BACKGROUND
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  <cit> .
wing patterns are also used in courtship and mate recognition, meaning they are both adaptive and contribute to genetic isolation and speciation  <cit> .
genetic crosses have shown that most phenotypic wing pattern and colour variation is controlled by a few mendelian loci  <cit> .
for example, in h. melpomene, genes in linkage group  <dig> control the yellow and white pattern elements  and genes in linkage group  <dig> control red pattern elements   <cit> .
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   <cit> .
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  <cit> .
key transcription factors are involved, such as notch, hedgehog and engrailed  <cit> , 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  <cit> .
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  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- <dig> nucleotides long, endogenously expressed non-coding rnas .
in animals, mature mirnas are derived from transcribed hairpin structures  of around 70- <dig> nucleotides in length that are processed by specialised proteins .
one strand of the resulting mirna duplex is incorporated into the rna-induced silencing complex .
post-transcriptional silencing is then mediated by binding of this complex to the 3' utr of the target messengerrna , which causes degradation or translational repression of the gene.
because the 5' seed sequence  that determines the mirna and mrna pairing is just seven nucleotides long in animals  <cit> , one mirna can have many potential targets and each mrna can be targeted by more than one mirna  <cit> .
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  <cit> .
one of the first mirnas to be discovered, let- <dig>  appears to act as an evolutionary conserved developmental timer, with temporal expression patterns being co-ordinated with progression to an adult fate  <cit> .
loss of let- <dig> function in drosophila leads to widespread defects during metamorphosis, including small wings  <cit> .
several other studies have shown a key role for mirnas in metamorphosis and many mirnas differ in their expression patterns across life stages  <cit> .
in the hemimetabolan insect blattella germanica, prevention of mirna processing by silencing of dicer- <dig> inhibits metamorphosis, with individuals retaining nymphoid features  <cit> .
newly emerged mirna genes have been detected in drosophila and these genes seem to be evolving adaptively and sometimes rapidly  <cit> .
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.
RESULTS
mirna characterisation in heliconius
a total of  <dig> , <dig> processed sequences were obtained from the small rna libraries developed for both heliconius races; with more reads being obtained for h. m. melpomene  than for h. m. rosina .
these data have been submitted to the ncbi gene expression omnibus   <cit>  under accession number gse <dig>  sequences were between  <dig> and  <dig> nucleotides in length, with  <dig> nucleotides being the most abundant length for both races .
a comparison of these sequences to mirnas housed in mirbase  revealed  <dig> different sequences with homology to previously identified mirnas.
<dig> % and  <dig> % of small rna sequences were identified as mirnas for h. m. melpomene and h. m. rosina respectively.
the remaining sequences are likely to include as yet unidentified heliconius mirnas and other types of small regulatory rnas.
a full list of detected mirnas, along with normalised abundances for each colour pattern race is given in additional file  <dig>  the sequences obtained for each race along with read counts are given in additional file  <dig>  and additional file  <dig> .
expression of mirnas
by far the most abundant mirna, mir- <dig>  accounts for > 50% of all sequenced mirnas in both colour pattern races.
altogether,  <dig> mirnas were present at more than five reads per million sequenced for at least one race and  <dig> had more than five reads per million for both races.
considering only these  <dig> mirnas,  <dig> had differences in abundance greater than 20% between the two races.
nine of these  were subject to northern blot analysis across time-staged pupal hindwing development .
for each race, hindwing tissue collected from two biological replicates at  <dig>   <dig> and  <dig> hours  post-pupation was compared to forewing and thorax tissue collected at the same time  and to a u <dig> non-coding small nuclear rna loading control.
in all cases the nine mirnas were expressed in the three tissue types, i.e.
none were either wing, or hindwing specific in their expression.
mir- <dig>  mir- <dig> and mir- <dig> showed a ubiquitous pattern of expression across stages and races.
mir- <dig> showed an upregulation in both races at  <dig> and  <dig> hours compared to  <dig> and this difference seems to be more pronounced in forewings than hindwings .
