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

Abstract

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.

Figure 1

Wild-type control.Time course illustrating individual variation in vitellogenin mRNA expression vs. vitellogenin protein levels in wild-type controls. Total RNA loaded for Northern blot was 6 μg. An equal hemolymph volume of 0.1 μl was loaded for SDS-PAGE. Apo-lipoprotein I [40] and ribosomic RNA were included as controls.

Figure 2

Targeted vitellogenin disruption. (a) Individual samples of workers injected as embryo and (b) workers injected as adults. All bees were sampled as 7 days old. Total RNA loaded was 6 μg, and an equal 0.1 μl hemolymph volume was loaded for SDS-PAGE. (c) Visualization of ~500 bp fragment in a worker injected as adult. Lane 1: Control. Lane 2: 6 μg total RNA. Lane 3: 20 μg total RNA. (d) Pooled samples of 10 workers each enriched with small RNA fragments. Two oligonucleotides (27 and 50 bp) were included as markers. Lane 1: Control. Lane 2: Adult workers injected with dsRNA at the preblastoderm stage. Lane 3: Intra-abdominally injected workers.

Figure 3

Sequence dependent Northern blot. Hybridization prepared with (a) AP4a5, and (b) AmR9. Lane 1: Control. Lane 2: Sample from an intra-abdominally injected worker, 6 μg total RNA. Lane 3: Original dsRNA template, 0.5 Ηg.