Eupyrene sperm migrates to spermatheca after apyrene sperm in the swallowtail butterfly, Papilio xuthus L. (Lepidoptera: Papilionidae)

When swallowtail butterflies, Papilio xuthus, are mated by the hand-pairing method, both types of sperm, eupyrene and apyrene sperm, are transferred from the male to the spermatheca via the spermatophore in the bursa copulatrix. This mechanism is demonstrated by two different kinds of experiments. The first set of experiments employed interrupted copulation, and the second set was examination of the sperm in the spermatophore and spermatheca after the termination of copulation. The sperm was transferred 30 min after the start of copulation. The eupyrene sperm was still in the bundle; the number of the bundles ranged from 9 to 108 (mean, 42.7; n = 27). The bundles were gradually released after the completion of copulation, and the free eupyrene spermatozoa then remained in the spermatophore at least 2 h before migrating to the spermatheca. On the other hand, about 160 000 apyrene spermatozoa were transferred to the spermatophore and remained there for more than 1 h. We observed 11 000 apyrene spermatozoa in the spermatheca 12 h after the completion of copulation, but most of this type of sperm disappeared shortly thereafter. In contrast, the eupyrene sperm arrived in the spermatheca more than 1 day after the completion of copulation and remained there at least 1 week. Therefore, these findings suggest that apyrene sperm migrate from the spermatophore to the spermatheca earlier than eupyrene sperm. Accordingly, if females mated multiply, the time difference might avoid the mixing of sperm. In addition, the predominance of sperm from the last mating session may occur not in the bursa copulatrix but in the spermatheca. Received: January 7, 2000 / Accepted: May 24, 2000     

Dynamics of eupyrene and apyrene sperm storage in ovipositing females of the swallowtail butterfly Papilio xuthus (Lepidoptera: Papilionidae)

A male swallowtail butterfly, Papilio xuthus, transfers both eupyrene and apyrene sperm during copulation, both of which migrate to the spermatheca via the spermatophore in the bursa copulatrix of the female. Because the spermatheca seems to remain constant in size during the female lifespan, the excess sperm migration may cause the spermatheca to overflow. Approximately 9000 eupyrene and 265 000 apyrene spermatozoa were transferred during a single copulation, and approximately 1000 eupyrene and 1100 apyrene spermatozoa successfully arrived in the spermatheca. The number of both types of spermatozoon decreased in the spermatheca after the onset of oviposition, and no eupyrene spermatozoa were found by 7 days after copulation, partly due to insemination. The spermathecal gland leading from the distal end of the spermatheca was gradually filled by eupyrene spermatozoa. Although the function of the gland remains unclear, the final destination of the sperm is likely to be the gland.     

Effects of saline intake on spermatophore and sperm ejaculation in the male swallowtail butterfly Papilio xuthus (Lepidoptera: Papilionidae)

Although sodium ions induce puddling behavior in males of some butterfly species, the role of sodium ions in male life history is unclear. Newly emerged laboratory‐reared males of the swallowtail butterfly Papilio xuthus were allocated to five adult diets: water (as a control) and 0.001, 0.01, 0.1 or 1 mol/L saline solution. Males preferred to take 0.1 mol/L saline solution. We attempted to hand‐pair males after two days of intake. Males on all diets, except those taking 1 mol/L saline solution, were likely to couple. Males taking 0.01 mol/L saline solution produced the largest spermatophore, although there were no significant differences in the number of eupyrene sperm bundles and apyrene spermatozoa between saline‐taking males and control males. Salt accumulation in the male body also had little effect on the number of both types of spermatozoon in the spermatophore.     

Interactions of environmental factors influencing pupal coloration in swallowtail butterfly Papilio xuthus

The swallowtail butterfly Papilio xuthus Linné [Lepidoptera: Papilionidae] exhibits pupal protective color polyphenism. Interactions of various environmental factors on pupal coloration were analyzed in non-diapausing individuals. Under sufficient light (200lux), most pupating larvae became green pupae when the surface of the pupation site was smooth, while they became brown when the surface was rough. Tactile signals are the positive environmental factors causing induction of the brown pupal coloration. In dark boxes, the induction of the brown pupal coloration was easily induced even on a smooth surface, suggesting that light suppresses induction of brown coloration. Different colors of pupation sites did not affect pupal coloration under sufficient light. Environmental factors received during a critical period both before girdling and after girdling affected pupal coloration. When tactile signals received from rough surfaces reach threshold levels during pupation, brown pupal coloration is determined. Larvae reared under a daily periodicity of natural light formed a girdle at midnight, subsequently, the prepupae received strong daylight the following day. Under natural light most larvae produced brown pupae on rough surfaces and green pupae on smooth surfaces.     

