Male and female contributions to diversity among birdwing butterfly images.

Machine learning (ML) newly enables tests for higher inter-species diversity in visible phenotype (disparity) among males versus females, predictions made from Darwinian sexual selection versus Wallacean natural selection, respectively. Here, we use ML to quantify variation across a sample of > 16,000 dorsal and ventral photographs of the sexually dimorphic birdwing butterflies (Lepidoptera: Papilionidae). Validation of image embedding distances, learnt by a triplet-trained, deep convolutional neural network, shows ML can be used for automated reconstruction of phenotypic evolution achieving measures of phylogenetic congruence to genetic species trees within a range sampled among genetic trees themselves. Quantification of sexual disparity difference (male versus female embedding distance), shows sexually and phylogenetically variable inter-species disparity. Ornithoptera exemplify high embedded male image disparity, diversification of selective optima in fitted multi-peak OU models and accelerated divergence, with cases of extreme divergence in allopatry and sympatry. However, genus Troides shows inverted patterns, including comparatively static male embedded phenotype, and higher female than male disparity - though within an inferred selective regime common to these females. Birdwing shapes and colour patterns that are most phenotypically distinctive in ML similarity are generally those of males. However, either sex can contribute majoritively to observed phenotypic diversity among species.

Monarch butterfly conservation through male germplasm cryopreservation.

Monarch butterfly (Danaus plexippus L.) populations have declined in North America. The International Union for Conservation of Nature (IUCN) recently classified the species as endangered, sparking public concern and conservation efforts. Our approach to conservation is through cryopreservation of germinal cells and tissue. The goal of this study was to develop a cryopreservation protocol for monarch spermatozoa to ensure successful long-term storage. Cryopreserved sperm cells would provide a reserve of monarch germplasm, which could be utilized in the event of population loss. In this study, sperm cell bundles collected from male monarch butterflies were cryopreserved in a cryoprotective medium and stored in liquid nitrogen. To determine the post-cryopreservation sperm cell viability, a subsample of preserved sperm bundles were thawed rapidly, and their viability was qualified using a sperm live/dead stain. We are presenting a protocol to preserve and store genetic material and viable sperm bundles of the monarch butterfly. To date, this is the first report of successful cryopreservation of monarch germplasm which sets the foundation for cryostorage and could be extensible to other vulnerable lepidopterans.

A trans-oceanic flight of over 4,200 km by painted lady butterflies.

The extent of aerial flows of insects circulating around the planet and their impact on ecosystems and biogeography remain enigmatic because of methodological challenges. Here we report a transatlantic crossing by Vanessa cardui butterflies spanning at least 4200 km, from West Africa to South America (French Guiana) and lasting between 5 and 8 days. Even more, we infer a likely natal origin for these individuals in Western Europe, and the journey Europe-Africa-South America could expand to 7000 km or more. This discovery was possible through an integrative approach, including coastal field surveys, wind trajectory modelling, genomics, pollen metabarcoding, ecological niche modelling, and multi-isotope geolocation of natal origins. The overall journey, which was energetically feasible only if assisted by winds, is among the longest documented for individual insects, and potentially the first verified transatlantic crossing. Our findings suggest that we may be underestimating transoceanic dispersal in insects and highlight the importance of aerial highways connecting continents by trade winds.

Bottom-up butterfly model with thorax-pitch control and wing-pitch flexibility.

