Hormonal control of seasonal morph determination in the swallowtail butterfly, Papilio xuthus L. (Lepidoptera: Papilionidae)

Seasonal morphs (spring and sumer forms) of Papilio xuthus L. are determined coincidentally with diapause and non-diapause in pupae by larval exposure to short days and long days respectively. The neuroendocrine principle underlying seasonal-morph determination was studied using surgical operations in P. xuthus.When recipient 0-day old or chilled diapause pupae were joined to donor 0-day old non-diapause pupae, the recipients developed into summer or intermediate morphs. When the same kinds of recipients used above were joined to 0-day old or chilled diapause pupae, there were no significant effects on the adult morph. In contrast, recipient non-diapause pupae all developed into summer morphs, regardless of groups of the type of donors.Furthermore, when the brain of 5th-instar larvae, pharate pupae or pupae, predetermined to be diapause, was transplanted into the abdomen of 0-day old, 30-day old or chilled diapause pupae, the recipients developed into summer or intermediate morphs.The results indicate that the brain of non-diapause pupae secretes a humoral factor producing the summer morph. In non-diapause pupae, the factor may be secreted at about the stage of larval-pupal ecdysis coincidentally with that of prothoracicotropic hormone.     

Presence of a cerebral factor showing summer-morph-producing hormone activity in the brain of the seasonal non-polyphenic butterflies Vanessa cardui, V. indica and Nymphalis xanthomelas japonica (Lepidoptera: Nymphalidae)

Three species of nymphalid butterflies, Vanessa cardui, V. indica and Nymphalis xanthomelas japonica , do not exhibit seasonal polyphenism in wing coloration. To determine whether seasonal non-polyphenic butterflies possess a cerebral factor affecting wing coloration, we used a Polygonia c-aureum female short-day pupal assay for detection of summer-morph-producing hormone (SMPH) activity in P. c-aureum. When 2% NaCl extracts of 25 brain-equivalents prepared from the pupal brains of V. cardui, V. indica or N. xanthomelas japonica were injected into Polygonia female short-day pupae, all recipients developed into summer-morph adults with dark-yellow wings, and the average grade score (AGS) of summer morphs showing SMPH activity was 3.8, 3.7 and 4.0, respectively. In contrast, when acetone or 80% ethanol extracts prepared from pupal brains were injected into Polygonia pupae, all recipients developed into autumn-morph adults with a dark-brown coloration and each exhibited an AGS of less than 0.5. Our results indicate that a cerebral factor showing SMPH activity is present in the pupal brain of seasonal non-polyphenic nymphalid butterflies, suggesting that a SMPH and cerebral factor showing SMPH activity occur widely among butterfly species. This finding will improve our understanding of the presence of cerebral factors showing interspecific actions of SHPH.     

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.     

Aristolochiaceae‐ and Asteraceae‐feeding by larvae of Papilio xuthus L. (Lepidoptera: Papilionidae) in Japan: A review

Our recent observations of Aristolochiaceae‐ and Asteraceae‐feeding by larvae of Papilio xuthus L. (Lepidoptera: Papilionidae), which is generally a typical Rutaceae‐feeding swallowtail, inspired us to survey published works describing its host range and aspects of its chemical ecology. Papilio xuthus larvae have been observed feeding on a total of 22 plant species other than members of Rutaceae, including those of Asarum (Aristolochiaceae) and Cosmos (Asteraceae). Most observation records and our current study indicated that Aristolochiaceae‐feeding by P. xuthus larvae was not due to oviposition error, but to larval movement to Asarum from adjacent rutaceous hosts after they had become unsustaining. Many larvae developed on Asarum to further stadia but we confirmed that some did not, indicating that Asarum was unsuitable for some individuals. According to previous and current observations, P. xuthus females oviposit directly on Cosmos and their larvae can develop to adults although, again, their performance on these plants is not always favorable. Host choice by swallowtail butterflies is determined both at the egg‐laying and larval‐feeding stages. Although adult P. xuthus use a mixture of unique secondary metabolites as their host‐location cue, larvae use primary nutrients as their major phagostimulants. Larval feeding on Asarum could suggest a reversion triggered by vestigial chemosensitivity to ancestral olfactory and/or gustatory cues, because several major clades of Papilionidae feed on Aristolochiaceae. Further studies on the phytochemical/chemosensory bases for these associations are needed if we are to understand the evolutionary pathway of host selection in P. xuthus, as indicated by these relatively unusual host‐seeking behaviors.     

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.     

