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.

Zygosity-based sex determination in a butterfly drives hypervariability of Masculinizer.

Nature has devised many ways of producing males and females. Here, we report on a previously undescribed mechanism for Lepidoptera that functions without a female-specific gene. The number of alleles or allele heterozygosity in a single Z-linked gene (BaMasc) is the primary sex-determining switch in Bicyclus anynana butterflies. Embryos carrying a single BaMasc allele develop into WZ (or Z0) females, those carrying two distinct alleles develop into ZZ males, while (ZZ) homozygotes initiate female development, have mismatched dosage compensation, and die as embryos. Consequently, selection against homozygotes has favored the evolution of spectacular allelic diversity: 205 different coding sequences of BaMasc were detected in a sample of 246 females. The structural similarity of a hypervariable region (HVR) in BaMasc to the HVR in Apis mellifera csd suggests molecular convergence between deeply diverged insect lineages. Our discovery of this primary switch highlights the fascinating diversity of sex-determining mechanisms and underlying evolutionary drivers.

A micro-RNA is the effector gene of a classic evolutionary hotspot locus.

In Lepidoptera (butterflies and moths), the genomic region around the gene cortex is a 'hotspot' locus, repeatedly used to generate intraspecific melanic wing color polymorphisms across 100-million-years of evolution. However, the identity of the effector gene regulating melanic wing color within this locus remains unknown. Here, we show that none of the four candidate protein-coding genes within this locus, including cortex, serve as major effectors. Instead, a micro-RNA (miRNA), mir-193, serves as the major effector across three deeply diverged lineages of butterflies, and its function is conserved in Drosophila. In Lepidoptera, mir-193 is derived from a gigantic long non-coding RNA, ivory, and it functions by directly repressing multiple pigmentation genes. We show that a miRNA can drive repeated instances of adaptive evolution in animals.

The molecular basis of scale development highlighted by a single-cell atlas of Bicyclus anynana butterfly pupal forewings.

Butterfly wings display a diversity of cell types, including large polyploid scale cells, yet the molecular basis of such diversity is poorly understood. To explore scale cell diversity at a transcriptomic level, we employ single-cell RNA sequencing of ∼5,200 large cells (>6 µm) from 22.5- to 25-h male pupal forewings of the butterfly Bicyclus anynana. Using unsupervised clustering, followed by in situ hybridization, immunofluorescence, and CRISPR-Cas9 editing of candidate genes, we annotate various cell types on the wing. We identify genes marking non-innervated scale cells, pheromone-producing glandular cells, and innervated sensory cell types. We show that senseless, a zinc-finger transcription factor, and HR38, a hormone receptor, determine the identity, size, and color of different scale cell types and are important regulators of scale cell differentiation. This dataset and the identification of various wing cell-type markers provide a foundation to compare and explore scale cell-type diversification across arthropod species.

Genome Assembly and Annotation of the Dark-Branded Bushbrown Butterfly Mycalesis mineus (Nymphalidae: Satyrinae).

We report a high-quality genome draft assembly of the dark-branded bushbrown, Mycalesis mineus, a member of the Satyrinae subfamily of nymphalid butterflies. This species is emerging as a promising model organism for investigating the evolution and development of phenotypic plasticity. Using 45.99 Gb of long-read data (N50 = 11.11 kb), we assembled a genome size of 497.4 Mb for M. mineus. The assembly is highly contiguous and nearly complete (96.8% of Benchmarking Universal Single-Copy Orthologs lepidopteran genes were complete and single copy). The genome comprises 38.71% of repetitive elements and includes 20,967 predicted protein-coding genes. The assembled genome was super-scaffolded into 28 pseudo-chromosomes using a closely related species, Bicyclus anynana, with a chromosomal-level genome as a template. This valuable genomic tool will advance both ongoing and future research focused on this model organism.

Tympanal ears mediate male-male competition, courtship and mating success in Bicyclus anynana butterflies.

The presence of intra-specific acoustic communication in diurnal butterflies is not well established. Here, we examined the function of the tympanal ear (Vogel's organ, VO) in the seasonally polyphenic butterfly Bicyclus anynana in the context of sexual signalling. We investigated how the VO and the flanking enlarged veins, which are suggested sound resonance chambers, scale with wing size across sexes and seasonal forms, and how disruptions to the VO alter courtship behaviour and mating outcomes. We found that males have VOs similar in size to females despite having smaller wings, and dry season (DS) male cubital and anal veins do not scale with the wing size. This suggests that the VO plays an important role in males and that cubital and anal veins in DS males may be tuned to amplify specific sound frequencies. Behavioural assays performed with deafened and hearing males of different seasonal forms, in pair and triad settings, showed that deafened DS males, but not wet season males, experienced lower mating success relative to their hearing counterparts. Our study documents a novel function for the wing tympanal membrane in mediating courtship and mating outcomes in diurnal butterflies.

