Transposable Element Insertions Are Associated with Batesian Mimicry in the Pantropical Butterfly Hypolimnas misippus.

Hypolimnas misippus is a Batesian mimic of the toxic African Queen butterfly (Danaus chrysippus). Female H. misippus butterflies use two major wing patterning loci (M and A) to imitate three color morphs of D. chrysippus found in different regions of Africa. In this study, we examine the evolution of the M locus and identify it as an example of adaptive atavism. This phenomenon involves a morphological reversion to an ancestral character that results in an adaptive phenotype. We show that H. misippus has re-evolved an ancestral wing pattern present in other Hypolimnas species, repurposing it for Batesian mimicry of a D. chrysippus morph. Using haplotagging, a linked-read sequencing technology, and our new analytical tool, Wrath, we discover two large transposable element insertions located at the M locus and establish that these insertions are present in the dominant allele responsible for producing mimetic phenotype. By conducting a comparative analysis involving additional Hypolimnas species, we demonstrate that the dominant allele is derived. This suggests that, in the derived allele, the transposable elements disrupt a cis-regulatory element, leading to the reversion to an ancestral phenotype that is then utilized for Batesian mimicry of a distinct model, a different morph of D. chrysippus. Our findings present a compelling instance of convergent evolution and adaptive atavism, in which the same pattern element has independently evolved multiple times in Hypolimnas butterflies, repeatedly playing a role in Batesian mimicry of diverse model species.

Whole-chromosome hitchhiking driven by a male-killing endosymbiont.

Neo-sex chromosomes are found in many taxa, but the forces driving their emergence and spread are poorly understood. The female-specific neo-W chromosome of the African monarch (or queen) butterfly Danaus chrysippus presents an intriguing case study because it is restricted to a single 'contact zone' population, involves a putative colour patterning supergene, and co-occurs with infection by the male-killing endosymbiont Spiroplasma. We investigated the origin and evolution of this system using whole genome sequencing. We first identify the 'BC supergene', a broad region of suppressed recombination across nearly half a chromosome, which links two colour patterning loci. Association analysis suggests that the genes yellow and arrow in this region control the forewing colour pattern differences between D. chrysippus subspecies. We then show that the same chromosome has recently formed a neo-W that has spread through the contact zone within approximately 2,200 years. We also assembled the genome of the male-killing Spiroplasma, and find that it shows perfect genealogical congruence with the neo-W, suggesting that the neo-W has hitchhiked to high frequency as the male-killer has spread through the population. The complete absence of female crossing-over in the Lepidoptera causes whole-chromosome hitchhiking of a single neo-W haplotype, carrying a single allele of the BC supergene and dragging multiple non-synonymous mutations to high frequency. This has created a population of infected females that all carry the same recessive colour patterning allele, making the phenotypes of each successive generation highly dependent on uninfected male immigrants. Our findings show how hitchhiking can occur between the physically unlinked genomes of host and endosymbiont, with dramatic consequences.

The gene cortex controls mimicry and crypsis in butterflies and moths.

The wing patterns of butterflies and moths (Lepidoptera) are diverse and striking examples of evolutionary diversification by natural selection. Lepidopteran wing colour patterns are a key innovation, consisting of arrays of coloured scales. We still lack a general understanding of how these patterns are controlled and whether this control shows any commonality across the 160,000 moth and 17,000 butterfly species. Here, we use fine-scale mapping with population genomics and gene expression analyses to identify a gene, cortex, that regulates pattern switches in multiple species across the mimetic radiation in Heliconius butterflies. cortex belongs to a fast-evolving subfamily of the otherwise highly conserved fizzy family of cell-cycle regulators, suggesting that it probably regulates pigmentation patterning by regulating scale cell development. In parallel with findings in the peppered moth (Biston betularia), our results suggest that this mechanism is common within Lepidoptera and that cortex has become a major target for natural selection acting on colour and pattern variation in this group of insects.

Karyotypes versus Genomes: The Nymphalid Butterflies Melitaea cinxia, Danaus plexippus, and D. chrysippus.