Hormonal control of seasonal-morph determination in the small copper butterfly, Lycaena phlaeas daimio Seitz.

Spring and summer morphs of Lycaena phlaeas daimio Seitz. are characterized by a wing colour of red and reddish brown, respectively.When newly ecdysed pupae destined to be summer or intermediate morphs (90 or 10%) by larval exposure to long days (long-day pupae) were decapitated or decerebrated, more than half of the operated pupae developed into intermediate and spring morphs (48 and 7%). But, in pupae destined to be spring, intermediate, or summer morphs (72, 22 or 4%) (short-day pupae) these operations did not produce any significant changes in the seasonal morph.Brains excised from newly ecdysed long-day pupae were transplanted into the abdomen of decapitated short-day pupae of the same age. The implants changed most recipients into summer and intermediate morphs (46 and 36%). However, when the brains of short-day pupae were used, no significant changes occurred in the seasonal morph.When long-day or short-day pupae were treated with 20-hydroxyecdysone just after pupation, they produced more reddish wings than those of the untreated or saline-treated controls. When the application was followed by chilling, already known to induce the reddish morph, the effects of both treatments are cumulative so that more reddish adults developed.The results indicate that the brain of long-day pupae secretes a factor causing the wing to be brownish. In the absence (or low titre) of the factor, most short-day pupae develop into spring or intermediate morphs. Furthermore, ecdysteroids make the wing more reddish, when applied to newly ecdysed pupae.     

Convergent evolution of neuroendocrine control of phenotypic plasticity in pupal colour in butterflies

Phenotypic plasticity in pupal colour occurs in three families of butterflies (the Nymphalidae, Papilionidae and Pieridae), typically in species whose pupation sites vary unpredictably in colour. In all species studied to date, larvae ready for pupation respond to environmental cues associated with the colour of their pupation sites and moult into cryptic light (yellow–green) or dark (brown–black) pupae. In nymphalids and pierids, pupal colour is controlled by a neuroendocrine factor, pupal melanization-reducing factor (PMRF), the release of which inhibits the melanization of the pupal cuticle resulting in light pupae. In contrast, the neuroendocrine factor controlling pupal colour in papilionid butterflies results in the production of brown pupae. PMRF was extracted from the ventral nerve chains of the peacock butterfly Inachis io (Nymphalidae) and black swallowtail butterfly Papilio polyxenes (Papilionidae). When injected into pre-pupae, the extracts resulted in yellow pupae in I. io but brown pupae in P. polyxenes. These results suggest that the same neuroendocrine factor controls the plasticity in pupal colour, but that plasticity in pupal colour in these species has evolved independently (convergently).     

Role of the brain in post-diapause adult development in the swallowtail, Papilio xuthus

The role of the brain in adult development was examined by brain removal in unchilled and chilled diapausing pupae of Papilio xuthus. Chilling was effective in shortening the pupal duration and synchronizing adult emergence, although photoperiod had little effect on diapause development. The brain played a role of shortening the pupal duration and synchronizing adult emergence in both unchilled and chilled individuals, although it was not essential for post-diapause adult development. The stimulus of low temperature was recorded even in the absence of the brain, because chilling shortened the pupal duration in brainless individuals. The brain showed activity which affected subsequent adult development in chilled pupae within one day after chilling in males. This period was less limited in females.     

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.     

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.     

Differences in pupal cold hardiness and larval food consumption between overwintering and non‐overwintering generations of the common yellow swallowtail,Papilio machaon (Lepidoptera: Papilionidae), from the Osaka population

To clarify differences in pupal cold hardiness and larval food consumption between overwintering and non‐overwintering generations of the common yellow swallowtail, Papilio machaon, we reared larvae from the Osaka population under photoperiods of 16 h light : 8 h dark (LD 16:8) (long day) or LD 12:12 (short day) at 20°C. We examined the relationship between food consumption and weight during the final larval stadium and pupae, and measured the pupal supercooling point (SCP). Although the ratio of assimilation to consumption did not differ significantly between photoperiods, the ratio of assimilation to pupal weight differed significantly between individuals reared under long and short days. All diapausing pupae were brown, whereas 56% of non‐diapausing pupae were green with the remainder brown. The mean pupal body length (L), dorsal width (W₁) and lateral width (W₂) were larger in non‐diapausing than in diapausing pupae, and the W₁/L and W₁/W₂ ratios differed significantly between non‐diapausing and diapausing pupae. SCP was approximately –20°C and did not differ among pupae 5, 15 and 30 days after pupation under long‐day conditions. However, under short‐day conditions, mean SCP gradually decreased, stabilizing at approximately –24 to –25°C by 30 days after pupation. After freezing, some diapausing pupae emerged as adults, whereas all non‐diapausing pupae died. Both egestion and assimilation were greater under long‐day conditions. The results revealed that pupae of this papilionid exhibit seasonal polyphenism in physiological and morphological traits. Energy from food appears to be expended on increasing cold hardiness in the overwintering generation and on reproduction in the non‐overwintering generation.     

