Transcription factors underlying wing margin color patterns and pupal cuticle markings in butterflies

Abstract

Background The diversity of butterfly color patterns can be attributed to a relatively small number of pattern elements that are homologous across Lepidoptera. Although genes involved in patterning some of these elements have been identified, the development of several major elements remains poorly understood. To identify genes underlying wing pupal cuticle markings and wing margin color patterns, we examined expression of the candidate transcription factors Engrailed/Invected (En/Inv), Distal-less (Dll), Cubitus interruptus (Ci), and Spalt in two nymphalids: Junonia coenia and Bicyclus anynana. Results We found that En/Inv, Dll, and Ci mark domains on the J. coenia last-instar forewing disc that closely correspond to the position and shape of pupal cuticle markings. We also found that Spalt demarcates wing margin color patterns in both J. coenia and B. anynana, and that CRISPR/Cas9 deletions in the spalt gene result in reduction and loss of wing margin color patterns in J. coenia. These data demonstrate a role for spalt in promoting wing margin color patterning, in addition to its previously described role in eyespot patterning. Conclusion Our observations support the model that a core set of regulatory genes are redeployed multiple times, and in multiple roles, during butterfly wing pattern development. Of these genes, spalt is of special interest as it plays a dual role in both eyespot and margin color pattern development.

Distal-lessandspaltare distal organisers of pierid wing patterns

“…Previous studies have shown that expression of both Dll and Sal proteins also correlate with patterns of different colour states on the wing. In 16-24h pupal wings, expression of Sal protein spatially maps to pale-colored non-eyespot marginal wing patterns of nymphalids (Reed et al, 2020) while both Dll and Sal proteins are expressed in silver scales along the wing margin in the lycaenid butterfly, Lycaeides melissa (Brunetti et al, 2001). Thus, both Dll and sal may be ancestral pattern organisers working within the distal part of the wing, operating independently of melanic fate.…”

“…In late larval wing discs of B. anynana , both Dll (green) and sal (orange) are co-expressed at high levels in the center of eyespots (Reed et al, 2020). However, in late larval wing discs of P. canidia, Dll and sal are not expressed in spot centers.…”

“…Collectively, our results suggest that pierid spots are unlikely homologs of patterns in the ‘border ocelli’ band but may be positional homologs of more distal pattern elements with respect to nymphalid eyespots located within the ‘ EIII’ or ‘ parafocal elements’ banding systems. Dll and sal knockout mutants in nymphalid butterflies showed a disruption to both submarginal and marginal pattern elements (EI-III) (Zhang & Reed, 2016; Connahs et al, 2019; Reed et al, 2020). Given the classification of pierid spots as part of the EIII band, we expected that knocking out Dll in P. canidia should also result in disruption or missing spot patterns.…”

“…Given that both Dll and sal proteins are associated with nymphalid eyespot foci, as well as other elements such a black scales and parafocal patterns along the wing margin of numerous nymphalid species (Monteiro et al, 2006;Zhang & Reed, 2016;Connahs et al, 2019;Reed et al, 2020), here we ask whether these transcription factors have a similar role in establishing melanic wing patterns in a species belonging to a more basal butterfly lineage, the Pieridae. We also ask whether the same candidate morphogens suspected of setting up eyespot centers in nymphalid butterfly larval wings might be setting up spot centers in pierid larval wings or defining the expression of sal or Dll in black scales, in pupal wings.…”

“…Both Dll and sal have also been implicated in the development of melanic color patterns in other areas of nymphalid wings, and sal in the larval integument of papilionids. Dll is required for the background brown color in B. anynana wings (Connahs et al, 2019), and both genes are required for the development of pattern elements along the parafocal, marginal, and submarginal wing bands of numerous nymphalid species (Zhang & Reed, 2016; Connahs et al, 2019; Reed et al, 2020). Aside from wings, sal is also expressed in melanic regions of eyespot patterns on the larval epidermis of Papilio xuthus (Futahashi et al, 2012).…”