The diversity in butterfly morphology has attracted many people around the world since ancient times. Despite morphological diversity, the wing and body kinematics of butterflies have several common features. In the present study, we constructed a bottom-up butterfly model, whose morphology and kinematics are simplified while preserving the important features of butterflies. The present bottom-up butterfly model is composed of two trapezoidal wings and a rod-shaped body with a thorax and abdomen. Its wings are flapped downward in the downstroke and backward in the upstroke by changing the geometric angle of attack (AOA). The geometric AOA is determined by the thorax-pitch and wing-pitch angles. The thorax-pitch angle is actively controlled by abdominal undulation, and the wing-pitch angle is passively determined because of a rotary spring representing the basalar and subalar muscles connecting the wings and thorax. We investigated the effectiveness of abdominal undulation for thorax-pitch control and how wing-pitch flexibility affects aerodynamic-force generation and thorax-pitch control, through numerical simulations using the immersed boundary-lattice Boltzmann method. As a result, the thorax-pitch angle perfectly follows the desired angle through abdominal undulation. In addition, there is an optimal wing-pitch flexibility that maximizes the flying speed in both the forward and upward directions, but the effect of wing-pitch flexibility on thorax-pitch control is not significant. Finally, we compared the flight behavior of the present bottom-up butterfly model with that of an actual butterfly. It was found that the present model does not reproduce reasonable body kinematics but can provide reasonable aerodynamics in butterfly flights.

Insect ecology: Tracking migratory butterflies through pollen.

Painted ladies are well-known migratory butterflies, but confirmation of the details of their swarming flights through Europe has evaded scientists until now. It was their role as pollinators, carrying pollen grains on their flights, that helped unlock the secrets of their migrations.

Wetland butterfly thriving in abandoned jungle: Neptis rivularis in the Czech Republic.

With ongoing insect declines, species expanding in distribution and abundance deserve attention, as understanding their success may help design conservation strategies for less successful species. Common causes of these successes include warmer climates, novel resources, and exploiting land use change, including land abandonment. These factors affect the nymphalid butterfly Neptis rivularis, developing on Spiraea spp. shrubs and reaching the north-western limits of its trans-Palearctic distribution in Central Europe. We combined mark-recapture, behaviour analysis, and distribution modelling to study N. rivularis in wetlands of the Trebonsko Protected Landscape (IUCN category V). The long-living adults (up to 4 weeks) spent a considerable amount of time searching for partners, ovipositing and nectaring at Spiraea shrubs, alternating this with stays in tree crowns, where they located cool shelters, spent nights, and presumably fed on honeydew. They formed high-density populations (310 adults/ha), exploiting high host plant abundance. They adhered to floodplains and to conditions of relatively mild winters. The ongoing Spiraea encroachment of abandoned alluvial grasslands is, thus, a transient situation, ultimately followed by forest encroachment. Rewilding the habitats by introducing native ungulates presents an opportunity to restore the disturbance regime of the sites. The increased resource supply combined with a warming climate has opened up temperate Europe to colonization by N. rivularis.

Pollen metabarcoding reveals the origin and multigenerational migratory pathway of an intercontinental-scale butterfly outbreak.

Migratory insects may move in large numbers, even surpassing migratory vertebrates in biomass. Long-distance migratory insects complete annual cycles through multiple generations, with each generation's reproductive success linked to the resources available at different breeding grounds. Climatic anomalies in these grounds are presumed to trigger rapid population outbreaks. Here, we infer the origin and track the multigenerational path of a remarkable outbreak of painted lady (Vanessa cardui) butterflies that took place at an intercontinental scale in Europe, the Middle East, and Africa from March 2019 to November 2019. Using metabarcoding, we identified pollen transported by 264 butterflies captured in 10 countries over 7 months and modeled the distribution of the 398 plants detected. The analysis showed that swarms collected in Eastern Europe in early spring originated in Arabia and the Middle East, coinciding with a positive anomaly in vegetation growth in the region from November 2018 to April 2019. From there, the swarms advanced to Northern Europe during late spring, followed by an early reversal toward southwestern Europe in summer. The pollen-based evidence matched spatiotemporal abundance peaks revealed by citizen science, which also suggested an echo effect of the outbreak in West Africa during September-November. Our results show that population outbreaks in a part of species' migratory ranges may disseminate demographic effects across multiple generations in a wide geographic area. This study represents an unprecedented effort to track a continuous multigenerational insect migration on an intercontinental scale.

Too hot to handle: temperature-induced plasticity influences pollinator behaviour and plant fitness.