for all other mirnas, expression patterns varied across developmental stage and were mirrored in hindwings and forewings of both races, but not thorax.
mir- <dig> and mir- <dig> were up-regulated at  <dig> and  <dig> hours compared to 72; mir- <dig> was up-regulated at  <dig> and  <dig> hours compared to 24; mir- <dig> was up-regulated at  <dig> hours compared to  <dig> and  <dig> and mir- <dig> was up-regulated at  <dig> hours compared to  <dig> and  <dig>
identification of mirnas in hmyb
when short rna sequence reads were compared to all available h. melpomene bac sequences  two mirnas were identified.
a comparison of these sequences to mirnas in mirbase identified them as hme-mir- <dig> and hme-mir- <dig>  mir- <dig> is a conserved mirna found in mammals, birds, fish and insects.
mir- <dig> is a newly described mirna, identified at this time only in b. mori  <cit> .
their predicted stem-loop structures are shown in figure  <dig> and an alignment of sequenced reads to the bac sequence, along with each read count are given in additional file  <dig>  interestingly, they are located just  <dig> bp apart in the hmyb region, in an intergenic location between genes hm00025; a putative member of the fizzy family, and hm00026; a putative homologue of poly-specific ribonuclease .
the butterfly hme-mir- <dig> has three mismatches with its closest sequence, found in b. mori.
sequence abundances per million reads calculated allowing for these three mismatches between our sequences and those of mature mirnas present in mirbase  were  <dig>  and  <dig>  for hme-mir- <dig> and  <dig>  and  <dig>  for hme-mir- <dig>  for h. m. melpomene and h. m. rosina respectively.
therefore both showed a  <dig> -fold increase in normalised abundance in h. m. melpomene compared to h. m. rosina.
northern blot analyses of these two mirnas show that they are both expressed in forewings and hindwings, with no expression detected in thorax .
there was no evidence that they were differentially expressed between colour pattern races, however both show an upregulation from  <dig> to  <dig> hours post-pupation.
a further search for predicted heliconius mirnas within the bac sequences did not identify any new mirnas, although this analysis did again identify the two mirnas described above as well as their star sequences .
discussion
characterisation of butterfly mirnas
here we identify the first mirnas in butterflies and characterise their expression in developing larval and pupal wings.
the size distribution of sequenced reads showed peaks at  <dig> nucleotides for both heliconius races.
average mirna length is  <dig> nucleotides in animals  <cit> .
using similar deep sequencing techniques, other studies on insects show small rna sequence size distributions with a peak at  <dig> nucleotides  <cit>  and mirna sequence sizes appear to peak at 20- <dig> nucleotides in b. mori  <cit> .
in our data the peak at  <dig> nucleotides is due to mir- <dig>  which is most highly abundant as a 23mer in both races.
if this mirna is excluded, both races show peaks at  <dig> nucleotides, with another peak at  <dig> nucleotides that possibly represents piwi-interacting rnas; pirnas .
sequences were identified as mirnas only if they shared homology with other mirnas deposited in mirbase, or if their precursor sequences were identified in heliconius genomic dna.
this approach identified  <dig> butterfly mirnas.
recently deep sequencing, or a combination of deep sequencing and bioinformatics has identified  <dig> mirnas in the pea aphid, acyrthosiphon pisum  <cit> , and  <dig> and  <dig> mirnas in aedes and culex mosquitoes respectively  <cit> .
two different studies of b. mori yielded  <dig> conserved and  <dig> novel mirnas  <cit>  and  <dig> mirnas, of which  <dig> are described as unique to this species  <cit> .
at this time,  <dig> drosophila melanogaster mirnas have been deposited in mirbase.
all of our identified mirnas show homology to other mirnas in mirbase, as a search of genomic dna sequences failed to reveal any predicted heliconius specific mirna precursor sequences.
