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

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

The 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.

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.

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.

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.

Wildlife-friendly garden practices increase butterfly abundance and species richness in urban and arable landscapes.

Insect declines are being reported worldwide and private, residential gardens could provide refugia for these species under increasing land use change. Interest in wildlife-friendly gardening has increased, but many management recommendations lack a scientific evidence-base. We used a large citizen science scheme, the Garden Butterfly Survey (GBS), with data from over 600 gardens across Great Britain (2016-2021) to determine how the surrounding landscape influences the abundance and species richness of butterflies in gardens and whether wildlife-friendly gardening practices, such as having long grass and providing nectar plants, benefit butterflies. First, we show that GBS provides reliable estimates of species abundances by comparing with results from standardised, long-term monitoring data. Garden size and surrounding land use had significant effects on butterfly abundance and richness in gardens, including positive relationships with garden size, woodland and arable farmland and negative relationships with urbanisation. Both the presence and area of long grass in gardens were positively related to higher butterfly richness and abundance, with the latter being driven by butterflies that use grasses as larval host plants. These effects differed depending on the surrounding landscape, such that long grass resulted in higher garden butterfly abundance in landscapes dominated by arable farming, and higher abundance and richness in highly urbanised areas. The presence of flowering ivy (Hedera spp.) in gardens resulted in higher abundance of Celastrina argiolus holly blue which uses ivy as a larval host, and of Vanessa atalanta red admiral and Polygonia c-album comma, which favour it as a nectar source. Our work provides evidence that undertaking simple wildlife-friendly garden practices can be beneficial for attracting butterflies, particularly in heavily modified areas. With over 728,000 ha of gardens in Great Britain, the cumulative effect of leaving areas of lawn uncut and providing nectar and larval host plants could be key for helping biodiversity.

Adaptive introgression of a visual preference gene.

Visual preferences are important drivers of mate choice and sexual selection, but little is known of how they evolve at the genetic level. In this study, we took advantage of the diversity of bright warning patterns displayed by Heliconius butterflies, which are also used during mate choice. Combining behavioral, population genomic, and expression analyses, we show that two Heliconius species have evolved the same preferences for red patterns by exchanging genetic material through hybridization. Neural expression of regucalcin1 correlates with visual preference across populations, and disruption of regucalcin1 with CRISPR-Cas9 impairs courtship toward conspecific females, providing a direct link between gene and behavior. Our results support a role for hybridization during behavioral evolution and show how visually guided behaviors contributing to adaptation and speciation are encoded within the genome.

Effects of climate change on Lepidoptera pollen loads and their pollination services in space and time.

Shifts in flowering time among plant communities as a result of climate change, including extreme weather events, are a growing concern. These plant phenological changes may affect the quantity and quality of food sources for specialized insect pollinators. Plant-pollinator interactions are threatened by habitat alterations and biodiversity loss, and changes in these interactions may lead to declines in flower visitors and pollination services. Most prior research has focused on short-term plant-pollinator interactions, which do not accurately capture changes in pollination services. Here, we characterized long-term plant-pollinator interactions and identified potential risks to specialized butterfly species due to habitat loss, fragmented landscapes, and changes in plant assemblages. We used 21 years of historical data from museum specimens to track the potential effects of direct and indirect changes in precipitation, temperature, monsoons, and wildfires on plant-pollinator mutualism in the Great Basin and Sierra Nevada. We found decreased pollen richness associated with butterflies within sites, as well as an increase in pollen grain abundance of drought-tolerant plants, particularly in the past 10 years. Moreover, increased global temperatures and the intensity and frequency of precipitation and wildfires were negatively correlated with pollen diversity. Our findings have important implications for understanding plant-pollinator interactions and the pollination services affected by global warming.

Viral Infection Induces Changes to the Metabolome, Immune Response and Development of a Generalist Insect Herbivore.

Host plant consumption and pathogen infection commonly influence insect traits related to development and immunity, which are ultimately reflected in the behavior and physiology of the insect. Herein, we explored changes in the metabolome of a generalist insect herbivore, Vanessa cardui (Lepidoptera: Nymphalidae), in response to both dietary variation and pathogen infection in order to gain insight into tritrophic interactions for insect metabolism and immunity. Caterpillars were reared on two different host plants, Plantago lanceolata (Plantaginaceae) and Taraxacum officinale (Asteraceae) and subjected to a viral infection by Junonia coenia densovirus (JcDV), along with assays to determine the insect immune response and development. Richness and diversity of plant and caterpillar metabolites were evaluated using a liquid chromatography-mass spectrometry approach and showed that viral infection induced changes to the chemical content of V. cardui hemolymph and frass dependent upon host plant consumption. Overall, the immune response as measured by phenoloxidase (PO) enzymatic activity was higher in individuals feeding on P. lanceolata compared with those feeding on T. officinale. Additionally, infection with JcDV caused suppression of PO activity, which was not host plant dependent. We conclude that viral infection combined with host plant consumption creates a unique chemical environment, particularly within the insect hemolymph. Whether and how these metabolites contribute to defense against viral infection is an open question in chemical ecology.