The number of sequenced lepidopteran genomes is increasing rapidly. However, the corresponding assemblies rarely represent whole chromosomes and generally also lack the highly repetitive W sex chromosome. Knowledge of the karyotypes can facilitate genome assembly and further our understanding of sex chromosome evolution in Lepidoptera. Here, we describe the karyotypes of the Glanville fritillary Melitaea cinxia (n = 31), the monarch Danaus plexippus (n = 30), and the African queen D. chrysippus (2n = 60 or 59, depending on the source population). We show by FISH that the telomeres are of the (TTAGG)n type, as found in most insects. M. cinxia and D. plexippus have "conventional" W chromosomes which are heterochromatic in meiotic and somatic cells. In D. chrysippus, the W is inconspicuous. Neither telomeres nor W chromosomes are represented in the published genomes of M. cinxia and D. plexippus. Representation analysis in sequenced female and male D. chrysippus genomes detected an evolutionarily old autosome-Z chromosome fusion in Danaus. Conserved synteny of whole chromosomes, so called "macro synteny", in Lepidoptera permitted us to identify the chromosomes involved in this fusion. An additional and more recent sex chromosome fusion was found in D. chrysippus by karyotype analysis and classical genetics. In a hybrid population between 2 subspecies, D. c. chrysippus and D. c. dorippus, the W chromosome was fused to an autosome that carries a wing colour locus. Thus, cytogenetics and the present state of genome data complement one another to reveal the evolutionary history of the species.

Chromosomal rearrangements maintain a polymorphic supergene controlling butterfly mimicry.

Supergenes are tight clusters of loci that facilitate the co-segregation of adaptive variation, providing integrated control of complex adaptive phenotypes. Polymorphic supergenes, in which specific combinations of traits are maintained within a single population, were first described for 'pin' and 'thrum' floral types in Primula and Fagopyrum, but classic examples are also found in insect mimicry and snail morphology. Understanding the evolutionary mechanisms that generate these co-adapted gene sets, as well as the mode of limiting the production of unfit recombinant forms, remains a substantial challenge. Here we show that individual wing-pattern morphs in the polymorphic mimetic butterfly Heliconius numata are associated with different genomic rearrangements at the supergene locus P. These rearrangements tighten the genetic linkage between at least two colour-pattern loci that are known to recombine in closely related species, with complete suppression of recombination being observed in experimental crosses across a 400-kilobase interval containing at least 18 genes. In natural populations, notable patterns of linkage disequilibrium (LD) are observed across the entire P region. The resulting divergent haplotype clades and inversion breakpoints are found in complete association with wing-pattern morphs. Our results indicate that allelic combinations at known wing-patterning loci have become locked together in a polymorphic rearrangement at the P locus, forming a supergene that acts as a simple switch between complex adaptive phenotypes found in sympatry. These findings highlight how genomic rearrangements can have a central role in the coexistence of adaptive phenotypes involving several genes acting in concert, by locally limiting recombination and gene flow.

Light pollution is associated with earlier tree budburst across the United Kingdom.

The ecological impact of night-time lighting is of concern because of its well-demonstrated effects on animal behaviour. However, the potential of light pollution to change plant phenology and its corresponding knock-on effects on associated herbivores are less clear. Here, we test if artificial lighting can advance the timing of budburst in trees. We took a UK-wide 13 year dataset of spatially referenced budburst data from four deciduous tree species and matched it with both satellite imagery of night-time lighting and average spring temperature. We find that budburst occurs up to 7.5 days earlier in brighter areas, with the relationship being more pronounced for later-budding species. Excluding large urban areas from the analysis showed an even more pronounced advance of budburst, confirming that the urban 'heat-island' effect is not the sole cause of earlier urban budburst. Similarly, the advance in budburst across all sites is too large to be explained by increases in temperature alone. This dramatic advance of budburst illustrates the need for further experimental investigation into the impact of artificial night-time lighting on plant phenology and subsequent species interactions. As light pollution is a growing global phenomenon, the findings of this study are likely to be applicable to a wide range of species interactions across the world.