however, only  <dig> mb of genomic bac clone sequence is available at this time.
the completion of the heliconius genome-sequencing project in the near future will facilitate the identification of further mirnas.
these could include butterfly or heliconius specific mirnas, or mirnas that were not detected in this study as they are not expressed in wings.
expression of butterfly mirnas in developing wings
the most highly abundant mirnas, mir- <dig>  mir- <dig> and mir- <dig> account for around 60% of detected mirnas in both races.
all three of these mirnas are also highly conserved and may have important roles in butterfly wing development.
remarkably, mir- <dig> represented 51% and 56% of all mirnas sequenced in h. m. melpomene and h. m. rosina respectively and was expressed in all tissues and developmental stages examined.
this mirna has been best studied in humans, where its dysregulation is implicated in breast, lung, colorectal and head and neck cancers  <cit> .
in drosophila it is expressed in a pair rule pattern of  <dig> stripes, in the anterior endoderm and the hindgut  <cit> .
mir- <dig> depleted embryos complete development, but have severe segmentation defects including abnormal cuticle patterns and a complete loss of alternating segments  <cit> .
in b. mori it shows highest expression during newly hatched larva and moulting larval stages  <cit>  leading to the suggestion that it controls epithelial metabolism during moulting.
the very high abundance of this mirna in heliconius during 5th larval instar and pupal developmental stages suggests a role in butterfly metamorphosis.
in the drosophila embryo, mir- <dig> is expressed in the central nervous system, brain and imaginal discs  <cit> .
expression continues into the adult stages and it is also expressed throughout the life cycle of b. mori  <cit>  and aedes and culex mosquitoes  <cit> .
it is the most highly expressed mirna in the mosquito a. aegypti  <cit> .
hence this mirna may have a general function in insects.
however, it shows a decreasing pattern of expression from  <dig> to  <dig> hours post-pupation in both races of heliconius  indicating it may possibly have a more specialised role in butterfly wing development.
mir- <dig> is preferentially and abundantly expressed in b. mori pupae compared to adults  <cit> .
in drosophila it appears to have a conserved affiliation with sensory organ differentiation and is expressed in peripheral sense organs  <cit> .
drosophila deletion mutants show a sporadic loss of sensory bristles, revealing the role of this mirna in ensuring the developmental 'robustness' of patterning processes .
intriguingly, this mirna increases in expression dramatically in heliconius between  <dig> and  <dig> hours post-pupation in both forewings and hindwings.
insect sensory bristles and lepidopteron wing scales are widely believed to be homologous structures  and share both a specification mechanism and a notch-mediated lateral inhibition mechanism of patterning  <cit> .
early scale-forming cell determination occurs  <dig> hours after pupation, hence the timing of expression of mir- <dig> in heliconius could be suggestive of a shared role in the developmental pathway of insect sensory organ bristle and lepidopteron wing scale cell patterning.
most of the mirnas examined in this study showed dynamic changes in expression pattern throughout pupal wing development, indicating that mirnas in general have an important role in this process.
changes were mirrored in forewings and hindwings of both colour pattern races  indicating a role for these mirnas in general wing development rather than in specific colour patterning.
this was in contrast to the sequencing results, where differences in normalised read counts of sequences between races indicated that some mirnas might be differentially regulated in wings with different colour patterns.
great care was taken to standardise the pool of rna used for sequencing each race, such that each pool contained the same amount of rna from each developmental stage represented.
however, 5th instar larval wings in particular are difficult to stage with a high degree of accuracy and development is both dynamic and very rapid.
therefore it seems likely that most differences in mirna abundances observed from the sequence data are due to differences in developmental staging.
future studies of the role of mirnas in wing colour patterning and pigmentation could investigate mirnas that are less abundantly expressed, using more sensitive methods for analysing changes in expression  and also examine specific areas of developing wings  to look for regional-specific up- or down-regulation of mirnas.
an understanding of the dynamics of mirna expression in adult wings would also require further work.