Rising minimum temperatures contribute to 50 years of occupancy decline among cold-adapted Arctic and boreal butterflies in North America.

Global climate change has been identified as a potential driver of observed insect declines, yet in many regions, there are critical data gaps that make it difficult to assess how communities are responding to climate change. Poleward regions are of particular interest because warming is most rapid while biodiversity data are most sparse. Building on recent advances in occupancy modeling of presence-only data, we reconstructed 50 years (1970-2019) of butterfly occupancy trends in response to rising minimum temperatures in one of the most under-sampled regions of North America. Among 90 modeled species, we found that cold-adapted species are far more often in decline compared with their warm-adapted, more southernly distributed counterparts. Furthermore, in a post hoc analysis using species' traits, we find that species' range-wide average annual temperature is the only consistent predictor of occupancy changes. Species with warmer ranges were most likely to be increasing in occupancy. This trend results in the majority of butterflies increasing in occupancy probability over the last 50 years. Our results provide the first look at macroscale butterfly biodiversity shifts in high-latitude North America. These results highlight the potential of leveraging the wealth of presence-only data, the most abundant source of biodiversity data, for inferring changes in species distributions.

Contrasting plant transcriptome responses between a pierce-sucking and a chewing herbivore go beyond the infestation site.

BACKGROUND: Plants have acquired a repertoire of mechanisms to combat biotic stressors, which may vary depending on the feeding strategies of herbivores and the plant species. Hormonal regulation crucially modulates this malleable defense response. Jasmonic acid (JA) and salicylic acid (SA) stand out as pivotal regulators of defense, while other hormones like abscisic acid (ABA), ethylene (ET), gibberellic acid (GA) or auxin also play a role in modulating plant-pest interactions. The plant defense response has been described to elicit effects in distal tissues, whereby aboveground herbivory can influence belowground response, and vice versa. This impact on distal tissues may be contingent upon the feeding guild, even affecting both the recovery of infested tissues and those that have not suffered active infestation. RESULTS: To study how phytophagous with distinct feeding strategies may differently trigger the plant defense response during and after infestation in both infested and distal tissues, Arabidopsis thaliana L. rosettes were infested separately with the chewing herbivore Pieris brassicae L. and the piercing-sucker Tetranychus urticae Koch. Moderate infestation conditions were selected for both pests, though no quantitative control of damage levels was carried out. Feeding mode did distinctly influence the transcriptomic response of the plant under these conditions. Though overall affected processes were similar under either infestation, their magnitude differed significantly. Plants infested with P. brassicae exhibited a short-term response, involving stress-related genes, JA and ABA regulation and suppressing growth-related genes. In contrast, T. urticae elicited a longer transcriptomic response in plants, albeit with a lower degree of differential expression, in particular influencing SA regulation. These distinct defense responses transcended beyond infestation and through the roots, where hormonal response, flavonoid regulation or cell wall reorganization were differentially affected. CONCLUSION: These outcomes confirm that the existent divergent transcriptomic responses elicited by herbivores employing distinct feeding strategies possess the capacity to extend beyond infestation and even affect tissues that have not been directly infested. This remarks the importance of considering the entire plant's response to localized biotic stresses.

Extreme heat reduces host and parasite performance in a butterfly-parasite interaction.

Environmental temperature fundamentally shapes insect physiology, fitness and interactions with parasites. Differential climate warming effects on host versus parasite biology could exacerbate or inhibit parasite transmission, with far-reaching implications for pollination services, biocontrol and human health. Here, we experimentally test how controlled temperatures influence multiple components of host and parasite fitness in monarch butterflies (Danaus plexippus) and their protozoan parasites Ophryocystis elektroscirrha. Using five constant-temperature treatments spanning 18-34°C, we measured monarch development, survival, size, immune function and parasite infection status and intensity. Monarch size and survival declined sharply at the hottest temperature (34°C), as did infection probability, suggesting that extreme heat decreases both host and parasite performance. The lack of infection at 34°C was not due to greater host immunity or faster host development but could instead reflect the thermal limits of parasite invasion and within-host replication. In the context of ongoing climate change, temperature increases above current thermal maxima could reduce the fitness of both monarchs and their parasites, with lower infection rates potentially balancing negative impacts of extreme heat on future monarch abundance and distribution.

