Evidence of a sex-linked diapause response in Papilio glaucus subspecies and their hybrids

ABSTRACT. Pupal diapause in the eastern tiger swallowtail. Papilio glaucus L., is obligatory or facultative depending on subspecies. The southern subspecies, P. g. glaucus has a facultative diapause, induced by a larval exposure to short photoperiods. In the northern subspecies. P.g. canadensis , diapause is obligatory, occurring in each generation. F*** hybrids inherit the diapause response of their father or respond differently from either parent. The incidence of diapause in hybrids was associated with sex, indicating that the diapause response may be determined by an X-linked gene.     

Poly modal emergence of the tiger swallowtail, Papilio glaucus (Lepidoptera: Papilionidae): source of a false second generation in central New York State

Abstract. 1. The tiger swallowtail butterfly, Papilio glaucus (L.) (Lepidoptera: Papilionidae), is commonly assumed to have a bivoltine life cycle in central New York State and other parts of the northeastern United States. The seasonal abundance of adults in Tompkins County, New York, shows a bimodal pattern in some years, and a skewed unimodal pattern in others. We studied the incidence of diapause, the potential for successful development of two generations in the field, and the dates of adult emergence from overwintering pupae to determine whether these seasonal patterns are the result of bivoltinism.
2. Insects from Tompkins County, reared as larvae under 16L:8D photoperiod, uniformly enter pupal diapause.
3. Comparison of the heat units required for successful development of two generations with the heat units available in the field during 11 years shows that emergence of non-diapausing individuals would occur too late in the summer to contribute to the second peak of butterfly abundance. Offspring of non-diapausing individuals could only rarely complete development before the end of the growth season.
4. Diapausing offspring of butterflies collected in early June and late July differed sufficiently in dates of emergence to account for the field pattern of seasonal abundance.
5. Electrophoretic studies revealed no evidence of genetic isolation between early and late emerging butterflies.
6. These results suggest that polymodal emergence of diapausing insects can masquerade as a bivoltine life cycle.     

Tarsal contact chemoreceptors of the black swallowtail butterfly Papilio polyxenes: responses to phytochemicals from host- and non-host plants

Abstract Tarsal contact chemoreceptors of the black swallowtail butterfly Papilio polyxenes (Lepidoptera: Papilionidae) were stimulated with leaf-surface extracts and ethanolic extracts of whole leaves of a host-plant (Daucus carota) and a non-host (Brassica oleracea). Both leaf extracts evoked large numbers of spikes but stimulated different receptor neurones. It is concluded that a large difference exists between the sensory responses to host and non-host extracts. Two chemicals, luteolin 7-0-(6"-0-malonyl)-β-D-glucopyranoside and trans- chlorogenic acid, known to be present in the host and known oviposi-tion stimulants for P.polyxenes , were also tested and shown to be active. No responses were found to luteolin 7-O-β-D-glucoside or to luteolin 7-O-β-D-glucuronide. These flavonoids occur in D.carota foliage, but do not stimulate oviposition.     

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.

     

Effects of plant odor on oviposition by the black swallowtail butterfly,Papilio polyxenes (Lepidoptera: Papilionidae)

Black swallowtail females laid more eggs on plant models treated with contact stimulants and volatiles from carrot leaves than on models treated only with contact stimulants. The volatiles enhanced landing rates and females alighted more frequently on artificial leaves treated with host volatiles than on adjacent control leaves. Volatiles from cabbage, a nonhost, inhibited landing rates on artificial leaves treated with carrot contact stimulants. Examination of antennae revealed two major types of sensilla, believed to be olfactory in function. Electroantennogram preparations responded more strongly to carrot volatiles than to cabbage volatiles and several shared responses at particular retention times to carrot volatile components eluting from a gas chromatograph. Our results are consistent with a long-standing hypothesis that behavioral responses to essential oil components characteristic of the larval food plants have facilitated host shifts in the genus Papilio.     

