Papilio Xuthus Research Articles

Immunolocalization suggests a role of the histamine-gated chloride channel PxHCLB in spectral opponent processing in butterfly photoreceptors.

Spectrally opponent responses, that is, wavelength-dependent inversions of response polarity, have been observed at the level of photoreceptors in butterflies. As inter-photoreceptor connections have been found in the butterfly Papilio xuthus, and histamine is the only neurotransmitter so far identified in insect photoreceptors, we hypothesize that histaminergic sign-inverting synapses exist in the lamina between different spectral receptors as a mechanism for spectral opponency as in the medulla of Drosophila. Here, we localized two histamine-gated chloride channels, PxHCLA (Drosophila Ort homolog) and PxHCLB (Drosophila HisCl1 homolog), in the visual system of Papilio xuthus by using specific antisera. The antiPxHCLA labeling was associated with the membrane of nonphotoreceptor cells that are postsynaptic to photoreceptors, while the antiPxHCLB labeling overlapped with photoreceptor axons, indicating that PxHCLB is expressed at inter-photoreceptor synapses: PxHCLB is likely involved in producing spectral opponency at the first visual synapses. Color processing in Papilio may appear earlier than previously hypothesized in insect visual systems.

Molecular cloning and characterization of C1 tetrahydrofolate (C1-THF) synthase in Bombyx mori, silkworm

Folate metabolism pathway is mainly associated with syntheses of nucleic acids and proteins as well as DNA methylation and repair. Cytoplasmic C1 tetrahydrofolate (C1-THF) synthase is a central enzyme in folate metabolism and plays an important role in C1-THF conversion. The full-length sequence of silkworm (Bombyx mori) cytoplasmic C1-THF synthase (BmC1-THF synthase) gene including an open reading frame (ORF) of 932 amino acid residues was cloned. Phylogenetic analysis indicated that BmC1-THF synthase shares 84% and 81% identity with C1-THF synthase of Papilio Xuthus and Danaus plexippus, respectively. Conserved sequence analysis showed that the 330 to 710 amino acid residues of BmC1-THF synthase were highly conserved among various organisms. Recombinant BmC1-THF synthase protein with a molecular weight of about 105.5 kDa was expressed in E. coli cells and identified using SDS-PAGE and western blot analysis. The expression of BmC1-THF synthase gene in eight silkworm tissues revealed that BmC1-THF synthase gene is widely expressed in various tissues of silkworm and highly expressed in the fat body. The expression characteristics of BmC1-THF synthase gene in response to oxidative stress were conducted under phoxim and high temperature (30 °C) exposure. The results showed that the transcriptional level of BmC1-THF synthase gene was upregulated by 3.435-fold and 6.845-fold after phoxim and high temperature (30 °C) exposure, respectively, indicating that BmC1-THF synthase gene may play a significant role in response of silkworm to oxidative stresses.

Biophysical Studies Reveal Key Interactions between Papiliocin-Derived PapN and Lipopolysaccharide in Gram-Negative Bacteria.

Papiliocin, isolated from the swallowtail butterfly (Papilio xuthus), is an antimicrobial peptide with high selectivity against gram-negative bacteria. We previously showed that the N-terminal helix of papiliocin (PapN) plays a key role in the antibacterial and anti-inflammatory activity of papiliocin. In this study, we measured the selectivity of PapN against multidrug-resistant gram-negative bacteria, as well as its anti-inflammatory activity. Interactions between Trp2 of PapN and lipopolysaccharide (LPS), which is a major component of the outer membrane of gram-negative bacteria, were studied using the Trp fluorescence blue shift and quenching in LPS micelles. Furthermore, using circular dichroism, we investigated the interactions between PapN and LPS, showing that LPS plays critical roles in peptide folding. Our results demonstrated that Trp2 in PapN was buried deep in the negatively charged LPS, and Trp2 induced the α-helical structure of PapN. Importantly, docking studies determined that predominant electrostatic interactions of positively charged arginine residues in PapN with phosphate head groups of LPS were key factors for binding. Similarly, hydrophobic interactions by aromatic residues of PapN with fatty acid chains in LPS were also significant for binding. These results may facilitate the development of peptide antibiotics with anti-inflammatory activity.

Non-linear amplification of graded voltage signals in the first-order visual interneurons of the butterfly Papilio xuthus.

