Butterfly Papilio Research Articles

Identification and in vivo functional analysis of furanocoumarin-responsive cytochrome P450s in a Rutaceae-feeding Papilio butterfly.

The Order Lepidoptera contains nearly 160,000 described species and most of them are specialist herbivores that use restricted plant species as hosts. Speciation that originated from host shift is one of the important factors for the diversification of Lepidoptera. Because plants prepare secondary metabolites for defense against herbivores, with varying profiles of the components among different plant taxa, the specialist herbivores need to be adapted to the toxic substances unique to their host plants. Swallowtail butterflies of the genus Papilio consist of over 200 species. Approximately 80% of them utilize Rutaceae plants, and among the remaining species, a specific subgroup uses phylogenetically distant Apiaceae plants as larval hosts. Rutaceae and Apiaceae commonly contain toxic secondary metabolites, furanocoumarins, and molecular phylogenetic studies support the concept that Apiaceae feeders were derived from Rutaceae feeders. Molecular mechanisms underlying furanocoumarin tolerance in Papilio butterflies have been investigated almost exclusively in an Apiaceae feeder by an in vitro assay. In contrast, there is little information regarding the Rutaceae feeders. Here, we focused on a Rutaceae feeder, Papilio xuthus, and identified two furanocoumarin-responsive cytochrome P450-6B (CYP6B) genes, of which one was an ortholog of a furanocoumarin-metabolizing enzyme identified in the Apiaceae-feeding Papilio while the other was previously unreported. We further conducted in vivo functional analysis using the CRISPR/Cas9 system, revealing a contribution of these CYP6Bs to furanocoumarin tolerance of P. xuthus larvae. Our findings suggest that co-option of furanocoumarin-metabolizing CYP6B enzymes at least partially contributed to the host shift from Rutaceae to Apiaceae in Papilio butterflies.

A Female-Biased Chemosensory Protein PxutCSP19 in the Antennae of Papilio xuthus Tuned to Host Volatiles and Insecticides.

Chemosensory protein (CSP) genes significantly enriched in the female antennae are potential molecular candidates for mediating female oviposition behaviors. In this study, we presented the interaction mechanisms of a female-antenna-biased PxutCSP19 in Papilio xuthus to 47 host volatiles, four biopesticides and 24 synthetic insecticides. Using a bioinformatics-based homology search, 22 genes orthologous to PxutCSP19 were identified from 22 other Papilio butterflies with high sequence identities to each other (73.20~98.72%). Multiple alignment analyses revealed a particularly extended N-terminus of Papilio CSP19s (an average of 154 residues) compared to insects' typical CSPs (approximately 120 residues). The expression profiles indicated that PxutCSP19 was significantly enriched in the female antennae, with a 31.81-fold difference relative to the male antennae. In ligand-binding assays, PxutCSP19 could strongly bind six host odorants with high affinities, ranging from dissociation constant (Ki) values of 20.44 ± 0.64 μM to 22.71 ± 0.73 μM. Notably, this protein was tuned to a monoterpenoid alcohol, linalool, which generally existed in the Rutaceae plants and elicited electrophysiological and behavioral activities of the swallowtail butterfly. On the other hand, PxutCSP19 was also capable of binding eight insecticides with stronger binding abilities (Ki < 12 μM) compared to host odorants. When an extended N-terminal region of PxutCSP19 was truncated into two different proteins, they did not significantly affect the binding of PxutCSP19 to ligands with high affinities, suggesting that this extended N-terminal sequences were not involved in the specificity of ligand recognition. Altogether, our study sheds light on the putative roles of PxutCSP19 enriched in the female antennae of P. xuthus in the perception of host volatiles and the sequestering of insecticides, and it complements the knowledge of butterfly CSPs in olfaction and insecticide resistance.

Motion-sensitive neurons activated by chromatic contrast in a butterfly visual system.

