A model for colour pattern formation in the butterfly wing of Papilio dardanus

The butterfly Papilio dardanus is well known for the spectacular phenotypic polymorphism in the female of the species. We show that numerical simulations of a reaction diffusion model on a geometrically accurate wing domain produce spatial patterns that are consistent with many of those observed on the butterfly. Our results suggest that the wing coloration is due to a simple underlying stripe-like pattern of some pigment-inducing morphogen. We focus on the effect of key factors such as parameter values for mode selection, threshold values which determine colour, wing shape and boundary conditions. The generality of our approach should allow us to investigate other butterfly species. The relationship between these key factors and gene activities is discussed in the context of recent biological advances.     

Local Enhancement in Mud-Puddling Swallowtail Butterflies (Battus philenor and Papilio glaucus)

Male butterflies aggregate at moist soil to acquire nutrients, a phenomenon termed “mud-puddling.” We studied the attraction of free-flying Papilio glaucus and Battus philenor swallowtails to dead decoys of those two species at artificial puddles moistened with NaCl solution. Both species landed preferentially at puddles with a decoy present rather than at unbaited puddles, demonstrating very strong local enhancement, a form of social facilitation. Papilio glaucus were attracted only to intraspecific decoys, whereas Battus philenor exhibited both intraspecific and interspecific attraction. Circular discs cut from the hindwings of male Battus were highly attractive to male Battus but completely unattractive to Papilio glaucus. The visual cues attractive to males in their search for salts differ between these two swallowtail species for unexplained reasons.     

No Evidence that Male Choice Contributes to the Maintenance of a Shared, Sex-Limited Trait in Mimetic and Non-mimetic Female Tiger Swallowtail Butterflies, Papilio glaucus

In animal species that have morphological polymorphisms maintained by unique or divergent selection pressures, understanding the preservation of shared traits is important for identifying the factors that are influencing overall evolutionary processes. In the Eastern tiger swallowtail butterfly, Papilio glaucus, females are dimorphic. One morph (‘dark-morph’) is mostly black and mimics the toxic pipevine swallowtail, Battus philenor. These females have large amounts of blue coloration on the dorsal hind wings that enhances their mimetic resemblance. Conversely, the alternate female type (‘yellow-morph’) is similar to males in coloration with the exception of extensive dorsal blue coloration, comparable to dark-morph females. Such coloration is almost completely absent in males. We examined dorsal blue coloration in P. glaucus to determine if mimetic resemblance in dark morphs is predominantly responsible for the maintenance of dorsal blue color in both female types, or whether mate recognition and/or sexual selection by males has a stronger influence on this trait. We measured the relative amount and variance of dorsal and ventral blue coloration in females of both color morphs, as well as males. We also compared these measurements to similar ones taken in the sister species, P. canadensis (which does not exhibit female dimorphism). Lastly, we investigated mate recognition and preferences of wild males. Our results suggest that mimetic resemblance may be more important than sexual selection for sustaining dorsal blue coloration in dark-morph females and that yellow-morphs could have elevated levels of blue due to currently unknown genetic associations. Although trait correlation between sexes is common, intrasexual trait correlation in a sex-limited, polymorphic species has not been frequently observed.     

Perturbation of the wing color pattern of a swallowtail butterfly,Papilio xuthus,induced by acid carboxypeptidase