Lamina monopolar cells (LMCs) are the first-order visual interneurons of insects and crustacea, primarily involved in achromatic vision. Here, we investigated morphological and electrophysiological properties of LMCs in the butterfly Papilio xuthus Using intracellular recording coupled with dye injection, we found two types of LMCs. Cells with roundish terminals near the distal surface of the medulla demonstrating no or small depolarizing spikes were classified as L1/2. Cells with elongated terminals deep in the medulla that showed prominent spiking were classified as L3/4. The majority of LMCs of both types had broad spectral sensitivities, peaking between 480 and 570 nm. Depending on the experimental conditions, spikes varied from small to action potential-like events, with their amplitudes and rates decreasing as stimulus brightness increased. When the eye was stimulated with naturalistic contrast-modulated time series, spikes were reliably triggered by high-contrast components of the stimulus. Spike-triggered average functions showed that spikes emphasize rapid membrane depolarizations. Our results suggest that spikes are mediated by voltage-activated Na+ channels, which are mainly inactivated at rest. Strong local minima in the coherence functions of spiking LMCs indicate that the depolarizing conductance contributes to the amplification of graded responses even when detectable spikes are not evoked. We propose that the information transfer strategies of spiking LMCs change with light intensity. In dim light, both graded voltage signals and large spikes are used together without mutual interference, as a result of separate transmission bandwidths. In bright light, signals are non-linearly amplified by the depolarizing conductance in the absence of detectable spikes.

Physiological responses of ionotropic histamine receptors, PxHCLA and PxHCLB, to neurotransmitter candidates in a butterfly, Papilio xuthus.

Histamine is the only known neurotransmitter released by arthropod photoreceptors. Synaptic transmission from photoreceptors to second-order neurons is mediated by the activation of histamine-gated chloride channels (HCLs). These histaminergic synapses have been assumed to be conserved among insect visual systems. However, our understanding of the channels in question has thus far been based on studies in flies. In the butterfly Papilio xuthus, we have identified two candidate histamine-gated chloride channels, PxHCLA and PxHCLB, and studied their physiological properties using a whole-cell patch-clamp technique. We studied the responses of channels expressed in cultured cells to histamine as well as to other neurotransmitter candidates, namely GABA, tyramine, serotonin, d-/l-glutamate and glycine. We found that histamine and GABA activated both PxHCLA and PxHCLB, while the other molecules did not. The sensitivity to histamine and GABA was consistently higher in PxHCLB than in PxHCLA. Interestingly, simultaneous application of histamine and GABA activated both PxHCLA and PxHCLB more strongly than either neurotransmitter individually; histamine and GABA may have synergistic effects on PxHCLs in the regions where they co-localize. Our results suggest that the physiological properties of the histamine receptors are basically conserved among insects, but that the response to GABA differs between butterflies and flies, implying variation in early visual processing among species.

Unique Temporal Expression of Triplicated Long-Wavelength Opsins in Developing Butterfly Eyes.

Following gene duplication events, the expression patterns of the resulting gene copies can often diverge both spatially and temporally. Here we report on gene duplicates that are expressed in distinct but overlapping patterns, and which exhibit temporally divergent expression. Butterflies have sophisticated color vision and spectrally complex eyes, typically with three types of heterogeneous ommatidia. The eyes of the butterfly Papilio xuthus express two green- and one red-absorbing visual pigment, which came about via gene duplication events, in addition to one ultraviolet (UV)- and one blue-absorbing visual pigment. We localized mRNAs encoding opsins of these visual pigments in developing eye disks throughout the pupal stage. The mRNAs of the UV and blue opsin are expressed early in pupal development (pd), specifying the type of the ommatidium in which they appear. Red sensitive photoreceptors first express a green opsin mRNA, which is replaced later by the red opsin mRNA. Broadband photoreceptors (that coexpress the green and red opsins) first express the green opsin mRNA, later change to red opsin mRNA and finally re-express the green opsin mRNA in addition to the red mRNA. Such a unique temporal and spatial expression pattern of opsin mRNAs may reflect the evolution of visual pigments and provide clues toward understanding how the spectrally complex eyes of butterflies evolved.