A pattern of two equally bright colours contains only chromatic contrast. Unlike in flies, such a pattern elicits strong optokinetic responses in the butterfly Papilio xuthus. To investigate the neural basis of chromatic motion vision, we performed single-cell electrophysiology. We found spiking neurons exhibiting direction-selective motion sensitivity in the second optic ganglion, the medulla. We analysed the response characteristics of these neurons using two-colour stripe patterns moving vertically. We systematically manipulated the intensities of the colours so that the set of presented patterns included an isoluminant condition for the butterfly. Moving patterns containing only chromatic contrast still elicited a response in the neurons. The neurons' sensitivity profile is similar to that of the behavioural responses. Post-recording dye injection revealed that the neurons have dendrites in the ventral lateral protocerebrum and axonal processes in the medulla, suggesting a feedback role. Presumably, the neurons contribute to subtracting wide-field motion to facilitate the detection of small moving targets. This article is part of the theme issue 'Understanding colour vision: molecular, physiological, neuronal and behavioural studies in arthropods'.

Genome-Wide Analysis of Odorant and Gustatory Receptors in Six Papilio Butterflies (Lepidoptera: Papilionidae).

Simple SummaryThe butterflies of the Lepidoptera are representatives of herbivorous insects. Host plant diversities may shape the evolution of chemosensory receptors in butterflies. However, little is known about whether the host plant range is correlated with the size differences of chemoreceptors as key molecular targets for butterfly–plant chemical interactions. Here, we characterized 381 odorant (ORs) and 328 gustatory (GRs) receptors in one Papilio generalist and five other Papilio specialists regarding their comprehensive annotation, gene structure, phylogenetics and expression profiles. Among six Papilio species, orthologous ORs or GRs shared conserved gene structure, but the differences in intron size frequencies were observed between the generalist and the specialists. Furthermore, expression profiles identified candidate OR and GR genes in antennae, foretarsi or reproductive tissues of P. xuthus. This study addresses, for the first time, the issue that the breath of host plants does not appear to result in the obvious expansions of ORs and GRs in Papilio butterflies and identifies candidate molecular targets associated with olfaction, oviposition or reproduction in P. xuthus.The chemical interactions of insects and host plants are shaping the evolution of chemosensory receptor gene families. However, the correlation between host range and chemoreceptor gene repertoire sizes is still elusive in Papilionidae. Here, we addressed the issue of whether host plant diversities are correlated with the expansions of odorant (ORs) or gustatory (GRs) receptors in six Papilio butterflies. By combining genomics, transcriptomics and bioinformatics approaches, 381 ORs and 328 GRs were annotated in the genomes of a generalist P. glaucus and five specialists, P. xuthus, P. polytes, P. memnon, P. machaon and P. dardanus. Orthologous ORs or GRs in Papilio had highly conserved gene structure. Five Papilio specialists exhibited a similar frequency of intron lengths for ORs or GRs, but which was different from those in the generalist. Phylogenetic analysis revealed 60 orthologous OR groups, 45 of which shared one-to-one relationships. Such a single gene in each butterfly also occurred in 26 GR groups. Intriguingly, bitter GRs had fewer introns than other GRs and clustered into a large clade. Focusing on the two chemoreceptor gene families in P. xuthus, most PxutORs (52/58) were expressed in antennae and 31 genes in reproductive tissues. Eleven out of 28 foretarsus-expressed PxutGRs were female-biased genes, as strong candidates for sensing oviposition stimulants. These results indicate that the host range may not shape the large-scale expansions of ORs and GRs in Papilio butterflies and identify important molecular targets involved in olfaction, oviposition or reproduction in P. xuthus.

Phenotypic Plasticity of the Mimetic Swallowtail Butterfly Papilio polytes: Color Pattern Modifications and Their Implications in Mimicry Evolution.

Butterfly wing color patterns are sensitive to environmental stress, such as temperature shock, and this phenotypic plasticity plays an important role in color pattern evolution. However, the potential contributions of phenotypic plasticity to mimicry evolution have not been evaluated. Here, we focused on the swallowtail butterfly Papilio polytes, which has nonmimetic and mimetic forms in females, to examine its plastic phenotypes. In the nonmimetic form, medial white spots and submarginal reddish spots in the ventral hindwings were enlarged by cold shock but were mostly reduced in size by heat shock. These temperature-shock-induced color pattern modifications were partly similar to mimetic color patterns, and nonmimetic females were more sensitive than males and mimetic females. Unexpectedly, injection of tungstate, a known modification inducer in nymphalid and lycaenid butterflies, did not induce any modification, but fluorescent brightener 28, another inducer discovered recently, induced unique modifications. These results suggest that phenotypic plasticity in nonmimetic females might have provided a basis of natural selection for mimetic color patterns during evolution.

