Lethal trap created by adaptive evolutionary response to an exotic resource.

Global transport of organisms by humans provides novel resources to wild species, which often respond maladaptively. Native herbivorous insects have been killed feeding on toxic exotic plants, which acted as 'ecological traps'1-4. We document a novel 'eco-evolutionary trap' stemming from the opposite effect; that is, high fitness on an exotic resource despite lack of adaptation to it. Plantago lanceolata was introduced to western North America by cattle-ranching. Feeding on this exotic plant released a large, isolated population of the native butterfly Euphydryas editha from a longstanding trade-off between maternal fecundity and offspring mortality. Because of this release-and despite a reduced insect developmental rate when feeding on this exotic-Plantago immediately supported higher larval survival than did the insects' traditional host, Collinsia parviflora 5 . Previous work from the 1980s documented an evolving preference for Plantago by ovipositing adults 6 . We predicted that if this trend continued the insects could endanger themselves, because the availability of Plantago to butterflies is controlled by humans, who change land management practices faster than butterflies evolve 6 . Here we report the fulfilment of this prediction. The butterflies abandoned Collinsia and evolved total dependence on Plantago. The trap was set. In 2005, humans withdrew their cattle, springing the trap. Grasses grew around the Plantago, cooling the thermophilic insects, which then went extinct. This local extinction could have been prevented if the population had retained partial use of Collinsia, which occupied drier microhabitats unaffected by cattle removal. The flush of grasses abated quickly, rendering the meadow once again suitable for Euphydryas feeding on either host, but no butterflies were observed from 2008 to 2012. In 2013-2014, the site was naturally recolonized by Euphydryas feeding exclusively on Collinsia, returning the system to its starting point and setting the stage for a repeat of the anthropogenic evolutionary cycle.

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.

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.

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.

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.

Genome-Wide Regulatory Adaptation Shapes Population-Level Genomic Landscapes in Heliconius.

Cis-regulatory evolution is an important engine of organismal diversification. Although recent studies have looked at genomic patterns of regulatory evolution between species, we still have a poor understanding of the magnitude and nature of regulatory variation within species. Here, we examine the evolution of regulatory element activity over wing development in three Heliconius erato butterfly populations to determine how regulatory variation is associated with population structure. We show that intraspecific divergence in chromatin accessibility and regulatory activity is abundant, and that regulatory variants are spatially clustered in the genome. Regions with strong population structure are highly enriched for regulatory variants, and enrichment patterns are associated with developmental stage and gene expression. We also found that variable regulatory elements are particularly enriched in species-specific genomic regions and long interspersed nuclear elements. Our findings suggest that genome-wide selection on chromatin accessibility and regulatory activity is an important force driving patterns of genomic divergence within Heliconius species. This work also provides a resource for the study of gene regulatory evolution in H. erato and other heliconiine butterflies.

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.

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.

Tolerance of Novel Toxins through Generalized Mechanisms: Simulating Gradual Host Shifts of Butterflies.

Organisms encounter a wide range of toxic compounds in their environments, from chemicals that serve anticonsumption or anticompetition functions to pollutants and pesticides. Although we understand many detoxification mechanisms that allow organisms to consume toxins typical of their diet, we know little about why organisms vary in their ability to tolerate entirely novel toxins. We tested whether variation in generalized stress responses, such as antioxidant pathways, may underlie variation in reactions to novel toxins and, if so, their associated costs. We used an artificial diet to present cabbage white butterfly caterpillars (Pieris rapae) with plant material containing toxins not experienced in their evolutionary history. Families that maintained high performance (e.g., high survival, fast development time, large body size) on diets containing one novel toxic plant also performed well when exposed to two other novel toxic plants, consistent with a generalized response. Variation in constitutive (but not induced) expression of genes involved in oxidative stress responses was positively related to performance on the novel diets. While we did not detect reproductive trade-offs of this generalized response, there was a tendency to have less melanin investment in the wings, consistent with the role of melanin in oxidative stress responses. Taken together, our results support the hypothesis that variation in generalized stress responses, such as genes involved in oxidative stress responses, may explain the variation in tolerance to entirely novel toxins and may facilitate colonization of novel hosts and environments.