Pupal colour polymorphism in Papilio machaon L. and the survival in the field of cryptic versus non-cryptic pupae

• 1 A field experiment was carried out in a natural habitat of Papilio machaon L. in southern Sweden to assess the evolutionary significance of pupal colour polymorphism.
• 2 Cryptic and non-cryptic pupae were planted in pairs in the vegetation, and exposed to predators.
• 3 The protective coloration conferred a selective advantage approximating 1.5 on the cryptic pupae of the summer generation. In the overwintering generation no difference could be detected between the predation of cryptic and non-cryptic pupae.
• 4 The adaptive fitness of protective coloration, as determined by the different rates of elimination of the colour morphs, was greater for the green pupae than for the brown ones.
• 5 Natural selection favours the evolution of a seasonal difference in the proportion of green and brown pupae in the summer and hibernating generations of P.machaon.

     

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.     

Phylogenetic relationships of three African swallowtail butterflies,Papilio dardanus,P.phorcas and P.constantinus: A cladistic analysis (Lepidoptera: Papilionidae)

Abstract. The well-known polymorphic mimetic African papilionid butterfly, Papilio dardanus Brown, is demonstrated by cladistic analysis to form a monophyletic group (the phorcas-group) with two other African swallowtails, P.phorcas Cramer and P.constantinus Ward. Within this group, dardanus and phorcas are shown to be sister-species, with constantinus as the stem. The supposed relationship of this group of three Rutaceae-feeding species to the five members of the Canellaceae/Lauraceae-feeding Papilio hesperus-group (including P.nobilis Rogenhofer) is questioned. The significance of this analysis is discussed with special reference to Trimen's original hypothesis on the evolution of mimetic patterns in female Papilio dardanus, the unique male patterns of phorcas and dardanus, and natural and laboratory-produced hybrids. A pathway modelling technique is developed to represent the possible course of evolution of the phorcas-group, in terms of speciation events and the accumulation and decay of the pattern polymorphisms.     

Response characteristics and occurrence of extraocular photoreceptors on lepidopteran genitalia

Summary Two excitatory photoreceptor sites (P1, P2) have been demonstrated on the genitalia in adults of both sexes of the Chinese yellow swallowtail butterflyPapilio xuthus. The light stimulus reaches sensory units through transparent windows in the overlying cuticle. Photoreceptor cells in the scaphium (P1) or papilla analis (P1) and both P2's respond similarly to light flashes with spike trains produced in afferent axons. All sites have spectral response curves which peak near 380 nm with a lower maximum at 450 nm. Afferent axons from P1 and P2 have ipsilateral terminal arborizations in the last abdominal ganglion; their somata are peripheral. Well developed in adults, this synthesis is absent in the mature larva ofPapilio xuthus. Comparative study of 22 other lepidopteran species in 10 families proves the presence of similar genital extraocular photoreception in all butterflies tested. It also occurs in skippers, but not in three other families of moths.Abbreviations EOP extraocular photoreception - P1 photoreceptor sites in the scaphium () and papilla analis () - P2 photoreceptor sites underneath P1     

The nature of migration in the red admiral butterfly Vanessa atalanta: evidence from the population ecology in its southern range

1. The migrant Vanessa atalanta (L.) occurs throughout Europe and North Africa. In autumn, populations emigrate from northern and central Europe to the Mediterranean region to overwinter. In the spring, the northern range is recolonised by migrants from the south. The dynamics of the species in the winter range is poorly known. 2. From 1994 to 1999, adults and immatures of V. atalanta were monitored all year round in Mediterranean habitats in north‐east Spain. 3. Data showed that the Catalonia lowlands is an area to which V. atalanta migrates to breed during the winter. Migrants arrive in October and early November and initiate a period of intensive breeding. Larval development occurs throughout the winter until a first annual generation of adults appears in early spring. 4. Most of the butterflies emerging in the spring emigrate and leave the area without breeding. The data suggest strongly that recolonisation of the northern range is by these butterflies not by wintering adults. Altitudinal migration also seems to be a common phenomenon, allowing a further summer generation of adults to occur at high elevations within the Mediterranean region. 5. The complex phenology of V. atalanta in its southern range has evolved as a strategy to track larval resources through space and time. Autumn migration coincides with the greatest availability of the main food plant, Urtica dioica L. Late spring migration occurs by the time food quality is decreasing.     