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.     

Properties of Orange-Pupa-Inducing Factor (OPIF) in the swallowtail butterfly, Papilio xuthus L

Diapause pupae of the swallowtail butterfly Papilio xuthus L. exhibit diapause-green, orange and brownish-orange color polymorphism. Development of orange pupae involves a neuroendocrine factor inducing orange pupa (Orange-Pupa-Inducing Factor, OPIF), which is secreted from the head-thoracic region during late pharate pupal stages, in particular from the ganglia of short-day animals located posteriorly from the second thoracic ganglion2 (TG2). This report describes certain properties of OPIF using bioassays involving ligated abdomens of short-day pharate pupae. Localization of OPIF in the central nervous system of short-day larvae indicated that it was present predominantly in TG2, thoracic ganglion3 (TG3) and abdominal ganglion1 (AG1) complexes. OPIF activity in TG(2,3)-AG1 complexes was over two times higher than in the more posteriorely located ganglia. The developmental profile of OPIF in last instar short-day larvae revealed that OPIF activity in larval ganglia posterior to TG2 became gradually higher as larval growth proceeded, suggesting that OPIF might be accumulated in TG(2,3)-AG(1-7) complexes as larvae prepare for pupal molting. Furthermore, ligated abdomens of short-day larvae developed into pupae of an orange type when a 2% NaCl extract containing OPIF prepared from TG(2,3)-AG(1-7) complexes of long-day larvae was injected into ligated abdomens of short-day pharate pupae, indicating that OPIF is also present in long-day larvae. Additionally, a biochemical investigation using gel filtration chromatography showed that the molecular weight of OPIF was about 10 kDa.     

The mitochondrial genome of the butterfly Papilio xuthus (Lepidoptera: Papilionidae) and related phylogenetic analyses

The nearly complete mitochondrial genome of the butterfly Papilio xuthus (Lepidoptera: Papilionidae) was sequenced for its nucleotide sequence of 13,964 bp. The genome has a typical gene order identical to other lepidopteran species. All tRNAs showed same stable canonical clover-leaf structure as those of other insects, except for tRNA^Ser (AGN), in which the dihydrouracil arm (DHU arm) could not form stable stem–loop structure. Anomalous initiation codons have been observed for the cox1 gene, where the ATTACG hexa-nucleotide was believed to be involved in the initiation signaling. Twelve mitochondrial protein-coding gene sequence data were used to infer the phylogenetic relationships among the insect orders. Even though the number of insect orders represented by complete mitochondrial genomes is still limited, several well-established relationships are evident in the phylogenetic analysis of the complete sequences. Monophyly of the Homometabola was not supported in this paper. Phylogenetic analyses of the available species of Bombycoidea, Pyraloidea, Papilionoidea and Tortricidea bolstered the current morphology-based hypothesis that Bombycoidea, Pyraloidea and Papilionoidea are monophyletic (Obtectomera). Bombycoidea (Bombyx mandarina and Antheraea pernyi) and Papilionoidea (P. xuthus and Coreana raphaelis) formed a sister group.     

Identification of odoriferous compounds from adults of a swallowtail butterfly, Papilio machaon (Lepidoptera: Papilionidae)

Adults, particularly males, of a papilionid butterfly, Papilio machaon hippocrates, emit a fairly strong scent perceivable by humans. We have identified a variety of volatile compounds (hydrocarbons, alcohols, aldehydes, ketones, esters, and so on) from the wings and bodies of both sexes of the butterfly. Male wings secreted n-dodecane, linalool and geranylacetone as major components together with small amounts of camphene, limonene, p-cymene, 2-phenylethanol, n-hexanal, n-decanal, isoamyl acetate, p-allylanisole, 2-pyrrolidone and other characteristic volatiles. The overall profile of volatile compounds detected from male body was quite different from that of the wings. Male body was devoid of camphene, 2-phenylethanol, n-hexanal but instead contained limonene, acetoin, a sesquiterpene hydrocarbon (C15H24, methyl n-octanoate, (E,E)-hepta-2,4-dienal, and another isomer of heptadienal as principal components, of which the last four compounds were specific to the body. All these substances seem to concurrently characterize the male odor. The chemical patterns of compounds found from female wings and body were essentially the same in quality as those of male wings and body, respectively, although their quantities in females were generally smaller than in males. Females, however, had a larger amount of acetamide than males. The chemical compositions of volatiles from the fore and hind wings of males were not greatly different from each other, and every component was considered to be present on all parts of the wings. This suggests that the scent-producing organs or scent-emitting pores are widely distributed on the whole wings. EAG responses of both sexes to 12 selected compounds identified from the butterfly were not strong at a dose of 1 microg, while both sexes showed relatively stronger responses to n-nonanal, methyl n-octanoate, D-limonene and linalool at a higher dose (10 microg). Although sexual difference in EAG response was not prominent, females appeared a little more sensitive, and n-nonanal and acetoin evoked significantly higher responses from females at 1 microg.     

