The evolution and diversification of oakleaf butterflies.

Oakleaf butterflies in the genus Kallima have a polymorphic wing phenotype, enabling these insects to masquerade as dead leaves. This iconic example of protective resemblance provides an interesting evolutionary paradigm that can be employed to study biodiversity. We integrated multi-omic data analyses and functional validation to infer the evolutionary history of Kallima species and investigate the genetic basis of their variable leaf wing patterns. We find that Kallima butterflies diversified in the eastern Himalayas and dispersed to East and Southeast Asia. Moreover, we find that leaf wing polymorphism is controlled by the wing patterning gene cortex, which has been maintained in Kallima by long-term balancing selection. Our results provide macroevolutionary and microevolutionary insights into a model species originating from a mountain ecosystem.

Epsin-Dependent Ligand Endocytosis Activates Notch by Force.

DSL ligands activate Notch by inducing proteolytic cleavage of the receptor ectodomain, an event that requires ligand to be endocytosed in signal-sending cells by the adaptor protein Epsin. Two classes of explanation for this unusual requirement are (1) recycling models, in which the ligand must be endocytosed to be modified or repositioned before it binds Notch and (2) pulling models, in which the ligand must be endocytosed after it binds Notch to exert force that exposes an otherwise buried site for cleavage. We demonstrate in vivo that ligands that cannot enter the Epsin pathway nevertheless bind Notch but fail to activate the receptor because they cannot exert sufficient force. This argues against recycling models and in favor of pulling models. Our results also suggest that once ligand binds receptor, activation depends on a competition between Epsin-mediated ligand endocytosis, which induces cleavage, and transendocytosis of the ligand by the receptor, which aborts the incipient signal.

Subcellular trafficking of FGF controls tracheal invasion of Drosophila flight muscle.

To meet the extreme oxygen demand of insect flight muscle, tracheal (respiratory) tubes ramify not only on its surface, as in other tissues, but also within T-tubules and ultimately surrounding every mitochondrion. Although this remarkable physiological specialization has long been recognized, its cellular and molecular basis is unknown. Here, we show that Drosophila tracheoles invade flight muscle T-tubules through transient surface openings. Like other tracheal branching events, invasion requires the Branchless FGF pathway. However, localization of the FGF chemoattractant changes from all muscle membranes to T-tubules as invasion begins. Core regulators of epithelial basolateral membrane identity localize to T-tubules, and knockdown of AP-1γ, required for basolateral trafficking, redirects FGF from T-tubules to surface, increasing tracheal surface ramification and preventing invasion. We propose that tracheal invasion is controlled by an AP-1-dependent switch in FGF trafficking. Thus, subcellular targeting of a chemoattractant can direct outgrowth to specific domains, including inside the cell.

Machine learning reveals the control mechanics of an insect wing hinge.

Insects constitute the most species-rich radiation of metazoa, a success that is due to the evolution of active flight. Unlike pterosaurs, birds and bats, the wings of insects did not evolve from legs1, but are novel structures that are attached to the body via a biomechanically complex hinge that transforms tiny, high-frequency oscillations of specialized power muscles into the sweeping back-and-forth motion of the wings2. The hinge consists of a system of tiny, hardened structures called sclerites that are interconnected to one another via flexible joints and regulated by the activity of specialized control muscles. Here we imaged the activity of these muscles in a fly using a genetically encoded calcium indicator, while simultaneously tracking the three-dimensional motion of the wings with high-speed cameras. Using machine learning, we created a convolutional neural network3 that accurately predicts wing motion from the activity of the steering muscles, and an encoder-decoder4 that predicts the role of the individual sclerites on wing motion. By replaying patterns of wing motion on a dynamically scaled robotic fly, we quantified the effects of steering muscle activity on aerodynamic forces. A physics-based simulation incorporating our hinge model generates flight manoeuvres that are remarkably similar to those of free-flying flies. This integrative, multi-disciplinary approach reveals the mechanical control logic of the insect wing hinge, arguably among the most sophisticated and evolutionarily important skeletal structures in the natural world.

The respiratory system influences flight mechanics in soaring birds.

