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A non-invasive method for silencing gene transcription in honeybees maintained under natural conditions

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Abstract

In the Apis mellifera post-genomic era, RNAi protocols have been used in functional approaches. However, sample manipulation and invasive methods such as injection of double-stranded RNA (dsRNA) can compromise physiology and survival. To circumvent these problems, we developed a non-invasive method for honeybee gene knockdown, using a well-established vitellogenin RNAi system as a model. Second instar larvae received dsRNA for vitellogenin (dsVg-RNA) in their natural diet. For exogenous control, larvae received dsRNA for GFP (dsGFP-RNA). Untreated larvae formed another control group. Around 60% of the treated larvae naturally developed until adult emergence when 0.5 μg of dsVg-RNA or dsGFP-RNA was offered while no larvae that received 3.0 μg of dsRNA reached pupal stages. Diet dilution did not affect the removal rates. Viability depends not only on the delivered doses but also on the internal conditions of colonies. The weight of treated and untreated groups showed no statistical differences. This showed that RNAi ingestion did not elicit drastic collateral effects. Approximately 90% of vitellogenin transcripts from 7-day-old workers were silenced compared to controls. A large number of samples are handled in a relatively short time and smaller quantities of RNAi molecules are used compared to invasive methods. These advantages culminate in a versatile and a cost-effective approach.

Introduction

With its genome sequenced, Apis mellifera has moved into the post-genomic era, providing a valuable database for comparative genomics and, remarkably, the opportunity to investigate and understand the molecular events underlying complex honeybee traits and sociality (The Honeybee Genome Sequencing Consortium, 2006). To this end, functional approaches are currently underway in many laboratories. Because of their reproductive characteristics, the application of mutational genetics is infeasible for honeybees. The reverse genetics technique of RNA interference (RNAi) has emerged as a powerful tool for understanding gene function even in non-model insect species. RNAi has been used to suppress the expression of target mRNA by using complementary double-stranded RNA (dsRNA) molecules (Fire et al., 1998). In the first RNAi protocol developed for honeybees, embryos were microinjected at the anterior pole in the preblastoderm stage with dsRNA derived from a 300 base pair (bp) stretch of the E30 homeobox motif. This successfully disrupted the protein expression of engrailed ortholog throughout the whole embryo (Beye et al., 2002). Despite their success, subsequent studies showed that a significant number of embryos were lost by handling or by the detrimental effects of the injection, a fact also observed when the same protocol was used to silence vitellogenin (Amdam et al., 2003) and csp5 (Maleszka et al., 2007) genes in honeybees. Tissue damage surrounding the site of injection may explain the mortality rates observed in the RNAi-injected larvae (experimental group) and also in buffer-injected larvae (control group) (Aronstein and Saldivar, 2005). Intra-abdominal injection in adult workers has been widely used (Amdam et al., 2003, Guidugli et al., 2005a, Nelson et al., 2007). However, adult workers are strongly sensitive to manipulation, confinement and other exogenous factors, activating stress mechanisms that affect gene expression, hormonal titers, behavior and survival (Pankiw and Page, 2003, Sullivan et al., 2003, Lin et al., 2004, Barron et al., 2007). In A. mellifera, in vitro feeding of dsRNA via voluntary feeding to the young larvae, maintained in incubators, has being successful to achieve gene knockdown (Aronstein et al., 2006, Patel et al., 2007). During these studies larvae were maintained at optimal temperature and humidity conditions, and were offered diet qualitatively and quantitatively controlled, but putative collateral effects cannot be completely discarded. To circumvent physiological or survival problems we developed a non-invasive method for A. mellifera gene knockdown where age-controlled larvae received a single dose of dsRNA offered in the natural diet. The vitellogenin gene that encodes a yolk precursor (for review see Nelson et al., 2007) was selected as a model because its knockdown does not impair the natural progression of development and there is a well-established RNAi protocol (Amdam et al., 2003, Guidugli et al., 2005a, Nelson et al., 2007, Patel et al., 2007). The dsRNA delivery in the diet was able to successfully down-regulate the targeted mRNA (vitellogenin) in honeybee workers without disturbing the larvae physically or physiologically, which developed until emergence under natural conditions in the original colony.

Section snippets

Material and methods

Samples of Africanized A. mellifera were provided by the Apiary of the Department of Genetics, University of São Paulo, at Ribeirão Preto, in Brazil, where the experiments were also performed. The synthesis of dsRNA for vitellogenin (dsVg-RNA) was based on the well-established silencing system described previously (Amdam et al., 2003). For exogenous control, another dsRNA derived from Green Fluorescence Protein (dsGFP-RNA) was also produced. Briefly, primers fused with T7 promoter sequence

Results and discussion

Here we present a non-invasive method for honeybee gene knockdown during development, by oral delivery of dsRNA. Vitellogenin mRNA silencing was used as a model. To achieve this, two doses of dsRNA (0.5 μg and 3.0 μg of dsVg-RNA or dsGFP-RNA) was mixed with the natural diet of the 2nd instar larva diet, the dsGFP-RNA was used as an exogenous control. Around 60% of the larvae that received 0.5 μg of dsVg-RNA or dsGFP-RNA (as controls) developed and had the cells capped at the expected time. All

Acknowledgements

The authors would like to thank Tiago C. Pereira for helpful RNAi discussions, Angel R. Barchuk and Navdeep S. Mutti for critically reading and comments, Luiz R. Aguiar and Vera L.C. Figueiredo for beekeeping and technical assistance, and Juliana R. Martins for assisting with figure preparation. We are also grateful to Alexandre S. Martinez, Anete P. Lourenço, Fernando H. Biase, Márcia M.G. Bitondi and Thomas D. Schmittgen for their advices in statistical analysis of qPCR data, and Maria Helena

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