Increased temperature can induce plastic changes in many plant traits. However, little is known about how these changes affect plant interactions with insect pollinators and herbivores, and what the consequences for plant fitness and selection are. We grew fast-cycling Brassica rapa plants at two temperatures (ambient and increased temperature) and phenotyped them (floral traits, scent, colour and glucosinolates). We then exposed plants to both pollinators (Bombus terrestris) and pollinating herbivores (Pieris rapae). We measured flower visitation, oviposition of P. rapae, herbivore development and seed output. Plants in the hot environment produced more but smaller flowers, with lower UV reflectance and emitted a different volatile blend with overall lower volatile emission. Moreover, these plants received fewer first-choice visits by bumblebees and butterflies, and fewer flower visits by butterflies. Seed production was lower in hot environment plants, both because of a reduction in flower fertility due to temperature and because of the reduced visitation of pollinators. The selection on plant traits changed in strength and direction between temperatures. Our study highlights an important mechanism by which global warming can change plant-pollinator interactions and negatively impact plant fitness, as well as potentially alter plant evolution through changes in phenotypic selection.

Evidence of plasticity, but not evolutionary divergence, in the thermal limits of a highly successful urban butterfly.

Despite the generally negative impact of urbanization on insect biodiversity, some insect species persist in urban habitats. Understanding the mechanisms underpinning the ability of insects to tolerate urban habitats is critical given the contribution of land-use change to the global insect decline. Compensatory mechanisms such as phenotypic plasticity and evolutionary change in thermal physiological traits could allow urban populations to persist under the altered thermal regimes of urban habitats. It is important to understand the contributions of plasticity and evolution to trait change along urbanization gradients as the two mechanisms operate under different constraints and timescales. Here, we examine the plastic and evolutionary responses of heat and cold tolerance (critical thermal maximum [CTmax] and critical thermal minimum [CTmin]) to warming among populations of the cabbage white butterfly, Pieris rapae, from urban and non-urban (rural) habitats using a two-temperature common garden experiment. Although we expected populations experiencing urban warming to exhibit greater CTmax and diminished CTmin through plastic and evolutionary mechanisms, our study revealed evidence only for plasticity in the expected direction of both thermal tolerance traits. We found no evidence of evolutionary divergence in either heat or cold tolerance, despite each trait showing evolutionary potential. Our results suggest that thermal tolerance plasticity contributes to urban persistence in this system. However, as the magnitude of the plastic response was low and comparable to other insect species, other compensatory mechanisms likely further underpin this species' success in urban habitats.

Selection drives divergence of eye morphology in sympatric Heliconius butterflies.

When populations experience different sensory conditions, natural selection may favor sensory system divergence, affecting peripheral structures and/or downstream neural pathways. We characterized the outer eye morphology of sympatric Heliconius butterflies from different forest types and their first-generation reciprocal hybrids to test for adaptive visual system divergence and hybrid disruption. In Panama, Heliconius cydno occurs in closed forests, whereas Heliconius melpomene resides at the forest edge. Among wild individuals, H. cydno has larger eyes than H. melpomene, and there are heritable, habitat-associated differences in the visual brain structures that exceed neutral divergence expectations. Notably, hybrids have intermediate neural phenotypes, suggesting disruption. To test for similar effects in the visual periphery, we reared both species and their hybrids in common garden conditions. We confirm that H. cydno has larger eyes and provide new evidence that this is driven by selection. Hybrid eye morphology is more H. melpomene-like despite body size being intermediate, contrasting with neural trait intermediacy. Overall, our results suggest that eye morphology differences between H. cydno and H. melpomene are adaptive and that hybrids may suffer fitness costs due to a mismatch between the peripheral visual structures and previously described neural traits that could affect visual performance.

Influence of Host Plants and Tending Ants on the Cuticular Hydrocarbon Profile of a Generalist Myrmecophilous Caterpillar.

