Ultrabithorax modifies a regulatory network of genes essential for butterfly eyespot development in a wing sector-specific manner.

Nymphalid butterfly species often have a different number of eyespots in forewings and hindwings, but how the hindwing identity gene Ultrabithorax (Ubx) drives this asymmetry is not fully understood. We examined a three-gene regulatory network for eyespot development in the hindwings of Bicyclus anynana butterflies and compared it with the same network previously described for forewings. We also examined how Ubx interacts with each of these three eyespot-essential genes. We found similar genetic interactions between the three genes in fore- and hindwings, but we discovered three regulatory differences: Antennapedia (Antp) merely enhances spalt (sal) expression in the eyespot foci in hindwings, but is not essential for sal activation, as in forewings; Ubx upregulates Antp in all hindwing eyespot foci but represses Antp outside these wing regions; and Ubx regulates sal in a wing sector-specific manner, i.e. it activates sal expression only in the sectors that have hindwing-specific eyespots. We propose a model for how the regulatory connections between these four genes evolved to produce wing- and sector-specific variation in eyespot number.

The Hox gene Antennapedia is essential for wing development in insects.

A long-standing view in the field of evo-devo is that insect forewings develop without any Hox gene input. The Hox gene Antennapedia (Antp), despite being expressed in the thoracic segments of insects, has no effect on wing development. This view has been obtained from studies in two main model species: Drosophila and Tribolium. Here, we show that partial loss of function of Antp resulted in reduced and malformed adult wings in Bombyx, Drosophila and Tribolium. Antp mediates wing growth in Bombyx by directly regulating the ecdysteriod biosynthesis enzyme gene (shade) in the wing tissue, which leads to local production of the growth hormone 20-hydroxyecdysone. Additional targets of Antp are wing cuticular protein genes CPG24, CPH28 and CPG9, which are essential for wing development. We propose, therefore, that insect wing development occurs in an Antp-dependent manner. This article has an associated 'The people behind the papers' interview.

Butterfly eyespots evolved via cooption of an ancestral gene-regulatory network that also patterns antennae, legs, and wings.

Butterfly eyespots are beautiful novel traits with an unknown developmental origin. Here we show that eyespots likely originated via cooption of parts of an ancestral appendage gene-regulatory network (GRN) to novel locations on the wing. Using comparative transcriptome analysis, we show that eyespots cluster most closely with antennae, relative to multiple other tissues. Furthermore, three genes essential for eyespot development, Distal-less (Dll), spalt (sal), and Antennapedia (Antp), share similar regulatory connections as those observed in the antennal GRN. CRISPR knockout of cis-regulatory elements (CREs) for Dll and sal led to the loss of eyespots, antennae, legs, and also wings, demonstrating that these CREs are highly pleiotropic. We conclude that eyespots likely reused an ancient GRN for their development, a network also previously implicated in the development of antennae, legs, and wings.

Haemolymph transfusions transfer heritable learned novel odour preferences to naive larvae of Bicyclus anynana butterflies.

The mechanisms whereby environmental experiences of parents are transmitted to their offspring to impact their behaviour and fitness are poorly understood. Previously, we showed that naive Bicyclus anynana butterfly larvae, whose parents fed on a normal plant feed but coated with a novel odour, inherited an acquired preference towards that odour, which had initially elicited avoidance in the naive parents. Here, we performed simple haemolymph transfusions from odour-fed and control-fed larvae to naive larval recipients. We found that larvae injected with haemolymph from odour-fed donors stopped avoiding the novel odour, and their naive offspring preferred the odour more, compared to the offspring of larvae injected with control haemolymph. These results indicate that factors in the haemolymph, potentially the odour molecule itself, play an important role in odour learning and preference transmission across generations. Furthermore, this mechanism of odour preference inheritance, mediated by the haemolymph, bypasses the peripheral odour-sensing mechanisms taking place in the antennae, mouthparts or legs, and may mediate food plant switching and diversification in Lepidoptera or more broadly across insects.

The genetic basis of wing spots in Pieris canidia butterflies.

