High level of novelty under the hood of convergent evolution.

Little is known about the extent to which species use homologous regulatory architectures to achieve phenotypic convergence. By characterizing chromatin accessibility and gene expression in developing wing tissues, we compared the regulatory architecture of convergence between a pair of mimetic butterfly species. Although a handful of color pattern genes are known to be involved in their convergence, our data suggest that different mutational paths underlie the integration of these genes into wing pattern development. This is supported by a large fraction of accessible chromatin being exclusive to each species, including the de novo lineage-specific evolution of a modular optix enhancer. These findings may be explained by a high level of developmental drift and evolutionary contingency that occurs during the independent evolution of mimicry.

Deep cis-regulatory homology of the butterfly wing pattern ground plan.

Butterfly wing patterns derive from a deeply conserved developmental ground plan yet are diverse and evolve rapidly. It is poorly understood how gene regulatory architectures can accommodate both deep homology and adaptive change. To address this, we characterized the cis-regulatory evolution of the color pattern gene WntA in nymphalid butterflies. Comparative assay for transposase-accessible chromatin using sequencing (ATAC-seq) and in vivo deletions spanning 46 cis-regulatory elements across five species revealed deep homology of ground plan-determining sequences, except in monarch butterflies. Furthermore, noncoding deletions displayed both positive and negative regulatory effects that were often broad in nature. Our results provide little support for models predicting rapid enhancer turnover and suggest that deeply ancestral, multifunctional noncoding elements can underlie rapidly evolving trait systems.

How butterfly wings got their pattern.

Gene regulatory elements play a crucial role in the pattern formation of butterfly wings.

Chemical and Molecular Composition of the Chrysalis Reveals Common Chitin-Rich Structural Framework for Monarchs and Swallowtails.

The metamorphosis of a caterpillar into a butterfly is an awe-inspiring example of how extraordinary functions are made possible through specific chemistry in nature's complex systems. The chrysalis exoskeleton is revealed and shed as a caterpillar transitions to butterfly form. We employed solid-state NMR to evaluate the chemical composition and types of biomolecules in the chrysalides from which Monarch and Swallowtail butterflies emerged. The chrysalis composition was remarkably similar between Monarch and Swallowtail. Chitin is the major polysaccharide component, present together with proteins and catechols or catechol-type linkages in each chrysalis. The high chitin content is comparable to the highest chitin-containing insect exoskeletons. Proteomics analyses indicated the presence of chitinases that could be involved in synthesis and remodeling of the chrysalis as well as cuticular proteins which play a role in the structural integrity of the chrysalis. The nearly identical 13C CPMAS NMR spectra of each chrysalis and similar structural proteins supports the presence of underlying design principles integrating chitin and protein partners to elaborate the chrysalis.

Evaluating toxicity of Varroa mite (Varroa destructor)-active dsRNA to monarch butterfly (Danaus plexippus) larvae.

Varroa mites (Varroa destructor) are parasitic mites that, combined with other factors, are contributing to high levels of honey bee (Apis mellifera) colony losses. A Varroa-active dsRNA was recently developed to control Varroa mites within honey bee brood cells. This dsRNA has 372 base pairs that are homologous to a sequence region within the Varroa mite calmodulin gene (cam). The Varroa-active dsRNA also shares a 21-base pair match with monarch butterfly (Danaus plexippus) calmodulin mRNA, raising the possibility of non-target effects if there is environmental exposure. We chronically exposed the entire monarch larval stage to common (Asclepias syriaca) and tropical (Asclepias curassavica) milkweed leaves treated with concentrations of Varroa-active dsRNA that are one- and ten-fold higher than those used to treat honey bee hives. This corresponded to concentrations of 0.025-0.041 and 0.211-0.282 mg/g leaf, respectively. Potassium arsenate and a previously designed monarch-active dsRNA with a 100% base pair match to the monarch v-ATPase A mRNA (leaf concentration was 0.020-0.034 mg/g) were used as positive controls. The Varroa mite and monarch-active dsRNA's did not cause significant differences in larval mortality, larval or pupal development, pupal weights, or adult eclosion rates when compared to negative controls. Irrespective of control or dsRNA treatment, larvae that consumed approximately 7500 to 10,500-mg milkweed leaf within 10 to 12 days had the highest pupal weights. The lack of mortality and sublethal effects following dietary exposure to dsRNA with 21-base pair and 100% base pair match to mRNAs that correspond to regulatory genes suggest monarch mRNA may be refractory to silencing by dsRNA or monarch dsRNase may degrade dsRNA to a concentration that is insufficient to silence mRNA signaling.