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.

Legacy of a Butterfly's Parental Microbiome in Offspring Performance.

An insect's phenotype can be influenced by the experiences of the parental generation. However, the effects of the parental symbiotic microbiome and host plant use on the offspring are unclear. We addressed this gap of knowledge by studying Pieris brassicae, a multivoltine butterfly species feeding on different brassicaceous plants across generations. We investigated how disturbance of the parental bacterial community by antibiotic treatment affects F1 larval traits. We tested the effects depending on whether F1 larvae are feeding on the same plant species as their parents or on a different one. The parental treatment alone had no impact on the biomass of F1 larvae feeding on the parental plant species. However, the parental treatment had a detrimental effect on F1 larval biomass when F1 larvae had a different host plant than their parents. This effect was linked to higher larval prophenoloxidase activity and greater downregulation of the major allergen gene (MA), a glucosinolate detoxification gene of P. brassicae Bacterial abundance in untreated adult parents was high, while it was very low in F1 larvae from either parental type, and thus unlikely to directly influence larval traits. Our results suggest that transgenerational effects of the parental microbiome on the offspring's phenotype become evident when the offspring is exposed to a transgenerational host plant shift.IMPORTANCE Resident bacterial communities are almost absent in larvae of butterflies and thus are unlikely to affect their host. In contrast, adult butterflies contain conspicuous amounts of bacteria. While the host plant and immune state of adult parental butterflies are known to affect offspring traits, it has been unclear whether also the parental microbiome imposes direct effects on the offspring. Here, we show that disturbance of the bacterial community in parental butterflies by an antibiotic treatment has a detrimental effect on those offspring larvae feeding on a different host plant than their parents. Hence, the study indicates that disturbance of an insect's parental microbiome by an antibiotic treatment shapes how the offspring individuals can adjust themselves to a novel host plant.

Genotype-environment interactions rule the response of a widespread butterfly to temperature variation.

Understanding how organisms adapt to complex environments is a central goal of evolutionary biology and ecology. This issue is of special interest in the current era of rapidly changing climatic conditions. Here, we investigate clinal variation and plastic responses in life history, morphology and physiology in the butterfly Pieris napi along a pan-European gradient by exposing butterflies raised in captivity to different temperatures. We found clinal variation in body size, growth rates and concomitant development time, wing aspect ratio, wing melanization and heat tolerance. Individuals from warmer environments were more heat-tolerant and had less melanised wings and a shorter development, but still they were larger than individuals from cooler environments. These findings suggest selection for rapid growth in the warmth and for wing melanization in the cold, and thus fine-tuned genetic adaptation to local climates. Irrespective of the origin of butterflies, the effects of higher developmental temperature were largely as expected, speeding up development; reducing body size, potential metabolic activity and wing melanization; while increasing heat tolerance. At least in part, these patterns likely reflect adaptive phenotypic plasticity. In summary, our study revealed pronounced plastic and genetic responses, which may indicate high adaptive capacities in our study organism. Whether this may help such species, though, to deal with current climate change needs further investigation, as clinal patterns have typically evolved over long periods.

Variation in Below-to Aboveground Systemic Induction of Glucosinolates Mediates Plant Fitness Consequences under Herbivore Attack.