Seasonal polyphenism in body size and juvenile development of the swallowtail butterfly Papilio xuthus (Lepidoptera: Papilionidae)

Seasonal polyphenism in adults may be a season-specific adaptation of the adult stage and/or a by-product of adaptive plasticity of the juvenile stages. The swallowtail butterfly Papilio xuthus L. exhibits seasonal polyphenism controlled by photoperiod. Adults emerging in spring from pupae that spend winter in diapause have smaller bodies than adults emerging in summer from pupae that do not undergo diapause. Pupal diapause is induced by short-day conditions typical of autumn. To explore the interactive effects of temperature and developmental pathways on the variation in adult body size in P. xuthus, we reared larvae at two temperatures (20°C, 25°C) under two photoperiods (12L : 12D and 16L : 8D). Pupal weight and adult forewing length were greater in the generation that did not undergo diapause and were greater at 25°C than at 20°C. Thus, body size differences were greatest between the individuals that were reared at the longer day length and higher temperature and did not undergo diapause and those that were reared at the shorter day length and lower temperature and did undergo diapause. Unlike in other Lepidoptera, larvae of individuals that undergo diapause had shorter developmental times and higher growth rates than those that did not undergo diapause. This developmental plasticity may enable this butterfly to cope with the unpredictable length of the growing season prior to the onset of winter. Our results indicate that there are unexplored variations in the life history strategy of multivoltine Lepidoptera.

Using electroretinograms and multi-model inference to identify spectral classes of photoreceptors and relative opsin expression levels.

Understanding how individual photoreceptor cells factor in the spectral sensitivity of a visual system is essential to explain how they contribute to the visual ecology of the animal in question. Existing methods that model the absorption of visual pigments use templates which correspond closely to data from thin cross-sections of photoreceptor cells. However, few modeling approaches use a single framework to incorporate physical parameters of real photoreceptors, which can be fused, and can form vertical tiers. Akaike’s information criterion (AICc) was used here to select absorptance models of multiple classes of photoreceptor cells that maximize information, given visual system spectral sensitivity data obtained using extracellular electroretinograms and structural parameters obtained by histological methods. This framework was first used to select among alternative hypotheses of photoreceptor number. It identified spectral classes from a range of dark-adapted visual systems which have between one and four spectral photoreceptor classes. These were the velvet worm, Principapillatus hitoyensis, the branchiopod water flea, Daphnia magna, normal humans, and humans with enhanced S-cone syndrome, a condition in which S-cone frequency is increased due to mutations in a transcription factor that controls photoreceptor expression. Data from the Asian swallowtail, Papilio xuthus, which has at least five main spectral photoreceptor classes in its compound eyes, were included to illustrate potential effects of model over-simplification on multi-model inference. The multi-model framework was then used with parameters of spectral photoreceptor classes and the structural photoreceptor array kept constant. The goal was to map relative opsin expression to visual pigment concentration. It identified relative opsin expression differences for two populations of the bluefin killifish, Lucania goodei. The modeling approach presented here will be useful in selecting the most likely alternative hypotheses of opsin-based spectral photoreceptor classes, using relative opsin expression and extracellular electroretinography.

Global Dosage Compensation Is Ubiquitous in Lepidoptera, but Counteracted by the Masculinization of the Z Chromosome.

While chromosome-wide dosage compensation of the X chromosome has been found in many species, studies in ZW clades have indicated that compensation of the Z is more localized and/or incomplete. In the ZW Lepidoptera, some species show complete compensation of the Z chromosome, while others lack full equalization, but what drives these inconsistencies is unclear. Here, we compare patterns of male and female gene expression on the Z chromosome of two closely related butterfly species, Papilio xuthus and Papilio machaon, and in multiple tissues of two moths species, Plodia interpunctella and Bombyx mori, which were previously found to differ in the extent to which they equalize Z-linked gene expression between the sexes. We find that, while some species and tissues seem to have incomplete dosage compensation, this is in fact due to the accumulation of male-biased genes and the depletion of female-biased genes on the Z chromosome. Once this is accounted for, the Z chromosome is fully compensated in all four species, through the up-regulation of Z expression in females and in some cases additional down-regulation in males. We further find that both sex-biased genes and Z-linked genes have increased rates of expression divergence in this clade, and that this can lead to fast shifts in patterns of gene expression even between closely related species. Taken together, these results show that the uneven distribution of sex-biased genes on sex chromosomes can confound conclusions about dosage compensation and that Z chromosome-wide dosage compensation is not only possible but ubiquitous among Lepidoptera.

The eyes and vision of butterflies.