Doublesex Controls Both Hindwing and Abdominal Mimicry Traits in the Female-Limited Batesian Mimicry of Papilio memnon.

Papilio butterflies are known to possess female-limited Batesian mimicry polymorphisms. In Papilio memnon, females have mimetic and non-mimetic forms, whereas males are monomorphic and non-mimetic. Mimetic females are characterized by color patterns and tails in the hindwing and yellow abdomens. Recently, an analysis of whole-genome sequences has shown that an approximately 160 kb region of chromosome 25 is responsible for mimicry and has high diversity between mimetic (A) and non-mimetic (a) alleles (highly diversified region: HDR). The HDR includes three genes, UXT, doublesex (dsx), and Nach-like, but the functions of these genes are unknown. Here, we investigated the function of dsx, a gene involved in sexual differentiation, which is expected to be functionally important for hindwing and abdominal mimetic traits in P. memnon. Expression analysis by reverse transcription quantitative PCR (RT-qPCR) and RNA sequencing showed that mimetic dsx (dsx-A) was highly expressed in the hindwings in the early pupal stage. In the abdomen, both dsx-A and dsx-a were highly expressed during the early pupal stage. When dsx was knocked down using small interfering RNAs (siRNAs) designed in the common region of dsx-A and dsx-a, a male-like pattern appeared on the hindwings of mimetic and non-mimetic females. Similarly, when dsx was knocked down in the abdomen, the yellow scales characteristic of mimetic females changed to black. Furthermore, when dsx-a was specifically knocked down, the color pattern of the hindwings changed, as in the case of dsx knockdown in non-mimetic females but not mimetic females. These results suggest that dsx-a is involved in color pattern formation on the hindwings of non-mimetic females, whereas dsx-A is involved in hindwing and abdominal mimetic traits. dsx was involved in abdominal and hindwing mimetic traits, but dsx expression patterns in the hindwing and abdomen were different, suggesting that different regulatory mechanisms may exist. Our study is the first to show that the same gene (dsx) regulates both the hindwing and abdominal mimetic traits. This is the first functional analysis of abdominal mimicry in butterflies.

Genomic architecture and functional unit of mimicry supergene in female limited Batesian mimic Papilio butterflies.

It has long been suggested that dimorphic female-limited Batesian mimicry of two closely related Papilio butterflies, Papilio memnon and Papilio polytes, is controlled by supergenes. Whole-genome sequencing, genome-wide association studies and functional analyses have recently identified mimicry supergenes, including the doublesex (dsx) gene. Although supergenes of both the species are composed of highly divergent regions between mimetic and non-mimetic alleles and are located at the same chromosomal locus, they show critical differences in genomic architecture, particularly with or without an inversion: P. polytes has an inversion, but P. memnon does not. This review introduces and compares the detailed genomic structure of mimicry supergenes in two Papilio species, including gene composition, repetitive sequence composition, breakpoint/boundary site structure, chromosomal inversion and linkage disequilibrium. Expression patterns and functional analyses of the respective genes within or flanking the supergene suggest that dsx and other genes are involved in mimetic traits. In addition, structural comparison of the corresponding region for the mimicry supergene among further Papilio species suggests three scenarios for the evolution of the mimicry supergene between the two Papilio species. The structural features revealed in the Papilio mimicry supergene provide insight into the formation, maintenance and evolution of supergenes. This article is part of the theme issue 'Genomic architecture of supergenes: causes and evolutionary consequences'.