Plants defend themselves against herbivore attack by constitutively producing toxic secondary metabolites, as well as by inducing them in response to herbivore feeding. Induction of secondary metabolites can cross plant tissue boundaries, such as from root to shoot. However, whether the potential for plants to systemically induce secondary metabolites from roots to shoots shows genetic variability, and thus, potentially, is under selection conferring fitness benefits to the plants is an open question. To address this question, we induced 26 maternal plant families of the wild species Cardamine hirsuta belowground (BG) using the wound-mimicking phytohormone jasmonic acid (JA). We measured resistance against a generalist (Spodoptera littoralis) and a specialist (Pieris brassicae) herbivore species, as well as the production of glucosinolates (GSLs) in plants. We showed that BG induction increased AG resistance against the generalist but not against the specialist, and found substantial plant family-level variation for resistance and GSL induction. We further found that the systemic induction of several GSLs tempered the negative effects of herbivory on total seed set production. Using a widespread natural system, we thus confirm that BG to AG induction has a strong genetic component, and can be under positive selection by increasing plant fitness. We suggest that natural variation in systemic induction is in part dictated by allocation trade-offs between constitutive and inducible GSL production, as well as natural variation in AG and BG herbivore attack in nature.

Seasonal phenotype-specific expression of microRNAs during metamorphosis in the European map butterfly Araschnia levana.

The European map butterfly (Araschnia levana) is a well-known example of seasonal polyphenism because the spring and summer imagoes exhibit distinct morphological phenotypes. The day length and temperature during larval and prepupal development determine whether spring or summer imagoes emerge after metamorphosis. Inspired by the fundamentally different transcriptomic profiles in prepupae developing from larvae exposed to long days or short days, we postulate that posttranscriptional epigenetic regulators such as microRNAs (miRNAs) may contribute to the epigenetic control of seasonal polyphenism in A. levana. To test this hypothesis, we used microarrays containing over 2,000 insect miRNAs to identify candidate regulators that are differentially expressed in last-instar larvae or pupae developing under long-day or short-day conditions. We used our transcriptomic database to identify potential 3'-untranslated regions of messenger RNAs to predict miRNA targets by considering both base pair complementarity and minimum free energy hybridization. This approach resulted in the identification of multiple targets of miRNAs that were differentially regulated in polyphenic morphs of A. levana including a candidate (miR-2856-3p) regulating the previously identified diapause bioclock protein gene. In conclusion, the expression profiling of miRNAs provided insights into their possible involvement in seasonal polyphenism of A. levana and offer an important resource for further studies.

Parasite-host specificity: A cross-infection study of the parasite Ophryocystis elektroscirrha.

Many parasites are constrained to only one or a few hosts, showing host specificity. It remains unclear why some parasites are specialists and other parasites are generalists. The parasite Ophryocystis elektroscirrha (OE) is a neogregarine protozoan thought to be restricted to monarch butterflies, Danaus plexippus (Nymphaliae) and D. gilippus. Recently, we found OE-like spores in other Lepidoptera, specifically in three noctuid moths: Helicoverpa armigera, H. assulta and H. punctigera, as well as another nymphalid, Parthenos sylvia. To our knowledge, this is the first report of OE-like parasite infections in species other than the genus Danaus. In sequencing 558 bp of 18S rRNA, we found the genetic similarity between OE from D. plexippus and OE-like parasite from the moths H. armigera and H. punctigera to be 95.2%. When we conducted cross-species infection experiments, we could not infect the moths with OE from D. plexippus, but OE-like parasite from H. armigera did infect D. plexippus and a closely related moth species Heliothis virescens. Interestingly, we did not find the OE-like parasite in the H. armigera population from Spain. Inter-population infection experiments with H. armigera demonstrated a higher sensitivity to OE-like infection in the population from Spain compared to the populations from Australia and China. These results suggest geographic variation in OE-like susceptibility and coevolution between parasite and host. Our findings give important new insights into the prevalence and host specificity of OE and OE-like parasites, and provide opportunities to study parasite transmission over spatial and temporal scales.

Single master regulatory gene coordinates the evolution and development of butterfly color and iridescence.