Ion channels as insecticide targets.

Ion channels remain the primary target of most of the small molecule insecticides. This review examines how the subunit composition of heterologously expressed receptors determines their insecticide-specific pharmacology and how the pharmacology of expressed receptors differs from those found in the insect nervous system. We find that the insecticide-specific pharmacology of some receptors, like that containing subunits of the Rdl encoded GABA receptor, can be reconstituted with very few of the naturally occurring subunits expressed. In contrast, workers have struggled even to express functional insect nicotinic acetylcholine receptors (nAChRs), and work has therefore often relied upon the expression of vertebrate receptor subunits in their place. We also examine the extent to which insecticide-resistance-associated mutations, such as those in the para encoded voltage-gated sodium channel, can reveal details of insecticide-binding sites and mode of action. In particular, we examine whether mutations are present in the insecticide-binding site and/or at sites that allosterically affect the drug preferred conformation of the receptor. We also discuss the ryanodine receptor as a target for the recently developed diamides. Finally, we examine the lethality of the genes encoding these receptor subunits and discuss how this might determine the degree of conservation of the resistance-associated mutations found.

Comparative genomics of the mimicry switch in Papilio dardanus.

The African Mocker Swallowtail, Papilio dardanus, is a textbook example in evolutionary genetics. Classical breeding experiments have shown that wing pattern variation in this polymorphic Batesian mimic is determined by the polyallelic H locus that controls a set of distinct mimetic phenotypes. Using bacterial artificial chromosome (BAC) sequencing, recombination analyses and comparative genomics, we show that H co-segregates with an interval of less than 500 kb that is collinear with two other Lepidoptera genomes and contains 24 genes, including the transcription factor genes engrailed (en) and invected (inv). H is located in a region of conserved gene order, which argues against any role for genomic translocations in the evolution of a hypothesized multi-gene mimicry locus. Natural populations of P. dardanus show significant associations of specific morphs with single nucleotide polymorphisms (SNPs), centred on en. In addition, SNP variation in the H region reveals evidence of non-neutral molecular evolution in the en gene alone. We find evidence for a duplication potentially driving physical constraints on recombination in the lamborni morph. Absence of perfect linkage disequilibrium between different genes in the other morphs suggests that H is limited to nucleotide positions in the regulatory and coding regions of en. Our results therefore support the hypothesis that a single gene underlies wing pattern variation in P. dardanus.

Pdl1 is a putative lipase that enhances Photorhabdus toxin complex secretion.

The Toxin Complex (TC) is a large multi-subunit toxin first characterized in the insect pathogens Photorhabdus and Xenorhabdus, but now seen in a range of pathogens, including those of humans. These complexes comprise three protein subunits, A, B and C which in the Xenorhabdus toxin are found in a 4:1:1 stoichiometry. Some TCs have been demonstrated to exhibit oral toxicity to insects and have the potential to be developed as a pest control technology. The lack of recognisable signal sequences in the three large component proteins hinders an understanding of their mode of secretion. Nevertheless, we have shown the Photorhabdus luminescens (Pl) Tcd complex has been shown to associate with the bacteria's surface, although some strains can also release it into the surrounding milieu. The large number of tc gene homologues in Pl make study of the export process difficult and as such we have developed and validated a heterologous Escherichia coli expression model to study the release of these important toxins. In addition to this model, we have used comparative genomics between a strain that releases high levels of Tcd into the supernatant and one that retains the toxin on its surface, to identify a protein responsible for enhancing secretion and release of these toxins. This protein is a putative lipase (Pdl1) which is regulated by a small tightly linked antagonist protein (Orf53). The identification of homologues of these in other bacteria, linked to other virulence factor operons, such as type VI secretion systems, suggests that these genes represent a general and widespread mechanism for enhancing toxin release in gram negative pathogens.

Penetrance interactions of colour pattern loci in the African Monarch and their implications for the evolution of dominance.