Natural pupation sites of swallowtail butterflies (Lepidoptera: Papilioninae): Papilio polyxenes Fabr., P.glaucus L. and Battus philenor (L.)

Abstract. 1. Natural pupation sites have been found in Papilio polyxenes and P.glaucus by releasing prepupal larvae marked with UV-fluorescent paint and locating them at night with a UV lamp, and in Battus philenor by searching a forest habitat where the larval foodplant is abundant.
2. P.polyxenes , a species of weedy habitats, pupates off the ground on a variety of substrates including grasses, weed stalks, posts, etc. The pupae may be green or brown, resembling the substrate.
3. P.glaucus , a species of forest habitats, pupates very close to the ground in the litter and has monomorphic brown pupae.
4. B.philenor , also a forest species, pupates on exposed surfaces (chiefly tree-trunks or cliffs) well off the ground. Its pupae may be brown or green, but the latter were found only on the slenderest twigs.
5. The results for polyxenes and glaucus support the generalization of Clarke & Sheppard (1972) that species of stable habitats are likely to have monomorphic pupae, while those of habitats in which available sites may not be so similar from one generation to the next will be dimorphic.
6. B.philenor is more problematical, but its tendency towards pupal monomorphism (brown) is logical in relation to its common pupation sites.     

Food utilization and antioxidant enzyme activities of black swallowtail in response to plant phototoxins

Phototoxic compounds are widely distributed among plant families; due to their ability to bind covalently to DNA or to react with oxygen and generate toxic oxyradicals, these compounds are toxic to a variety of herbivorous organisms. Black swallowtail (Papilio polyxenes) larvae feed exclusively on phototoxic host plants in the Apiaceae and Rutaceae. In this study, we examined the toxicity of four phototoxins—three furanocoumarins and one β-carboline alkaloid—to P. polyxenes, as well as the inducibility of antioxidant enzyme defenses in response to these phototoxins. Neither the furanocoumarins nor the β-carboline alkaloid demonstrated any toxic effect on digestive efficiencies of P. polyxenes in the presence of light; harmine, the alkaloid, did significantly reduce growth and consumption rates. None of the compounds had a significant effect on antioxidant enzyme levels. These findings contrast with those reported in earlier studies for Trichoplusia ni, a generalist noctuid sensitive to both furanocoumarins and β-carboline alkaloids. Greater detoxicative metabolic capabilities, coupled with substantially higher constitutive levels of antioxidant enzyme activity, likely explain at least in part the absence of induced antioxidant enzyme responses in the specialist feeder on phototoxic plants. © 1993 Wiley-Liss, Inc.     

Characterization of 17 polymorphic microsatellite loci in the Anise swallowtail,Papilio zelicaon (Lepidoptera: Papilionidae), and their amplification in related species

Fifteen polymorphic dinucleotide and two trinucleotide microsatellite loci were identified in the Anise swallowtail, Papilio zelicaon, from DNA genomic libraries enriched for simple sequence repeats. Allele numbers varied from eight to 29, with an excess of homozygotes observed for nine loci. This homozygosity is a feature of other lepidopteran microsatellites and is probably due to null alleles. Sixteen markers were amplified successfully in other representatives of Papilio with 11 loci retaining polymorphism in at least one species. These results suggest that the microsatellites reported here may be appropriate for measuring population genetic structure in a number of Papilio species.     