The optix gene has been implicated in butterfly wing pattern adaptation by genetic association, mapping, and expression studies. The actual developmental function of this gene has remained unclear, however. Here we used CRISPR/Cas9 genome editing to show that optix plays a fundamental role in nymphalid butterfly wing pattern development, where it is required for determination of all chromatic coloration. optix knockouts in four species show complete replacement of color pigments with melanins, with corresponding changes in pigment-related gene expression, resulting in black and gray butterflies. We also show that optix simultaneously acts as a switch gene for blue structural iridescence in some butterflies, demonstrating simple regulatory coordination of structural and pigmentary coloration. Remarkably, these optix knockouts phenocopy the recurring "black and blue" wing pattern archetype that has arisen on many independent occasions in butterflies. Here we demonstrate a simple genetic basis for structural coloration, and show that optix plays a deeply conserved role in butterfly wing pattern development.

Characterization of Limonoids Isolated from the Fruits of Melia toosendan and Their Antifeedant Activity against Pieris rapae.

The fruits of Melia toosendan Sieb. et Zucc. (Meliaceae) are a source of bioactive limonoids that can be used as effective pesticides. In this study, two novel limonoids, 6-acetylsendanal and 6-ketocinamodiol, were isolated together with fourteen known compounds, namely four protolimonoids, six trichilin-class limonoids, and four C-seco limonoids. The structures of the new compounds were determined by extensive spectroscopic analyses (HR-ESI-MS, UV, IR, 1D and 2D NMR). The bioassay results revealed that eleven of the extracted limonoids exhibited interesting antifeedant activities against the larvae of Pieris rapae with AFC50 values in the range of 0.11-1.79 mm. Particularly, mesendanin H, with an AFC50 value of 0.11 mm, exhibited a higher activity than the positive control toosendanin. Information on new bioactive limonoids may provide further insight into M. toosendan as a source of bioactive components.

Genomic sequencing of Troides aeacus nucleopolyhedrovirus (TraeNPV) from golden birdwing larvae (Troides aeacus formosanus) to reveal defective Autographa californica NPV genomic features.

BACKGROUND: The golden birdwing butterfly (Troides aeacus formosanus) is a rarely observed species in Taiwan. Recently, a typical symptom of nuclear polyhedrosis was found in reared T. aeacus larvae. From the previous Kimura-2 parameter (K-2-P) analysis based on the nucleotide sequence of three genes in this isolate, polh, lef-8 and lef-9, the underlying virus did not belong to any known nucleopolyhedrovirus (NPV) species. Therefore, this NPV was provisionally named "TraeNPV". To understand this NPV, the nucleotide sequence of the whole TraeNPV genome was determined using next-generation sequencing (NGS) technology. RESULTS: The genome of TraeNPV is 125,477 bp in length with 144 putative open reading frames (ORFs) and its GC content is 40.45%. A phylogenetic analysis based on the 37 baculoviral core genes suggested that TraeNPV is a Group I NPV that is closely related to Autographa californica nucleopolyhedrovirus (AcMNPV). A genome-wide analysis showed that TraeNPV has some different features in its genome compared with other NPVs. Two novel ORFs (Ta75 and Ta139), three truncated ORFs (pcna, he65 and bro) and one duplicated ORF (38.7 K) were found in the TraeNPV genome; moreover, there are fewer homologous regions (hrs) than there are in AcMNPV, which shares eight hrs within the TraeNPV genome. TraeNPV shares similar genomic features with AcMNPV, including the gene content, gene arrangement and gene/genome identity, but TraeNPV lacks 15 homologous ORFs from AcMNPV in its genome, such as ctx, host cell-specific factor 1 (hcf-1), PNK/PNL, vp15, and apsup, which are involved in the auxiliary functions of alphabaculoviruses. CONCLUSIONS: Based on these data, TraeNPV would be clarified as a new NPV species with defective AcMNPV genomic features. The precise relationship between TraeNPV and other closely related NPV species were further investigated. This report could provide comprehensive information on TraeNPV for evolutionary insights into butterfly-infected NPV.