it seems likely that different mirnas would be involved than those expressed during wing development, however once scale cells are developed and pigmented the wing tissue may not have high levels of transcriptional activity .
mirnas in hmyb
two mirnas, hme-mir- <dig> and hme-mir- <dig>  and their star sequences were discovered in the hmyb region.
these are located together in an intergenic region 276bp downstream of the stop codon of parn  and may be co-transcribed.
indeed, their sequences are both represented at a  <dig>  fold increase in abundance in h. m. melpomene.
the other flanking gene  shows differences in splicing that associate with wing colour pattern and is a possible candidate gene for the hmyb switch locus.
in contrast to the nine other mirnas examined by northern blot  these mirnas did not appear to be expressed in thorax tissue.
this implies a wing-specific role.
the northern blots show no evidence for differential expression of the two mirnas between colour pattern races.
furthermore, both are expressed in forewings and hindwings indicating that their expression is not specific to the yellow hindwing bar pattern encoded by hmyb.
however, they are strongly up-regulated between  <dig> and  <dig> hours in wings, when patterning and pigmentation processes are specified.
a role for these two mirnas in wing colour patterning will be the subject of future research.
CONCLUSIONS
here we present the first butterfly mirnas and investigate their role in wing development.
several conserved mirnas are highly expressed in heliconius pupal wings and one, mir- <dig>  could be involved in wing scale cell patterning in a developmental mechanism that is shared with insect sensory organ bristle patterning.
other mirnas show dynamic changes in expression in developing pupal wings, indicating an important role for mirnas in this process.
this study did not detect changes in expression of mirnas associated with wing colour pattern, however two mirnas were detected in the hmyb region that contains colour pattern switch loci.
these mirnas are located close together near a gene that associates with colour pattern and are up-regulated in wings between  <dig> and  <dig> hours post-pupation.
future work will investigate possible roles for these and other mirnas in wing colour patterning.

 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. genetic crosses have shown that most phenotypic wing pattern and colour variation is controlled by a few mendelian loci. for example, in h. melpomene, genes in linkage group control the yellow and white pattern elements and genes in linkage group control red pattern elements. 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. 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. key transcription factors are involved, such as notch, hedgehog and engrailed, 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. 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 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- nucleotides long, endogenously expressed non-coding rnas. in animals, mature mirnas are derived from transcribed hairpin structures of around 70- nucleotides in length that are processed by specialised proteins. one strand of the resulting mirna duplex is incorporated into the rna-induced silencing complex. post-transcriptional silencing is then mediated by binding of this complex to the 3' utr of the target messengerrna, which causes degradation or translational repression of the gene. because the 5' seed sequence 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- 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- 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. in the hemimetabolan insect blattella germanica, prevention of mirna processing by silencing of dicer- 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. 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.  mirna characterisation in heliconius a total of, processed sequences were obtained from the small rna libraries developed for both heliconius races; with more reads being obtained for h. m. melpomene than for h. m. rosina. these data have been submitted to the ncbi gene expression omnibus under accession number gse sequences were between and nucleotides in length, with nucleotides being the most abundant length for both races. a comparison of these sequences to mirnas housed in mirbase revealed different sequences with homology to previously identified mirnas. % and % of small rna sequences were identified as mirnas for h. m. melpomene and h. m. rosina respectively. the remaining sequences are likely to include as yet unidentified heliconius mirnas and other types of small regulatory rnas. a full list of detected mirnas, along with normalised abundances for each colour pattern race is given in additional file the sequences obtained for each race along with read counts are given in additional file and additional file. expression of mirnas by far the most abundant mirna, mir- accounts for > 50% of all sequenced mirnas in both colour pattern races. altogether, mirnas were present at more than five reads per million sequenced for at least one race and had more than five reads per million for both races. considering only these mirnas, had differences in abundance greater than 20% between the two races. nine of these were subject to northern blot analysis across time-staged pupal hindwing development. for each race, hindwing tissue collected from two biological replicates at and hours post-pupation