Scoring the penetrance of heterozygotes in complex phenotypes, like colour pattern, is difficult and complicates the analysis of systems in which dominance is incomplete or evolving. The African Monarch (Danaus chrysippus) represents an example where colour pattern heterozygotes, formed in the contact zone between the different subspecies, show such intermediate dominance. Colour pattern in this aposematic butterfly is controlled by three loci A, B and C. The B and C loci are closely linked in a B/C supergene and significant interaction of B and C phenotypes is therefore expected via linkage alone. The A locus, however, is not linked to B/C and is found on a different chromosome. To study interactions between these loci we generated colour pattern heterozygotes by crossing males and females bearing different A and B/C genotypes, collected from different parts of Africa. We derived a novel scoring system for the expressivity of the heterozygotes and, as predicted, we found significant interactions between the genotypes of the closely linked B and C loci. Surprisingly, however, we also found highly significant interactions between C and the unlinked A locus, modifications that generally increased the resemblance of heterozygotes to homozygous ancestors. In contrast, we found no difference in the penetrance of any of the corresponding heterozygotes from crosses conducted either in allopatry or sympatry, in reciprocal crosses of males and females, or in the presence or absence of endosymbiont mediated male-killing or its associated neoW mediated sex-linkage of colour pattern. Together, this data supports the idea that the different colour morphs of the African Monarch meet transiently in the East African contact zone and that genetic modifiers act to mask inappropriate expression of colour patterns in the incorrect environments.

Eyespot variation and field temperature in the Meadow Brown butterfly.

Since the classic work of E.B. Ford, explanations for eyespot variation in the Meadow Brown butterfly have focused on the role of genetic polymorphism. The potential role of thermal plasticity in this classic example of natural selection has therefore been overlooked. Here, we use large daily field collections of butterflies from three sites, over multiple years, to examine whether field temperature is correlated with eyespot variation, using the same presence/absence scoring as Ford. We show that higher developmental temperature in the field leads to the disappearance of the spots visible while the butterfly is at rest, explaining the historical observation that hindwing spotting declines across the season. Strikingly, females developing at 11°C have a median of six spots and those developing at 15°C only have three. In contrast, the large forewing eyespot is always present and scales with forewing length. Furthermore, in contrast to the smaller spots, the size of the large forewing spot is best explained by calendar date (days since 1st March) rather than the temperature at pupation. As this large forewing spot is involved in startling predators and/or sexual selection, its constant presence is therefore likely required for defence, whereas the disappearance of the smaller spots over the season may help with female crypsis. We model annual total spot variation with phenological data from the UK and derive predictions as to how spot patterns will continue to change, predicting that female spotting will decrease year on year as our climate warms.

Shedding light on moths: shorter wavelengths attract noctuids more than geometrids.

With moth declines reported across Europe, and parallel changes in the amount and spectra of street lighting, it is important to understand exactly how artificial lights affect moth populations. We therefore compared the relative attractiveness of shorter wavelength (SW) and longer wavelength (LW) lighting to macromoths. SW light attracted significantly more individuals and species of moth, either when used alone or in competition with LW lighting. We also found striking differences in the relative attractiveness of different wavelengths to different moth groups. SW lighting attracted significantly more Noctuidae than LW, whereas both wavelengths were equally attractive to Geometridae. Understanding the extent to which different groups of moth are attracted to different wavelengths of light will be useful in determining the impact of artificial light on moth populations.

A serine proteinase homologue, SPH-3, plays a central role in insect immunity.

Numerous vertebrate and invertebrate genes encode serine proteinase homologues (SPHs) similar to members of the serine proteinase family, but lacking one or more residues of the catalytic triad. These SPH proteins are thought to play a role in immunity, but their precise functions are poorly understood. In this study, we show that SPH-3 (an insect non-clip domain-containing SPH) is of central importance in the immune response of a model lepidopteran, Manduca sexta. We examine M. sexta infection with a virulent, insect-specific, Gram-negative bacterium Photorhabdus luminescens. RNA interference suppression of bacteria-induced SPH-3 synthesis severely compromises the insect's ability to defend itself against infection by preventing the transcription of multiple antimicrobial effector genes, but, surprisingly, not the transcription of immune recognition genes. Upregulation of the gene encoding prophenoloxidase and the activity of the phenoloxidase enzyme are among the antimicrobial responses that are severely attenuated on SPH-3 knockdown. These findings suggest the existence of two largely independent signaling pathways controlling immune recognition by the fat body, one governing effector gene transcription, and the other regulating genes encoding pattern recognition proteins.