Hybrid dynamics in a species group of swallowtail butterflies

Hybrid zones provide unique natural laboratories for studying mechanisms of evolution. But identification and classification of hybrid individuals (F1, F2, backcross, etc.) can be complicated by real population changes over time as well as by use of different marker types, both of which challenge documentation of hybrid dynamics. Here, we use multiple genetic markers (mitochondrial DNA, microsatellites and genomewide single nucleotide polymorphisms) to re‐examine population structure in a hybrid zone between two species of swallowtail butterflies in western Canada, Papilio machaon and P. zelicaon. Our aim was to test whether their hybrid dynamics remain the same as found 30 years ago using morphology and allozymes, and we compared different genetic data sets as well as alternative hybrid identification and classification methods. Overall, we found high differentiation between the two parental species, corroborating previous research from the 1980s. We identified fewer hybrid individuals in the main zone of hybridization in recent years, but this finding depended on the genetic markers considered. Comparison of methods with simulated data sets generated from our data showed that single nucleotide polymorphisms were more powerful than microsatellites for both hybrid identification and classification. Moreover, substantial variation among comparisons underlined the value of multiple markers and methods for documenting evolutionarily dynamic systems.     

Microsatellite markers for the hybridizing tiger swallowtails, Papilio glaucus and P. canadensis, and their applicability to historic specimens and congeneric species

We report the development of microsatellite markers for the sister species Papilio glaucus and P. canadensis (Papilionidae: Lepidoptera). All 16 markers displayed a high degree of variation in both species, ranging from eight to 24 alleles per locus. Substantial heterozygote deficits were observed for several loci, indicating the presence of null alleles. All markers were successfully used to genotype dried samples from a historical collection. Cross-species amplification with six additional Papilio species showed that most loci can be used to study genetic variation in other closely related species of tiger swallowtails.     

Independent evolution of sexual dimorphism and female‐limited mimicry in swallowtail butterflies (Papilio dardanus and Papilio phorcas)

Several species of swallowtail butterflies (genus Papilio) are Batesian mimics that express multiple mimetic female forms, while the males are monomorphic and nonmimetic. The evolution of such sex‐limited mimicry may involve sexual dimorphism arising first and mimicry subsequently. Such a stepwise scenario through a nonmimetic, sexually dimorphic stage has been proposed for two closely related sexually dimorphic species: Papilio phorcas, a nonmimetic species with two female forms, and Papilio dardanus, a female‐limited polymorphic mimetic species. Their close relationship indicates that female‐limited polymorphism could be a shared derived character of the two species. Here, we present a phylogenomic analysis of the dardanus group using 3964 nuclear loci and whole mitochondrial genomes, showing that they are not sister species and thus that the sexually dimorphic state has arisen independently in the two species. Nonhomology of the female polymorphism in both species is supported by population genetic analysis of engrailed, the presumed mimicry switch locus in P. dardanus. McDonald–Kreitman tests performed on SNPs in engrailed showed the signature of balancing selection in a polymorphic population of P. dardanus, but not in monomorphic populations, nor in the nonmimetic P. phorcas. Hence, the wing polymorphism does not balance polymorphisms in engrailed in P. phorcas. Equally, unlike in P. dardanus, none of the SNPs in P. phorcas engrailed were associated with either female morph. We conclude that sexual dimorphism due to female polymorphism evolved independently in both species from monomorphic, nonmimetic states. While sexual selection may drive male–female dimorphism in nonmimetic species, in mimetic Papilios, natural selection for protection from predators in females is an alternative route to sexual dimorphism.     

Immunochemical analysis of cytochrome P450 monooxygenase diversity in the black swallowtail caterpillar,Papilio polyxenes

Antisera to nineteen cytochrome P450 monooxygenases were assayed for cross-reactivity with microsomal and mitochondrial proteins of the black swallowtail caterpillar, Papilio polyxenes (Lepidoptera:Papilionidae), on Western blots. Antisera to P450s from a bacterium, an insect and three mammals showed selective cross-reactivity with seven bands in the P450 molecular weight range of 45–60 kDa. Of the five putative P450s identified in midgut microsomes, only two were induced in larvae reared on a diet supplemented with high levels of xanthotoxin, a host plant constituent metabolized by P450 in the black swallowtail. The results confirm that insect P450s share conserved epitopes with P450s from other taxa and provide further evidence of the selective induction of P450 isozymes by xanthotoxin in black swallowtail larvae.     

