Larval RNAi in Tribolium (Coleoptera) for analyzing adult development
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We report here on the use of RNA interference (RNAi) to create pupal and adult loss-of-function phenotypes in the red flour beetle, Tribolium castaneum, by injection of double-stranded RNA (dsRNA) into late instar larvae (we refer to this method as larval RNAi). RNAi is well-established as a useful method to mimic loss-of-function phenotypes in many organisms including insects. However, with a few exceptions (such as in the fruit fly Drosophila melanogaster), RNAi analysis has usually been limited to studies of embryogenesis. Here we demonstrate that injection of green fluorescent protein (GFP) dsRNA into the larval body cavity can inhibit GFP expression beginning shortly after injection and continuing through pupal and adult stages. RNAi analysis of the Tc-achaete-scute-homolog (Tc-ASH) revealed that larval RNAi can induce morphological defects in adult beetles, and also that larval RNAi affects the entire body rather than being localized near the site of injection. The larval RNAi technique will be useful to analyze gene functions in post-embryonic development, giving us the opportunity to study the molecular basis of adult morphological diversity in various organisms.
KeywordsRNAi Injection Larval body cavity Post-embryonic stage Tribolium castaneum (red flour beetle)
We thank G. Bucher, M. Weber and M. Klingler for the pu11 line and S. Wheeler for the Tc-ASH cDNA. We also thank T. Shippy for critical reading of the manuscript, S. Brown, R. Beeman, and all the Manhattan beetle lab members for helpful discussion. This work was supported by the international Human Frontier Science Program Organization (Long-term Fellow) and the National Science Foundation.
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