was compared to forewing and thorax tissue collected at the same time and to a u non-coding small nuclear rna loading control. in all cases the nine mirnas were expressed in the three tissue types, i.e. none were either wing, or hindwing specific in their expression. mir- mir- and mir- showed a ubiquitous pattern of expression across stages and races. mir- showed an upregulation in both races at and hours compared to and this difference seems to be more pronounced in forewings than hindwings. for all other mirnas, expression patterns varied across developmental stage and were mirrored in hindwings and forewings of both races, but not thorax. mir- and mir- were up-regulated at and hours compared to 72; mir- was up-regulated at and hours compared to 24; mir- was up-regulated at hours compared to and and mir- was up-regulated at hours compared to and identification of mirnas in hmyb when short rna sequence reads were compared to all available h. melpomene bac sequences two mirnas were identified. a comparison of these sequences to mirnas in mirbase identified them as hme-mir- and hme-mir- mir- is a conserved mirna found in mammals, birds, fish and insects. mir- is a newly described mirna, identified at this time only in b. mori. their predicted stem-loop structures are shown in figure and an alignment of sequenced reads to the bac sequence, along with each read count are given in additional file interestingly, they are located just bp apart in the hmyb region, in an intergenic location between genes hm00025; a putative member of the fizzy family, and hm00026; a putative homologue of poly-specific ribonuclease. the butterfly hme-mir- has three mismatches with its closest sequence, found in b. mori. sequence abundances per million reads calculated allowing for these three mismatches between our sequences and those of mature mirnas present in mirbase were and for hme-mir- and and for hme-mir- for h. m. melpomene and h. m. rosina respectively. therefore both showed a -fold increase in normalised abundance in h. m. melpomene compared to h. m. rosina. northern blot analyses of these two mirnas show that they are both expressed in forewings and hindwings, with no expression detected in thorax. there was no evidence that they were differentially expressed between colour pattern races, however both show an upregulation from to hours post-pupation. a further search for predicted heliconius mirnas within the bac sequences did not identify any new mirnas, although this analysis did again identify the two mirnas described above as well as their star sequences. discussion characterisation of butterfly mirnas here we identify the first mirnas in butterflies and characterise their expression in developing larval and pupal wings. the size distribution of sequenced reads showed peaks at nucleotides for both heliconius races. average mirna length is nucleotides in animals. using similar deep sequencing techniques, other studies on insects show small rna sequence size distributions with a peak at nucleotides and mirna sequence sizes appear to peak at 20- nucleotides in b. mori. in our data the peak at nucleotides is due to mir- which is most highly abundant as a 23mer in both races. if this mirna is excluded, both races show peaks at nucleotides, with another peak at nucleotides that possibly represents piwi-interacting rnas; pirnas. sequences were identified as mirnas only if they shared homology with other mirnas deposited in mirbase, or if their precursor sequences were identified in heliconius genomic dna. this approach identified butterfly mirnas. recently deep sequencing, or a combination of deep sequencing and bioinformatics has identified mirnas in the pea aphid, acyrthosiphon pisum, and and mirnas in aedes and culex mosquitoes respectively. two different studies of b. mori yielded conserved and novel mirnas and mirnas, of which are described as unique to this species. at this time, drosophila melanogaster mirnas have been deposited in mirbase. all of our identified mirnas show homology to other mirnas in mirbase, as a search of genomic dna sequences failed to reveal any predicted heliconius specific mirna precursor sequences. however, only mb of genomic bac clone sequence is available at this time. the completion of the heliconius genome-sequencing project in the near future will facilitate the identification of further mirnas. these could include butterfly or heliconius specific mirnas, or mirnas that were not detected in this study as they are not expressed in wings. expression of butterfly mirnas in developing wings the most highly abundant mirnas, mir- mir- and mir- account for around 60% of detected mirnas in both races. all three of these mirnas are also highly conserved and may have important roles in butterfly wing development. remarkably, mir- represented 51% and 56% of all mirnas sequenced in h. m. melpomene and h. m. rosina respectively and was expressed in all tissues and developmental stages examined. this mirna has been best studied in humans, where its dysregulation is implicated in breast, lung, colorectal and head and neck cancers. in drosophila it is expressed in a pair rule pattern of stripes, in the anterior endoderm and the hindgut. mir- depleted embryos complete development, but have severe segmentation defects including abnormal cuticle patterns and a complete loss of alternating segments. in b. mori it shows highest expression during newly hatched larva and moulting larval stages leading to the suggestion