Butterflies use colour vision when searching for flowers. Unlike the trichromatic retinas of humans (blue, green and red cones; plus rods) and honeybees (ultraviolet, blue and green photoreceptors), butterfly retinas typically have six or more photoreceptor classes with distinct spectral sensitivities. The eyes of the Japanese yellow swallowtail (Papilio xuthus) contain ultraviolet, violet, blue, green, red and broad-band receptors, with each ommatidium housing nine photoreceptor cells in one of three fixed combinations. The Papilio eye is thus a random patchwork of three types of spectrally heterogeneous ommatidia. To determine whether Papilio use all of their receptors to see colours, we measured their ability to discriminate monochromatic lights of slightly different wavelengths. We found that Papilio can detect differences as small as 1-2nm in three wavelength regions, rivalling human performance. We then used mathematical modelling to infer which photoreceptors are involved in wavelength discrimination. Our simulation indicated that the Papilio vision is tetrachromatic, employing the ultraviolet, blue, green and red receptors. The random array of three ommatidial types is a common feature in butterflies. To address the question of how the spectrally complex eyes of butterflies evolved, we studied their developmental process. We have found that the development of butterfly eyes shares its molecular logic with that of Drosophila: the three-way stochastic expression pattern of the transcription factor Spineless determines the fate of ommatidia, creating the random array in Papilio.

국내 나비온실의 식물 식재현황 분석과 적용방안 연구

This study analyzed the relationship between butterflies and plants. We examined 8 butterfly greenhouse. Butterflies are 5 families, 22 species found in 8 locations. Papilio xuthus, Pieris rapae, Papilio bianor etc. was expected to be introduced in the garden. Plants were identified in the 8 greenho...

Molecular logic behind the three-way stochastic choices that expand butterfly colour vision.

Butterflies rely on color vision extensively to adapt to the natural world. Most species express a broad range of color sensitive Rhodopsins in three stochastically distributed types of ommatidia (unit eyes)1–3. The retinas of Drosophila deploy just two main types, where fate is controlled by the binary stochastic decision to express the transcription factor Spineless (Ss) in R7 photoreceptors4. We investigated how butterflies instead generate three stochastically distributed ommatidial types, resulting in a more diverse retinal mosaic that provides the basis for additional color comparisons and an expanded range of color vision. We show that the Japanese Yellow Swallowtail (Papilio xuthus, Papilionidae) and the Painted Lady (Vanessa cardui, Nymphalidae) have a second R7-like photoreceptor in each ommatidium. Independent stochastic expression of Ss in each R7-like cell results in expression of a Blue (Ss-ON) or a UV (Ss-OFF) Rhodopsin. In Papilio, these choices of Blue/Blue, Blue/UV, or UV/UV in the two R7s are coordinated with expression of additional Rhodopsins in the remaining photoreceptors, and together define the three types of ommatidia. Knocking out ss using CRISPR/Cas95,6 leads to the loss of the Blue fate in R7-like cells and transforms retinas into homogeneous fields of UV/UV-type ommatidia, with all corresponding features. Hence, the three possible outcomes of Ss expression define the three ommatidial types in butterflies. This developmental strategy allowed the deployment of an additional red-sensitive Rhodopsin in Papilio, allowing for the evolution of expanded color vision with a richer variety of receptors7,8. This surprisingly simple mechanism that makes use of two binary stochastic decisions coupled with local coordination may prove to be a general means of generating an increased diversity of developmental outcomes.

Characterization of a Digestive α-Amylase in the Midgut of Pieris brassicae L. (Lepidoptera: Pieridae).

The current study deals with a digestive α-amylase in the larvae of Pieris brassicae L. through purification, enzymatic characterization, gene expression, and in vivo effect of a specific inhibitor, Acarbose. Although α-amylase activity was the highest in the whole gut homogenate of larvae but compartmentalization of amylolytic activity showed an equal activity in posterior midgut (PM) and anterior midgut (AM). A three step purification using ammonium sulfate, Sepharyl G-100 and DEAE-Cellulose Fast flow revealed an enzyme with a specific activity of 5.18 U/mg, recovery of 13.20, purification fold of 19.25 and molecular weight of 88 kDa. The purified α-amylase had the highest activity at optimal pH and temperature of 8 and 35°C. Also, the enzyme had Vmax values of 4.64 and 3.02 U/mg protein and Km values of 1.37 and 1.74% using starch and glycogen as substrates, respectively. Different concentrations of acarbose, ethylenediamine tetraacetic acid, and ethylene glycol-bis (β-aminoethylether) N, N, N′, N′-tetraacetic acid significantly decreased activity of the purified α-amylase. The 4th instar larvae of P. brassicae were fed on the treated leaves of Raphanus sativus L. with 0.22 mM of Acarbose to find in vivo effects on nutritional indices, α-amylase activity, and gene expression. The significant differences were only found in conversion efficiency of digested food, relative growth rate, and metabolic cost of control and fed larvae on Acarbose. Also, amylolytic activity significantly decreased in the treated larvae by both biochemical and native-PAGE experiments. Results of RT-PCR revealed a gene with 621 bp length responsible for α-amylase expression that had 75% identity with Papilio xuthus and P. polytes. Finally, qRT-PCR revealed higher expression of α-amylase in control larvae compared to acarbose-fed ones.