Papilio butterfly vs. hawkmoth pollination explains floral syndrome dichotomy in a clade of Lilium

Abstract Pollination niche shifts can drive remarkable floral divergence between closely related plant species. The Leucolirion clade of Lilium contains species with either tepal-recurved or trumpet-shaped flowers. The tepal-recurved flowers are bright orange and might be pollinated by butterflies and/or birds. The trumpet-shaped flowers are mostly pale and strongly fragrant and might permit visitation by a variety of hawkmoths. Lilium leucanthum has trumpet-shaped flowers, and some populations of this species show dark coloration on the floral outer surface, suggesting pollination by mammals. We identified pollinators and examined the dependence of reproduction on pollinators by floral visitor observations, pollen load analysis and pollination experiments. We also analysed floral traits to contrast the two floral syndromes involving different lepidopteran groups. The tepal-recurved lilies are specialized on a group of Papilio butterflies for pollination with pollen predominantly attached to the hindwings. The trumpet-shaped flowers are almost exclusively pollinated by hawkmoths, including diverse species with proboscises of different lengths. No mammal visitation was found to the populations of L. leucanthum with dark outer surfaces of flowers. Self-incompatibility prevails throughout the clade, including the populations in which pollinators were scarce. The butterfly- and hawkmoth-pollinated species display contrasting floral syndromes. Our findings confirmed that the dichotomy in floral syndrome in the Leucolirion clade is associated with Papilio butterfly vs. hawkmoth pollination, whereas intraspecific variation in colour of the floral outer surface of L. leucanthum many need a non-pollinator explanation.

Integrated Analysis of Transcriptome and Proteome to Reveal Pupal Color Switch in Papilio xuthus Butterflies.

Pupal color polyphenism in Papilio butterflies, including green, intermediate, or brown, is an excellent study system for understanding phenotypic plasticity. Previous studies suggested that development of brown pupae may be controlled by a hormone called pupal-cuticle-melanizing-hormone (PCMH) which is synthesized and secreted from brain-suboesophageal ganglion and prothoracic ganglion complexes (Br-SG-TG1) during the pre-pupa stage. However, detailed molecular mechanisms of neuroendocrine regulation in pupal color development remain unknown. In this study, we integrated the expression profiles of transcriptome and proteome at pre-pupa stages [2 h after gut purge (T1) and 3 h after forming the garter around the body (T2)] and pigmentation stages [10 h after ecdysis (T3) and 24 h after ecdysis (T4)] to identify important genes and pathways underlying the development of green and brown pupa in the swallowtail butterfly Papilio xuthus. Combined comparisons of each developmental stage and each tissue under green and brown conditions, a total of 1042 differentially expressed genes (DEGs) and 430 different abundance proteins (DAPs) were identified. Weighted gene co-expression network analysis (WGCNA) and enrichment analysis indicate that these DEGs were mainly related to oxidation-reduction, structural constituent of cuticle, and pigment binding. Soft clustering by Mfuzz and enrichment analysis indicate that these DAPs are mainly involved in tyrosine metabolism, insect hormone biosynthesis, and melanogenesis. By homologous alignment, we further identified those genes encoding neuropeptides (51), GPCRs (116), G-proteins (8), cuticular proteins (226), chitinases (16), and chitin deacetylases (8) in the whole genome of P. xuthus and analyzed their expression profiles. Although we identified no gene satisfying with hypothesized expression profile of PCMH, we found some genes in the neuropeptide cascade showed differentially expressed under two pupal color conditions. We also found that Toll signaling pathway genes, juvenile hormone (JH) related genes, and multiple cuticular proteins play important roles in the formation of selective pupal colors during the prepupal-pupal transition. Our data also suggest that both green and brown pupa include complex pigment system that is regulated by genes involved in black, blue, and yellow pigments. Our results provide important insights into the evolution of pupal protective colors among swallowtail butterflies.

Visual and olfactory sensory responses of the butterfly Papilio maackii during foraging and courtship

AbstractColors and chemicals are the most important cues for butterflies to localize mating partners and food. In this study, we used artificial flowers with different colors and plastic butterfly wing mimics to separate the impact of colors from other cues on foraging and courtship of the butterfly Papilio maackii. We found that males relied more on visual cues during foraging and courtship, but females responded more to olfactory signals. Males visited artificial flowers more frequently than females. However, when honey was applied on artificial flowers, the frequency of female's flower visits increased and was much higher than that of male's, suggesting that females may have stronger olfactory than visual senses. During courtship, females were passive, whereas males were very active in chasing females and driving away other males. Similar to courtship among butterflies in a natural population, males showed recognition ability to butterfly wing mimics, indicating that males could accurately identify mates using colors only without any chemical cues. We also analyzed volatiles and identified five types of male‐specific chemicals while no female‐specific chemicals were identified, suggesting that females might have used these male‐specific volatiles to recognize and accepted males during courtship. Pollination of the plant Primula sieboldii mainly depends on P. maackii, and the plant has flowers with purple and mauve colors. Coincidently, P. maackii showed color preference to blue and purple, suggesting that the butterfly and plants might have coevolved during the long course of interactions.