In myrmecophilous organisms, which live in symbiosis with ants, cuticular hydrocarbons (CHCs) play a pivotal role in interspecific communication and defense against chemical-oriented predators. Although these interactions form complex information webs, little is known about the influence of biotic environmental factors on the CHC profiles of myrmecophiles. Here, we analyzed the effect of different host plants and tending ants on the larval CHC profile of Synargis calyce (Lepidoptera: Riodinidae), a polyphagous species with facultative myrmecophily. Groups of caterpillars were fed individually with three host plant species (without tending ants), and with two tending ant species. Through gas chromatography analysis, we compared the cuticular profiles of treatments and found a high similarity between plants and caterpillars (65-82%), but a low similarity between caterpillars and their tending ants (30 - 25%). Cluster analysis showed that caterpillars, ants, and plants form distinct groups, indicating that S. calyce caterpillars have their own chemical profile. These results are similar to those observed for Lycaenidae caterpillars indicating that there is functional convergence in the chemical strategies used by myrmecophilous caterpillar species with similar ecology. Also, the results suggest that the cuticular compounds of S. calyce are primarily influenced by their host plants rather than their tending ants. Thus, we propose that these caterpillars present a trade-off between camouflage and directly informing their presence to ants, maintaining their unique chemical profile, though slightly affected by biotic environmental factors.

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.

Bee and butterfly records indicate diversity losses in western and southern North America, but extensive knowledge gaps remain.

Pollinator losses threaten ecosystems and food security, diminishing gene flow and reproductive output for ecological communities and impacting ecosystem services broadly. For four focal families of bees and butterflies, we constructed over 1400 ensemble species distribution models over two time periods for North America. Models indicated disproportionally increased richness in eastern North America over time, with decreases in richness over time in the western US and southern Mexico. To further pinpoint geographic areas of vulnerability, we mapped records of potential pollinator species of conservation concern and found high concentrations of detections in the Great Lakes region, US East Coast, and southern Canada. Finally, we estimated asymptotic diversity indices for genera known to include species that visit flowers and may carry pollen for ecoregions across two time periods. Patterns of generic diversity through time mirrored those of species-level analyses, again indicating a decline in pollinators in the western U.S. Increases in generic diversity were observed in cooler and wetter ecoregions. Overall, changes in pollinator diversity appear to reflect changes in climate, though other factors such as land use change may also explain regional shifts. While statistical methods were employed to account for unequal sampling effort across regions and time, improved monitoring efforts with rigorous sampling designs would provide a deeper understanding of pollinator communities and their responses to ongoing environmental change.

Rapid spread of a vertically transmitted symbiont induces drastic shifts in butterfly sex ratio.

The causes and consequences of sex-ratio dynamics constitutes a pivotal subject in evolutionary biology1. Under conditions of evolutionary equilibrium, the male-to-female ratio tends to be approximately 1:1; however, this equilibrium is susceptible to distortion by selfish genetic elements exemplified by driving sex chromosomes and cytoplasmic elements2,3. Although previous studies have documented instances of these genetic elements distorting the sex ratio, studies specifically tracking the process with which these distorters spread within populations, leading to a transition from balanced parity to a skewed, female-biased state, are notably lacking. Herein, we present compelling evidence documenting the rapid spread of the cytoplasmic endosymbiont Wolbachia within a localized population of the pierid butterfly Eurema hecabe (Figure 1A). This spread resulted in a shift in the sex ratio from near parity to an exceedingly skewed state overwhelmingly biased toward females, reaching 93.1% within a remarkably brief period of 4 years.

The actin cytoskeleton plays multiple roles in structural colour formation in butterfly wing scales.

