Disruption of vitellogenin gene function in adult honeybees by intra-abdominal injection of double-stranded RNA

BACKGROUND: The ability to manipulate the genetic networks underlying the physiological and behavioural repertoires of the adult honeybee worker (Apis mellifera) is likely to deepen our understanding of issues such as learning and memory generation, ageing, and the regulatory anatomy of social systems in proximate as well as evolutionary terms. Here we assess two methods for probing gene function by RNA interference (RNAi) in adult honeybees. RESULTS: The vitellogenin gene was chosen as target because its expression is unlikely to have a phenotypic effect until the adult stage in bees. This allowed us to introduce dsRNA in preblastoderm eggs without affecting gene function during development. Of workers reared from eggs injected with dsRNA derived from a 504 bp stretch of the vitellogenin coding sequence, 15% had strongly reduced levels of vitellogenin mRNA. When dsRNA was introduced by intra-abdominal injection in newly emerged bees, almost all individuals (96 %) showed the mutant phenotype. An RNA-fragment with an apparent size similar to the template dsRNA was still present in this group after 15 days. CONCLUSION: Injection of dsRNA in eggs at the preblastoderm stage seems to allow disruption of gene function in all developmental stages. To dissect gene function in the adult stage, the intra-abdominal injection technique seems superior to egg injection as it gives a much higher penetrance, it is much simpler, and it makes it possible to address genes that are also expressed in the embryonic, larval or pupal stages

A member of the short-chain dehydrogenase/reductase (SDR) superfamily is a target of the ecdysone response in honey bee (Apis mellifera) caste development

Many aspects in caste polyphenism result from hormonally controlled differential gene expression. A DDRT-PCR screen for ecdysteroid-regulated genes in ovaries revealed a set of ESTs coding for metabolic enzymes. For a cDNA encoding a short-chain dehydrogenase/reductase (SDR) we obtained the complete coding sequence (246 amino acids), revealing the protein motifs typical of insect SDRs. Its initially high expression in early fifth-instar larvae vanished in prepupae. Expression levels in worker larvae were higher than in queen larvae, suggesting negative regulation by the caste-specific ecdysteroid titer. This finding was confirmed by in vitro exposure of competent worker ovaries to makisterone A. In contrast to whole body RNA extracts, two SDR transcripts were detected in the ovaries. Both had their expression downregulated by makisterone A. Hormonal regulation and tissue-specific expression pattern makes this SDR an interesting enzyme for comparative molecular studies on social insect caste polyphenisms