Acquired preferences for a novel food odor do not become stronger or stable after multiple generations of odor feeding in Bicyclus anynana butterfly larvae.

Many herbivorous insects have specific host-plant preferences, and it is unclear how these preferences evolved. Previously, we found that Bicyclus anynana larvae can learn to prefer novel food odors from eating leaves with those odors and transmit those learned preferences to the next generation. It is uncertain whether such acquired odor preferences can increase across generations of repeated odor feeding and be maintained even in the absence of odor. In this study, we fed larvae with novel banana odor-coated leaves (odor-fed larvae) for five consecutive generations, without selection on behavioral choices, and measured how larval innate preferences changed over time. Then, we removed the odor stimulus from a larval subgroup, while the other group continued to be odor-fed. Our results show that larvae learned to prefer the novel odor within a generation of odor feeding and transmitted the learned preference to the next generation, as previously found. Odor-fed larvae preferred odor significantly more compared to control larvae across five generations of repeated odor or control feeding. However, this led neither to increased odor preference, nor its stabilization. This suggests that when butterfly larvae feed on a new host, a preference for that novel food plant may develop and be transmitted to the next generation, but this preference lasts for a single generation and disappears once the odor stimulus is removed.

Correction: Detection and measurement of butterfly eyespot and spot patterns using convolutional neural networks.

[This corrects the article DOI: 10.1371/journal.pone.0280998.].

Lepidopteran prolegs are novel traits, not leg homologs.

Lepidopteran larvae have both thoracic legs and abdominal prolegs, yet it is unclear whether these are serial homologs. A RNA-seq analysis with various appendages of Bicyclus anynana butterfly larvae indicated that the proleg transcriptome resembles the head-horn transcriptome, a novel trait in the lepidoptera, but not a thoracic leg. Under a partial segment abdominal-A (abd-A) knockout, both thoracic leg homologs (pleuropodia) and prolegs developed in the same segment, arguing that both traits are not serial homologs. Further, three of the four coxal marker genes, Sp5, Sp6-9, and araucan, were absent from prolegs, but two endite marker genes, gooseberry and Distal-less, were expressed in prolegs, suggesting that prolegs may be using a modular endite gene-regulatory network (GRN) for their development. We propose that larval prolegs are novel traits derived from the activation of a pre-existing modular endite GRN in the abdomen using abd-A, the same Hox gene that still represses legs in more lateral positions.

Multi-scale dissection of wing transparency in the clearwing butterfly Phanus vitreus.

Optical transparency is rare in terrestrial organisms, and often originates through loss of pigmentation and reduction in scattering. The coloured wings of some butterflies and moths have repeatedly evolved transparency, offering examples of how they function optically and biologically. Because pigments are primarily localized in the scales that cover a colourless wing membrane, transparency has often evolved through the complete loss of scales or radical modification of their shape. Whereas bristle-like scales have been well documented in glasswing butterflies, other scale modifications resulting in transparency remain understudied. The butterfly Phanus vitreus achieves transparency while retaining its scales and exhibiting blue/cyan transparent zones. Here, we investigate the mechanism of wing transparency in P. vitreus by light microscopy, focused ion beam milling, microspectrophotometry and optical modelling. We show that transparency is achieved via loss of pigments and vertical orientation in normal paddle-like scales. These alterations are combined with an anti-reflective nipple array on portions of the wing membrane being more exposed to light. The blueish coloration of the P. vitreus transparent regions is due to the properties of the wing membrane, and local scale nanostructures. We show that scale retention in the transparent patches might be explained by these perpendicular scales having hydrophobic properties.

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.

Detection and measurement of butterfly eyespot and spot patterns using convolutional neural networks.

Butterflies are increasingly becoming model insects where basic questions surrounding the diversity of their color patterns are being investigated. Some of these color patterns consist of simple spots and eyespots. To accelerate the pace of research surrounding these discrete and circular pattern elements we trained distinct convolutional neural networks (CNNs) for detection and measurement of butterfly spots and eyespots on digital images of butterfly wings. We compared the automatically detected and segmented spot/eyespot areas with those manually annotated. These methods were able to identify and distinguish marginal eyespots from spots, as well as distinguish these patterns from less symmetrical patches of color. In addition, the measurements of an eyespot's central area and surrounding rings were comparable with the manual measurements. These CNNs offer improvements of eyespot/spot detection and measurements relative to previous methods because it is not necessary to mathematically define the feature of interest. All that is needed is to point out the images that have those features to train the CNN.

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.

Butterfly eyespots exhibit unique patterns of open chromatin.

