Specific developmental gene silencing in the honey bee using a homeobox motif

Abstract

Manipulating the expression of genes in species that are not currently used as genetic models will provide comparative insights into the evolution of gene functions. However the experimental tools in doing so are limited in species that have not served as models for genetic studies. We have examined the effects of double stranded RNA (dsRNA) in the honey bee, an insect with considerably basic scientific interest. dsRNA derived from a 300 bp stretch of the E30 homeobox motif was injected into honey bee embryos at the anterior pole in the preblastoderm stage. We found that the dsRNA fragment successfully disrupted the protein expression of the target gene throughout the whole embryo. The disruption caused deficient phenotypes similar to known loss of function mutants of Drosophila engrailed, whereas embryos injected with nonsense dsRNA showed no abnormalities. We show that the large size of the honey bee egg (D: 0.3 mm, L: 1.6 mm) and the long preblastoderm stage (11-12 h) can be exploited to generate embryos with partial disruption of gene function, which may provide an elegant alternative to classical chimeric analyses. This is the first report of targeted disruption of gene function in the honey bee, and the results prove that the chosen target gene is a functional ortholog to engrailed in Drosophila.

RNA Interference with Special Reference to Combating Viruses of Crustacea

“…To date, RNAi related events have been recognised in almost all eukaryotic animals including algae [202], yeast [151], protozoans [11,27,94,118], plants [43,196], insects [2,17,26,77,100], fish [29,131] and in mammals [38,48,121,198].…”

“…Following the 1998 demonstration of dsRNA-mediated RNAi in C. elegans [45], the use of RNA silencing as a nucleic acid-based antiviral immunity in invertebrates has been established in a number of arthropod species including the fruit fly [80], honeybee [17] and mosquitoes [2]. Furthermore, RNAi inhibition of viral replication has been demonstrated in vitro for a variety of viruses in plants and mammals [12,38,48,55,121].…”

Downregulation of vitellogenin gene activity increases the gustatory responsiveness of honey bee workers (Apis mellifera)

“…Thus, the two injected groups described in Section 2.1 received 2 μl dsRNA solution (5 μg dsRNA per μl nuclease free water) against the vitellogenin gene (i.e., "Vg-RNAi" treatment) or derived from GFP sequence (i.e., "GFPControls"), respectively. GFP-derived dsRNA does not share close homology with Apis mellifera genes, and close homology is required to trigger RNAi [19][20][21][22][23]. Therefore, injections of GFP-derived dsRNA allowed us to control for effects stemming from the full injection procedure; including handling, wounding, and transfer of dsRNA template.…”

Order, Disorder, Death: Lessons from a Superorganism

“…The bee is an established model in systems theory (Mitchell, 2003;Page and Erber, 2002), behavioral ecology (Seeley, 1995), neurobiology (Farooqui et al, 2004;Humphries et al, 2003;Menzel, 1979;Scheiner et al, 2001), and aging (Amdam and Page, 2005;Omholt and Amdam, 2004;Seehuus et al, 2006). It has a rising position in molecular research that is fueled by an emerging availability of functional genomic tools Beye et al, 2002;Omholt et al, 1995;Yu et al, 1997) and a newly annotated genome sequence (www.hgsc.bcm.tmc.edu).…”