Spots in pierid butterflies and eyespots in nymphalid butterflies are likely non-homologous wing colour pattern elements, yet they share a few features in common. Both develop black scales that depend on the function of the gene spalt, and both might have central signalling cells. This suggests that both pattern elements may be sharing common genetic circuitry. Hundreds of genes have already been associated with the development of nymphalid butterfly eyespot patterns, but the genetic basis of the simpler spot patterns on the wings of pierid butterflies has not been investigated. To facilitate studies of pierid wing patterns, we report a high-quality draft genome assembly for Pieris canidia, the Indian cabbage white. We then conducted transcriptomic analyses of pupal wing tissues sampled from the spot and non-spot regions of P. canidia at 3-6 h post-pupation. A total of 1352 genes were differentially regulated between wing tissues with and without the black spot, including spalt, Krüppel-like factor 10, genes from the Toll, Notch, TGF-ß, and FGFR signalling pathways, and several genes involved in the melanin biosynthetic pathway. We identified 14 genes that are up-regulated in both pierid spots and nymphalid eyespots and propose that spots and eyespots share regulatory modules despite their likely independent origins.

A transcriptomic atlas underlying developmental plasticity of seasonal forms of Bicyclus anynana butterflies.

Organisms residing in regions with alternating seasons often develop different phenotypes, or forms, in each season. These forms are often adaptations to each season and result from an altered developmental response to specific environmental cues such as temperature. While multiple studies have examined form-specific gene expression profiles in a diversity of species, little is known about how environments and developmental transitions, cued by hormone pulses, alter post-transcriptional patterns. In this study, we examine how gene expression, alternative splicing, and miRNA-mediated gene silencing in Bicyclus anynana butterfly hindwing tissue, varies across two rearing temperatures at four developmental timepoints. These timepoints flank two temperature-sensitive periods that coincide with two pulses of the insect hormone 20E. Our results suggest that developmental transitions, coincident with 20E pulses, elicit a greater impact on all these transcriptomic patterns than rearing temperatures per se. More similar transcriptomic patterns are observed pre-20E pulses than those observed post-20E pulses. We also found functionally distinct sets of differentially expressed and differentially spliced genes in the seasonal forms. Furthermore, around 10% of differentially expressed genes are predicted to be direct targets of, and regulated by, differentially expressed miRNAs between the seasonal forms. Many differentially expressed genes, miRNAs, or differentially spliced genes potentially regulate eyespot size plasticity, and we validated the differential splicing pattern of one such gene, daughterless. We present a comprehensive and interactive transcriptomic atlas of the hindwing tissue of both seasonal forms of B. anynana throughout development, a model organism of seasonal plasticity.

Molecular mechanisms underlying simplification of venation patterns in holometabolous insects.

How mechanisms of pattern formation evolve has remained a central research theme in the field of evolutionary and developmental biology. The mechanism of wing vein differentiation in Drosophila is a classic text-book example of pattern formation using a system of positional information, yet very little is known about how species with a different number of veins pattern their wings, and how insect venation patterns evolved. Here, we examine the expression pattern of genes previously implicated in vein differentiation in Drosophila in two butterfly species with more complex venation Bicyclus anynana and Pieris canidia We also test the function of some of these genes in B. anynana We identify both conserved as well as new domains of decapentaplegic, engrailed, invected, spalt, optix, wingless, armadillo, blistered and rhomboid gene expression in butterflies, and propose how the simplified venation in Drosophila might have evolved via loss of decapentaplegic, spalt and optix gene expression domains, via silencing of vein-inducing programs at Spalt-expression boundaries, and via changes in expression of vein maintenance genes.

Evolution of modular and pleiotropic enhancers.

Cis-regulatory elements (CREs), or enhancers, are segments of noncoding DNA that regulate the spatial and temporal expression of nearby genes. Sometimes, genes are expressed in more than one tissue, and this can be driven by two main types of CREs: tissue-specific "modular" CREs, where different CREs drive expression of the gene in the different tissues, or by "pleiotropic" CREs, where the same CRE drives expression in the different tissues. In this perspective, we will discuss some of the ways (i) modular and pleiotropic CREs might originate; (ii) propose that modular CREs might derive from pleiotropic CREs via a process of duplication, degeneration, and complementation (the CRE-DDC model); and (iii) propose that hotspot loci of evolution are associated with the origin of modular CREs belonging to any gene in a regulatory network.

Distinguishing serial homologs from novel traits: Experimental limitations and ideas for improvements.

One of the central but yet unresolved problems in evolutionary biology concerns the origin of novel complex traits. One hypothesis is that complex traits derive from pre-existing gene regulatory networks (GRNs) reused and modified to specify a novel trait somewhere else in the body. This simple explanation encounters problems when the novel trait that emerges in a body is in a region that is known to harbor a latent or repressed trait that has been silent for millions of years. Is the novel trait merely a re-emerged de-repressed trait or a truly novel trait that emerged via a novel deployment of an old GRN? A couple of new studies sided on opposite sides of this question when investigating the origin of horns in dung beetles and helmets in treehoppers that develop in the first thoracic segment (T1) of their bodies, a segment known to harbor a pair of repressed/modified wings in close relatives. Here, I point to some key limitations of the experimental approaches used and highlight additional experiments that could be done in future to resolve the developmental origin of these and other traits.