that it controls epithelial metabolism during moulting. the very high abundance of this mirna in heliconius during 5th larval instar and pupal developmental stages suggests a role in butterfly metamorphosis. in the drosophila embryo, mir- is expressed in the central nervous system, brain and imaginal discs. expression continues into the adult stages and it is also expressed throughout the life cycle of b. mori and aedes and culex mosquitoes. it is the most highly expressed mirna in the mosquito a. aegypti. hence this mirna may have a general function in insects. however, it shows a decreasing pattern of expression from to hours post-pupation in both races of heliconius indicating it may possibly have a more specialised role in butterfly wing development. mir- is preferentially and abundantly expressed in b. mori pupae compared to adults. in drosophila it appears to have a conserved affiliation with sensory organ differentiation and is expressed in peripheral sense organs. drosophila deletion mutants show a sporadic loss of sensory bristles, revealing the role of this mirna in ensuring the developmental'robustness' of patterning processes. intriguingly, this mirna increases in expression dramatically in heliconius between and hours post-pupation in both forewings and hindwings. insect sensory bristles and lepidopteron wing scales are widely believed to be homologous structures and share both a specification mechanism and a notch-mediated lateral inhibition mechanism of patterning. early scale-forming cell determination occurs hours after pupation, hence the timing of expression of mir- in heliconius could be suggestive of a shared role in the developmental pathway of insect sensory organ bristle and lepidopteron wing scale cell patterning. most of the mirnas examined in this study showed dynamic changes in expression pattern throughout pupal wing development, indicating that mirnas in general have an important role in this process. changes were mirrored in forewings and hindwings of both colour pattern races indicating a role for these mirnas in general wing development rather than in specific colour patterning. this was in contrast to the sequencing results, where differences in normalised read counts of sequences between races indicated that some mirnas might be differentially regulated in wings with different colour patterns. great care was taken to standardise the pool of rna used for sequencing each race, such that each pool contained the same amount of rna from each developmental stage represented. however, 5th instar larval wings in particular are difficult to stage with a high degree of accuracy and development is both dynamic and very rapid. therefore it seems likely that most differences in mirna abundances observed from the sequence data are due to differences in developmental staging. future studies of the role of mirnas in wing colour patterning and pigmentation could investigate mirnas that are less abundantly expressed, using more sensitive methods for analysing changes in expression and also examine specific areas of developing wings to look for regional-specific up- or down-regulation of mirnas. an understanding of the dynamics of mirna expression in adult wings would also require further work. it seems likely that different mirnas would be involved than those expressed during wing development, however once scale cells are developed and pigmented the wing tissue may not have high levels of transcriptional activity. mirnas in hmyb two mirnas, hme-mir- and hme-mir- and their star sequences were discovered in the hmyb region. these are located together in an intergenic region 276bp downstream of the stop codon of parn and may be co-transcribed. indeed, their sequences are both represented at a fold increase in abundance in h. m. melpomene. the other flanking gene shows differences in splicing that associate with wing colour pattern and is a possible candidate gene for the hmyb switch locus. in contrast to the nine other mirnas examined by northern blot these mirnas did not appear to be expressed in thorax tissue. this implies a wing-specific role. the northern blots show no evidence for differential expression of the two mirnas between colour pattern races. furthermore, both are expressed in forewings and hindwings indicating that their expression is not specific to the yellow hindwing bar pattern encoded by hmyb. however, they are strongly up-regulated between and hours in wings, when patterning and pigmentation processes are specified. a role for these two mirnas in wing colour patterning will be the subject of future research.  here we present the first butterfly mirnas and investigate their role in wing development. several conserved mirnas are highly expressed in heliconius pupal wings and one, mir- could be involved in wing scale cell patterning in a developmental mechanism that is shared with insect sensory organ bristle patterning. other mirnas show dynamic changes in expression in developing pupal wings, indicating an important role for mirnas in this process. this study did not detect changes in expression of mirnas associated with wing colour pattern, however two mirnas were detected in the hmyb region that contains colour pattern switch loci. these mirnas are located close together near a gene that associates with colour pattern and are up-regulated in wings between and hours post-pupation. future work will investigate possible roles for these and other mirnas in wing colour patterning. 