Vivid structural colours in butterflies are caused by photonic nanostructures scattering light. Structural colours evolved for numerous biological signalling functions and have important technological applications. Optically, such structures are well understood, however insight into their development in vivo remains scarce. We show that actin is intimately involved in structural colour formation in butterfly wing scales. Using comparisons between iridescent (structurally coloured) and non-iridescent scales in adult and developing H. sara, we show that iridescent scales have more densely packed actin bundles leading to an increased density of reflective ridges. Super-resolution microscopy across three distantly related butterfly species reveals that actin is repeatedly re-arranged during scale development and crucially when the optical nanostructures are forming. Furthermore, actin perturbation experiments at these later developmental stages resulted in near total loss of structural colour in H. sara. Overall, this shows that actin plays a vital and direct templating role during structural colour formation in butterfly scales, providing ridge patterning mechanisms that are likely universal across lepidoptera.

Phenotypic plasticity in tropical butterflies is linked to climatic seasonality on a macroevolutionary scale.

Phenotypic plasticity can be adaptive in fluctuating environments by providing rapid environment-phenotype matching and this applies particularly in seasonal environments. African Bicyclus butterflies have repeatedly colonized seasonal savannahs from ancestral forests around the late Miocene, and many species now exhibit seasonal polyphenism. On a macroevolutionary scale, it can be expected that savannah species will exhibit higher plasticity because of experiencing stronger environmental seasonality than forest species. We quantified seasonality using environmental niche modeling and surveyed the degree of plasticity in a key wing pattern element (eyespot size) using museum specimens. We showed that species occurring in highly seasonal environments display strong plasticity, while species in less seasonal or aseasonal environments exhibit surprisingly variable degrees of plasticity, including strong to no plasticity. Furthermore, eyespot size plasticity has a moderate phylogenetic signal and the ancestral Bicyclus likely exhibited some degree of plasticity. We propose hypotheses to explain the range of plasticity patterns seen in less seasonal environments and generate testable predictions for the evolution of plasticity in Bicyclus. Our study provides one of the most compelling cases showing links between seasonality and phenotypic plasticity on a macroevolutionary scale and the potential role of plasticity in facilitating the colonization of novel environments.

Blind measurements did not confirm effects of forest fragmentation on fluctuating asymmetry of a tropical butterfly Morpho helenor.

Re-evaluation of photographs of the tropical butterfly Morpho helenor from a previous study (Pignataro et al. 2023) revealed that its conclusion regarding increased wing fluctuating asymmetry in forest edge habitats compared to forest interior habitats could not be replicated. This discrepancy likely arises from (i) original measurements not being conducted blindly, (ii) insufficient photograph quality hindering accurate landmark selection, and (iii) a lack of detailed description of the measurement protocol. The likelihood of false positive discoveries within the published data concerning the impacts of environmental stress on the fluctuating asymmetry of plants and animals is probably higher than previously assumed.

The quantitative genetic basis of variation in sexual versus non-sexual butterfly wing colouration: autosomal, Z-linked, and maternal effects.

Viability indicator traits are expected to be integrated extensively across the genome yet sex-limited to ensure that any benefits are sexually concordant. Understanding how such expectations are accommodated requires elucidating the quantitative genetic architecture of candidate traits in and across the sexes. Here we applied an animal modelling approach to partition the autosomal, allosomal, and direct maternal bases of variation in sexual versus non-sexual dorsal wing colouration in the butterfly Eurema hecabe. The sexual colour trait-coherently scattered ultraviolet that is under strong directional selection due to female choice-is brighter and more expansive in males, and overlays non-sexual pigmentary yellow markings that otherwise dominate both wing surfaces in each sex. Our modelling estimated high and sexually equivalent autosomal variances for ultraviolet reflectance (furnishing h2 ~ 0.58 overall and ~0.75 in males), accompanied by smaller but generally significant Z-linked and maternal components. By contrast, variation in non-sexual yellow was largely attributed to Z-linked sources. Intersexual genetic correlations based upon the major source of variation in each trait were high and not different from 1.0, implying regulation by a pool of genes common to each sex. An expansive autosomal basis for ultraviolet is consistent with its hypothesized role as a genome-wide viability indicator and ensures that both sons and daughters will inherit their father's attractiveness.