Porcupine/Wntless-dependent trafficking of the conserved WntA ligand in butterflies.

Wnt ligands are key signaling molecules in animals, but little is known about the evolutionary dynamics and mode of action of the WntA orthologs, which are not present in the vertebrates or in Drosophila. Here we show that the WntA subfamily evolved at the base of the Bilateria + Cnidaria clade, and conserved the thumb region and Ser209 acylation site present in most other Wnts, suggesting WntA requires the core Wnt secretory pathway. WntA proteins are distinguishable from other Wnts by a synapomorphic Iso/Val/Ala216 amino-acid residue that replaces the otherwise ubiquitous Thr216 position. WntA embryonic expression is conserved between beetles and butterflies, suggesting functionality, but the WntA gene was lost three times within arthropods, in podoplean copepods, in the cyclorrhaphan fly radiation, and in ensiferan crickets and katydids. Finally, CRISPR mosaic knockouts (KOs) of porcupine and wntless phenocopied the pattern-specific effects of WntA KOs in the wings of Vanessa cardui butterflies. These results highlight the molecular conservation of the WntA protein across invertebrates, and imply it functions as a typical Wnt ligand that is acylated and secreted through the Porcupine/Wntless secretory pathway.

Assessing the Role of Developmental and Environmental Factors in Chemical Defence Variation in Heliconiini Butterflies.

Chemical defences in animals are both incredibly widespread and highly diverse. Yet despite the important role they play in mediating interactions between predators and prey, extensive differences in the amounts and types of chemical compounds can exist between individuals, even within species and populations. Here we investigate the potential role of environment and development on the chemical defences of warningly coloured butterfly species from the tribe Heliconiini, which can both synthesize and sequester cyanogenic glycosides (CGs). We reared 5 Heliconiini species in captivity, each on a single species-specific host plant as larvae, and compared them to individuals collected in the wild to ascertain whether the variation in CG content observed in the field might be the result of differences in host plant availability. Three of these species were reared as larvae on the same host plant, Passiflora riparia, to further test how species, sex, and age affected the type and amount of different defensive CGs, and how they affected the ratio of synthesized to sequestered compounds. Then, focusing on the generalist species Heliconius numata, we specifically explored variation in chemical profiles as a result of the host plant consumed by caterpillars and their brood line, using rearing experiments carried out on two naturally co-occurring host plants with differing CG profiles. Our results show significant differences in both the amount of synthesized and sequestered compounds between butterflies reared in captivity and those collected in the field. We also found a significant effect of species and an effect of sex in some, but not all, species. We show that chemical defences in H. numata continue to increase throughout their life, likely because of continued biosynthesis, and we suggest that variation in the amount of synthesized CGs in this species does not appear to stem from larval host plants, although this warrants further study. Interestingly, we detected a significant effect of brood lines, consistent with heritability influencing CG concentrations in H. numata. Altogether, our results point to multiple factors resulting in chemical defence variation in Heliconiini butterflies and highlight the overlooked effect of synthesis capabilities, which may be genetically determined to some extent.

Genotype-environment interaction reveals varied developmental responses to unpredictable host phenology in a tropical insect.

Understanding the genetic architecture of life history plasticity may inform resilience under environmental change, but relatively little is known for the inhabitants of unpredictable wet-dry tropical environments. Here, I explore the quantitative genetics of juvenile growth and development relative to hostplant phenology in the butterfly Eurema hecabe. Wet season generations of this species breed explosively on leguminous annuals whereas dry season generations subsist at low density upon an alternative perennial host. The wet-to-dry season transition is temporally unpredictable and marked by widespread host defoliation, forcing a large cohort of stranded larvae to either pupate prematurely or prolong development in the hope of renewed foliage production. A split-brood experiment demonstrated greater performance on high quality annual as opposed to perennial host foliage and a marked decline under the stressed conditions faced by stranded wet season larvae. Genetic variances for rates of growth and development were equivalent among high quality treatments but strikingly elevated under resource stress, and the associated cross-environment genetic correlations were indistinguishable from zero. The results demonstrate genotype-environment interaction involving both rank order and variance scale, thereby revealing genetic variance for norms of reaction that may reflect variable risk aversion given an unpredictable tropical host phenology.

