Neotropical butterflies of the genus Heliconius provide striking examples of both divergence and convergence in their wing colour patterns. Distributed throughout the tropical forests of central and southern America, they signal their distastefulness to predators through brightly coloured wings. Many species take part in Müllerian mimicry 'rings', where multiple species converge in wing pattern and thereby benefit through protection from predators . Wing patterns are also used in courtship and mate recognition, meaning they are both adaptive and contribute to genetic isolation and speciation [2, 3]. Genetic crosses have shown that most phenotypic wing pattern and colour variation is controlled by a few Mendelian loci [4, 5]. For example, in H. melpomene, genes in linkage group 15 control the yellow and white pattern elements (HmYb/Sb/N) and genes in linkage group 18 control red pattern elements (HmB/D) [6–8]. The HmYb locus, which controls the presence or absence of a hindwing yellow bar, is orthologous to Cr in the mimetic species H. erato. The loci are found in the same genomic location in these two species and interestingly, also in that of the P supergene locus of H. numata (which controls a polymorphic whole-wing patterning system) . This suggests that in Heliconius the same genetic loci are involved in the repeated evolution of adaptive traits.
In other butterfly species, such as Bicyclus anynana, conserved developmental pathways appear to have been co-opted to a role in development of wing pattern elements like eyespots [10, 11]. Key transcription factors are involved, such as Notch, Hedgehog and Engrailed [12–14], which have possibly evolved their new role through cis -regulatory changes. The developmental basis of wing colour patterning in Heliconius has yet to be elucidated. Positional cloning and sequencing of the HmYb and HmB/D loci and their orthologous loci in H. erato have revealed genes that have not been implicated previously in butterfly wing patterning [15–17]. Work is ongoing to further identify the switch genes within the HmYb and HmB/D regions using population genetics and gene expression approaches. Genetic changes at these switch genes among different colour pattern races are likely to involve cis -regulatory or coding sequence changes, changes to post-transcriptional control or a combination of these. MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression that have been particularly implicated in the fine-tuning of transient and complicated developmental processes. Therefore, they could have a role in the regulation and development of wing patterning that occurs during larval/pupal transitional stages in butterflies.
miRNAs are 19-25 nucleotides long, endogenously expressed non-coding RNAs (for a review see ). In animals, mature miRNAs are derived from transcribed hairpin structures (pre-miRNAs) of around 70-90 nucleotides in length that are processed by specialised proteins (Dicer proteins). One strand of the resulting miRNA duplex is incorporated into the RNA-induced silencing complex (RISC). Post-transcriptional silencing is then mediated by binding of this complex to the 3' UTR of the target messengerRNA (mRNA), which causes degradation or translational repression of the gene. Because the 5' seed sequence (at nucleotide positions 2-8) that determines the miRNA and mRNA pairing is just seven nucleotides long in animals , one miRNA can have many potential targets and each mRNA can be targeted by more than one miRNA .
Some miRNAs are remarkably conserved across distant orders, suggesting conserved evolutionary functions. For example, over half of Caenorhabditis elegans miRNAs share sequence homology with those found in human and Drosophila . One of the first miRNAs to be discovered, let-7, appears to act as an evolutionary conserved developmental timer, with temporal expression patterns being co-ordinated with progression to an adult fate . Loss of let-7 function in Drosophila leads to widespread defects during metamorphosis, including small wings . Several other studies have shown a key role for miRNAs in metamorphosis and many miRNAs differ in their expression patterns across life stages [24–27]. In the hemimetabolan insect Blattella germanica, prevention of miRNA processing by silencing of Dicer-1 inhibits metamorphosis, with individuals retaining nymphoid features . Newly emerged miRNA genes have been detected in Drosophila and these genes seem to be evolving adaptively and sometimes rapidly [29, 30]. Hence, in addition to performing conserved roles, miRNAs could also be involved in the fine-tuning of gene expression patterns underlying the evolution of novel phenotypic traits.
In this study we generated and sequenced small RNA libraries from mixed larval and pupal wings of two colour pattern races of H. melpomene; H. m. rosina, which has the yellow hindwing bar encoded by the HmYb locus and H. m. melpomene, which does not. Our aims were to characterise the first miRNAs in Heliconius butterflies, to examine differences in expression between two colour pattern races and to identify miRNAs encoded within the HmYb region and elsewhere in the genome.