Genomic hotspots for adaptation: the population genetics of Müllerian mimicry in the Heliconius melpomene clade.

Wing patterning in Heliconius butterflies is a longstanding example of both Müllerian mimicry and phenotypic radiation under strong natural selection. The loci controlling such patterns are "hotspots" for adaptive evolution with great allelic diversity across different species in the genus. We characterise nucleotide variation, genotype-by-phenotype associations, linkage disequilibrium, and candidate gene expression at two loci and across multiple hybrid zones in Heliconius melpomene and relatives. Alleles at HmB control the presence or absence of the red forewing band, while alleles at HmYb control the yellow hindwing bar. Across HmYb two regions, separated by approximately 100 kb, show significant genotype-by-phenotype associations that are replicated across independent hybrid zones. In contrast, at HmB a single peak of association indicates the likely position of functional sites at three genes, encoding a kinesin, a G-protein coupled receptor, and an mRNA splicing factor. At both HmYb and HmB there is evidence for enhanced linkage disequilibrium (LD) between associated sites separated by up to 14 kb, suggesting that multiple sites are under selection. However, there was no evidence for reduced variation or deviations from neutrality that might indicate a recent selective sweep, consistent with these alleles being relatively old. Of the three genes showing an association with the HmB locus, the kinesin shows differences in wing disc expression between races that are replicated in the co-mimic, Heliconius erato, providing striking evidence for parallel changes in gene expression between Müllerian co-mimics. Wing patterning loci in Heliconius melpomene therefore show a haplotype structure maintained by selection, but no evidence for a recent selective sweep. The complex genetic pattern contrasts with the simple genetic basis of many adaptive traits studied previously, but may provide a better model for most adaptation in natural populations that has arisen over millions rather than tens of years.

Genomic hotspots for adaptation: the population genetics of Müllerian mimicry in Heliconius erato.

Wing pattern evolution in Heliconius butterflies provides some of the most striking examples of adaptation by natural selection. The genes controlling pattern variation are classic examples of Mendelian loci of large effect, where allelic variation causes large and discrete phenotypic changes and is responsible for both convergent and highly divergent wing pattern evolution across the genus. We characterize nucleotide variation, genotype-by-phenotype associations, linkage disequilibrium (LD), and candidate gene expression patterns across two unlinked genomic intervals that control yellow and red wing pattern variation among mimetic forms of Heliconius erato. Despite very strong natural selection on color pattern, we see neither a strong reduction in genetic diversity nor evidence for extended LD across either patterning interval. This observation highlights the extent that recombination can erase the signature of selection in natural populations and is consistent with the hypothesis that either the adaptive radiation or the alleles controlling it are quite old. However, across both patterning intervals we identified SNPs clustered in several coding regions that were strongly associated with color pattern phenotype. Interestingly, coding regions with associated SNPs were widely separated, suggesting that color pattern alleles may be composed of multiple functional sites, conforming to previous descriptions of these loci as "supergenes." Examination of gene expression levels of genes flanking these regions in both H. erato and its co-mimic, H. melpomene, implicate a gene with high sequence similarity to a kinesin as playing a key role in modulating pattern and provides convincing evidence for parallel changes in gene regulation across co-mimetic lineages. The complex genetic architecture at these color pattern loci stands in marked contrast to the single casual mutations often identified in genetic studies of adaptation, but may be more indicative of the type of genetic changes responsible for much of the adaptive variation found in natural populations.

Transposable elements and xenobiotic resistance.