The complete mitochondrial genome of Papilio xuthus (Lepidoptera: Papilionidae) and implications for Papilioninae taxonomy

The complete mitochondrial genome of Papilio xuthus (Lepidoptera: Papilionidae) was determined in this study. The mitochondrial genome is a circular molecule of 15 359 and contains 37 genes including 13 protein-coding genes (PCGs), 22 transfer RNA genes, two ribosomal RNA genes and one control region. The nucleotide composition of the A. chinensis mitogenome is strongly biased toward A + T nucleotides (80.45%). Nine protein-coding genes and 14 tRNA genes are encoded on the H strand, and the other four protein-coding genes and eight tRNA genes are encoded on the L strand. The gene order and the orientation of their mitogenomes were similar to all know Papilionidae species. Finally, the phylogenetic relationships of 11 Papilionidae species were reconstructed based on complete mitochondrial genome using the Bayesian inference (BI) and the maximum-likelihood (ML) method. These molecular-based phylogenies support the traditional morphologically based view of relationships within the Papilionidae.

Wavelength discrimination in the hummingbird hawkmoth Macroglossum stellatarum

Despite the strong relationship between insect vision and the spectral properties of flowers, the visual system has been studied in detail in only a few insect pollinator species. For instance, wavelength discrimination thresholds have been determined in two species only: the honeybee (Apis mellifera) and the butterfly Papilio xuthus. Here, we present the wavelength discrimination thresholds (Δλ) for the hawkmoth Macroglossum stellatarum. We compared the data with those found for the honeybee, the butterfly P. xuthus and the predictions of a colour discrimination model. After training moths to feed from a rewarded disc illuminated with a monochromatic light, we tested them in a dual-choice situation, in which they had to choose between light of the training wavelength and a novel unrewarded wavelength. To characterise the Δλ function, we decreased the difference between wavelengths in subsequent tests. We also varied the light intensity to test its effect on the discrimination capacity. In agreement with the predictions of the model, we found two expected minima of discrimination where photoreceptor sensitivities overlap, as well as a third, minor, unpredicted minimum around the peak of the blue photoreceptor. Macroglossum stellatarum is capable of discriminating between lights with a wavelength difference of 1-2 nm. These discrimination minima are similar to those found for the tetrachromatic P. xuthus, and are better than those of the honeybee. The moth is also capable of using achromatic information to discriminate between lights of long wavelengths.

Molecular cloning of a novel cecropin-like peptide gene from the swallowtail butterfly, Papilio xuthus

A new cecropin-like antimicrobial peptide (Px-CLP) gene was isolated from the immunechallenged larvae of the swallowtail butterfly, Papilio xuthus, by employing annealing control primer (ACP)-based GeneFishing PCR. The full-length cDNA of Px-CLP is 310 nucleotides encoding a 70 amino acid precursor that contains a putative 22-residue signal peptide, a 4-residue propeptide, a presumed 37-residue mature peptide, and an uncommon 7-residue acidic pro-region at the C-terminus. The deduced amino acid sequence of Px-CLP showed significant identities with other Lepidopteran cecropin D type peptides. RT-PCR revealed that the Px-CLP transcript was detected at significant level after injection with bacterial lipopolysaccharide (LPS). The peptides with or without C-terminal acidic sequence region were synthesized on-solid phage and submitted to antibacterial activity assay. The synthetic 37-mer peptide (Px-CLPa), which removed C-terminal acidic sequence region, was showed exclusively antibacterial activity against E. coli ML35; meanwhile, a 44-mer peptide (Px-CLPb) with C-terminal acidic peptide region was not active. This result suggests that Px-CLP is produced as a larger precursor containing a C-terminal pro-region that is subsequently removed by C-terminal modification.

