Characterisation and expression of microRNAs in developing wings of the neotropical butterfly Heliconius melpomene

Abstract

Background: 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 (miRNAs) 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.

Results: We sequenced small RNA libraries from two colour pattern races and detected 142 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-193 and hme-miR-2788; located 2380 bp apart in an intergenic region. These two miRNAs are expressed in wings and show an upregulation between 24 and 72 hours post-pupation, indicating a potential role in butterfly wing development. A search for miRNAs in all available H. melpomene BAC sequences (~2.5 Mb) did not reveal any other miRNAs and no novel miRNAs were predicted.

Conclusions: 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.

Figure 1

Size distribution of sequenced small RNAs in Heliconius melpomene melpomene (black bars) and H. m. rosina (yellow bars). % of sequenced reads for each nucleotide size class are given both with (a) and without (b) the most abundant miRNA, miR-31.

Figure 2

Northern blots of nine Heliconius miRNAs in hindwing tissue from two colour pattern races collected at 24, 48 and 72 hours post-pupation. Blots were also run for forewing and thorax tissue collected at the same time and all gels were run twice (shown in Additional file 4). Probes are compared to a U6 non-coding small nuclear RNA loading control.

Figure 3

Predicted stem-loop structures for two miRNAs, hme-miR-193 and hme-miR-2788 found at the HmYb/Sb locus. miRNA sequence is given in green with the star sequence shown in pink.

Figure 4

Northern blots of a. hme-miR-193 and b. hme-miR-2788 in forewing, hindwing and thorax tissue from two colour pattern races collected at 24, 48 and 72 hours post-pupation. Probes are compared to a U6 non-coding small nuclear RNA loading control.