Pyrethroid Exposure Reduces Growth and Development of Monarch Butterfly (Lepidoptera: Nymphalidae) Caterpillars.

Insecticide exposure has been identified as a contributing stressor to the decline in the North American monarch butterfly Danaus plexippus L. (Lepidoptera: Nymphalidae) population. Monarch toxicity data are currently limited and available data focuses on lethal endpoints. This study examined the 72-h toxicity of two pyrethroid insecticides, bifenthrin and ß-cyfluthrin, and their effects on growth and diet consumption. The toxicity of bifenthrin to caterpillars was lower than ß-cyfluthrin after 72 h. Survival was the most sensitive endpoint for bifenthrin, but diet consumption and caterpillar growth were significantly reduced at sublethal levels of ß-cyfluthrin. Using AgDRIFT spray drift assessment, the aerial application of bifenthrin or ß-cyfluthrin is predicted to pose the greatest risk to fifth-instar caterpillars, with lethal insecticide deposition up to 28 m for bifenthrin and up to 23 m for ß-cyfluthrin from treated edges of fields. Low boom ground applications are predicted to reduce distances of lethal insecticide exposure to 2 m from the treated field edge for bifenthrin and ß-cyfluthrin. Growth and survival of fifth-instar monarch caterpillars developing within the margins of a treated field may be significantly impacted following foliar applications of bifenthrin or ß-cyfluthrin. These findings provide evidence that pyrethroid insecticides commonly used for soybean pest control are a potential risk to monarch caterpillars in agricultural landscapes.

Hox genes are essential for the development of eyespots in Bicyclus anynana butterflies.

The eyespot patterns found on the wings of nymphalid butterflies are novel traits that originated first in hindwings and subsequently in forewings, suggesting that eyespot development might be dependent on Hox genes. Hindwings differ from forewings in the expression of Ultrabithorax (Ubx), but the function of this Hox gene in eyespot development as well as that of another Hox gene Antennapedia (Antp), expressed specifically in eyespots centers on both wings, are still unclear. We used CRISPR-Cas9 to target both genes in Bicyclus anynana butterflies. We show that Antp is essential for eyespot development on the forewings and for the differentiation of white centers and larger eyespots on hindwings, whereas Ubx is essential not only for the development of at least some hindwing eyespots but also for repressing the size of other eyespots. Additionally, Antp is essential for the development of silver scales in male wings. In summary, Antp and Ubx, in addition to their conserved roles in modifying serially homologous segments along the anterior-posterior axis of insects, have acquired a novel role in promoting the development of a new set of serial homologs, the eyespot patterns, in both forewings (Antp) and hindwings (Antp and Ubx) of B. anynana butterflies. We propose that the peculiar pattern of eyespot origins on hindwings first, followed by forewings, could be due to an initial co-option of Ubx into eyespot development followed by a later, partially redundant, co-option of Antp into the same network.

Thermal tolerance and environmental persistence of a protozoan parasite in monarch butterflies.

Many parasites have external transmission stages that persist in the environment prior to infecting a new host. Understanding how long these stages can persist, and how abiotic conditions such as temperature affect parasite persistence, is important for predicting infection dynamics and parasite responses to future environmental change. In this study, we explored environmental persistence and thermal tolerance of a debilitating protozoan parasite that infects monarch butterflies. Parasite transmission occurs when dormant spores, shed by adult butterflies onto host plants and other surfaces, are later consumed by caterpillars. We exposed parasite spores to a gradient of ecologically-relevant temperatures for 2, 35, or 93 weeks. We tested spore viability by feeding controlled spore doses to susceptible monarch larvae, and examined relationships between temperature, time, and resulting infection metrics. We also examined whether distinct parasite genotypes derived from replicate migratory and resident monarch populations differed in their thermal tolerance. Finally, we examined evidence for a trade-off between short-term within-host replication and long-term persistence ability. Parasite viability decreased in response to warmer temperatures over moderate-to-long time scales. Individual parasite genotypes showed high heterogeneity in viability, but differences did not cluster by migratory vs. resident monarch populations. We found no support for a negative relationship between environmental persistence and within-host replication, as might be expected if parasites invest in short-term reproduction at the cost of longer-term survival. Findings here indicate that dormant spores can survive for many months under cooler conditions, and that heat dramatically shortens the window of transmission for this widespread and virulent butterfly parasite.

