Papilio aegeus Donovan (Lepidoptera: Papilionidae) host plant range evaluated experimentally on ancient angiosperms

Abstract  Chemical similarities among ancient Angiosperms presumably played a role in the ecological and evolutionary diversification of the swallowtail butterflies (Papilionidae). The abilities of neonate larvae of the Citrus swallowtail, Papilio (= Princeps ) aegeus (from Queensland, Australia), to eat, survive and grow on leaves (a choice of young and old) of 34 plant species from families of ancient Angiosperms; 8 Rutaceae, 3 Magnoliaceae, 13 Lauraceae, 3 Monimiaceae, 1 Aristolochiaceae, 2 Apiaceae, 1 Sapotaceae, 1 Winteraceae and 2 Annonaceae were tested. It was apparent that there is genetic variation in populations of Rutaceae-specialised Australian P. aegeus for acceptance, consumption and larval growth, reflecting differential suitability of some native Australian Lauraceae species as food plants (as well as certain Winteraceae, Monimiaceae and non-Australian Magnoliaceae, Lauraceae and Annonaceae). No consumption or survival of P. aegeus was seen on Aristolochia elegans (Aristolochiaceae) or Pouteria australis (Sapotaceae) despite literature records alluding to this possibility. The Rutaceae specialist P. aegeus appears to have the fundamental detoxification capabilities for processing many existing species of the basal Angiosperm families, without having direct ancestors that historically had fed on them.     

Species‐specific coordinated gene expression and trans‐regulation of larval color pattern in three swallowtail butterflies

SUMMARY The diversity of butterfly larval color pattern has been attracted to people since Darwin's time; however, its molecular mechanisms still remain largely unknown. Larval body markings often differ completely between closely related species under natural selection. The final instar larvae of the swallowtail butterflies Papilio xuthus and Papilio polytes show a green camouflage pattern, whereas those of Papilio machaon show a warning color pattern, although P. xuthus and P. machaon are closely related species. To identify the genes that contribute to species divergence, we compared the expression pattern of eight pigment‐associated genes between three Papilio species. The spatial expression pattern of melanin‐related genes coincided with the species‐specific cuticular markings. We newly found that the combination of bilin‐binding protein and yellow‐related gene (YRG) correlated perfectly with larval blue, yellow, and green coloration. To distinguish whether the interspecific differences in pigment‐associated genes are caused by cis‐regulatory changes or distribution differences in trans‐regulatory proteins, we compared species‐specific mRNA expression in an F1 hybrid specimen. Px‐YRG and Pp‐YRG showed a similar expression pattern, suggesting that the change in expression of YRG is caused mainly by changes in the distribution of trans‐regulatory proteins. Our findings shed light on the gene regulatory networks for butterfly larval color pattern.     

Caterpillar color patterns are determined by a two‐phase melanin gene prepatterning process: new evidence from tan and laccase2

SUMMARY The larval color patterns in Lepidoptera exhibit splendid diversity, and identifying the genes responsible for pigment distribution is essential to understanding color‐pattern evolution. The swallowtail butterfly, Papilio xuthus, is a good candidate for analyzing marking‐associated genes because its body markings change dramatically at the final molt. Moreover, the silkworm Bombyx mori is most suitable for identification of lab‐generated color mutants because genome information and many color mutants are available. Here, we analyzed the expression pattern of 10 melanin‐related genes in P. xuthus, and analyzed whether these genes were responsible for Bombyx larval color mutants. We found that seven genes correlated strongly with the stage‐specific larval cuticular markings of P. xuthus, suggesting that, compared with Drosophila, more genes showed marking specificity in lepidopteran larvae. We newly found that the expression of both tan and laccase2 is strongly correlated with the larval black markings in both P. xuthus and B. mori. The results of F2 linkage analysis and mutant analysis strongly suggest that tan is the responsible gene for Bombyx larval color mutant rouge, and that tan is important in emphasizing black markings of lepidopteran larvae. Detailed comparison of temporal and spatial expression patterns showed that larval cuticular markings were regulated at two different phases. Marking‐specific expression of oxidizing enzymes preceded the marking‐specific expression of melanin synthesis enzymes at mRNA level, which is the reverse of the melanin synthesis step.     