Cover Caption

Butterflies are considered bioindicators of environmental change. We evaluated the combined effects of climate and land‐use interactions on butterfly diversity in tropical rainforest and savanna ecosystems of southwestern China. The results showed that increasing land‐use intensity not only caused a dramatic decrease in butterfly alpha diversity but also significantly simplified butterfly species composition, and finally revealed interactions between human‐modified habitat change and climate change shaped butterfly diversity in both ecosystems. Our study has important implications for biodiversity conservation under the current era of rapid human‐induced habitat loss and climate change (see pages 1109‐1120). The photo is provided by Bai‐Ge Miao, which the butterfly Papilio memnon is visiting the Ixora casei ‐Super king‐ flowers.

New distributional data of the protected butterfly Papilio alexanor Esper, 1800 in north-western Italy and some ecological observations (Lepidoptera Papilionidae)

New distributional data of the protected butterfly Papilio alexanor Esper, 1800 in north-western Italy and some ecological observations (Lepidoptera Papilionidae)

Butterflies (Lepidoptera: Rhopalocera) of the undivided Midnapore District, West Bengal, India: a preliminary report

A butterfly study in the biodiversity rich biogeography transition zone of the undivided Midnapore District of West Bengal was carried out from March 2014 to March 2017. A total of 98 species of butterflies under five families, 19 subfamilies, and 70 genera were documented by the study. Maximum species diversity was found in Nymphalidae family with 31 species (31.63%), followed by Lycaenidae 24 species (24.48%), Hesperiidae-17 (17.34%), Pieridae 16 (16.32%), and the least by Papilionidae 10 (10.28%). Among the families, the highest common species (n=10) was recorded under the family Nymphalidae and maximum rare species (n=4) under the family Lycaenidae. Highest species richness has been recorded in post-monsoon season (97 species) followed by monsoon (78 species), winter (66 species), and pre-monsoon (47 species). During the study, five species of butterflies (Castalius rosimon, Neptis jumbah, Discophora sondaica, Lethe europa, and Papilio clytia) under Schedule-I, three species (Euchrysops cnejus, Mahathala ameria, and Cepora nerissa) under Schedule-II and three species (Baoris farri, Hyarotis adrastus, and Euploea core) under Schedule-IV were legally protected under the Indian Wildlife (Protection) Act, 1972. A good species to genera ratio (1.4: 1) along with 10 newly recorded species and their range extension provide information for better understanding of the ecology and distribution pattern of the butterfly fauna. The information of the study will thus help to develop conservation strategies for management of the unique bio-geographical transitional zone.

Diversification and selection pattern of CYP6B genes in Japanese Papilio butterflies and their association with host plant spectra.

Herbivorous insects are thought to have evolved counteradaptations to conquer chemical defenses in their host plants in a stepwise co-evolutionary process. Papilio butterflies use CYP6B gene family members to metabolize furanocoumarins in their Rutaceae or Apiaceae host plants. CYP6Bs have functionally diverged among Papilio species to be able to metabolite diverse types of furanocoumarins in their host plants. In this study, we examined the diversification and selection patterns of CYP6B among nine Papilio species in Japan (eight Rutaceae specialists and one Apiaceae specialist) and their association with host plant spectra and furanocoumarin profiles. We compared host plant spectrum of eight Rutaceae feeding Papilio species and also performed a furanocoumarin profiling of their host plants. In addition, we reconstructed CYP6B gene phylogeny and performed selection analysis based on the transcriptome data of those nine Papilio species. Among Rutaceae-feeding Papilio species, host plant spectrum differences were correlated with their furanocoumarin profiles. However, all tested Papilio species had similar duplicated sets of CYP6B, with no apparent lineage-specific or host plant-specific pattern of CYP6B diversification. Selection analysis showed a signature of positive selection on a CYP6B branch. The positively selected sites located at predicted substrate recognition sites and we also found that these CYP6B genes were observed only in Rutaceae-feeding species. These findings indicate that most CYP6B diversification occurred in ancestral species of these Papilio species, possibly in association with specific host plant chemical defenses and subsequent gene loss due to host specialization. These processes would have shaped the complex diversification patterns of the CYP6B gene family in Papilio butterflies. Our results also show potentially important CYP6B clades among Papilio species which likely to have diverged functions and associated with host plant phytochemicals in ancestral Papilio species.