Molecular mechanisms underlying simplification of venation patterns in holometabolous insects.

How mechanisms of pattern formation evolve has remained a central research theme in the field of evolutionary and developmental biology. The mechanism of wing vein differentiation in Drosophila is a classic text-book example of pattern formation using a system of positional information, yet very little is known about how species with a different number of veins pattern their wings, and how insect venation patterns evolved. Here, we examine the expression pattern of genes previously implicated in vein differentiation in Drosophila in two butterfly species with more complex venation Bicyclus anynana and Pieris canidia We also test the function of some of these genes in B. anynana We identify both conserved as well as new domains of decapentaplegic, engrailed, invected, spalt, optix, wingless, armadillo, blistered and rhomboid gene expression in butterflies, and propose how the simplified venation in Drosophila might have evolved via loss of decapentaplegic, spalt and optix gene expression domains, via silencing of vein-inducing programs at Spalt-expression boundaries, and via changes in expression of vein maintenance genes.

Host Plant Choice Determined by Reproductive Interference between Closely Related Butterflies.

AbstractA number of empirical studies have concluded that reproductive interference (RI) contributes to parapatric species distributions or sexual exclusion. However, the possibility that divergent host plant use in phytophagous insects is due to sexual exclusion has seldom been considered. Here, we present evidence that RI is responsible for different host plant use by two Pierid butterfly species, Pieris napi and Pieris melete. When a novel host species was introduced about 50 years ago, two Pierid butterfly species at first used both the ancestral host species and the novel one. Subsequently, P. napi shifted to use only the novel host, while P. melete shifted to specialize on the ancestral host. To explain these patterns, we investigated whether the two host species differ in suitability for larval growth and survival. Additionally, we tested whether RI occurred between the two butterfly species using large outdoor field cages. Courtship of females by conspecific and heterospecific males reduces the number of eggs laid by approximately half. However, RI is asymmetric and would generate selection on P. melete females to evolve to avoid the more suitable host species preferred by P. napi. Thus, our study suggests that sexual exclusion can explain the shift in host plant use by these two butterfly species.

Genetic Variation Influences Tolerance to a Neonicotinoid Insecticide in 3 Butterfly Species.

Neonicotinoid pesticides harm nontarget insects, but their sublethal effects on butterflies are understudied. We exposed larvae of 3 butterfly species (Pieris rapae, Colias philodice, and Danaus plexippus) to low levels of the neonicotinoid imidacloprid in their host plants and followed individuals to adulthood. Imidacloprid altered adult body size, especially in female monarchs, but its effects varied across maternal families, highlighting the importance of considering genetic variation in ecotoxicological testing. Environ Toxicol Chem 2020;39:2228-2236. © 2020 SETAC.

Eradicating the large white butterfly from New Zealand eliminates a threat to endemic Brassicaceae.

In May 2010 the large white butterfly, Pieris brassicae L. (Lepidoptera: Pieridae), was discovered to have established in New Zealand. It is a Palearctic species that-due to its wide host plant range within the Brassicaceae-was regarded as a risk to New Zealand's native brassicas. New Zealand has 83 native species of Brassicaceae including 81 that are endemic, and many are threatened by both habitat loss and herbivory by other organisms. Initially a program was implemented to slow its spread, then an eradication attempt commenced in November 2012. The P. brassicae population was distributed over an area of approximately 100 km2 primarily in urban residential gardens. The eradication attempt involved promoting public engagement and reports of sightings, including offering a bounty for a two week period, systematically searching gardens for P. brassicae and its host plants, removing host plants, ground-based spraying of insecticide to kill eggs and larvae, searching for pupae, capturing adults with nets, and augmenting natural enemy populations. The attempt was supported by research that helped to progressively refine the eradication strategy and evaluate its performance. The last New Zealand detection of P. brassicae occurred on 16 December 2014, the eradication program ceased on 4 June 2016 and P. brassicae was officially declared eradicated from New Zealand on 22 November 2016, 6.5 years after it was first detected and 4 years after the eradication attempt commenced. This is the first species of butterfly ever to have been eradicated worldwide.