Hypodermal injection of Toughmac-E, a digestive mixture composed of nine digestive components, or Molsin induced perturbation of the wing color pattern in 0- to 2-day-old pupae of Papilio xuthus, but had no effect on prepupae or 3- to 4-day-old pupae. The effective component in Toughmac-E was identified as Molsin, an acid carboxypeptidase of Aspergillus saitoi which specifically liberates tyrosine and phenylalanine from the C-terminal residues of proteins. The pattern perturbation occurred in either side of the fore- and hindwings of both sexes. When this enzyme was administered, stronger melanization than in the normal wings was found in the whole wings of most butterflies, but in other butterflies, the yellow region was enlarged. These findings suggest that the pattern perturbation was caused by changes in the levels of melanin and papiliochromes in scales. Melanin is a black pigment and papiliochromes are yellow pigments; their common precursor is dopamine. The normal pattern is formed by a predetermined balance of melanin and papiliochromes, whereas the deposit of an excess amount of tyrosine and/or phenylalanine disturbs this balance and results in perturbation of the color pattern. Administration of L-dopa or dopamine had no effect on the wing pattern. When the activity of an endogenous acid carboxypeptidase similar to Molsin appears in the early pupa, the summed activities of the endogenous and exogenous acid carboxypeptidases must induce a pattern perturbation. The relations between the endogenous acid carboxypeptidase and its probable substrate, the reserve protein, and the physiological roles of these relations in the regulation, utilization and excretion of amino acids are discussed.     

Host plant alteration of detoxication activity in Papilio glaucus glaucus

Host plant alteration of activity of midgut detoxication enzymes was measured in larvae of the eastern tiger swallowtail, Papilio glaucus glaucus L. (Lepidoptera: Papilionidae). Larvae were reared from hatching to the fifth stadium on leaves of black cherry (Prunus serotina Ehrh.), tulip tree (Liriodendron tulipifera L.), paper birch (Betula papyrifera Marsh.), white ash (Fraxinus americana L.), or basswood (Tilia americana L.). O-Demethylase activity varied up to 16-fold, microsomal esterase activity varied up to 6-fold, and microsomal glutathione transferase varied up to almost 5-fold among larvae fed the various host plants. In contrast, cytochrome c reductase, soluble esterase, and soluble glutathione transferase activities varied only 1.8-, 2.1-, and 2.2-fold, respectively. Overall detoxication enzyme activity was highest in larvae fed tulip tree and lowest in larvae fed basswood. Induction varied qualitatively and quantitatively among the enzyme systems assayed. Some patterns of induction were related to allelochemical profiles of the plants and nutritional status of the larvae.

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Résumé La modification des principaux systèmes de détoxification de l'intestin moyen par la plante consommée a été évaluée chez les chenilles de P. glaucus glaucus. Les chenilles ont été élevées dès l'éclosion sur feuilles de Prunus serotina Ehrh., Liriodendron tulipifera L., Betula papyrifera Marsh., Fraxinus americana L. et Tilia americana L. Les chenilles du cinquième stade (de 3 à 6 jours) ont été pesées, dessécheées et leur intestin moyen extrait pour préparation enzymatique. L'activité polysubstrate monooxygénase (PSMO) a été déterminée par des dosages à la cytochrome c réductase et à la O-déméthylase, l'activité estérase a été mesurée par dosage au 1-naphthyl acétate et la glutathion transférase a été mesurée par des dosages au 1-chloro-2,4-dinitrobenzène (CDNB) et au 2,4-dichloro-1-nitrobenzène (DCNB).L'ordre de performance de développement des chenilles a été P. serotina > L. tulipifera > B. papyrifera > F. americana > T. americana. Les activités PSMO les plus fortes ont été observées chez les chenilles élevées sur L. tulipifera, et les plus faibles sur celles qui ont consommé T. americana. L'activité des estérases solubles a été la plus élevée chez les chenilles ayant consommé L. tulipifera, et la plus faible chez les chenilles nourries de T. americana. L'activité glutathion transférase soluble a été la plus forte chez les chenilles élevées sur B. papyrifera, et la plus faible sur les chenilles élevées sur P. serotina, tandis que l'activité glutathion transférase microsomale a été la plus forte chez les chenilles élevées sur D. tulipifera, et la plus faible chez les chenilles èlevées sur T. americana (dosage au CDBN). L'activité totale de détoxification enzymatique élevée des chenilles nourries de L. tulipifera provenait probablement de l'abondance de différentes substances allélochimiques chez cette espèce; la faible activité totale chez les larves nourries de T. americana a pu provenir de leur basse valeur alimentaire.