Divergence in larval responses to food plants between temperate and tropical populations of the black swallowtail butterfly

ABSTRACT.

• 1 Larvae of the black swallowtail butterfly, Papilio polyxenes Fabr., from New York and Costa Rica were reared under the same environmental conditions on Daucus carota L. and Spananthe paniculata Jacq. (Umbelliferae), which are the host plants most commonly used by the two populations, respectively. Feeding rates, growth rates and feeding efficiencies of the fifth instar larvae were measured by standard techniques.
• 2 Larvae from each population grew faster on the host-plant species from their region of origin than did larvae from the other population, though differences on S.paniculata were not significant. On D.carota the faster growth by New York larvae was attributable to greater efficiency. On S.paniculata, by contrast, the somewhat faster growth by Costa Rican larvae seemed to have resulted chiefly from faster feeding. The two populations have thus come to differ in metabolic and, probably, behavioural responses to host plants.
• 3 S.paniculata was a superior host plant for larvae of both populations, even though the total energy, nitrogen and water content of its foliage was similar to that of D.carota.
• 4 These results are consistent with the hypothesis that insects in the two populations have diverged in adaptation to their host plants following curtailment of gene flow between them as the species colonized Central America from the north.

     

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.     

Tyramine from the leaves of wild parsnip: a stimulant and synergist for oviposition by the black swallowtail butterfly

Abstract .Oviposition stimulants from the foliage of wild parsnip, Pastinaca sativa (Apiaceae), were isolated by column chromatography and HPLC and tested in bioassay experiments with hand-held female black swallowtail butterflies, Papilio polyxenes (Lepidoptera: Papilionidae). Two of the stimulants were identified as tyramine and trans -chlorogenic acid. A combination of tyramine, trans -chlorogenic acid and an active neutral fraction was needed to elicit a significant oviposition response. These results are discussed in the context of previous research on the oviposition stimulants of swallowtail butterflies and on the significance of tyramine as a neuromodulator of physiological processes in invertebrates.     

Larval developmental rates of three putative subspecies of tiger swallowtail butterflies,Papilio glaucus,and their hybrids in relation to temperature

Summary The eastern tiger swallowtail butterfly, Papilio glaucus L. has been presumed to be comprised of three parapatric subspecies (P. g. canadensis R & J, across Canada and the northern U.S.; P. g. glaucus L., in the eastern half of the U.S.; and P. g. australis Maynard, in Florida). Populations from 27° north latitude in Florida to 50° north latitude in Manitoba, Canada and from 75° west longitude in Pennsylvania to 96° west longitide in Canada were tested by laboratory larval feeding studies at various constant temperatures ranging from 16°–28° C. These data indicate that populations of Papilio glaucus show generally similar developmental responses to temperature, regardless of their geographic origin. Limited differences do exist between some populations studied, involving such traits as upper threshold temperature and differences between individual developmental rate curves. No consistent differences were noted between the three putative subspecies in their developmental response under these experimental conditions. In Wisconsin, the twenty year average of total season degree day accumulations above a base temperature (developmental threshold) of 10° C delineates a relatively narrow geographical region across the center of the state for which the bivoltine potential reaches its northernmost limits. This zone also closely corresponds to the southernmost limit of the P. g. canadensis subspecies (generally believed to be obligately univoltine).     

Carotenoids and bile pigments in Danaid and swallowtail butterflies

The carotenoids found in adult kite swallowtails ( Graphium spp.) and the Danaid glassy tigers ( Ideopsis, Tirumala and Parantica ), butterflies with exposed blue/green pigmented wing membranes (which contain pterobilins) and a modified scale vestiture, were compared with those of related species of Papilios and Danaids with concealed wing membranes an an entire scale vestiture. It was found that both the kite swallowtails contained only lutein, and even lacked β-carotene, the first known instance among butterflies. This was presumably due to selective storage by the larva since the food-plant was found to be rich in varied carotenoids. A possible link between the high concentration of pterobilins in the wing membrane and the lack of carotenoids is discussed.     

