Parental RNAi in Tribolium (Coleoptera)

RNA interference (RNAi) allows for rapid and straightforward analysis of gene function. RNAi can be applied with special ease in Caenorhabditis elegans where injection of dsRNA into the body cavity or application of dsRNA via ingestion leads to gene inactivation in offspring embryos [1,2]. This parental RNAi effect made possible efficient genome-wide functional screens in this organism [3]. Here we show for the flour beetle Tribolium castaneum that injection of double stranded RNA (dsRNA) into the mother's haemocoel results in knock down of zygotic genes in offspring embryos ('parental RNAi'). This suggests that transfer across cell boundaries is an ancient feature of the RNAi pathway, and opens up new applications in functional genomics and in the study of developmental evolution. We tested parental RNAi in Tribolium for two genes of known function, the leg gene Distalless (Tc'Dll) [4] and the homeotic gene maxillopedia (mxp), a Tribolium homologue of proboscipedia (pb) [5]. In addition, we applied this method to investigate the function of a newly isolated segmentation gene, the Tribolium giant homologue (Tc'gt). For injection, female Tribolium pupae were affixed to microscope slides with a drop of rubber cement (Fixogum, Marabu) at their posterior abdomen. Using a simple micromanipulator setup, approximately 0.15 µ µl of dsRNA solution (0.2–2 mg/ml in injection buffer [1]) were injected per pupa, at a ventro-lateral position between abdominal segments 3 and 4. After completion of metamorphosis, these females were mated to untreated males, and eggs were harvested in weekly time intervals. For all three genes, nearly all offspring larvae (665 of 669) displayed gene specific phenotypes one week after injection. Buffer injected females and dsRNA injected males gave rise to normal offspring. For Tc'Dll and mxp, the phenotypes obtained by parental RNAi (Figure 1B,C,E) were similar to those of partial or complete loss of function mutants [4,5]. This shows that parental RNAi faithfully reproduces reduction or inactivation of gene function in Tribolium. As neither Tc'Dll nor mxp are maternally provided, it is evident that parental RNAi can knockdown zygotic gene expression in offspring embryos. In the case of Tc'giant, parental RNAi phenotypes were obtained similar to those generated by injecting embryos with dsRNA or with morpholino oligonucleotides designed to inhibit translation of Tc'giant mRNA (not shown). The congruence of these data also shows that the observed parental Tc'gt phenotype is a gene-specific effect. Parental RNAi is highly efficient in Tribolium: in all three experiments nearly 100% …