A study on selection of Butterfly and Plant Species for Butterfly Gardening

Various types of parks such as ecological park, dragonfly park, etc. are tried to design in order to resolve different environmental issues in urban areas. Parks are considered organism habitats that not only improve the biodiversity but also give people a chance to observe living organisms in urban area. This study was conducted to select basic materials for planning a butterfly garden through reference reviews. The following 21 species of butterflies belonged to 1 order, 4 families, and 16 genera were selected: Phengaris teleius, Pseudozizeeria maha, Thecla betulae, Argynnis hyperbius, Dichorragia nesimachus, Hestina assimilis, Polygonia caureum, Sasakia charonda, Vanessa cardui, Vanessa indica, Boloria selene, Byasa alcinous, Papilio machaon, Papilio macilentus, Papilio protenor, Papilio xuthus, Papilio bianor, Sericinus montela, Colias erate, Eurema hecabe, and Pieris rapae. 85 species belonged to 22 families, 59 genera, 74 species, 10 varieties, and 1 subspecies were selected for host and nectar plants for butterflies selected above. These results would be expected to useful in butterfly gardening. After that, we think it is necessary to be applied these study results in field. This study requires a test in the butterfly garden. The test results will be make the butterfly-plant matrix.

Complete mitochondrial genome and phylogenetic analysis of the Asian swallowtail, Papilio xuthus

The Asian swallowtail, Papilio xuthus, a member of the Papilioniniis family, is one of the most popular swallowtail. Here, we first assembled the complete mitochondrial genome of P. xuthus. The mitogenome is 15 359 bp in length, including 13 protein-coding genes (PCGs), 22 transfer RNA genes, and 2 ribosomal RNA genes. The structure of non-coding control region was also analyzed. The gene content and the order are in accord with the common Invertebrate form. Phylogenetic tree was constructed based on the complete mitogenomes of P. xuthus and closely related nine swallowtail species to assess their phylogenic relationship and evolution. The complete mitochondrial genome data would be useful for further studies of P. xuthus.

Habitat specificity of butterflies along urban environmental gradients in Tama City, Tokyo

AbstractButterfly assemblages were monitored by transect counts in a riverine area along Tamagawa River (RIV), a residential area on the plain (RES1), a residential area on the hill (RES2), the core area of the city (COR), the Tama Experimental Station of Forestry and Forest Products Research Institute (TES) and Tokyo Metropolitan Sakuragaoka Park (MSP) in Tama City, Tokyo, in 2005. The butterfly assemblages in forest‐dominated TES and MSP were more species‐rich than those in the other areas. The assemblage in the grassland‐dominated RIV was characterized by the highest abundance of individuals. Species richness and abundance were lowest in COR. Ordination of the areas by detrended correspondence analysis placed RIV, MSP and TES in increasing order of scores along axis 1, and RES1, RES2 and COR had higher scores along axis 2 than RIV, TES and MSP. In axis 1, grassland species had low scores and forest species high scores; the two groups were generally separated, coinciding with Tanaka's classification. However, the scores for two “forest species”, Papilio xuthus and Ypthima argus, were low and those for two “grassland species”, Potanthus flavus and Anthocharis scolymus, were high, challenging the validity of the classification. Most species recorded in this study were “seminatural type”, with relatively few “natural type” species, according to Sunose's classification. Most “urban type” species occurred in several different habitats and were not specific to highly human‐dominated RES1, RES2 and COR.

Outbred genome sequencing and CRISPR/Cas9 gene editing in butterflies.

Butterflies are exceptionally diverse but their potential as an experimental system has been limited by the difficulty of deciphering heterozygous genomes and a lack of genetic manipulation technology. Here we use a hybrid assembly approach to construct high-quality reference genomes for Papilio xuthus (contig and scaffold N50: 492 kb, 3.4 Mb) and Papilio machaon (contig and scaffold N50: 81 kb, 1.15 Mb), highly heterozygous species that differ in host plant affiliations, and adult and larval colour patterns. Integrating comparative genomics and analyses of gene expression yields multiple insights into butterfly evolution, including potential roles of specific genes in recent diversification. To functionally test gene function, we develop an efficient (up to 92.5%) CRISPR/Cas9 gene editing method that yields obvious phenotypes with three genes, Abdominal-B, ebony and frizzled. Our results provide valuable genomic and technological resources for butterflies and unlock their potential as a genetic model system.