lastname: Aleixo
authors.affiliation: Departamento de Biologia Celular, Tecidual e do Desenvolvimento, Instituto de Ciências Biomédicas, Universidade Federal de Alfenas, Alfenas, Minas Gerais, 37130-000, Brazil. barchuk@unifal-mg.edu.br.
authors.forename: Angel R
authors.initials: AR
authors.lastname: Barchuk
authors.affiliation: Departamento de Genética, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, 14049-900, Brazil. anadurvalina@usp.br.
authors.forename: Ana D
authors.initials: AD
authors.lastname: Bomtorin
authors.affiliation: Department of Entomology, Center for Pollinator Research, Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, 16802, Pennsylvania, USA. cmgrozinger@psu.edu.
authors.forename: Christina M
authors.initials: CM
authors.lastname: Grozinger
authors.affiliation: Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, 14040-901, Brazil. zlpsimoe@usp.br.
authors.forename: Zilá L P
authors.initials: ZL
authors.lastname: Simões
keywords: Apis mellifera; GFP; RNAi; dsRNA; honeybee; off-target effects