Activation of butterfly eyespots by Distal-less is consistent with a reaction-diffusion process.

Eyespots on the wings of nymphalid butterflies represent colorful examples of pattern formation, yet the developmental origins and mechanisms underlying eyespot center differentiation are still poorly understood. Using CRISPR-Cas9 we re-examine the function of Distal-less (Dll) as an activator or repressor of eyespots, a topic that remains controversial. We show that the phenotypic outcome of CRISPR mutations depends upon which specific exon is targeted. In Bicyclus anynana, exon 2 mutations are associated with both missing and ectopic eyespots, and also exon skipping. Exon 3 mutations, which do not lead to exon skipping, produce only null phenotypes, including missing eyespots, lighter wing coloration and loss of scales. Reaction-diffusion modeling of Dll function, using Wnt and Dpp as candidate morphogens, accurately replicates these complex crispant phenotypes. These results provide new insight into the function of Dll as a potential activator of eyespot development, scale growth and melanization, and suggest that the tuning of Dll expression levels can generate a diversity of eyespot phenotypes, including their appearance on the wing.This article has an associated 'The people behind the papers' interview.

The yellow gene regulates behavioural plasticity by repressing male courtship in Bicyclus anynana butterflies.

Seasonal plasticity in male courtship in Bicyclus anynana butterflies is due to variation in levels of the steroid hormone 20E (20-hydroxyecdysone) during pupation. Wet season (WS) males have high levels of 20E and become active courters. Dry season (DS) males have lower levels of 20E and reduced courtship rates. However, WS courtship rates can be achieved if DS male pupae are injected with 20E at 30% of pupation. Here, we investigated the genes involved in male courtship plasticity and examined whether 20E plays an organizational role in the pupal brain that later influences the sexual behaviour of adults. We show that DS pupal brains have a sevenfold upregulation of the yellow gene relative to the WS brains, and that knocking out yellow leads to increased male courtship. We find that injecting 20E into DS pupa reduced yellow expression although not significantly. Our results show that yellow is a repressor of the neural circuity for male courtship behaviour in B. anynana. 20E levels experienced during pupation could play an organizational role during pupal brain development by regulating yellow expression, however, other factors might also be involved. Our findings are in striking contrast to Drosophila where yellow is required for male courtship.

Scleral pigmentation leads to conspicuous, not cryptic, eye morphology in chimpanzees.

Gaze following has been argued to be uniquely human, facilitated by our depigmented, white sclera [M. Tomasello, B. Hare, H. Lehmann, J. Call, J. Hum. Evol. 52, 314-320 (2007)]-the pale area around the colored iris-and to underpin human-specific behaviors such as language. Today, we know that great apes show diverse patterns of scleral coloration [J. A. Mayhew, J. C. Gómez, Am. J. Primatol. 77, 869-877 (2015); J. O. Perea García, T. Grenzner, G. Hesková, P. Mitkidis, Commun. Integr. Biol. 10, e1264545 (2016)]. We compare scleral coloration and its relative contrast with the iris in bonobos, chimpanzees, and humans. Like humans, bonobos' sclerae are lighter relative to the color of their irises; chimpanzee sclerae are darker than their irises. The relative contrast between the sclera and iris in all 3 species is comparable, suggesting a perceptual mechanism to explain recent evidence that nonhuman great apes also rely on gaze as a social cue.

Doublesex Mediates the Development of Sex-Specific Pheromone Organs in Bicyclus Butterflies via Multiple Mechanisms.