Interactions between Papilionidae and ancient Australian Angiosperms: evolutionary specialization or ecological monophagy?

The evolution of host range for insect herbivores involves many behavioral, physiological, and biochemical adaptations that often lead to locally specialized populations or species. Such specialization may be constrained by ecological factors (e.g., local host availability) or by evolutionary factors (e.g., phylogenetic divergence in behavioral, physiological, or biochemical detoxification enzymes; and potential inabilities to return to ancestral hosts). While insect adaptations to new hosts can be rapid, ancient detoxification systems may persist in some lineages of swallowtail butterflies (Papilionidae) for millions of years. Here, we test various species of specialized species/populations of Papilionidae (Lepidoptera) from North America and from Australia on an array of Australian host plant families in order to determine whether the current feeding constraints reflect loss of capabilities to recognize and use hosts other than their current (local) favorites. We selected two species of Lauraceae specialists (Papilio troilus L. and Papilio palamedes Drury) from North America and one locally specialized population of Papilio glaucus L. that only uses one plant species in the Magnoliaceae in Florida. We also examined three species/populations of Australian swallowtails for comparison, including the Monimiaceae‐specialized Graphium macleayanum moggana L. E. Couchman, the Rutaceae‐specialized Papilio aegeus Donovan, and the Annonaceae‐specialized Graphium eurypylus L. Our aim was to determine whether neonate larvae of these six specialists could survive on any plants other than their currently favored species. While the Lauraceae specialists could use nothing else and were thus evolutionarily constrained, the Magnoliaceae‐, Rutaceae‐, and Monimiaceae specialists all had common abilities to accept, feed, and grow on plants in the Lauraceae, Monimiaceae, Magnoliaceae, and Rutaceae families. Even the Annonaceae specialist was discovered using Magnoliaceae in the field, suggesting existence here also of both flexiblity in preferences and detoxification abilities and ‘ecological monophagy’.     

The effect of host plant on larval survivorship of the Alaskan swallowtail butterfly (Papilio machaon aliaska)

The Alaskan swallowtail butterfly, Papilio machaon aliaska (Lepidoptera: Papilionidae), uses three plant species as host plants. Cnidium cnidiifolium (Turcz.) Schischk. belongs to the family Apiaceae, the ancestral host‐plant family of the P. machaon group. Artemisia arctica Less. and Petasites frigidus (L.) Franch, in contrast, belong to the distantly related Asteraceae family and were colonized relatively recently by this group of butterflies. Previous work has shown that larval survival is highest on the novel host plants when natural enemies are present in the field. Here I examine whether P. m. aliaska fitness varied when individuals were reared on the three host plants in a common environment, free of environmental and ecological complications such as predation. I collected 12 P. m. aliaska females and measured their reproductive success when their progeny were reared on each of the three host plants. I assessed larval fitness on each of the hosts by recording the percentage pupation and percentage emergence as well as by measuring pupal mass. I found that larvae reared on the ancestral host plant, C. cnidiifolium, had higher fitness than did larvae reared on either of the novel host plants, A. arctica or P. frigidus. Larvae reared on C. cnidiifolium were more likely to pupate, achieved a greater pupal mass, and had higher emergence rates than larvae reared on the novel hosts. I interpret these results to mean that, in the absence of predation, the ancestral host plant is a better host for P. m. aliaska larvae than either novel host and I contend that this result does not appear to support the hypothesis that P. m. aliaska populations in central Alaska are divided into host races.     