Heterodynamic processes in Coccinella septempunctata L. (Coccinellidae: Coleoptera): a mini review

Among ladybirds, diapause has evidently been most studied in Coccinella septempunctata L. adults. Coccinella septempunctata (C‐7), the seven spot, has attracted attention both in Europe and the USA for being heterogeneous as to the induction of diapause. A polyphenic character of diapause is a prominent feature in C. septempunctata and this phenomenon generally and often generates voltinism heterogeneity within populations. A greater part of the C‐7 population shows an obligatory univoltine cycle, whereas a smaller proportion is facultatively polyvoltine. Coccinella s. bruckii in Japan is bivoltine, in which the first generation of adults aestivate while the second generation hibernates. This paper reviews this heterodynamic cycle and its importance in the life history of C‐7, as reported from different regions of the world. Heterogeneous voltinism and diapause smooth the progress of a portion of C‐7 populations to take benefit of aphids well into late summer and autumn. Understanding such variation and plasticity in the life history of this species can help in organizing proper biological control attempts using it as a biocontrol agent.     

Seasonal selection and resource dynamics in a seasonally polyphenic butterfly

Seasonal polyphenisms are widespread in nature, yet the selective pressures responsible for their evolution remain poorly understood. Previous work has largely focussed either on the developmental regulation of seasonal polyphenisms or putative ‘top‐down’ selective pressures such as predation that may have acted to drive phenotypic divergence. Much less is known about the influence of seasonal variation in resource availability or seasonal selection on optimal resource allocation. We studied seasonal variation in resource availability, uptake and allocation in Araschnia levana L., a butterfly species that exhibits a striking seasonal colour polyphenism consisting of predominantly orange ‘spring form’ adults and black‐and‐white ‘summer form’ adults. ‘Spring form’ individuals develop as larvae in the late summer, enter a pupal diapause in the fall and emerge in the spring, whereas ‘summer form’ individuals develop directly during the summer months. We find evidence for seasonal declines in host plant quality, and we identify similar reductions in resource uptake in late summer, ‘spring form’ larvae. Further, we report shifts in the body composition of diapausing ‘spring form’ pupae consistent with a physiological cost to overwintering. However, these differences do not translate into detectable differences in adult body composition. Instead, we find minor seasonal differences in adult body composition consistent with augmented flight capacity in ‘summer form’ adults. In comparison, we find much stronger signatures of sex‐specific selection on patterns of resource uptake and allocation. Our results indicate that resource dynamics in A. levana are shaped by seasonal fluctuations in host plant nutrition, climatic conditions and intraspecific interactions.     

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.     

Survivorship and fecundity of the radiate and non-radiate morphs of Groundsel,Senecio vulgaris L., raised in pure stand and mixture

British populations of Senecio vulgaris frequently contain two common capitulum morphs (radiate and non-radiate) and one rare intermediate morph. The radiate morph shows a higher maternal rate of intermorph outcrossing than the non-radiate morph and due to the ‘cost of outcrossing’ should decline in frequency, ultimately to be lost from a population. To determine whether the radiate morph exhibits some inherent advantage in fitness to offset the ‘cost of outcrossing’, a comparison was made of the survivorship and fecundity of the radiate and non-radiate morphs raised in pure stands and 1 : 1 mixture at three planting dates (autumn 1983, and spring and autumn 1984). Plants in stands established in spring 1984 were harvested in late August 1984, while plants in stands established in autumn overwintered before being harvested the following summer. In spring planted stands, the two morphs exhibited equivalent survivorships, while the fecundity of the non-radiate morph tended to be greater than the radiate morph. In autumn planted stands, survivorship and Net Reproductive Output (survivorship × fecundity) of the non-radiate morph was greater than that of the radiate morph in mixture, and also in pure stands established in 1983. In no instance was the Net Reproductive Output of either morph significantly greater in mixture than in pure stand. Density had a contrasting effect on morph survivorship and fecundity in the spring and autumn 1984 planted stands. Whereas, in spring stands, fecundity was subject to compensating density dependent regulation while survivorship was density independent, the opposite trend was observed in autumn planted stands. It is concluded that under the conditions of the experiments, the radiate morph exhibited no fitness advantage which might offset the inherent disadvantage it suffers in natural polymorphic populations due to the ‘cost of outcrossing’.