Female Behaviour Drives Expression and Evolution of Gustatory Receptors in Butterflies

<div><p>Secondary plant compounds are strong deterrents of insect oviposition and feeding, but may also be attractants for specialist herbivores. These insect-plant interactions are mediated by insect gustatory receptors (<i>Grs</i>) and olfactory receptors (<i>Ors</i>). An analysis of the reference genome of the butterfly <i>Heliconius melpomene</i>, which feeds on passion-flower vines (<i>Passiflora</i> spp.), together with whole-genome sequencing within the species and across the <i>Heliconius</i> phylogeny has permitted an unprecedented opportunity to study the patterns of gene duplication and copy-number variation (CNV) among these key sensory genes. We report <i>in silico</i> gene predictions of 73 <i>Gr</i> genes in the <i>H. melpomene</i> reference genome, including putative CO₂, sugar, sugar alcohol, fructose, and bitter receptors. The majority of these <i>Grs</i> are the result of gene duplications since <i>Heliconius</i> shared a common ancestor with the monarch butterfly or the silkmoth. Among <i>Grs</i> but not <i>Ors</i>, CNVs are more common within species in those gene lineages that have also duplicated over this evolutionary time-scale, suggesting ongoing rapid gene family evolution. Deep sequencing (∼1 billion reads) of transcriptomes from proboscis and labial palps, antennae, and legs of adult <i>H. melpomene</i> males and females indicates that 67 of the predicted 73 <i>Gr</i> genes and 67 of the 70 predicted <i>Or</i> genes are expressed in these three tissues. Intriguingly, we find that one-third of all <i>Grs</i> show female-biased gene expression (n = 26) and nearly all of these (n = 21) are <i>Heliconius</i>-specific <i>Grs</i>. In fact, a significant excess of <i>Grs</i> that are expressed in female legs but not male legs are the result of recent gene duplication. This difference in <i>Gr</i> gene expression diversity between the sexes is accompanied by a striking sexual dimorphism in the abundance of gustatory sensilla on the forelegs of <i>H. melpomene</i>, suggesting that female oviposition behaviour drives the evolution of new gustatory receptors in butterfly genomes.</p>