Population dynamics of the swallowtail butterfly,Papilio xuthus L., in a deforested area

The long-term population dynamics of the swallowtail butterfly, Papilio xuthus, were studied by means of life table analysis in a deforested area, where the host tree, Zanthoxylum ailanthoides, was growing. The number of eggs laid on host trees in the deforested area decreases as secondary succession progresses. When the trees were classified into three groups, i. e. tall, medium and short, according to their height relative to the surrounding vegetation, less eggs were laid on tall than on short host trees. Life tables for the natural populations in each generation were developed on the basis of mean values for 6 years. Eggs and larvae in early stages were attacked chiefly by small- and middle-sized predators, such as ants, spiders, bugs and orthopterids, while later stage larvae were attacked chiefly by birds or Polistes wasps. An inchneumonid wasp was the most important mortality factor in the pupal stage. Key-factor analysis was tested on the life table data of each immature stage. The late larval stage was a key stage, and most mortality factors were considered to operate independently. The test for density dependency did not show any tendencies. The analysis of variance in two-way classifications was carried out for the difference of survival rate among the tree groups. It was suggested that tall host trees were unsuitable for P. xuthus. The construction of life tables for artificially inoculated populations on large host trees showed that high physiological death rate were characteristic of such trees in comparison with natural populations on smaller trees. The characteristics of the population dynamics of P. xuthus in the deforested area are compared among some kinds of the habitat, and the interrelationship between P. xuthus and its host plant are discussed.     

Transplantation in relation to pigment formation of the integument in the larvae of the swallowtail, Papilio xuthus L

In the swallowtail, Papilio xuthus L., a switchover in the pigmentary system of epidermis occurs at the last larval molt: viz., from reddish brown xanthommatin to bluish green bile pigment. By transplantation of integument between the larvae of different developmental stages, a control by some humoral factor of the pigmentary system was revealed. But the transplantation of brain or of several combinations of known endocrine systems failed to affect the switchover of the pigmentary system at the last larval molt.     

Coincidental intraguild predation by caterpillars on spider mites

Intraguild predation (IGP) is defined as the killing and eating of prey species by a predator that also can utilize the resources of the prey. It is mainly reported among carnivores that share common herbivorous prey. However, a large chewing herbivore could prey upon sedentary and/or micro herbivores in addition to utilizing a host plant. To investigate such coincidental IGP, we observed the behavioral responses of the polyphagous mite Tetranychus kanzawai Kishida (Acari: Tetranychidae) when its host plant Cayratia japonica (Thunb.) Gagnep. (Vitaceae) was attacked by hornworms, Theretra japonica Boisduval (Sphingidae) and T. oldenlandiae Fabricius (Sphingidae). We also examined an interaction between the oligophagous mite Panonychus citri McGregor (Acari: Tetranychidae) and caterpillars of the swallowtail Papilio xuthus L. (Papilionidae) that share citrus plants as their main food source. Although all T. kanzawai and some active stage P. citri tried to escape from the coincidental IGP, some were consumed together with eggs, quiescent mites, and host plant leaves, suggesting that coincidental IGP occurs on spider mites in the wild. Moreover, neither hornworms nor swallowtail caterpillars distinguished between spider mite-infested and uninfested leaves, suggesting that the mite-infested leaves do not discourage caterpillar feeding. The reasons that the mites have no effective defense against coincidental IGP other than escaping are discussed.     

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.     

Effect of larval brain extracts derived from three swallowtail butterflies,Papilio helenus L., P. machaon L. and P. memnon L., on seasonal morph development of P. xuthus L. (Lepidoptera: Papilionidae)