     

Oviposition site selection: an aid to rapid growth and development in the tiger swallowtail butterfly,Papilio glaucus

Summary Female tiger swallowtail butterflies oviposited on the tips of branches exposed to sunlight. Over 50 percent of the eggs had westward exposures, yet only 7.6% faced northward. Over 97% of the eggs were laid below 3 m. Larvae with sunny exposures developed 15 to 35% faster than those lacking radiant input. Leaf curling aided caterpillars in attaining elevated body temperatures. Caterpillars with western exposures had lower survivorship. This oviposition pattern and the thermal behavior of the larvae allow completion of two broods per season in an area where growth-degree-hours are sometimes insufficient for a second brood.     

Differential toxicity of a phenolic glycoside from quaking aspen to Papilio glaucus butterfly subspecies,hybrids and backcrosses

Summary Papilio glaucus subspecies, hybrids and backcrosses exhibit greatly different abilities to use quaking aspen (Populus tremuloides) and other members of the Salicaceae as host plants. This study was conducted to test the hypotheses that phenolic glycosides account for the differences in larval performance, and that differential performance is correlated with differential larval esterase activities. To test the hypotheses we conducted first instar survival trials and fourth (penultimate) instar feeding trials with tremulacin, a phenolic glycoside. We also conducted assays of -glucosidase, esterase, and glutathione transferase activities, using midgut enzyme preparations from fifth instars. First instar survival on the tremulacin treated diet generally improved with a higher proportion of Papilio glaucus canadensis genes in the genotype, although survival in one backcross treatment was surprisingly low. Penultimate instars of P.g. glaucus and P.g. australis fed tremulacin treated black cherry leaves experienced a severe reduction in growth rate relative to larvae fed control leaves. This seriously suppressed growth was partially due to reduced consumption rates and reduced conversion efficiencies, however, approximate digestibility was not affected. In contrast, P. g. canadensis and hybrids showed no differences in growth rates between tremulacin treated and control leaves. Reciprocal backcrosses of hybrids with P. g. glaucus resulted in slightly suppressed growth on treated versus control leaves. The results suggest that after a certain threshold, increased proportions of P. g. glaucus genes resulted in poorer growth performance with tremulacin in the diet. Soluble esterase activities generally increased with the proportion of Papilio glaucus canadensis genes in the genotype, and paralleled overall trends in larval survival and feeding performance. We conclude that phenolic glycosides such as tremulacin are responsible for differential performance of Papilio glaucus subspecies, hybrids and backcrosses fed plants in the Salicaceae, and that detoxification of phenolic glycosides by midgut esterase explains why some Papilio glaucus genotypes can effectively utilize these plants.     

Histopathological effects of tannins on the midgut epithelium of Papilio polyxenes and Papilio glaucus

The effects of dietary tannin on midgut epithelial structures were compared in two closely related species of swallowtail caterpillars — Papilio polyxenes, a species restricted to the tannin-free Umbelliferae, and P. glaucus, which feeds on tanniniferous tree species in several families. The effects of tannin ingestion were compared in second and fifth instar caterpillars. While large numbers of lesions were found in the guts of P. polyxenes ingesting tannins, only one small lesion was found in any P. glaucus ingesting tannins. No such lesions could be found in larvae of either species raised on tannin-free leaves. The observed histopathological changes, discussed in detail, resemble those previously reported in several species of Lepidoptera in response to a variety of substances and may represent a general degenerative response to the presence of toxins in the gut.

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Résumé L'étude à porté sur les effets de tanins dans l'alimentation sur les structures épithéliales de l'intestin moyen des chenilles de second et cinquième stades, de deux espèces très voisines: Papilio polyxenes-espèce ne consommant que des ombellifères sans tanins-et P. glaucus qui s'alimente sur des arbres contenant des tanins et appartenant à plusieurs familles. Alors qu'après ingestion de tanins, de nombreuses lésions ont été observées sur l'intestin de P. polyxenes, seulement quelques petites lésions ont été trouvées sur l'intestin de P. glaucus. Aucune lésion de même type n'a été observée dans les chenilles de ces espèces élevées sur feuilles sans tanin. Les modifications histopathologiques, discutées en détail, correspondent à la mortalité induite par le tanin due à une septicémie bactérienne et peut représenter une réponse dégénérative à la présence de substances toxiques dans l'intestin.