Perfect mimicry between Heliconius butterflies is constrained by genetics and development.

Müllerian mimicry strongly exemplifies the power of natural selection. However, the exact measure of such adaptive phenotypic convergence and the possible causes of its imperfection often remain unidentified. Here, we first quantify wing colour pattern differences in the forewing region of 14 co-mimetic colour pattern morphs of the butterfly species Heliconius erato and Heliconius melpomene and measure the extent to which mimicking colour pattern morphs are not perfectly identical. Next, using gene-editing CRISPR/Cas9 KO experiments of the gene WntA, which has been mapped to colour pattern diversity in these butterflies, we explore the exact areas of the wings in which WntA affects colour pattern formation differently in H. erato and H. melpomene. We find that, while the relative size of the forewing pattern is generally nearly identical between co-mimics, the CRISPR/Cas9 KO results highlight divergent boundaries in the wing that prevent the co-mimics from achieving perfect mimicry. We suggest that this mismatch may be explained by divergence in the gene regulatory network that defines wing colour patterning in both species, thus constraining morphological evolution even between closely related species.

Multiple roles forlaccase2 in butterfly wing pigmentation, scale development, and cuticle tanning.

Lepidopteran wing scales play important roles in a number of functions including color patterning and thermoregulation. Despite the importance of wing scales, however, we still have a limited understanding of the genetic mechanisms that underlie scale patterning, development, and coloration. Here, we explore the function of the phenoloxidase-encoding gene laccase2 in wing and scale development in the nymphalid butterfly Vanessa cardui. Somatic deletion mosaics of laccase2 generated by CRISPR/Cas9 genome editing presented several distinct mutant phenotypes. Consistent with the work in other nonlepidopteran insect groups, we observed reductions in melanin pigmentation and defects in cuticle formation. We were also surprised, however, to see distinct effects on scale development including complete loss of wing scales. This study highlights laccase2 as a gene that plays multiple roles in wing and scale development and provides new insight into the evolution of lepidopteran wing coloration.

Sexual differences in age-dependent survival and life span of adults in a natural butterfly population.

Adult survival and longevity in insects are key life-history traits, but their variation between sexes and individuals in natural populations is largely unexplored. Sexual divergence in senescence, the decline in survival with age is also poorly understood. Based on an intensive mark-recapture dataset of the butterfly Polyommatus daphnis, we aimed to assess whether adult survival is age-dependent, and to estimate life span distribution and abundance of males and females using Cormack-Jolly-Seber and Jolly-Seber models. Female survival slightly increased with date of emergence and slightly decreased with age, while male survival considerably declined with age. Mean life span of females (12.7 days) was ~50% higher than that of males (8.5 days), but two times higher if only the oldest 5% of each sex was considered (39 vs.19 days). Abundance of females (358 ± 14) and males (359 ± 11) was similar, but peak abundance of males preceded that of females by 11 days. Our results suggest that senescence is much more rapid in males than in females in this butterfly, which is in agreement with sexual selection theory. We also conclude that estimating life span distributions provides much more valuable information on the demography of natural populations than simply reporting the mean life span.

Morphology and histology of vom Rath's organ in brush-footed butterflies (Lepidoptera: Nymphalidae).

Vom Rath's organ, located at the distal end of the third segment of the labial palp, is one of the recognized synapomorphies of Lepidoptera (Insecta). Information about the structural and histological morphology of this organ is sparse. The structure of vom Rath's organ in four species of Nymphalidae, three frugivorous: Fountainea ryphea (Charaxinae: Anaeini), Morpho helenor achillaena (Satyrinae: Morphini) and Hamadryas epinome (Biblidinae: Ageroniini), and the nectarivorous species Aeria olena (Danainae: Ithomiini) is described by means of scanning electron microscopy and histology. The species showed significant differences in the cavity shape, setal morphology and arrangement, opening shape and location, associated with the organization of cell groups, type of axon, and degree of development. These differences do not seem to be related to feeding habit. No cell groups were found in Actinote thalia (Heliconiinae: Acraeini) and Heliconius erato phyllis (Heliconiinae: Heliconiini), and for the first time the absence of vom Rath's organ is documented in the clade Ditrysia. A terminology is proposed to improve understanding of the organ morphology, with an extensive analysis of the previous descriptions.