apterous A specifies dorsal wing patterns and sexual traits in butterflies

Abstract

Butterflies have evolved different colour patterns on their dorsal and ventral wing surfaces to serve different signalling functions, yet the developmental mechanisms controlling surface-specific patterning are still unknown. Here, we mutate both copies of the transcription factor apterous in Bicyclus anynana butterflies using CRISPR/Cas9 and show that apterous A, expressed dorsally, functions both as a repressor and modifier of ventral wing colour patterns, as well as a promoter of dorsal sexual ornaments in males. We propose that the surface-specific diversification of wing patterns in butterflies proceeded via the co-option of apterous A or its downstream effectors into various gene regulatory networks involved in the differentiation of discrete wing traits. Further, interactions between apterous and sex-specific factors such as doublesex may have contributed to the origin of sexually dimorphic surface-specific patterns. Finally, we discuss the evolution of eyespot number diversity in the family Nymphalidae within the context of developmental constraints due to apterous regulation.

Keywords: apterous; butterfly wing patterns; developmental constraints; dorsal–ventral differentiation; eyespot repression.

Figure 1.

Dorsal-ventral surface-specific variation in butterflies. (a) Dorsal (left) and ventral (right) surfaces of Morpho menelaus and Panacea regina illustrating striking variation in colour and patterns between surfaces. (b) Dorsal (left) and ventral (right) surfaces of a male and female Bicyclus anynana. The regions boxed in red are expanded in c. (c) Magnified view of the androconial organs present only in males. Top: forewing ventral androconia with a characteristic teardrop shape surrounded by silver scales. The scales on the corresponding dorsal forewing surface are completely brown. Bottom: hindwing dorsal androconia, also surrounded by silver scales, along with two patches of hair-pencils. These traits are absent from the ventral hindwing. (Online version in colour.)

Figure 2.

apA mRNA localization in developing wing discs of Bicyclus anynana. (a) apA expression is uniform across the epidermis but absent in future dorsal eyespot centres of hindwings (n = 5). (b) apA expression is absent in the future dorsal eyespot centre of the wild-type forewing (left) (n = 3) and also in the additional eyespot centre in the B. anynana ‘Spotty’ mutant (right) (n = 7). (c) Male wings (left) (28 h after pupation) showing upregulated dorsal apA expression in the hair-pencil regions. Inset shows the hair-pencils in adult male B. anynana. Female wings (right) (25 h after pupation) show no upregulation of apA in corresponding regions of the dorsal surface. (d) Cross-sectional view of a developing wing disc showing dorsal-specific apA expression (left side of the cross section). Scale bar, 20 µm. (Online version in colour.)

Figure 3.

CRISPR/Cas9 mosaic wing pattern phenotypes of apA knockouts. (a) Top: regions of the apA gene in B. anynana targeted using the CRISPR/Cas9 system. Bottom: sequences of the homeodomain and LIM domain regions of mutant individuals compared with the wild-type sequence in bold. Blue is the region targeted and the PAM sequence is in red. Deletions are indicated with ‘-’. (b) A subset of the CRISPR/Cas9 apA mutant phenotypes observed in B. anynana. The left column shows the wild-type (WT) dorsal and ventral surfaces for male forewings and hindwings. M9-12 (top): the dorsal forewing of a mutant male highlighting some of the ventral-like phenotypes and defects. The boxed regions are expanded to show the appearance of ventral-like white band and silver scales. M9-3: dorsal forewing surface of a mutant female resembling the ventral surface. M9-27: mutant with the ventral teardrop shape forewing androconial organ appearing on the dorsal surface (red arrow). WT dorsal forewing androconia is shown for comparison. M9-12 (bottom): a mutant dorsal hindwing with the appearance of all seven eyespots (red arrows), normally only seen on the ventral surface. The boxed regions are expanded to show the loss of silver scales associated with the dorsal hindwing androconia and improper development of hair-pencils. WT hair-pencil is shown for comparison. M9-2: mosaic phenotype (left) on the dorsal surface with ventral-like light coloured scales. Clones are indicated with a dashed white line. Corresponding region of the other wing of the same individual (right) shows no mosaicism. M235-11: a dorsal hindwing of a mutant with the width of the gold ring resembling that of ventral eyespots. (Online version in colour.)

Figure 4.

The role of apterous in surface-specific wing patterning in B. anynana and evolution of serial homologues in butterflies. (a) A schematic of the different functions of apA on the dorsal surface of B. anynana. apA acts as a repressor of ventral traits such as the white transversal band, forewing androconia, hindwing eyespots and the outer perimeter of the gold ring, and acts as an activator of hindwing hair-pencils and silver scales. (b) Different modes of serial homologue evolution involving the co-option of a (fin) gene network to a novel body location [18], repression of the ancestrally repeated (wing) network in a subset of body segments (adapted from [19]), repression followed by de-repression of the (limb) network in certain body segments [5] and de-repression of a never-expressed (eyespot) network at a novel body location. (Online version in colour.)