Discussion
This study presents the first functional analysis directed at elucidating the cis‐regulatory modularity of genes in Lepidoptera. Colour divergence in this insect order is thought to be the product of the regulatory evolution of genes coding for pigmentation and those controlling them (Jiggins et al., 2017). Although we have considerably increased our understanding of colour divergence, the functional evaluation of the underlying regulatory structures remains poorly understood. Here, we generated several transgenic lines and have provided functional validation of cis‐regulatory modularity in the yellow gene. Colour divergence of insects is based on various pigmentation in several tissues. Pigmentation genes have been found to be used recursively for tissue‐specific pigmentation, and these multiple uses place a pleiotropic constraint on protein‐coding sequence evolution. Based on our results, we suggest that the cis‐regulatory modularity of the pigmentation gene yellow has been a means to bypass pleiotropic constraints and facilitate gene expression and colour divergence.
The present study showed that many silkmoth larva body parts are pigmented through the expression and use of the yellow gene (Fig. 1). Nevertheless, we were not able to locate the CREs responsible for regulating the pigmentation of certain body parts (ie the head and the epidermis; Fig. 2). There are a few possible explanations for this result: (1) the CREs for these body parts are very distant from the genomic regions we investigated (22 kb in total); (2) expression of the yellow gene in these body parts requires the combinatorial regulation of several distinct genomic regions; or (3) the regulatory regions controlling yellow gene expression must be longer (eg ~100 kb) than those investigated in this study (~5 kb). Transposition efficiency has been shown to significantly decrease with an increase in the length of piggyBac (Ding et al., 2005). In any case, the complete identification of yellow CREs will thus require further investigation, and may require the construction of transgenic lines with longer genomic regions using bacteriophage phiC31 integrase (Yonemura et al., 2013). In Drosophila, phiC31‐mediated transgenesis integrates DNA fragments up to 133 kb long from Bacterial Artificial Chromosome clones (Venken et al., 2006). Although the present study did not completely identify all possible CREs controlling yellow gene expression in silkmoth larvae, it clearly demonstrates that cis‐regulatory modularity is involved in the regulation of yellow gene expression.
Interestingly, we found that a long genomic region is required to regulate yellow gene expression in B. mori, whereas CREs are usually restricted to ~500–2000 bp in other insect species (eg D. melanogaster, T. castaneum, A. gambiae; Cande et al., 2009). Furthermore, as transcription factor binding sites are several tens of base pairs long, it is reasonable to assume that CREs would be a few hundred base pairs long as they are collections of binding sites (Spitz and Furlong, 2012). One possible reason for the apparently longer genomic region required to regulate yellow gene expression in B. mori could be the difference in genome size between B. mori and D. melanogaster: the genome of B. mori (482 Mb) is three times larger than that of D. melanogaster (175 Mb) [cf. T. castaneum (166 Mb) and A. gambiae (265 Mb)]. The numerous repetitive sequences (eg transposons) in the B. mori genome account, in part, for its large size (Mita et al., 2004; Osanai‐Futahashi et al., 2008), as has been observed in other lepidopterans (Papa et al., 2008). As previously suggested, numerous repetitive sequences may facilitate the extraordinary colour and pattern divergence seen in Lepidoptera.
How did the CREs of the yellow gene evolve? Previous studies have offered two contrasting hypotheses: (1) de novo evolution and (2) inheritance from a common ancestor (Rebeiz et al., 2011). Whereas the yellow gene CREs have rapid evolutionary turnovers (Kalay and Wittkopp, 2010), it has been proposed that the yellow cis‐regulatory modules are conserved and inherited with some modifications (Gompel et al., 2005; Jeong et al., 2006; Prud’homme et al., 2006; Werner et al., 2010). Our experimental findings may support the de novo evolution hypothesis because no CRE sequence conservation was observed and the reciprocal tests demonstrated no reporter expression between B. mori and D. melanogaster. Furthermore, as there were no previous reports indicating yellow gene expression in the trachea of Drosophila larvae, and Drosophila larvae do not have thoracic legs, the results of our experiments did not substantiate the hypothesis that the three CREs in B. mori evolved from a common ancestor of the two species. Nevertheless, the results of this study do not completely rule out the possibility that the yellow CREs were inherited from a common ancestor and experienced substantial sequence turnovers until their sequence homology became undetectable. Because of its rapid turnover rate, yellow CREs have a limitation for tracing their origins between distantly related insects, such as B. mori and D. melanogaster. Comparison amongst B. mori and other lepidopterans may provide additional evidence for seeking the origins of B. mori yellow CREs.
The practical application of new technologies increases our understanding of the regulatory mechanisms underlying colour pattern divergence. Transgenic techniques have also been applied to construct transgenic strains in other lepidopteran species (Marcus et al., 2004; Haghighat‐Khah et al., 2015). Although previous studies have not investigated the regulatory region of pigmentation genes in lepidopteran insects, the regulatory regions of upstream regulatory genes that govern pattern formation, including transcription factors such as distal‐less (Carroll et al., 1994; Brakefield et al., 1996; Zhang and Reed, 2016), optix (Reed et al., 2011), cortex (Nadeau et al., 2016 ); and morphogens such as wingless (Martin and Reed, 2010; Yamaguchi et al., 2013) and wntA (Martin et al., 2012; Gallant et al., 2014; Martin and Reed, 2014) were suggested. Previous implications on such regulatory regions have only been supported by empirical evidence derived from sequence‐based observational methods such as Genome‐wide association study (Nadeau et al., 2016) and chromatin immunoprecipitation sequencing (Lewis et al., 2016). The integration of transgenic technologies demonstrated in the present study into sequence‐informed approaches would provide systematic insights for understanding regulatory mechanisms of lepidopteran colour patterns. As these technologies progress further, they can be exploited in other aspects of lepidopteran evo‐devo research [eg the identification of CREs that define the nymphalid ground plan (NGP) of moth and butterfly wing patterns (ngpCREs)]. We hope that the methods used in the present study can be applied to other nonmodel systems to further elucidate the regulatory mechanisms that drive evolutionary important traits.