Adults of the three papilionid butterflies, Papilio helenus L., Papilio machaon L. and Papilio memnon L., exhibit seasonal diphenism comprising spring and summer morphs. To elucidate the physiological mechanism underlying seasonal morph development in papilionid butterflies, we investigated whether a cerebral factor showing summer‐morph‐producing hormone (SMPH) activity is present in the brain of three Papilio species using an assay system with chilled male short‐day pupae of P. xuthus L. When 2% NaCl extracts derived from 20 larval brains of the three species were injected into abdomens of chilled male short‐day pupae of P. xuthus, all recipients destined to develop into spring‐morph adults developed into summer‐ and intermediate‐morph adults. On the other hand, all recipients injected with distilled water as a control developed into spring‐morph adults. These results indicate that a cerebral factor showing SMPH activity is present in the larval brain of the three Papilio species. Additionally, all recipients injected with 2% NaCl extracts derived from 20 adult brains of Bombyx mori L. also developed into summer‐ and intermediate‐morph adults. The results revealed that SMPH or a cerebral factor showing SMPH activity is widely distributed among lepidopteran insects.     

Melanin-synthesis enzymes coregulate stage-specific larval cuticular markings in the swallowtail butterfly, <Emphasis Type="Italic">Papilio xuthus</Emphasis>

Like the adult wing, butterfly larvae are unique in their coloring. However, the molecular mechanisms underlying the formation of insect larval color patterns are largely unknown. The larva of the swallowtail butterfly Papilio xuthus changes its color pattern markedly during the 4th ecdysis. We investigated its cuticular color pattern, which is thought to be composed of melanin and related pigments derived from tyrosine. We cloned three enzymes involved in the melanin-synthesis pathway in P. xuthus: tyrosine hydroxylase (TH), dopa decarboxylase (DDC), and ebony. Whole-mount in situ hybridization showed that the expression of both TH and DDC is strongly correlated with the black markings. ebony is strongly expressed only in the reddish-brown area. The expression pattern of each enzyme coincides with the cuticular color pattern of the subsequent instar. We also investigated the uptake of melanin precursors into cultured integument. Inhibition of either TH or DDC activity prevents in vitro pigmentation completely. Addition of dopamine to integuments in the presence of TH inhibitor causes overall darkening without specific markings. From these results, specific larval cuticular color patterns are regulated by stage-specific colocalization of enzymes in epidermal cells rather than by the differential uptake of melanin precursors into individual epidermal cells. Epidermal cells expressing TH and DDC, but not ebony, produce the black cuticle, and epidermal cells expressing TH, DDC, and ebony produce the reddish-brown cuticle.     

Soldier bug predation on swallowtail caterpillars (Lepidoptera: Papilionidae): Circumvention of defensive chemistry

All larvae in the family Papilionidae possess osmeteria, eversible glands that release defensive chemicals upon disturbance. These chemicals have been shown to be repellent to ants, mantids, and other predaceous arthropods with chewing mouthparts. In this study, we demonstrate that the pentatomid Podisus maculiventris,an important predator of the black swallowtail Papilio polyxenes,is capable of piercing the body wall of the caterpillar and consuming body fluids without eliciting osmeterial eversion. Tenebrio molitorlarvae coated with osmeterial secretions of Papilio polyxeneswere universally rejected by Podisus maculiventris,suggesting that the predator is in fact repelled by the swallowtail defensive chemistry. By circumventing the defensive behavior of the caterpillar, the pentatomid is able to utilize an otherwise chemically unsuitable prey species.     

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.     

Regulatory mechanisms in phenotypic plasticity of diapause and nondiapause pupal colouration of the swallowtail butterfly Papilio machaon

Nondiapause pupae of Papilio machaon L. exhibit pupal colour diphenism comprising green–yellow and brown–white types. To understand the regulatory mechanism underlying the control of pupal colouration in P. machaon, the effect of environmental cues on diapause and nondiapause pupal colouration is investigated. When larvae reared under short‐day and long‐day conditions are allowed to pupate in sites with a smooth surface and a yellow background colour, all diapause pupae exhibit a brown–white type and 89.5% of nondiapause pupae exhibit a green–yellow type, respectively. With rough‐surface pupation sites, all diapause pupae exhibit brown–white and intermediate types, whereas a large proportion of nondiapause pupae exhibit brown–white and intermediate types, although some exhibit a green–yellow type. When extracts prepared from the head‐thoracic and thoracic‐abdominal regions of larval central nervous systems are injected into the ligated abdomens of P. machaon short‐day pharate pupae, all recipients exhibit a brown–white colouration. Furthermore, when each extract is injected into the ligated abdomen of Papilio xuthus L. short‐day pharate pupae with orange‐pupa‐inducing factor activity, recipients injected with the head‐thoracic extract exhibit the brown type, whereas those injected with the thoracic‐abdominal extract exhibit an orange colour. The results indicate that the response to the environmental cues of pupation site in P. machaon changes according to the photoperiodic conditions experienced during larval stages, and that at least two hormonal factors producing brown–white pupae are located in the larval central nervous system, with the secretion of these factors being regulated by the recognition of environmental cues in long‐day larvae.     