     

Divergence in the ovipositional behavior of the Papilio glaucus group

This study contrasts the ovipositional profiles of four members of the Papilio glaucus group, P. glaucus , P. multicaudatus , P. canadensis , and P. rutulus. We used seven choice oviposition bioassays containing leaves from hosts in seven plant families utilized by members of the P. glaucus group. Specifically, we contrast the overall ovipositional profiles of these species and their acceptance of a host in a novel plant family ( Populus tremuloides : Salicaceae) and a host in a putatively ancestral host plant family ( Liriodendron tulipifera : Magnoliaceae). Significant differences were observed between the ovipositional profiles of P. glaucus and P. multicaudatus relative to each other and to P. canadensis and P. rutulus. In contrast, no significant differences were observed between the ovipositional profiles of P. canadensis and P. rutulus , which were also the only species that accepted P. tremuloides. Unlike the acceptance of P. tremuloides , the acceptance of L. tulipifera was present throughout the group despite the inability of the larvae of most species in the group to utilize this host. These results support the prediction of the "hierarchical threshold model" that ancestral host plants are likely to be retained in the ovipositional hierarchy while novel hosts should only be accepted by derived populations.     

Origins of the regional feeding abilities in the tiger swallowtail butterfly: ecological monophagy and the Papilio glaucus australis subspecies in Florida

Summary The Highlands County, Florida populations of the eastern tiger swallowtail butterfly, Papilio glaucus L., represent a putative subspecies, P. g. australis Maynard. The only foodplant ever reported in southern half of Florida is sweetbay, Magnolia virginiana L. (Magnoliaceae). In fact, there are apparently no other reported foodplants for the polyphagous tiger swallowtail species which exist in southern Florida. These ecologically monophagous P. g. australis populations were studied in order to determine whether or not significant behavioral and biochemical adaptations in the larvae are detectable as a result of the ecological specialization on sweetbay. These results suggest an emphatic yes. On sweetbay, neonate larval survival of these Florida populations is the best of any other population tested from across North America. In addition, penultimate instar larval growth rates are significantly faster on sweet bay than any other foodplant tested. Furthermore, these biochemical adaptations to sweetbay by P. g. australis in Florida are accompanied by a significantly poorer neonate survival and growth performance on northern foodplants (most notably the Salicaceae and Betulaceae). Reciprocal inabilities of the northern subspecies, P. g. canadensis, and the western P. rutulus to utilize sweetbay are described, as well as the reciprocal inabilities of two congeneric and sympatric (Florida) species, Papilio palamedes and Papilio troilus, that have specialized on another bay (redbay, Persea borbonia; of the Lauraceae). It remains uncertain to what degree such apparent negative genetic correlations in foodplant adaptations represent a driving force in the formation of host races or species, especially since so little is known about oviposition preferences of Papilio glaucus across North America. In Florida we have an apparently strict ecological monophagy coupled with a degree of reproductive isolation that has resulted in its tentative classification as a subspecies, P. g. australis. The role of foodplants in the speciation process can be significant even for P. glaucus, the most polyphagous of all 563 species of world Papilionidae. It remains uncertain whether the significant differences in detoxication and biochemical processing abilities observed in this study were evolved in parapatry or allopatry. Disjunct populations of P. alexiares in Mexico retain some ability to detoxify sweetbay, as do some individuals in most P. glaucus glaucus populations tested from ten different states north of Florida.     