Pollination of the Orchid Habenaria rhodocheila by the Swallowtail Butterfly Papilio helenus in Subtropical Evergreen Broad-Leaved Forests in Southern China

Habenaria rhodocheila is different from other species in the genus as it has bright yellow-orange, orange-red or red floral colors and grows in subtropical evergreen broad-leaved forests. It is listed as a level II protected plant in China. This study focused on details of the pollination mechanism, including floral structure, the pollinator and its behavior, the pollination process, and fruit set in its natural environment compared to controlled artificial pollination. Our results showed that the flowers of H. rhodocheila are self-compatible, but rely on a pollinating agent due to the lack of an automatic self-pollination mechanism. At three sites the flowers of H. rhodocheila were frequently pollinated by a species of the swallowtail butterfly Papilio helenus L. During pollination, the butterfly inserts its proboscis into the spur aperture. The base of the proboscis then contacts the viscidia and the pollinarium sticks to the insect, followed by contact between pollinia and stigmas. The length of the proboscis of P. helenus is comparable to, but slightly shorter than the length of the floral spur. The pollination activity generally occurs between 9:30 AM to 1:30 PM daily and is especially frequent between 10:30 AM and 11:30 AM. These results suggest that the similarity between the length of the butterfly's proboscis and the length of the orchid's spur is a major factor contributing to the efficient pollination of H. rhodocheila in its natural habitat. Most species in Habenaria are moth-pollinated, whereas our research has shown that H. rhodocheila in subtropical evergreen broad-leaved forests in China is specifically adapted to butterfly pollination by P. helenus.

Ecological significance of puddling as a behavioural phenomenon in the life history of butterfly Papilio polytes Linn. (Lepidoptera: Papilionidae)

• Male abundance in presence of female can enhance the puddling in male P. polytes. • The onset of puddling is directly regulated by variation in the temperature. • The fecundity and longevity of imago are completely unaffected by puddling. • The outcomes of present study elucidate the behavioral significance of puddling. • The results provide the empirical evidences for preexisting hypotheses on puddling. Puddling in butterflies is an essential physiological phenomenon, by which they gain mineral nutrients required for their reproductive success and flight. The preference given to the puddling by butterflies has been analysed by two hypotheses: butterflies puddle when there is a scarcity of nutritional resource in one hypothesis and competition with other males to increase their probability of mating in the other. In the present investigation, an attempt has been made to verify these hypotheses through experimental evidences in the butterfly Papilio polytes , in which only males are known to puddle. Our investigation revealed the significance of puddling as an alternative source of nutrition in the absence of nectar. However, increase in the number of males in a given vicinity with constant number of females enhances the puddling activity in males. This shows the importance of puddling as a distinct competitive behaviour by which male improves their chances of mating. Additionally, in both cases the puddling is drastically regulated by the environmental temperature. Males prefer to puddle at a temperature ranging from 26 °C to 28 °C, which is considered as optimum for puddling activity. Further, the fecundity and longevity of imago are completely unaffected by puddling activity. All these results are in good agreement with the existing hypotheses and show the behavioural significance of puddling in butterflies.

Retinal organization and visual abilities for flower foraging in swallowtail butterflies.