Morphology,life cycle and management of two invasive subspecies of Papilio demoleus (Lepidoptera: Papilionidae): A review

Papilio demoleus L., also known as the citrus butterfly or chequered swallowtail, is a common species in the Asia-Pacific region, with two of its subspecies (P. d. demoleus and P. d. malayanus) posing significant threats to the citrus industry in Asia and other regions of the world. Its predominant natural host plants are the Rutaceae for the invasive northern subspecies and Fabaceae for the southern subspecies. The morphology and development of this species are reasonably well studied, although further information is required on its natural mortality rates and environmental tolerance in order to accurately predict its future potential spread throughout the Middle East and Central Americas. Synthetic pyrethroids such as deltamethrin are highly effective for the control of P. demoleus larvae. However, better control is likely to be found using an integrated approach to pest management, incorporating techniques such as biocontrol agents, microbial pesticides and phytopesticides, in conjunction with synthetic pesticides where required.     

Host plant exodus and larval wandering behaviour in a butterfly: diapause generation larvae wander for longer periods than do non‐diapause generation larvae

1. Prior to pupation, lepidopteran larvae enter a wandering phase lasting up to 30 h before choosing a pupation site. Because stillness is important for concealment, this behaviour calls for an adaptive explanation. 2. The explanation most likely relates to the need to find a suitable pupation substrate, especially in terms of shelter from predation, and given that many predators and parasitoids use host plants as prey‐location cues, mortality probably decreases with distance from the host plant. Hence, remaining on the host includes a long‐term risk, while moving away from the host introduces an increased risk during locomotion. 3. Bivoltine species that overwinter in the pupal stage produce two kinds of pupae; non‐diapausing pupae from which adults emerge after 1–2 weeks, or diapausing pupae that overwinter with adults emerging after 8–10 months. 4. Given the hypothesis of distance‐from‐host‐plant‐related predation, this should select for phenotypic plasticity with larvae in the diapausing generation having a longer wandering phase than larvae under direct development, if there is a trade‐off between mortality during the wandering phase and accumulated mortality during winter. 5. Here this prediction is tested by studying the duration of the wandering period in larvae of the partially bivoltine swallowtail butterfly, Papilio machaon, under both developmental pathways. 6. The results are in agreement with the predictions and show that the larval wandering phase is approximately twice as long under diapause development. The authors suggest that the longer duration of the wandering phase in the diapause generation is a general phenomenon in Lepidoptera.     

Significance of a new field oviposition record for Graphium eurypylus (L.) (Lepidoptera: Papilionidae) on Michelia champaca (Magnoliaceae)

Abstract  Phytochemical similarities among ancient Angiosperms presumably played a role in the ecological and evolutionary diversification of the swallowtail butterflies (Papilionidae). Host family feeding specialisation is typical of most Papilionidae species, but field records of oviposition are rare for most swallowtail butterflies. It is even more uncommon to witness oviposition and larval feeding on new host plant species, especially in plant families not previously reported for the butterfly species. Oviposition by a female on a new host, or even on a toxic plant, may represent ancestral behaviour (with a loss of larval acceptance, detoxification or processing abilities) or novel behaviour (providing genetic variation for a potential expansion of host range, or host shift). We document the oviposition, larval use and pupation of the Annonaceae specialised and geographically widespread Graphium eurypylus on a Magnoliaceae species, all under field conditions in Queensland, Australia. This is the first time such field observations of oviposition and larval feeding on Michelia champaca (Magnoliaceae) have been documented anywhere for this species.     