The subpectoral diverticulum (SPD) is an extension of the respiratory system in birds that is located between the primary muscles responsible for flapping the wing1,2. Here we survey the pulmonary apparatus in 68 avian species, and show that the SPD was present in virtually all of the soaring taxa investigated but absent in non-soarers. We find that this structure evolved independently with soaring flight at least seven times, which indicates that the diverticulum might have a functional and adaptive relationship with this flight style. Using the soaring hawks Buteo jamaicensis and Buteo swainsoni as models, we show that the SPD is not integral for ventilation, that an inflated SPD can increase the moment arm of cranial parts of the pectoralis, and that pectoralis muscle fascicles are significantly shorter in soaring hawks than in non-soaring birds. This coupling of an SPD-mediated increase in pectoralis leverage with force-specialized muscle architecture produces a pneumatic system that is adapted for the isometric contractile conditions expected in soaring flight. The discovery of a mechanical role for the respiratory system in avian locomotion underscores the functional complexity and heterogeneity of this organ system, and suggests that pulmonary diverticula are likely to have other undiscovered secondary functions. These data provide a mechanistic explanation for the repeated appearance of the SPD in soaring lineages and show that the respiratory system can be co-opted to provide biomechanical solutions to the challenges of flight and thereby influence the evolution of avian volancy.

Hybrid speciation driven by multilocus introgression of ecological traits.

Hybridization allows adaptations to be shared among lineages and may trigger the evolution of new species1,2. However, convincing examples of homoploid hybrid speciation remain rare because it is challenging to demonstrate that hybridization was crucial in generating reproductive isolation3. Here we combine population genomic analysis with quantitative trait locus mapping of species-specific traits to examine a case of hybrid speciation in Heliconius butterflies. We show that Heliconius elevatus is a hybrid species that is sympatric with both parents and has persisted as an independently evolving lineage for at least 180,000 years. This is despite pervasive and ongoing gene flow with one parent, Heliconius pardalinus, which homogenizes 99% of their genomes. The remaining 1% introgressed from the other parent, Heliconius melpomene, and is scattered widely across the H. elevatus genome in islands of divergence from H. pardalinus. These islands contain multiple traits that are under disruptive selection, including colour pattern, wing shape, host plant preference, sex pheromones and mate choice. Collectively, these traits place H. elevatus on its own adaptive peak and permit coexistence with both parents. Our results show that speciation was driven by introgression of ecological traits, and that speciation with gene flow is possible with a multilocus genetic architecture.

Nuclear position and local acetyl-CoA production regulate chromatin state.

Histone acetylation regulates gene expression, cell function and cell fate1. Here we study the pattern of histone acetylation in the epithelial tissue of the Drosophila wing disc. H3K18ac, H4K8ac and total lysine acetylation are increased in the outer rim of the disc. This acetylation pattern is controlled by nuclear position, whereby nuclei continuously move from apical to basal locations within the epithelium and exhibit high levels of H3K18ac when they are in proximity to the tissue surface. These surface nuclei have increased levels of acetyl-CoA synthase, which generates the acetyl-CoA for histone acetylation. The carbon source for histone acetylation in the rim is fatty acid ß-oxidation, which is also increased in the rim. Inhibition of fatty acid ß-oxidation causes H3K18ac levels to decrease in the genomic proximity of genes involved in disc development. In summary, there is a physical mark of the outer rim of the wing and other imaginal epithelia in Drosophila that affects gene expression.

Cytoskeletal tension inhibits Hippo signaling through an Ajuba-Warts complex.

Mechanical forces have been proposed to modulate organ growth, but a molecular mechanism that links them to growth regulation in vivo has been lacking. We report that increasing tension within the cytoskeleton increases Drosophila wing growth, whereas decreasing cytoskeletal tension decreases wing growth. These changes in growth can be accounted for by changes in the activity of Yorkie, a transcription factor regulated by the Hippo pathway. The influence of myosin activity on Yorkie depends genetically on the Ajuba LIM protein Jub, a negative regulator of Warts within the Hippo pathway. We further show that Jub associates with α-catenin and that its localization to adherens junctions and association with α-catenin are promoted by cytoskeletal tension. Jub recruits Warts to junctions in a tension-dependent manner. Our observations delineate a mechanism that links cytoskeletal tension to regulation of Hippo pathway activity, providing a molecular understanding of how mechanical forces can modulate organ growth.

The atypical cadherin fat directly regulates mitochondrial function and metabolic state.