Plant defensive responses to insect eggs are inducible by general egg-associated elicitors.

Egg deposition by herbivorous insects is well known to elicit defensive plant responses. Our study aimed to elucidate the insect and plant species specificity of these responses. To study the insect species specificity, we treated Arabidopsis thaliana with egg extracts and egg-associated secretions of a sawfly (Diprion pini), a beetle (Xanthogaleruca luteola) and a butterfly (Pieris brassicae). All egg extracts elicited salicylic acid (SA) accumulation in the plant, and all secretions induced expression of plant genes known to be responsive to the butterfly eggs, among them Pathogenesis-Related (PR) genes. All secretions contained phosphatidylcholine derivatives, known elicitors of SA accumulation and PR gene expression in Arabidopsis. The sawfly egg extract did not induce plant camalexin levels, while the other extracts did. Our studies on the plant species specificity revealed that Solanum dulcamara and Ulmus minor responded with SA accumulation and cell death to P. brassicae eggs, i.e. responses also known for A. thaliana. However, the butterfly eggs induced neoplasms only in S. dulcamara. Our results provide evidence for general, phosphatidylcholine-based, egg-associated elicitors of plant responses and for conserved plant core responses to eggs, but also point to plant and insect species-specific traits in plant-insect egg interactions.

Regional differences in thermoregulation between two European butterfly communities.

Understanding how different organisms cope with changing temperatures is vital for predicting future species' distributions and highlighting those at risk from climate change. As ectotherms, butterflies are sensitive to temperature changes, but the factors affecting butterfly thermoregulation are not fully understood. We investigated which factors influence thermoregulatory ability in a subset of the Mediterranean butterfly community. We measured adult thoracic temperature and environmental temperature (787 butterflies; 23 species) and compared buffering ability (defined as the ability to maintain a consistent body temperature across a range of air temperatures) and buffering mechanisms to previously published results from Great Britain. Finally, we tested whether thermoregulatory ability could explain species' demographic trends in Catalonia. The sampled sites in each region differ climatically, with higher temperatures and solar radiation but lower wind speeds in the Catalan sites. Both butterfly communities show nonlinear responses to temperature, suggesting a change in behaviour from heat-seeking to heat avoidance at approximately 22°C. However, the communities differ in the use of buffering mechanisms, with British populations depending more on microclimates for thermoregulation compared to Catalan populations. Contrary to the results from British populations, we did not find a relationship between region-wide demographic trends and butterfly thermoregulation, which may be due to the interplay between thermoregulation and the habitat changes occurring in each region. Thus, although Catalan butterfly populations seem to be able to thermoregulate successfully at present, evidence of heat avoidance suggests this situation may change in the future.

Morphology and spectral sensitivity of long visual fibers and lamina monopolar cells in the butterfly Papilio xuthus.

Extensive analysis of the flower-visiting behavior of a butterfly, Papilio xuthus, has indicated complex interaction between chromatic, achromatic, and motion cues. Their eyes are spectrally rich with six classes of photoreceptors, respectively sensitive in the ultraviolet, violet, blue, green, red, and broad-band wavelength regions. Here, we studied the anatomy and physiology of photoreceptors and second-order neurons of P. xuthus, focusing on their spectral sensitivities and projection terminals to address where the early visual integration takes place. We thus found the ultraviolet, violet, and blue photoreceptors and all second-order neurons terminate in the distal region of the second optic ganglion, the medulla. We identified five types of second-order neurons based on the arborization in the first optic ganglion, the lamina, and the shape of the medulla terminals. Their spectral sensitivity is independent of the morphological types but reflects the combination of pre-synaptic photoreceptors. The results indicate that the distal medulla is the most plausible region for early visual integration.

Thirty-six years of butterfly monitoring, snow cover, and plant productivity reveal negative impacts of warmer winters and increased productivity on montane species.

Climate change is contributing to declines of insects through rising temperatures, altered precipitation patterns, and an increasing frequency of extreme events. The impacts of both gradual and sudden shifts in weather patterns are realized directly on insect physiology and indirectly through impacts on other trophic levels. Here, we investigated direct effects of seasonal weather on butterfly occurrences and indirect effects mediated by plant productivity using a temporally intensive butterfly monitoring dataset, in combination with high-resolution climate data and a remotely sensed indicator of plant primary productivity. Specifically, we used Bayesian hierarchical path analysis to quantify relationships between weather and weather-driven plant productivity on the occurrence of 94 butterfly species from three localities distributed across an elevational gradient. We found that snow pack exerted a strong direct positive effect on butterfly occurrence and that low snow pack was the primary driver of reductions during drought. Additionally, we found that plant primary productivity had a consistently negative effect on butterfly occurrence. These results highlight mechanisms of weather-driven declines in insect populations and the nuances of climate change effects involving snow melt, which have implications for ecological theories linking topographic complexity to ecological resilience in montane systems.