future work will examine the expression of these mirnas in different colour pattern races and identify mirna targets among wing patterning genes. heliconius butterflies are an excellent system for studies of adaptive convergent and divergent phenotypic traits. we sequenced small rna libraries from two colour pattern races and detected heliconius mirnas with homology to others found in mirbase. wing colour patterns are used as signals to both predators and potential mates and are inherited in a mendelian manner. micrornas are important post-transcriptional regulators of gene expression that have key roles in many biological processes, including development. these candidates were tested further using northern blots, showing that differences in expression were primarily due to developmental stage rather than colour pattern. a search for mirnas in all available h. melpomene bac sequences did not reveal any other mirnas and no novel mirnas were predicted. for this reason we characterised mirnas in developing butterfly wings and examined differences in their expression between colour pattern races. several highly abundant mirnas were differentially represented in the libraries between colour pattern races. assembly of sequenced reads to the hmyb region identified hme-mir- and hme-mir-2788; located bp apart in an intergenic region. two mirnas were located in the hmyb region and were expressed in developing pupal wings. in heliconius melpomene, the yellow hindwing bar is controlled by the hmyb locus. mirnas could act as regulators of genes involved in wing development, patterning and pigmentation. these two mirnas are expressed in wings and show an upregulation between and hours post-pupation, indicating a potential role in butterfly wing development. some show differences in expression across developing pupal stages and may have important functions in butterfly wing development.
 heliconius butterflies are an excellent system for studies of adaptive convergent and divergent phenotypic traits. wing colour patterns are used as signals to both predators and potential mates and are inherited in a mendelian manner. the underlying genetic mechanisms of pattern formation have been studied for many years and shed light on broad issues, such as the repeatability of evolution. in heliconius melpomene, the yellow hindwing bar is controlled by the hmyb locus. micrornas are important post-transcriptional regulators of gene expression that have key roles in many biological processes, including development. mirnas could act as regulators of genes involved in wing development, patterning and pigmentation. for this reason we characterised mirnas in developing butterfly wings and examined differences in their expression between colour pattern races.  we sequenced small rna libraries from two colour pattern races and detected heliconius mirnas with homology to others found in mirbase. several highly abundant mirnas were differentially represented in the libraries between colour pattern races. these candidates were tested further using northern blots, showing that differences in expression were primarily due to developmental stage rather than colour pattern. assembly of sequenced reads to the hmyb region identified hme-mir- and hme-mir-2788; located bp apart in an intergenic region. these two mirnas are expressed in wings and show an upregulation between and hours post-pupation, indicating a potential role in butterfly wing development. a search for mirnas in all available h. melpomene bac sequences did not reveal any other mirnas and no novel mirnas were predicted.  here we describe the first butterfly mirnas and characterise their expression in developing wings. some show differences in expression across developing pupal stages and may have important functions in butterfly wing development. two mirnas were located in the hmyb region and were expressed in developing pupal wings. future work will examine the expression of these mirnas in different colour pattern races and identify mirna targets among wing patterning genes. 