Papilio butterflies' ability to forage for flowers relies upon multiple visual cues such as color, brightness, and motion. Papilio learns the color of rewarding flowers and detects it at a distance. Its color vision is based on four photoreceptor classes: UV, blue, green, and red, providing sensitive wavelength discrimination. These four receptor classes also contribute to the perception of brightness and polarization. Papilio's motion vision is based on a different set of receptors: green, red, and broad band. This implies that two visual pathways exist in Papilio. The contribution of several receptor classes not only for chromatic vision but also achromatic vision likely enhances the butterfly's ability to detect flowers in complex visual environments.

Molecular Mechanisms Underlying Pupal Protective Color Switch in Papilio polytes Butterflies

Mimicry is a survival strategy in which organisms deceive their predators by imitating other organisms or the surrounding environment. One example of this involves pupal color polymorphism, which is widely observed in butterflies and moths. It has been suggested that the pupal colors of Papilio butterflies are selected according to the tactile stimulation experienced before pupation. Specifically, larvae crawling on smooth leaves become green pupae, but those crawling on rough stems become brown pupae. These protective colors fit with the surrounding environment. However, the detailed molecular mechanisms underlying pupal polymorphism have generally remained unknown. To reveal these mechanisms, we first established control over the green and brown pupal coloration in Papilio polytes under laboratory conditions and clarified temporal and spatial changes of pupal pigments under both conditions. We also analyzed the expression of coloration genes during the pre-pupal stage and at pupation in the epidermis under the green and brown conditions, by RNA sequencing and quantitative RT-PCR. These analyses revealed that the brown pupal color is regulated mainly by melanin synthesis genes, tyrosine hydroxylase (TH) and laccase 2. In contrast, the green pupal color was suggested to be formed mainly through the expression of both multiple bilin binding protein (BBP)-related genes responsible for blue pigmentation and multiple juvenile hormone binding protein (JHBP)-related genes responsible for yellow pigmentation. Electroporation-mediated RNAi showed that the knockdown of TH or laccase 2 blocked the brown pupal coloration, and that the knockdown of BBP- or JHBP-related genes caused yellow or blue coloration in the green-conditioned pupae, respectively, supporting the above hypothesis. We here report how genes involved in the pupal coloration of P. polytes are regulated, which sheds light on the evolutionary process of pupal protective colors among Lepidoptera.

First Report on Blue Mold Parasitism on Butterfly (Papilio polytes) Egg

The fungal (Penicillium) infection, on the eggs of butterfly Papilio polytes (Lepidoptera: Papilionidae), was reported for the first time at the urban settings, Kolkata, India. In course of rearing P. polytes, the eggs were observed to be vulnerable to the fungal attack. The eggs that oviposited on the leaves of Citrus limon were found to be infested with the fungi, identified as Penicillium sp., through further microbial culture. In all instances, the growth of Penicillium on the eggs restrained further development with the absence of larval emergence. The infestation by Penicillium on butterfly eggs caused disruption of development and eventual shrinking.

What's in a band? The function of the color and banding pattern of the Banded Swallowtail.

Butterflies have evolved a diversity of color patterns, but the ecological functions for most of these patterns are still poorly understood. The Banded Swallowtail butterfly, Papilio demolion demolion, is a mostly black butterfly with a greenish‐blue band that traverses the wings. The function of this wing pattern remains unknown. Here, we examined the morphology of black and green‐blue colored scales, and how the color and banding pattern affects predation risk in the wild. The protective benefits of the transversal band and of its green‐blue color were tested via the use of paper model replicas of the Banded Swallowtail with variations in band shape and band color in a full factorial design. A variant model where the continuous transversal green‐blue band was shifted and made discontinuous tested the protective benefit of the transversal band, while grayscale variants of the wildtype and distorted band models assessed the protective benefit of the green‐blue color. Paper models of the variants and the wildtype were placed simultaneously in the field with live baits. Wildtype models were the least preyed upon compared with all other variants, while gray models with distorted bands suffered the greatest predation. The color and the continuous band of the Banded Swallowtail hence confer antipredator qualities. We propose that the shape of the band hinders detection of the butterfly's true shape through coincident disruptive coloration; while the green color of the band prevents detection of the butterfly from its background via differential blending. Differential blending is aided by the green‐blue color being due to pigments rather than via structural coloration. Both green and black scales have identical structures, and the scales follow the Bauplan of pigmented scales documented in other Papilio butterflies.