Chemosensory detection of glucosinolates as token stimuli for specialist insects on brassicaceous plants: discovery and impact.

In this paper, we take a historical perspective by going back to Verschaffelt's landmark study published in 1910, in which he found that glucosinolates were used as token stimuli by larvae of Pieris butterflies, specialist feeders on plants in the family Brassicaceae. This classic discovery provided key evidence for Fraenkel (Science 129:1466-1470, 1959) to elaborate on the function of secondary plant substances and for Ehrlich and Raven (Evolution 18:586-608, 1964) to put forward the hypothesis of insect-plant coevolution. The discovery by Schoonhoven (Kon Nederl Akad Wetensch Amsterdam Proc Ser C70:556-568, 1967) of taste neurons highly sensitive to glucosinolates in Pieris brassicae was an important milestone in elucidating the chemosensory basis of host-plant specialization. The molecular basis of glucosinolate sensitivity was elucidated recently (Yang et al., PLoS Genet 17, 2021) paving the way to unravel the evolution of gustatory receptors tuned to glucosinolates that are crucial for host-plant selection of Pieris butterflies. We propose a hypothetical model for the evolution of labeled-line neurons tuned to token stimuli.

Multiple axes of visual system diversity in Ithomiini, an ecologically diverse tribe of mimetic butterflies.

The striking structural variation seen in arthropod visual systems can be explained by the overall quantity and spatio-temporal structure of light within habitats coupled with developmental and physiological constraints. However, little is currently known about how fine-scale variation in visual structures arises across shorter evolutionary and ecological scales. In this study, we characterise patterns of interspecific (between species), intraspecific (between sexes) and intraindividual (between eye regions) variation in the visual system of four ithomiine butterfly species. These species are part of a diverse 26-million-year-old Neotropical radiation where changes in mimetic colouration are associated with fine-scale shifts in ecology, such as microhabitat preference. Using a combination of selection analyses on visual opsin sequences, in vivo ophthalmoscopy, micro-computed tomography (micro-CT), immunohistochemistry, confocal microscopy and neural tracing, we quantify and describe physiological, anatomical and molecular traits involved in visual processing. Using these data, we provide evidence of substantial variation within the visual systems of Ithomiini, including: (i) relaxed selection on visual opsins, perhaps mediated by habitat preference, (ii) interspecific shifts in visual system physiology and anatomy, and (iii) extensive sexual dimorphism, including the complete absence of a butterfly-specific optic neuropil in the males of some species. We conclude that considerable visual system variation can exist within diverse insect radiations, hinting at the evolutionary lability of these systems to rapidly develop specialisations to distinct visual ecologies, with selection acting at the perceptual, processing and molecular level.

The cost of movement: assessing energy expenditure in a long-distant ectothermic migrant under climate change.

Migration is an energetically taxing phenomenon as animals move across vast, heterogeneous landscapes where the cost of transport is impacted by permissible ambient conditions. In this study, we assessed the energetic demands of long-distance migration in a multigenerational ectothermic migrant, the monarch butterfly (Danaus plexippus). We tested the hypotheses that temperature-dependent physiological processes reduce energy reserves faster during migration than previously estimated, and that increasing climatic temperatures resulting from the climate crisis will intensify baseline daily energy expenditure. First, we reared monarchs under laboratory conditions to assess energy and mass conversion from fifth instar to adult stages, as a baseline for migratory adult mass and ontogenetic shifts in metabolic rate from larvae to adult. Then, using historical tag-recapture data, we estimated the movement propensity and migratory pace of autumn migrants using computer simulations and subsequently calculated energy expenditure. Finally, we estimated the energy use of monarchs based on these tag-recapture data and used this information to estimate daily energy expenditure over a 57 year period. We found support for our two hypotheses, noting that incorporating standard metabolic rate into estimates of migratory energy expenditure shows higher energy demand and that daily energy expenditure has been gradually increasing over time since 1961. Our study shows the deleterious energetic consequences under current climate change trajectories and highlights the importance of incorporating energetic estimates for understanding migration by small, ectothermic migrants.