The Bicyclus lineage of satyrid butterflies exhibits male-specific traits, the scent organs, used for chemical communication during courtship. These organs consist of tightly packed brush-like scales (hair-pencils) that rub against scent patches to disperse pheromones, but the evolution and molecular basis of these organ's male-limited development remains unknown. Here, we examine the evolution of the number and location of the scent patches and hair-pencils within 53 species of Bicyclus butterflies, and the involvement of the sex determinant gene doublesex (dsx) in scent organ development in Bicyclus anynana using CRISPR/Cas9. We show that scent patches and hair-pencils arose via multiple, independent gains, in a correlated manner. Further, an initially nonsex-specific Dsx protein expression pattern in developing wing discs becomes male-specific and spatially refined to areas that develop the scent patches. Functional perturbations of dsx show that this gene activates patch development in males whereas hair-pencils develop in both sexes without Dsx input. Dsx in females is, instead, required to repress hair-pencils whereas Dsx in males regulates minor aspects of its development. These findings suggest that the patches and hair-pencils evolve as correlated composite organs presumably due to their functional integration. Divergence in the function of dsx isoforms occurred across the sexes, where the male isoform promotes patch development in males and the female isoform represses hair-pencil development in females, both leading to the development of male-limited traits. Furthermore, evolution in number of patches in males is due to the evolution of spatial regulation of dsx.

Predation favours Bicyclus anynana butterflies with fewer forewing eyespots.

There are fewer eyespots on the forewings versus hindwings of nymphalids but the reasons for this uneven distribution remain unclear. One possibility is that, in many butterflies, the hindwing covers part of the ventral forewing at rest and there are fewer forewing sectors to display eyespots (covered eyespots are not continuously visible and are less likely to be under positive selection). A second explanation is that having fewer forewing eyespots confers a selective advantage against predators. We analysed wing overlap at rest in 275 nymphalid species with eyespots and found that many have exposed forewing sectors without eyespots: i.e. wing overlap does not constrain the forewing from having the same number or more eyespots than the hindwing. We performed two predation experiments with mantids to compare the relative fitness of and attack damage patterns on two forms of Bicyclus anynana butterflies, both with seven hindwing eyespots, but with two (in wild-type) or four (in Spotty) ventral forewing eyespots. Spotty experienced more intense predation on the forewings, were shorter-lived and laid fewer eggs. These results suggest that predation pressure limits forewing eyespot number in B. anynana. This may occur if attacks on forewing eyespots have more detrimental consequences for flight than attacks on hindwing eyespots.

Sex Differences in 20-Hydroxyecdysone Hormone Levels Control Sexual Dimorphism in Bicyclus anynana Wing Patterns.

In contrast to the important role of hormones in the development of sexual traits in vertebrates (Cox RM, Stenquist DS, Calsbeek R. 2009. Testosterone, growth and the evolution of sexual size dimorphism. J Evol Biol. 22(8):1586-1598.), the differentiation of these traits in insects is attributed almost exclusively to cell-autonomous mechanisms controlled by members of the sex determination pathway (Verhulst EC, van de Zande L. 2015. Double nexus - doublesex is the connecting element in sex determination. Brief Funct Genomics 14(6):396-406.), such as doublesex. Although hormones can shape the development of sexual traits in insects, variation in hormone levels are not conclusively known to cause dimorphism in these traits (Prakash A, Monteiro A. 2016. Molecular mechanisms of secondary sexual trait development in insects. Curr Opin Insect Sci. 17:40-48.). Here, we show that butterflies use sex-specific differences in 20-hydroxyecdysone hormone titers to create sexually dimorphic wing ornaments. Females of the dry season (DS) form of Bicyclus anynana display a larger sexual ornament on their wings than males, whereas in the wet season form both sexes have similarly sized ornaments (Prudic KL, Jeon C, Cao H, Monteiro A. 2011. Developmental plasticity in sexual roles of butterfly species drives mutual sexual ornamentation. Science 331(6013):73-75.). High levels of circulating 20-hydroxyecdysone during larval development in DS females and wet season forms cause proliferation of the cells fated to give rise to this wing ornament, and results in sexual dimorphism in the DS forms. This study advances our understanding of how the environment regulates sex-specific patterns of plasticity of sexual ornaments and conclusively shows that hormones can play a role in the development of secondary sexual traits in insects, just like they do in vertebrates.

Male Bicyclus anynana Butterflies Choose Females on the Basis of Their Ventral UV-Reflective Eyespot Centers.

Butterflies often use their dorsal and ventral wing color patterns for distinct signaling functions. Color patterns on hidden dorsal wing surfaces are often used in sexual signaling, while exposed ventral patterns are often used to ward off predator attacks. At rest, however, part of the ventral forewings are often hidden by the hindwings, allowing individuals to also use the patterns on this wing surface for sexual signaling. Here, we test this hypothesis in Bicyclus anynana (Butler, Lepidoptera, Nymphalidae) butterflies by first determining the degree of sexual dimorphism in ventral forewing patterns, focusing on the eyespots, from both wet and dry season forms, and then testing the role of the larger ventral forewing eyespots of dry season females in male mate choice. We also test male investment in reproduction. We show that ventral forewing UV-reflective eyespot centers, in addition to dorsal forewing eyespot centers previously examined in this species, play a role in sexual signaling as males preferentially mated with females with their ventral eyespot centers intact instead of blocked with black paint. This male preference, however, did not translate into a detectable higher reproductive investment via a single mating toward ornamented females. This study provides an example of how ventral forewing patterns, often hidden by hindwings, are used in sexual communication, in this case by females to attract males.

