Embryonic RNAi analysis in the firebrat, Thermobia domestica : Distal-less is required to form caudal filament

Ametabolous insects show direct development to the adult stage with almost no morphological changes. These insects are indispensable for understanding several of the most important aspects in development in insects, such as evolution of wing and metamorphosis. However, previous studies have concentrated on holometabolous or hemimetabolous insects based on the availability of well-established genetic analysis tools. Since the genetic analysis in ametabolous insects may be key to understanding molecular mechanism underlying insect evolution, gene functional analysis tools in ametabolous insects are needed. Here, we applied RNA interference (RNAi) as a gene functional analysis tool in the ametabolous insect, the firebrat, Thermobia domestica (Fig. 1A). RNAi is a posttranscriptional gene silencing process that allows rapid analysis of unknown gene function (Agrawal et al., 2003). In this process, double-stranded RNA (dsRNA) is degraded into small RNAs of ~20 to 30 nucleotides and is further unwound into single-stranded small RNAs. The target single-stranded RNA (guide strand) is loaded onto the nuclease complex, designated RISC (RNA-induced silencing complex), and then the RISC cleaves specific mRNA depending on the sequence of the guide strand (Siomi and Siomi, 2009). Since the first discovery of RNAi in Caenorhabditis elegans (Fire et al., 1998), this technique has been developed in various eukaryotic organisms and has been applied as a powerful reverse-genetic analysis tool not only in model system, but also in non-model systems (Agrawal et al., 2003). We tested the utility of RNAi using dsRNA based on the sequence of Distal-less (Dll) homolog from Thermobia. Dll encodes a transcription factor that includes a homeodomain as a DNA-binding domain (Cohen et al., 1989) and has a surprisingly well-conserved sequence among arthropods (Panganiban et al., 1995; Prpic and Tautz, 2003; Niimi et al., 2005). In Drosophila, Dll protein is required for the formation of ventral appendages, legs, antennae, mouthparts, genitalia, and analia (Cohen and Jürgens, 1989; Gorfinkiel et al., 1997, 1999; Campbell and Tomlinson, 1998; Moreno and Morata, 1999). This function of appendage formation has also been reported in other insects and non-insect arthropods, such as the spider mite, Tetranychus urticae (Khila and Grbić, 2007), the spider, Cupiennius saler (Schoppmeier and Damen, 2001), the milkweed bug, Oncopeltus fasciatus (Angelini and Kaufman, 2004), the ladybird beetle, Harmonia axyridis (Niimi et al., 2005), and the red flour beetle, Tribolium castaneum (Beermann et al., 2001; Bucher et al., 2002; Suzuki et al., 2009). Since Dll loss-of-function phenotype appears obvious truncations of the distal regions of appendages, it is a good marker to examine the utility of RNAi method. Corresponding to its function, Dll expression in the distal end of limbs has been observed in a wide range of organisms (Panganiban et al., 1995, 1997; Grenier et al., 1997). Remarkably, Dll expression in the caudal filament of Thermobia was reported by Rogers et al. (2002). In the caudal region of Thermobia, there are two types of outgrowth: cercus and caudal filament. Although these outgrowths have similar morphology (Fig. 1B), the developmental origin is different. The cauAmetabolous insects are important for understanding the mechanism of insect evolution based on their phylogenetic position. Thus, the development and application of an effective gene functional analysis using the RNA interference (RNAi) method is an important step in research on ametabolous insects. We tested RNAi utility in the firebrat, Thermobia domestica (Zygentoma, Lepismatidae) by focusing on the homeobox gene, Distal-less (Dll), based on its conserved sequence and obvious loss-of-function phenotype. Thermobia nymphs that were injected with Dll double-stranded RNA at an early embryonic stage displayed truncated appendages, and thus, we concluded that the RNAi method is useful for analyzing gene function in Thermobia. Remarkably, Dll RNAi induced truncation of the caudal appendage, cerci and the caudal non-appendage outgrowth, caudal filament. It is known that although these two caudal structures look similar, they have different origins. Our data suggests that these two types of outgrowths may be formed by similar developmental program, at least with respect to Dll, despite their different origins and that Dll even plays a role in a non-appendage structure.