Does group feeding by toxic prey confer a defensive benefit? Aristolochic acid content,group size and survival of first‐instar pipevine swallowtail (Battus philenor L.) larvae

1. Aggregative feeding of larvae is widespread in the Lepidoptera, and many hypotheses have been proposed to explain the adaptive significance of this trait. Group feeding occurs disproportionately more in species with aposematic, chemically defended larvae compared with species with cryptic, non‐chemically defended larvae, consistent with the hypothesis that group feeding provides an enhanced aposematic signal to natural enemies. Most species characterised as having chemically defended larvae are cryptic during the first instar, when they are most highly aggregated and most vulnerable to predation. 2. The benefits of group feeding in terms of decreased predation were explored for first‐instar larvae of the pipevine swallowtail, Battus philenor, a species that sequesters aristolochic acids from its Aristolochia host plant and exhibits aposematism in later instars and as adults. We found that groups of larvae with experimentally enhanced aristolochic acid content had significantly lower survivorship due to predation both in the field and in laboratory experiments compared with groups of larvae without enhanced chemical defence. 3. A laboratory experiment found that aristolochic acid does not deter the generalist predator Hippodamia convergens. 4. No evidence was found that was consistent with the hypothesis that group feeding and increased sequestered chemical defence interact to decrease rates of larval mortality in non‐aposematic, first‐instar larvae. Future research on chemical defence, aposematism, and aggregative feeding should continue to appreciate that particular chemical defences and feeding behaviours are not universally effective against all natural enemies.     

Interspecific differences among swallowtail larvae (Lepidoptera: Papilionidae) in susceptibility to aristolochic acids and berberine

Abstract. 1. The larvae of four swallowtail butterfly species, Papilio polyxenes Fabr. in the Papilionini, Parides bunichus (Hübner) and Battus polydamas (L.) in the Troidini, and Eurytides marcellus (Cramer) in the Graphiini, were grown on host leaves to which test compounds had been added.
2. The test chemicals are biosynthetically related. Berberine, a 1-benzyl-tetrahydroisoquinoline-related (1-BTIQ) alkaloid, is found in many rutaceous hosts of the Papilionini; laudanosine, a simpler 1-BTIQ alkaloid, is found in the Papaveraceae, plants on which no papilionids feed; aristolochic acids are found only in the Aristolochiaceae, hosts of the Troidini.
3. Swallowtail larvae can tolerate the compounds characteristic of their host plants, but not chemicals found in the hosts of other papilionid groups. Diets with laudanosine did not affect the test species.
4. Our results support the contention that plant secondary chemicals, such as berberine and aristolochic acids, are important feeding barriers, and are partially responsible for host specificity in swallowtails.     

Enemy‐free space and the host range of a lichenivorous moth: a field experiment

According to the enemy‐free space hypothesis (EFS), parasites and predators create a selective force for a specialization on a host that assures better protection against natural enemies than other potential hosts. Few studies have found support for EFS and none of them have covered the whole larval period in natural conditions. I studied the growth and survival of lichen‐feeding moth larvae on five lichen species with and without their natural enemies in natural conditions covering the whole larval period. All the three following EFS predictions gained support. First, natural enemies caused significant mortality of larvae. Second, when natural enemies were present, larval survival was highest on preferred Ramalina lichens. Third, larvae attained higher mass on non‐preferred Parmelia sulcata than on Ramalina species, indicating fecundity cost to feed on Ramalina species instead of P. sulcata. EFS for C. lichenaria larvae on Ramalina species is likely a consequence of shrubby appearance of Ramalina species which provide better larval protection from predation than other hosts.