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.

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.

Female spider aggression is associated with genetic underpinnings of the nervous system and immune response to pathogens.

Identifying the genetic architecture underlying phenotypic variation in natural populations and assessing the consequences of polymorphisms for individual fitness are fundamental goals in evolutionary and molecular ecology. Consistent between-individual differences in behaviour have been documented for a variety of taxa. Dissecting the genetic basis of such behavioural differences is however a challenging endeavour. The molecular underpinnings of natural variation in aggression remain elusive. Here, we used comparative gene expression (transcriptome analysis and RT-PCR), genetic association analysis and pharmacological experiments to gain insight into the genetic basis of aggression in wild-caught jumping spiders (Portia labiata). We show that spider aggression is associated with a putative viral infection response gene, BTB/POZ domain-containing protein 17 (BTBDH), in addition to a putative serotonin receptor 1A (5-HT1A) gene. Spider aggression varies with virus loads, and BTBDH is upregulated in docile spiders and exhibits a genetic variant associated with aggression. We also identify a putative serotonin receptor 5-HT1A gene upregulated in docile P. labiata. Individuals that have been treated with serotonin become less aggressive, but individuals treated with a nonselective serotonin receptor antagonist (methiothepin) also reduce aggression. Further, we identify the genetic variants in the 5-HT1A gene that are associated with individual variation in aggression. We therefore conclude that co-evolution of the immune and nervous systems may have shaped the between-individual variation in aggression in natural populations of jumping spiders.

Transcriptome analysis of the painted lady butterfly, Vanessa cardui during wing color pattern development.

BACKGROUND: Butterfly wing color patterns are an important model system for understanding the evolution and development of morphological diversity and animal pigmentation. Wing color patterns develop from a complex network composed of highly conserved patterning genes and pigmentation pathways. Patterning genes are involved in regulating pigment synthesis however the temporal expression dynamics of these interacting networks is poorly understood. Here, we employ next generation sequencing to examine expression patterns of the gene network underlying wing development in the nymphalid butterfly, Vanessa cardui. RESULTS: We identified 9, 376 differentially expressed transcripts during wing color pattern development, including genes involved in patterning, pigmentation and gene regulation. Differential expression of these genes was highest at the pre-ommochrome stage compared to early pupal and late melanin stages. Overall, an increasing number of genes were down-regulated during the progression of wing development. We observed dynamic expression patterns of a large number of pigment genes from the ommochrome, melanin and also pteridine pathways, including contrasting patterns of expression for paralogs of the yellow gene family. Surprisingly, many patterning genes previously associated with butterfly pattern elements were not significantly up-regulated at any time during pupation, although many other transcription factors were differentially expressed. Several genes involved in Notch signaling were significantly up-regulated during the pre-ommochrome stage including slow border cells, bunched and pebbles; the function of these genes in the development of butterfly wings is currently unknown. Many genes involved in ecdysone signaling were also significantly up-regulated during early pupal and late melanin stages and exhibited opposing patterns of expression relative to the ecdysone receptor. Finally, a comparison across four butterfly transcriptomes revealed 28 transcripts common to all four species that have no known homologs in other metazoans. CONCLUSIONS: This study provides a comprehensive list of differentially expressed transcripts during wing development, revealing potential candidate genes that may be involved in regulating butterfly wing patterns. Some differentially expressed genes have no known homologs possibly representing genes unique to butterflies. Results from this study also indicate that development of nymphalid wing patterns may arise not only from melanin and ommochrome pigments but also the pteridine pigment pathway.

Spatial and temporal regulation of Wnt signaling pathway members in the development of butterfly wing patterns.

Wnt signaling members are involved in the differentiation of cells associated with eyespot and band color patterns on the wings of butterflies, but the identity and spatio-temporal regulation of specific Wnt pathway members remains unclear. Here, we explore the localization and function of Armadillo/ß-catenin dependent (canonical) and Armadillo/ß-catenin independent (noncanonical) Wnt signaling in eyespot and band development in Bicyclus anynana by localizing Armadillo (Arm), the expression of all eight Wnt ligand and four frizzled receptor transcripts present in the genome of this species and testing the function of some of the ligands and receptors using CRISPR-Cas9. We show that distinct Wnt signaling pathways are essential for eyespot and band patterning in butterflies and are likely interacting to control their active domains.

Tropical forests are not flat: how mountains affect herbivore diversity.

Ecologists debate whether tropical insect diversity is better explained by higher plant diversity or by host plant species specialization. However, plant-herbivore studies are primarily based in lowland rainforests (RF) thus excluding topographical effects on biodiversity. We examined turnover in Eois (Geometridae) communities across elevation by studying elevational transects in Costa Rica and Ecuador. We found four distinct Eois communities existing across the elevational gradients. Herbivore diversity was highest in montane forests (MF), whereas host plant diversity was highest in lowland RF. This was correlated with higher specialization and species richness of Eois/host plant species we found in MF. Based on these relationships, Neotropical Eois richness was estimated to range from 313 (only lowland RF considered) to 2034 (considering variation with elevation). We conclude that tropical herbivore diversity and diet breadth covary significantly with elevation and urge the inclusion of montane ecosystems in host specialization and arthropod diversity estimates.

Geographic variation in host-specificity and parasitoid pressure of an herbivore (geometridae) associated with the tropical genus piper (piperaceae).

