Gene expression underlying adaptive variation in Heliconius wing patterns: non-modular regulation of overlapping cinnabar and vermilion prepatterns

Abstract

Geographical variation in the mimetic wing patterns of the butterfly Heliconius erato is a textbook example of adaptive polymorphism; however, little is known about how this variation is controlled developmentally. Using microarrays and qPCR, we identified and compared expression of candidate genes potentially involved with a red/yellow forewing band polymorphism in H. erato. We found that transcripts encoding the pigment synthesis enzymes cinnabar and vermilion showed pattern- and polymorphism-related expression patterns, respectively. cinnabar expression was associated with the forewing band regardless of pigment colour, providing the first gene expression pattern known to be correlated with a major Heliconius colour pattern. In contrast, vermilion expression changed spatially over time in red-banded butterflies, but was not expressed at detectable levels in yellow-banded butterflies, suggesting that regulation of this gene may be involved with the red/yellow polymorphism. Furthermore, we found that the yellow pigment, 3-hydroxykynurenine, is incorporated into wing scales from the haemolymph rather than being synthesized in situ. We propose that some aspects of Heliconius colour patterns are determined by spatio-temporal overlap of pigment gene transcription prepatterns and speculate that evolutionary changes in vermilion regulation may in part underlie an adaptive colour pattern polymorphism.

Figure 1

Wing colour patterns of two races of H. erato: (a) H. erato petiverana and (b) H. erato cyrbia, (c) their sister species H. himera and (d,e) F1 progeny of the interspecific crosses. Pigment development in H. erato petiverana pupal (f–k) forewings and (l–o) hindwings. (f) In early pupae, the wing epithelia are very thin and scales are undeveloped. Scales fated to become (g,l) yellow or red mature first, with red-fated scales progressively darkening from (g) orange to (h) red, and (l–n) yellow-fated scales remaining colourless. (i,j) After the red pigment has matured, melanic scales develop in a ‘wave’ emanating from the centre of the wing. (n) There is a period after the ommochrome and melanin pigments have fully developed that the yellow pigment 3-OHK is undetectable in the wing. (o) Within a few hours before adult emergence 3-OHK appears in yellow-fated scales.

Figure 2

(a,b) Early ommochrome development (cyrbia, himera, respectively) and (c,d) late melanin development (cyrbia, himera, respectively). Quantitative RT-PCR comparison of (i–iv) vermilion and (v–viii) cinnabar transcript abundance during pigment development in (i,v,iii,vii) H. e. cyrbia and (ii,vi,iv,viii) H. himera. Transcription levels are presented relative to control gene expression and error bars represent the standard deviation. Synthesis of red pigment occurs when the transcription of both genes spatio-temporally coincides in the forewing band (i,v).

Figure 3

The yellow pigment 3-OHK is stored in the fat bodies, circulated in the haemolymph and taken up from the haemolymph into yellow scales. The absorbance spectrum of (a) pure 3-OHK is similar to (b) fat body extract and (c) haemolymph, suggesting that 3-OHK is a major component of both. (d) There is a rapid increase of 3-OHK in the haemolymph (solid line) and the fat bodies (broken line) of late-stage pupae, as inferred from spectrophotometric analysis of the 380 nm absorbance peak. ¹⁴C-3-OHK injected into pupal haemolymph is incorporated specifically into yellow scales. (e) At a stage shortly preceding the appearance of yellow in the H. himera forewing band, (f) only low levels of ¹⁴C-3-OHK are incorporated around the edges of the band. At an intermediate stage of yellow development, when 3-OHK is observed only in the anterior portion of the forewing band, high levels of (h) ¹⁴C-3-OHK incorporation are perfectly correlated with (g) yellow pigmentation. (i–l) No incorporation of ¹⁴C-3-OHK was detected in red hindwing scales.

Figure 4

(a) A hypothetical model of ommochrome synthesis in butterfly wing scales based on the work from the Drosophila eye. Tryptophan, the ommochrome precursor, is thought to be taken up into cells by the karmoisin transporter. Tryptophan is then processed by several enzymes, including those encoded by vermilion and cinnabar into 3-OHK, which is then transported into granules by the scarlet/white heterodimer. 3-OHK is processed into xanthommatin in a granule, where a binding protein (XBP) is thought to modulate the redox state of xanthommatin, thereby determining whether the pigment will appear more orange or red in hue. 3-OHK can be taken up directly into scales, and we have previously speculated that a binding protein (BP) may be required to stabilize this molecule in a pigment granule. See Reed & Nagy (2005) for a detailed discussion. (b) A working model of pigment regulation through the overlap of vermilion and cinnabar prepatterns. Red pigment is synthesized at the time and place transcription of vermilion and cinnabar (shown in red) overlap.