Fat (Ft) cadherins are enormous cell adhesion molecules that function at the cell surface to regulate the tumor-suppressive Hippo signaling pathway and planar cell polarity (PCP) tissue organization. Mutations in Ft cadherins are found in a variety of tumors, and it is presumed that this is due to defects in either Hippo signaling or PCP. Here, we show Drosophila Ft functions in mitochondria to directly regulate mitochondrial electron transport chain integrity and promote oxidative phosphorylation. Proteolytic cleavage releases a soluble 68 kDa fragment (Ft(mito)) that is imported into mitochondria. Ft(mito) binds directly to NADH dehydrogenase ubiquinone flavoprotein 2 (Ndufv2), a core component of complex I, stabilizing the holoenzyme. Loss of Ft leads to loss of complex I activity, increases in reactive oxygen species, and a switch to aerobic glycolysis. Defects in mitochondrial activity in ft mutants are independent of Hippo and PCP signaling and are reminiscent of the Warburg effect.

Bridging two insect flight modes in evolution, physiology and robophysics.

Since taking flight, insects have undergone repeated evolutionary transitions between two seemingly distinct flight modes1-3. Some insects neurally activate their muscles synchronously with each wingstroke. However, many insects have achieved wingbeat frequencies beyond the speed limit of typical neuromuscular systems by evolving flight muscles that are asynchronous with neural activation and activate in response to mechanical stretch2-8. These modes reflect the two fundamental ways of generating rhythmic movement: time-periodic forcing versus emergent oscillations from self-excitation8-10. How repeated evolutionary transitions have occurred and what governs the switching between these distinct modes remain unknown. Here we find that, despite widespread asynchronous actuation in insects across the phylogeny3,6, asynchrony probably evolved only once at the order level, with many reversions to the ancestral, synchronous mode. A synchronous moth species, evolved from an asynchronous ancestor, still preserves the stretch-activated muscle physiology. Numerical and robophysical analyses of a unified biophysical framework reveal that rather than a dichotomy, these two modes are two regimes of the same dynamics. Insects can transition between flight modes across a bridge in physiological parameter space. Finally, we integrate these two actuation modes into an insect-scale robot11-13 that enables transitions between modes and unlocks a new self-excited wingstroke strategy for engineered flight. Together, this framework accounts for repeated transitions in insect flight evolution and shows how flight modes can flip with changes in physiological parameters.

Birds can transition between stable and unstable states via wing morphing.

Birds morph their wing shape to accomplish extraordinary manoeuvres1-4, which are governed by avian-specific equations of motion. Solving these equations requires information about a bird's aerodynamic and inertial characteristics5. Avian flight research to date has focused on resolving aerodynamic features, whereas inertial properties including centre of gravity and moment of inertia are seldom addressed. Here we use an analytical method to determine the inertial characteristics of 22 species across the full range of elbow and wrist flexion and extension. We find that wing morphing allows birds to substantially change their roll and yaw inertia but has a minimal effect on the position of the centre of gravity. With the addition of inertial characteristics, we derived a novel metric of pitch agility and estimated the static pitch stability, revealing that the agility and static margin ranges are reduced as body mass increases. These results provide quantitative evidence that evolution selects for both stable and unstable flight, in contrast to the prevailing narrative that birds are evolving away from stability6. This comprehensive analysis of avian inertial characteristics provides the key features required to establish a theoretical model of avian manoeuvrability.

Novel flight style and light wings boost flight performance of tiny beetles.

Flight speed is positively correlated with body size in animals1. However, miniature featherwing beetles can fly at speeds and accelerations of insects three times their size2. Here we show that this performance results from a reduced wing mass and a previously unknown type of wing-motion cycle. Our experiment combines three-dimensional reconstructions of morphology and kinematics in one of the smallest insects, the beetle Paratuposa placentis (body length 395 µm). The flapping bristled wings follow a pronounced figure-of-eight loop that consists of subperpendicular up and down strokes followed by claps at stroke reversals above and below the body. The elytra act as inertial brakes that prevent excessive body oscillation. Computational analyses suggest functional decomposition of the wingbeat cycle into two power half strokes, which produce a large upward force, and two down-dragging recovery half strokes. In contrast to heavier membranous wings, the motion of bristled wings of the same size requires little inertial power. Muscle mechanical power requirements thus remain positive throughout the wingbeat cycle, making elastic energy storage obsolete. These adaptations help to explain how extremely small insects have preserved good aerial performance during miniaturization, one of the factors of their evolutionary success.

Morphogen gradient scaling by recycling of intracellular Dpp.