Neurons innervating the lamina in the butterfly, Papilio xuthus

The butterfly Papilio xuthus has compound eyes with three types of ommatidia. Each type houses nine spectrally heterogeneous photoreceptors (R1–R9) that are divided into six spectral classes: ultraviolet, violet, blue, green, red, and broad-band. Analysis of color discrimination has shown that P. xuthus uses the ultraviolet, blue, green, and red receptors for foraging. The ultraviolet and blue receptors are long visual fibers terminating in the medulla, whereas the green and red receptors are short visual fibers terminating in the lamina. This suggests that processing of wavelength information begins in the lamina in P. xuthus, unlike in flies. To establish the anatomical basis of color discrimination mechanisms, we examined neurons innervating the lamina by injecting Neurobiotin into this neuropil. We found that in addition to photoreceptors and lamina monopolar cells, three distinct groups of cells project fibers into the lamina. Their cell bodies are located (1) at the anterior rim of the medulla, (2) between the proximal surface of the medulla and lobula plate, and (3) in the medulla cell body rind. Neurobiotin injection also labeled distinct terminals in medulla layers 1, 2, 3, 4 and 5. Terminals in layer 4 belong to the long visual fibers (R1, 2 and 9), while arbors in layers 1, 2 and 3 probably correspond to terminals of three subtypes of lamina monopolar cells, respectively. Immunocytochemistry coupled with Neurobiotin injection revealed their transmitter candidates; neurons in (1) and a subset of neurons in (2) are immunoreactive to anti-serotonin and anti-γ-aminobutyric acid, respectively.     

Polarization-based brightness discrimination in the foraging butterfly, Papilio xuthus

The human eye is insensitive to the angular direction of the light e-vector, but several animal species have the ability to discriminate differently polarized lights. How the polarization is detected is often unclear, however. Egg-laying Papilio butterflies have been shown to see false colours when presented with differently polarized lights. Here we asked whether this also holds in foraging butterflies. After training individuals to feed on nectar in front of an unpolarized spectral light, we carried out three dual-choice tests, where the discrimination of (i) the spectral content, (ii) the light intensity, and (iii) the e-vector orientation were investigated. In the first test, the butterflies selected the trained spectrum irrespective of its intensity, and in the second test they chose the light with the higher intensity. The result of the e-vector discrimination test was very similar to that of the second test, suggesting that foraging butterflies discriminate differently polarized lights as differing in brightness rather than as differing in colour. Papilio butterflies are clearly able to use at least two modes of polarization vision depending on the behavioural context.     

Spectral organization of the eye of a butterfly,Papilio

Journal of Comparative Physiology A - This review outlines our recent studies on the spectral organization of butterfly compound eyes, with emphasis on the Japanese yellow swallowtail butterfly,...     

Repeated Reticulate Evolution in North American Papilio machaon Group Swallowtail Butterflies

Hybridization between distinct populations or species is increasingly recognized as an important process for generating biodiversity. However, the interaction between hybridization and speciation is complex, and the diverse evolutionary outcomes of hybridization are difficult to differentiate. Here we characterize potential hybridization in a species group of swallowtail butterflies using microsatellites, DNA sequences, and morphology, and assess whether adaptive introgression or homoploid hybrid speciation was the primary process leading to each putative hybrid lineage. Four geographically separated hybrid populations were identified in the Papilio machaon species group. One distinct mitochondrial DNA clade from P. machaon was fixed in three hybrid taxa (P. brevicauda, P. joanae, and P. m. kahli), while one hybrid swarm (P. zelicaon x machaon) exhibited this hybrid mtDNA clade as well as widespread parental mtDNA haplotypes from both parental species. Microsatellite markers and morphology showed variable admixture and intermediacy, ranging from signatures of prolonged differential introgression from the paternal species (P. polyxenes/P. zelicaon) to current gene flow with both parental species. Divergences of the hybrid lineages dated to early- to mid-Pleistocene, suggesting that repeated glaciations and subsequent range shifts of parental species, particularly P. machaon hudsonianus, facilitated initial hybridization. Although each lineage is distinct, P. joanae is the only taxon with sufficient evidence (ecological separation from parental species) to define it as a homoploid hybrid species. The repetition of hybridization in this group provides a valuable foundation for future research on hybridization, and these results emphasize the potential for hybridization to drive speciation in diverse ways.     