Furanocoumarin metabolism in Papilio polyxenes: biochemistry,genetic variability,and ecological significance

The ubiquitous occurrence of series of biosynthetically related plant secondary compounds within individual species has given rise to the suggestion that such multiplicity is adaptive; one possible mechanism that would serve to maintain such within-plant diversity is analog synergism. In a series of experiments, we provide evidence that synergism may account for the presence of multiple structurally related furanocoumarins in apiaceous plants. The black swallowtail, Papilio polyxenes, feeds exclusively on plant species containing furanocoumarins. Growth of larvae fed parsley leaves treated with both xanthotoxin and angelicin, two furanocoumarins that co-occur widely in swallowtail hostplants, was significantly slower than that of larvae fed leaves with an equimolar concentration of either xanthotoxin or angelicin. A multivariate combination of growth, food consumption and frass excretion differed significantly between larvae fed leaves treated with both xanthotoxin and angelicin and larvae fed leaves treated with angelicin alone. In addition, we measured rates of in vitro cytochrome P450-mediated metabolism of three furanocoumarins — bergapten, xanthotoxin, and angelicin. While bergapten and xanthotoxin, both linear furanocoumarins, were metabolized at similar rates (8.07 and 9.86 nmoles/min/g fw caterpillar, respectively), angelicin, an angular furanocoumarin, was metabolized more slowly (2.76 nmoles/min/g fw caterpillar). When all three furanocoumarins were assayed together, overall rates of metabolism were significantly reduced, suggesting substrate inhibition. Thus, the pattern of growth of larvae is consistent with the pattern of in vitro metabolism and is evidence in support of analog synergism. In a separate experiment, metabolism of xanthotoxin and angelicin individually and together were compared in six maternal families. Again, angelicin was metabolized more slowly than xanthotoxin and each furanocoumarin inhibited metabolism of the other. That significant family effects were found for rates of metabolism and for the ratio of moles of angelicin metabolized for each mole of xanthotoxin metabolized raises the possibility that genetic variation exists for the rate and specificity of metabolism and suggests that insect herbivores may be able to adapt to analog synergism.     

Segregation of F-2 interspecific hybrid growth performance and wing color patterns relative to parental species in the Papilio machaon species group （Lepidoptera： Papilionidae）

A strange Papilio female specimen with a prominent yellow wing band type very much like P. brevicauda （and also seen in P. machaon, and P. zelicaon） was captured in the forested mountains of southwest Vermont, USA. Despite its brevicauda-like appearance, it had mt-DNA of P. polyxenes. In eastern North America the P. brevicauda （Maritime Provinces of Canada） is currently believed to reflect previous （ancient） hybridization between ancestral P. machaon and P. polyxenes. It is uncertain whether the brevicauda-like wing trait features in the odd Vermont female may be a result of some introgression of P. machaon or P. brevicauda with P. polyxenes in eastern North America as seen also in the Ozark Mountains with P. joanae. Nonetheless, the significance of wing trait analyses in relation to hybrid zones with P. polyxenes remains relevant. The segregation of wing color patterns and hostplant use abilities （efficiencies and growth rates） were examined for laboratory hand-paired F-2 hybrids of P. zelicaon ~ Papilio polyxenes relative to the parental species and primary hybrids. The black adult color pattern characterizing P. polyxenes is presumed to be inherited as a simple autosomal dominant. These hybrids were examined to determine： （i） how F-1 primary hybrid offspring perform relative to the parental types in survival, growth rates, and growth efficiencies; and （ii） whether these larval traits and the adult color patterns might be linked with wing morphology in the F-2 hybrids. While the F-1 hybrids grew as well as the P. polyxenes parent （P. zelicaon was significantly less efficient and slower in growth than either of these）, we could not convincingly determine whether the growth performances of the F-2 segregants were linked with the parental wing patterns.