Transposable elements or TEs are well known drivers of adaptive change in plants and animals but their role in insecticide resistance remains poorly documented. This review examines the potential role of transposons in resistance and identifies key areas where our understanding remains unclear. Despite well-known model systems such as upregulation of Drosophila Cyp6g1, many putative examples lack functional validation. The potential types of transposon-associated changes that could lead to resistance are reviewed, including changes in up-regulation, message stability, loss of function and alternative splicing. Where potential mechanisms appear absent from the resistance literature examples are drawn from other areas of biology. Finally, ways are suggested in which transgenic expression could be used to validate the biological significance of TE insertion. In the absence of such functional expression studies many examples of the association of TEs and resistance genes therefore remain as correlations.

Lepidopteran Synteny Units reveal deep chromosomal conservation in butterflies and moths.

DNA is compacted into individual particles or chromosomes that form the basic units of inheritance. However, different animals and plants have widely different numbers of chromosomes. This means that we cannot readily tell which chromosomes are related to which. Here, we describe a simple technique that looks at the similarity of genes on each chromosome and thus gives us a true picture of their homology or similarity through evolutionary time. We use this new system to look at the chromosomes of butterflies and moths or Lepidoptera. We term the associated synteny units, Lepidopteran Synteny Units (LSUs). Using a sample of butterfly and moth genomes from across evolutionary time, we show that LSUs form a simple and reliable method of tracing chromosomal homology back through time. Surprisingly, this technique reveals that butterfly and moth chromosomes show conserved blocks dating back to their sister group the Trichoptera. As Lepidoptera have holocentric chromosomes, it will be interesting to see if similar levels of synteny are shown in groups of animals with monocentric chromosomes. The ability to define homology via LSU analysis makes it considerably easier to approach many questions in chromosomal evolution.

African Queens find mates when males are rare.

In butterflies and moths, male-killing endosymbionts are transmitted from infected females via their eggs, and the male progeny then perish. This means that successful transmission of the parasite relies on the successful mating of the host. Paradoxically, at the population level, parasite transmission also reduces the number of adult males present in the final population for infected females to mate with. Here we investigate if successful female mating when males are rare is indeed a likely rate-limiting step in the transmission of male-killing Spiroplasma in the African Monarch, Danaus chrysippus. In Lepidoptera, successful pairings are hallmarked by the transfer of a sperm-containing spermatophore from the male to the female during copulation. Conveniently, this spermatophore remains detectable within the female upon dissection, and thus, spermatophore counts can be used to assess the frequency of successful mating in the field. We used such spermatophore counts to examine if altered sex ratios in the D. chrysippus do indeed affect female mating success. We examined two different field sites in East Africa where males were often rare. Surprisingly, mated females carried an average of 1.5 spermatophores each, regardless of male frequency, and importantly, only 10-20% remained unmated. This suggests that infected females will still be able to mate in the face of either Spiroplasma-mediated male killing and/or fluctuations in adult sex ratio over the wet-dry season cycle. These observations may begin to explain how the male-killing mollicute can still be successfully transmitted in a population where males are rare.

Overexpression of the UDP-glycosyltransferase UGT34A23 confers resistance to the diamide insecticide chlorantraniliprole in the tomato leafminer, Tuta absoluta.

The tomato leafminer, Tuta absoluta, is an invasive crop pest that has evolved resistance to many of the insecticides used for its control. To facilitate the investigation of the underpinning mechanisms of resistance in this species we generated a contiguous genome assembly using long-read sequencing data. We leveraged this genomic resource to investigate the genetic basis of resistance to the diamide insecticide chlorantraniliprole in Spanish strains of T. absoluta that exhibit high levels of resistance to this insecticide. Transcriptomic analyses revealed that, in these strains, resistance is not associated with previously reported target-site mutations in the diamide target-site, the ryanodine receptor, but rather is associated with the marked overexpression (20- to >100-fold) of a gene encoding a UDP-glycosyltransferase (UGT). Functional expression of this UGT, UGT34A23, via ectopic expression in Drosophila melanogaster demonstrated that it confers strong and significant resistance in vivo. The genomic resources generated in this study provide a powerful resource for further research on T. absoluta. Our findings on the mechanisms underpinning resistance to chlorantraniliprole will inform the development of sustainable management strategies for this important pest.