The extraordinary diversity of tropical herbivores may be linked to hostplant specialization driven in part by variation in pressure from natural enemies. We quantified levels of host-specificity and parasitoid attack for the specialist herbivore, Eois (Geometridae). The goals of this research were to examine: 1) whether Eois are specialized on the genus Piper (Piperaceae) and if hostplant specialization varies geographically; 2) whether Eois are equally vulnerable to parasitoid attack across different geographic regions and by the same parasitoid families; and 3) whether parasitism levels vary with precipitation and elevation. Based on over 15,000 rearings, we found Eois caterpillars feeding exclusively on Piper. However, we did not detect geographic differences in host-specificity; each Eois species fed on an average of two Piper species. Parasitism levels of Eois varied significantly with climate and topography; Eois were most vulnerable to parasitoid attack in moist versus dry and wet forests and at low versus high elevations. The diversity of parasitoid families reared from Eois was greater in Ecuador and Costa Rica than in Panama, where parasitoids were primarily in the family Braconidae. The quantitative evidence for host-specificity provides support for the hypothesis that Eois are specialized on Piper. Our results also reveal that Eois are exposed to a mosaic of potential selective pressures due to variation in parasitoid attack over a large spatial scale.

A key to new world distatrix mason (Hymenoptera: Braconidae), with descriptions of six new reared neotropical species.

Six new species of the genus Distatrix Mason from Central and South America, D. loretta, D. xanadon, D. vigilis, D. pitillaensis, D. pandora Grinter, n. sp., and D. antirrheae Whitfield & Grinter, n. sp., are described from large-scale caterpillar inventory endeavors, mostly from the larvae of geometrid moths. Biological information and diagnostic features that distinguish these species from other previously described Distatrix, especially those from the Neotropical region, are provided; and the first key to New World species is presented. The new discoveries expand our knowledge of the World's widespread Distatrix fauna by about a third, suggesting that similar survey efforts in other poorly sampled regions will reveal numerous additional undescribed species.

The early stages and natural history of Antirrhea adoptive porphyrosticta (Watkins, 1928) in eastern ecuador (Lepidoptera: Nymphalidae: Morphinae).

Here we describe the immature stages and ecological associations of Antirrhea adoptiva porphyrosticta Watkins, 1928 (Lepidoptera:Nymphalidae:Morphinae). The cloud forest bamboo, Chusquea scandens Kunth (Bambusoidea: Poaceae), serves as the larval food plant for this butterfly in eastern Ecuador, the first hostplant record for Antirrhea outside the family Arecaceae. The larvae of A. adoptiva porphyrosticta are superficially similar to those of other Antirrhea species. We also provide observations on adult and larval behavior. Caterpillars of this butterfly species are parasitized by tachinid flies, as well as by Ichneumonidae and a newly described braconid wasp.

A high-coverage draft genome of the mycalesine butterfly Bicyclus anynana.

The mycalesine butterfly Bicyclus anynana, the "Squinting bush brown," is a model organism in the study of lepidopteran ecology, development, and evolution. Here, we present a draft genome sequence for B. anynana to serve as a genomics resource for current and future studies of this important model species. Seven libraries with insert sizes ranging from 350 bp to 20 kb were constructed using DNA from an inbred female and sequenced using both Illumina and PacBio technology; 128 Gb of raw Illumina data was filtered to 124 Gb and assembled to a final size of 475 Mb (∼×260 assembly coverage). Contigs were scaffolded using mate-pair, transcriptome, and PacBio data into 10 800 sequences with an N50 of 638 kb (longest scaffold 5 Mb). The genome is comprised of 26% repetitive elements and encodes a total of 22 642 predicted protein-coding genes. Recovery of a BUSCO set of core metazoan genes was almost complete (98%). Overall, these metrics compare well with other recently published lepidopteran genomes. We report a high-quality draft genome sequence for Bicyclus anynana. The genome assembly and annotated gene models are available at LepBase (http://ensembl.lepbase.org/index.html).

Physiological Perturbation Reveals Modularity of Eyespot Development in the Painted Lady Butterfly, Vanessa cardui.

Butterfly eyespots are complex morphological traits that can vary in size, shape and color composition even on the same wing surface. Homology among eyespots suggests they share a common developmental basis and function as an integrated unit in response to selection. Despite strong evidence of genetic integration, eyespots can also exhibit modularity or plasticity, indicating an underlying flexibility in pattern development. The extent to which particular eyespots or eyespot color elements exhibit modularity or integration is poorly understood, particularly following exposure to novel conditions. We used perturbation experiments to explore phenotypic correlations among different eyespots and their color elements on the ventral hindwing of V. cardui. Specifically, we identified which eyespots and eyespot features are most sensitive to perturbation by heat shock and injection of heparin-a cold shock mimic. For both treatments, the two central eyespots (3 + 4) were most affected by the experimental perturbations, whereas the outer eyespot border was more resistant to modification than the interior color elements. Overall, the individual color elements displayed a similar response to heat shock across all eyespots, but varied in their response to each other. Graphical modeling also revealed that although eyespots differ morphologically, regulation of eyespot size and colored elements appear to be largely integrated across the wing. Patterns of integration, however, were disrupted following heat shock, revealing that the strength of integration varies across the wing and is strongest between the two central eyespots. These findings support previous observations that document coupling between eyespots 3 + 4 in other nymphalid butterflies.