Morphogen gradients are fundamental to establish morphological patterns in developing tissues1. During development, gradients scale to remain proportional to the size of growing organs2,3. Scaling is a universal gear that adjusts patterns to size in living organisms3-8, but its mechanisms remain unclear. Here, focusing on the Decapentaplegic (Dpp) gradient in the Drosophila wing disc, we uncover a cell biological basis behind scaling. From small to large discs, scaling of the Dpp gradient is achieved by increasing the contribution of the internalized Dpp molecules to Dpp transport: to expand the gradient, endocytosed molecules are re-exocytosed to spread extracellularly. To regulate the contribution of endocytosed Dpp to the spreading extracellular pool during tissue growth, it is the Dpp binding rates that are progressively modulated by the extracellular factor Pentagone, which drives scaling. Thus, for some morphogens, evolution may act on endocytic trafficking to regulate the range of the gradient and its scaling, which could allow the adaptation of shape and pattern to different sizes of organs in different species.

Accommodating unobservability to control flight attitude with optic flow.

Attitude control is an essential flight capability. Whereas flying robots commonly rely on accelerometers1 for estimating attitude, flying insects lack an unambiguous sense of gravity2,3. Despite the established role of several sense organs in attitude stabilization3-5, the dependence of flying insects on an internal gravity direction estimate remains unclear. Here we show how attitude can be extracted from optic flow when combined with a motion model that relates attitude to acceleration direction. Although there are conditions such as hover in which the attitude is unobservable, we prove that the ensuing control system is still stable, continuously moving into and out of these conditions. Flying robot experiments confirm that accommodating unobservability in this manner leads to stable, but slightly oscillatory, attitude control. Moreover, experiments with a bio-inspired flapping-wing robot show that residual, high-frequency attitude oscillations from flapping motion improve observability. The presented approach holds a promise for robotics, with accelerometer-less autopilots paving the road for insect-scale autonomous flying robots6. Finally, it forms a hypothesis on insect attitude estimation and control, with the potential to provide further insight into known biological phenomena5,7,8 and to generate new predictions such as reduced head and body attitude variance at higher flight speeds9.

Local Ecdysone synthesis in a wounded epithelium sustains developmental delay and promotes regeneration in Drosophila.

Regenerative ability often declines as animals mature past embryonic and juvenile stages, suggesting that regeneration requires redirection of growth pathways that promote developmental growth. Intriguingly, the Drosophila larval epithelia require the hormone ecdysone (Ec) for growth but require a drop in circulating Ec levels to regenerate. Examining Ec dynamics more closely, we find that transcriptional activity of the Ec-receptor (EcR) drops in uninjured regions of wing discs, but simultaneously rises in cells around the injury-induced blastema. In parallel, blastema depletion of genes encoding Ec biosynthesis enzymes blocks EcR activity and impairs regeneration but has no effect on uninjured wings. We find that local Ec/EcR signaling is required for injury-induced pupariation delay following injury and that key regeneration regulators upd3 and Ets21c respond to Ec levels. Collectively, these data indicate that injury induces a local source of Ec within the wing blastema that sustains a transcriptional signature necessary for developmental delay and tissue repair.

Organ transformation by environmental disruption of protein integrity and epigenetic memory in Drosophila.

Despite significant progress in understanding epigenetic reprogramming of cells, the mechanistic basis of "organ reprogramming" by (epi-)gene-environment interactions remained largely obscure. Here, we use the ether-induced haltere-to-wing transformations in Drosophila as a model for epigenetic "reprogramming" at the whole organism level. Our findings support a mechanistic chain of events explaining why and how brief embryonic exposure to ether leads to haltere-to-wing transformations manifested at the larval stage and on. We show that ether interferes with protein integrity in the egg, leading to altered deployment of Hsp90 and widespread repression of Trithorax-mediated establishment of active H3K4me3 chromatin marks throughout the genome. Despite this global reduction, Ubx targets and wing development genes preferentially retain higher levels of H3K4me3 that predispose these genes for later up-regulation in the larval haltere disc, hence the wing-like outcome. Consistent with compromised protein integrity during the exposure, the penetrance of bithorax transformations increases by genetic or chemical reduction of Hsp90 function. Moreover, joint reduction in Hsp90 and trx gene dosage can cause bithorax transformations without exposure to ether, supporting an underlying epistasis between Hsp90 and trx loss-of-functions. These findings implicate environmental disruption of protein integrity at the onset of histone methylation with altered epigenetic regulation of developmental patterning genes. The emerging picture provides a unique example wherein the alleviation of the Hsp90 "capacitor function" by the environment drives a morphogenetic shift towards an ancestral-like body plan. The morphogenetic impact of chaperone response during a major setup of epigenetic patterns may be a general scheme for organ transformation by environmental cues.