Factors affecting equal catchability in two swallowtail butterflies, Papilio polyxenes and P.glaucus

Abstract. 1. Recapture probabilities were analysed for individually marked black swallowtails, Papilio polyxenes F., and tiger swallowtails, P.glaucus L. (Lepidoptera: Papilionidae).
2. Recapture rates differed with sex and behaviour at time of capture for P.polyxenes , but not with age. For P.glaucus , only differences related to behaviour at time of capture were significant.
3. Black swallowtail males that were not physically restrained in identification had a recapture probability (73%) that was significantly higher than the 45% for males that were captured and handled. The higher rate was also exhibited by released, laboratory-reared males.
4. The capture effect was due to increased dispersal out of the areas and not to avoidance of capture or the capture site.     

Mitochondrial DNA Sequence Variation of the Swallowtail Butterfly, Papilio xuthus, and the Cabbage Butterfly, Pieris rapae

We analyzed a portion of mitochondrial COI gene sequences (658 bp) to investigate the genetic diversity and geographic variation of the swallowtail butterfly, Papilio xuthus L. (Lepidoptera: Papilionidae), and the cabbage butterfly, Pieris rapae L. (Lepidoptera: Pieridae). Papilio xuthus showed a moderate level of sequence divergence (0.91% at maximum) in 15 haplotypes, whereas Pi. rapae showed a moderate to high level of sequence divergence (1.67% at maximum) in 30 haplotypes, compared with other relevant studies. Analyses of population genetic structure showed that most populations are not genetically differentiated in both species. The distribution pattern of both species appears to be consistent with category IV of the phylogeographic pattern sensu Avise: a phylogenetic continuity, an absence of regional isolation of mtDNA clones, and extensive distribution of close clones. The observed pattern of genetic diversity and geographic variation of the two butterfly species seem to reflect the abundant habitats, abundant host plants, and flying abilities in connection with the lack of historical biogeographic barriers.     

Elements of butterfly wing patterns

The color patterns on the wings of butterflies are unique among animal color patterns in that the elements that make up the overall pattern are individuated. Unlike the spots and stripes of vertebrate color patterns, the elements of butterfly wing patterns have identities that can be traced from species to species, and typically across genera and families. Because of this identity it is possible to recognize homologies among pattern elements and to study their evolution and diversification. Individuated pattern elements evolved from non-individuated precursors by compartmentalization of the wing into areas that became developmentally autonomous with respect to color pattern formation. Developmental compartmentalization led to the evolution of serially repeated elements and the emergence of serial homology. In these compartments, serial homologues were able to acquire site-specific developmental regulation and this, in turn, allowed them to diverge morphologically. Compartmentalization of the wing also reduced the developmental correlation among pattern elements. The release from this developmental constraint, we believe, enabled the great evolutionary radiation of butterfly wing patterns. During pattern evolution, the same set of individual pattern elements is arranged in novel ways to produce species-specific patterns, including such adaptations as mimicry and camouflage.     

Purification and Characterization of Biliverdin-binding Protein from Larval Hemolymph of the Swallowtail Butterfly,Papilio xuthus L.

The biliverdin-binding protein from the larval hemolymph of the swallowtail butterfly, Papilio xuthus L., was purified and characterized. The crude biliverdin-binding protein, obtained by ammonium sulfate fractionation, was purified in two steps, the first one by gel filtration chromatography and the second one by ion-exchange chromatography. The molecular mass of the purified protein was analyzed by SDS-polyacrylamide gel electrophoresis and estimated to be 21 kDa. The N-amino terminal sequence of P. xuthus biliverdin-binding protein analyzed up to the 19th residue showed that 42% of the amino acid sequence are sequence similarity to the bilin-binding protein from Pieris brassicae. These results suggest that the P. xuthus biliverdin-binding protein belongs to the insecticyanin-type.