Background: How the precise spatial regulation of genes is correlated with spatial variation in chromatin accessibilities is not yet clear. Previous studies that analysed chromatin from homogenates of whole-body parts of insects found little variation in chromatin accessibility across those parts, but single-cell studies of Drosophila brains showed extensive spatial variation in chromatin accessibility across that organ. In this work we studied the chromatin accessibility of butterfly wing tissue fated to differentiate distinct colors and patterns in pupal wings of Bicyclus anynana. Methods: We dissected small eyespot and adjacent control tissues from 3h pupae and performed ATAC-Seq to identify the chromatin accessibility differences between different sections of the wings. Results: We observed that three dissected wing regions showed unique chromatin accessibilities. Open chromatin regions specific to eyespot color patterns were highly enriched for binding motifs recognized by Suppressor of Hairless (Su(H)), Krüppel (Kr), Buttonhead (Btd) and Nubbin (Nub) transcription factors. Genes in the vicinity of the eyespot-specific open chromatin regions included those involved in wound healing and SMAD signal transduction pathways, previously proposed to be involved in eyespot development. Conclusions: We conclude that eyespot and non-eyespot tissue samples taken from the same wing have distinct patterns of chromatin accessibility, possibly driven by the eyespot-restricted expression of potential pioneer factors, such as Kr.

Colour patterns: Predicting patterns without knowing the details.

A bewildering array of colour patterns occurs across animals. New work studying five lizard species reveals that reaction-diffusion models can be remarkably predictive of future adult skin patterning, even though molecular details are unknown. This has implications for understanding how complex patterns evolve.

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.

Ecological factors are likely drivers of eye shape and colour pattern variations across anthropoid primates.

External eye appearance across primate species is diverse in shape and colouration, yet we still lack an explanation for the drivers of such diversity. Here we quantify substantial interspecific variation in eye shape and colouration across 77 primate species representing all extant genera of anthropoid primates. We reassess a series of hypotheses aiming to explain ocular variation in horizontal elongation and in colouration across species. Heavier body weight and terrestrial locomotion are associated with elongated eye outlines. Species living closer to the equator present more pigmented conjunctivae, suggesting photoprotective functions. Irises become bluer in species living further away from the equator, adding to existing literature supporting a circadian clock function for bluer irises. These results shift the current focus from communicative, to ecological factors in driving variation in external eye appearance in anthropoid primates. They also highlight the possibility that similar ecological factors contributed to selection for blue eyes in ancestral human populations living in northern latitudes.

Antennapedia and optix regulate metallic silver wing scale development and cell shape in Bicyclus anynana butterflies.

Butterfly wing scales can develop intricate cuticular nanostructures that produce silver colors, but the underlying genetic and physical basis of such colors is mostly unexplored. Here, we characterize different types of wild-type silver scales in Bicyclus anynana butterflies and show that the varying thickness of the air layer between two cuticular laminas is most important for producing silvery broadband reflectance. We then address the function of five genes-apterous A, Ultrabithorax, doublesex, Antennapedia, and optix-in silver scale development by examining crispants with either ectopic gains or losses of silver scales. Simultaneous transformations of three parameters-loss of the upper lamina, increased lower lamina thickness, and increased pigmentation-occur when silver scales become brown and vice versa when brown scales become silver. Antennapedia and optix are high-level regulators of different silver scale types and determine cell shape in both sexes. Moreover, Antennapedia is involved in determining ridge and crossrib orientation.

Distal-less and spalt are distal organisers of pierid wing patterns.

Two genes, Distal-less (Dll) and spalt (sal), are known to be involved in establishing nymphalid butterfly wing patterns. They function in several ways: in the differentiation of the eyespot's central signalling cells, or foci; in the differentiation of the surrounding black disc; in overall scale melanisation (Dll); and in elaborating marginal patterns, such as parafocal elements. However, little is known about the functions of these genes in the development of wing patterns in other butterfly families. Here, we study the expression and function of Dll and sal in the development of spots and other melanic wing patterns of the Indian cabbage white, Pieris canidia, a pierid butterfly. In P. canidia, both Dll and Sal proteins are expressed in the scale-building cells at the wing tips, in chevron patterns along the pupal wing margins, and in areas of future scale melanisation. Additionally, Sal alone is expressed in the future black spots. CRISPR knockouts of Dll and sal showed that each gene is required for the development of melanic wing pattern elements, and repressing pteridine granule formation, in the areas where they are expressed. We conclude that both genes likely play ancestral roles in organising distal butterfly wing patterns, across pierid and nymphalid butterflies, but are unlikely to be differentiating signalling centres in pierids black spots. The genetic and developmental mechanisms that set up the location of spots and eyespots are likely distinct in each lineage.