Pervasive mimicry in flight behavior among aposematic butterflies.

Flight was a key innovation in the adaptive radiation of insects. However, it is a complex trait influenced by a large number of interacting biotic and abiotic factors, making it difficult to unravel the evolutionary drivers. We investigate flight patterns in neotropical heliconiine butterflies, well known for mimicry of their aposematic wing color patterns. We quantify the flight patterns (wing beat frequency and wing angles) of 351 individuals representing 29 heliconiine and 9 ithomiine species belonging to ten color pattern mimicry groupings. For wing beat frequency and up wing angles, we show that heliconiine species group by color pattern mimicry affiliation. Convergence of down wing angles to mimicry groupings is less pronounced, indicating that distinct components of flight are under different selection pressures and constraints. The flight characteristics of the Tiger mimicry group are particularly divergent due to convergence with distantly related ithomiine species. Predator-driven selection for mimicry also explained variation in flight among subspecies, indicating that this convergence can occur over relatively short evolutionary timescales. Our results suggest that the flight convergence is driven by aposematic signaling rather than shared habitat between comimics. We demonstrate that behavioral mimicry can occur between lineages that have separated over evolutionary timescales ranging from <0.5 to 70 My.

What one genus of showy moths can say about migration, adaptation, and wing pattern.

The Ornate Moth, Utetheisa ornatrix, has served as a model species in chemical ecology studies for decades. Like in the widely publicized stories of the Monarch and other milkweed butterflies, the Ornate Moth and its relatives are tropical insects colonizing whole continents assisted by their chemical defenses. With the recent advances in genomic techniques and evo-devo research, it is becoming a model for studies in other areas, from wing pattern development to phylogeography, from toxicology to epigenetics. We used a genomic approach to learn about Utetheisa's evolution, detoxification, dispersal abilities, and wing pattern diversity. We present an evolutionary genomic analysis of the worldwide genus Utetheisa, then focusing on U. ornatrix. Our reference genome of U. ornatrix reveals gene duplications in the regions possibly associated with detoxification abilities, which allows them to feed on toxic food plants. Finally, comparative genomic analysis of over 100 U. ornatrix specimens from the museum with apparent differences in wing patterns suggest the potential roles of cortex and lim3 genes in wing pattern formation of Lepidoptera and the utility of museum-preserved collection specimens for wing pattern research.

Functional constraints on the number and shape of flight feathers.

As a fundamental ecological aspect of most organisms, locomotor function significantly constrains morphology. At the same time, the evolution of novel locomotor abilities has produced dramatic morphological transformations, initiating some of the most significant diversifications in life history. Despite significant new fossil evidence, it remains unclear whether volant locomotion had a single or multiple origins in pennaraptoran dinosaurs and the volant abilities of individual taxa are controversial. The evolution of powered flight in modern birds involved exaptation of feathered surfaces extending off the limbs and tail yet most studies concerning flight potential in pennaraptorans do not account for the structure and morphology of the wing feathers themselves. Analysis of the number and shape of remex and rectrix feathers across a large dataset of extant birds indicates that the number of remiges and rectrices and the degree of primary vane asymmetry strongly correlate with locomotor ability revealing important functional constraints. Among these traits, phenotypic flexibility varies reflected by the different rates at which morphological changes evolve, such that some traits reflect the ancestral condition, whereas others reflect current locomotor function. While Mesozoic birds and Microraptor have remex morphologies consistent with extant volant birds, that of anchiornithines deviate significantly providing strong evidence this clade was not volant. The results of these analyses support a single origin of dinosaurian flight and indicate the early stages of feathered wing evolution are not sampled by the currently available fossil record.

Accurate staging of chick embryonic tissues via deep learning of salient features.

Recent work shows that the developmental potential of progenitor cells in the HH10 chick brain changes rapidly, accompanied by subtle changes in morphology. This demands increased temporal resolution for studies of the brain at this stage, necessitating precise and unbiased staging. Here, we investigated whether we could train a deep convolutional neural network to sub-stage HH10 chick brains using a small dataset of 151 expertly labelled images. By augmenting our images with biologically informed transformations and data-driven preprocessing steps, we successfully trained a classifier to sub-stage HH10 brains to 87.1% test accuracy. To determine whether our classifier could be generally applied, we re-trained it using images (269) of randomised control and experimental chick wings, and obtained similarly high test accuracy (86.1%). Saliency analyses revealed that biologically relevant features are used for classification. Our strategy enables training of image classifiers for various applications in developmental biology with limited microscopy data.