Interacting Effects of Eyespot Number and Ultraviolet Reflectivity on Predation Risk in Bicyclus anynana (Lepidoptera: Nymphalidae).

Small marginal eyespots on lepidopteran wings are conspicuous elements that attract a predator's attention to deflect attacks away from the body, but the role of ultraviolet (UV) reflectivity at the center of these patterns and variation in eyespot number in altering the function of eyespots remains unclear. Here, we performed a field-based predation experiment with artificial prey items based on the appearance of squinting bush brown butterflies Bicyclus anynana (Butler, 1879). We tested how two visual properties of the wing pattern affect predation risk: i) the number of eyespots on the ventral forewing surface-two or four; and ii) the UV reflectivity of eyespot centers-normal (where the UV reflectivity of the centers contrasts strongly with that of the darker surrounding ring) or blocked (where this contrast is reduced). In total, 807 prey items were deployed at two sites. We found a significant interaction between the number of ventral forewing eyespots and UV reflectivity in the eyespot centers: in items with fewer eyespots, blocking UV resulted in increased predation risk whereas in items with more eyespots, blocking UV resulted in decreased predation risk. If higher predation of paper models can be equated with higher levels of wing margin/eyespot conspicuity, these results demonstrate that UV reflectivity is an important factor in making eyespots more conspicuous to predators and suggest that the fitness of particular butterfly eyespot number variants may depend on the presence or absence of UV in their centers and on the ability of local predator guilds to detect UV.

Wingless is a positive regulator of eyespot color patterns in Bicyclus anynana butterflies.

Eyespot patterns of nymphalid butterflies are an example of a novel trait yet, the developmental origin of eyespots is still not well understood. Several genes have been associated with eyespot development but few have been tested for function. One of these genes is the signaling ligand, wingless, which is expressed in the eyespot centers during early pupation and may function in eyespot signaling and color ring differentiation. Here we tested the function of wingless in wing and eyespot development by down-regulating it in transgenic Bicyclus anynana butterflies via RNAi driven by an inducible heat-shock promoter. Heat-shocks applied during larval and early pupal development led to significant decreases in wingless mRNA levels and to decreases in eyespot size and wing size in adult butterflies. We conclude that wingless is a positive regulator of eyespot and wing development in B. anynana butterflies.

apterous A specifies dorsal wing patterns and sexual traits in butterflies.

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.

Differential Expression of Ecdysone Receptor Leads to Variation in Phenotypic Plasticity across Serial Homologs.

Bodies are often made of repeated units, or serial homologs, that develop using the same core gene regulatory network. Local inputs and modifications to this network allow serial homologs to evolve different morphologies, but currently we do not understand which modifications allow these repeated traits to evolve different levels of phenotypic plasticity. Here we describe variation in phenotypic plasticity across serial homologous eyespots of the butterfly Bicyclus anynana, hypothesized to be under selection for similar or different functions in the wet and dry seasonal forms. Specifically, we document the presence of eyespot size and scale brightness plasticity in hindwing eyespots hypothesized to vary in function across seasons, and reduced size plasticity and absence of brightness plasticity in forewing eyespots hypothesized to have the same function across seasons. By exploring the molecular and physiological causes of this variation in plasticity across fore and hindwing serial homologs we discover that: 1) temperature experienced during the wandering stages of larval development alters titers of an ecdysteroid hormone, 20-hydroxyecdysone (20E), in the hemolymph of wet and dry seasonal forms at that stage; 2) the 20E receptor (EcR) is differentially expressed in the forewing and hindwing eyespot centers of both seasonal forms during this critical developmental stage; and 3) manipulations of EcR signaling disproportionately affected hindwing eyespots relative to forewing eyespots. We propose that differential EcR expression across forewing and hindwing eyespots at a critical stage of development explains the variation in levels of phenotypic plasticity across these serial homologues. This finding provides a novel signaling pathway, 20E, and a novel molecular candidate, EcR, for the regulation of levels of phenotypic plasticity across body parts or serial homologs.