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.     

The effect of ejaculate mass on female reproductive output in the european swallowtail butterfly,Papilio machaon (L.) (Lepidoptera: Papilionidae)

Previously, we showed that virgin males of Papilio machaondeliver ejaculates that are twice as big as any ejaculates they transfer at later matings. Here, we investigate the consequences of these two size classes of ejaculates on female reproductive output and demonstrate that females that received one small ejaculate laid as many eggs, fertilized the same proportion of eggs, and lived as long as those that had received one big ejaculate. However, females that received big ejaculates laid heavier eggs, but only between the twelfth and the fifteenth days of egg-laying. We conclude that male-derived nutrients appear to have a limited effect on female reproductive output in P. machaonand that the large size of ejaculates delivered by male butterflies are determined primarily by selection on males to produce longer refractory periods in females.     

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.     

Oviposition preference of swallowtail butterfly,Papilio polytes (Lepidoptera: Papilionidae) on four Rutaceae (Sapindales) host plant species

Abstract Oviposition preference and egg deposition site selection by the butterfly, Papilio polytes L. (Lepidoptera: Papilionidae) on four rutaceous host plants, Citrus aurantofolia (Christm.) Swing., Citrus hystrix DC., Citrus reticulata Blanco, and Murraya koenigii (L.) Sprengel, were assessed in field cages measuring 2 × 2 × 2 m. Simultaneous two-choice and four-choice oviposition tests of whole host plants were conducted. The mean total number of eggs laid per plant on C. reticulata in the two-choice test was significantly higher (P < 0.01) than those on C. aurantifolia, C. hystrix, and M. koenigii. Among the three other host plants, C. aurantifolia was preferred over C. hystrix, and M. koenigii (P < 0.01) and C. hystrix was preferred over M. koenigii (P < 0.01). In the four-choice test, C. reticulata was highly preferred and significantly different from C. hystrix and M. koenigii. However, no oviposition preference was detected with C. aurantifolia. Among the various plant parts in the two- and four-choice tests, eggs on leaves of each plant were the highest, followed by numbers on stems, and negligible numbers on pots. These numbers on leaves of C. reticulata and C. aurantifolia were not significantly different (P > 0.05), but differed significantly (P < 0.01) from those on leaves of C. hystrix and M. koenigii. The quantitative trend of egg-laying on stems was very similar to that observed for the leaves. Papilio polytes showed strong preference to lay eggs on the underside of leaves of all host plants than on the upper side or on the petiole. More eggs were laid on the upper side of each host plant than on its petiole. The four host plants in descending order of preference were C. reticulata≥C. aurantifolia > C. hystrix > M. koenigii. Although M. koenigii was the least preferred, it has the potential to serve as an alternative host plant for P. polytes which can be manipulated when necessary, to alleviate the infestation of this pest to the citrus industry.     

Pupation site preference and environmentally cued pupal colour dimorphism in the swallowtail butterfly Papilio polyxenes Fabr. (Lepidoptera: Papilionidae)

Environmentally cued polymorphisms are hypothesized to evolve when the environment is coarsegrained and different genotypes are unable to choose the habitats in which they are most fit. In Papilio polyxenes , which has an environmentally cued pupal colour dimorphism, there is genetic variation in both tendency to produce brown or green pupae and preference for green- or brown-inducing pupation sites, but the two traits are not correlated.     

A novel retinol-binding protein in the retina of the swallowtail butterfly, Papilio xuthus.

Retinoid-binding proteins are indispensable for visual cycles in both vertebrate and invertebrate retinas. These proteins stabilize and transport hydrophobic retinoids in the hydrophilic environment of plasma and cytoplasm, and allow regeneration of visual pigments. Here, we identified a novel retinol-binding protein in the eye of a butterfly, Papilio xuthus. The protein that we term Papilio retinol-binding protein (Papilio RBP) is a major component of retinal soluble proteins and exclusively binds 3-hydroxyretinol, and emits fluorescence peaking at 480 nm under ultraviolet (UV) illumination. The primary structure, deduced from the nucleotide sequence of the cDNA, shows no similarity to any other lipophilic ligand-binding proteins. The molecular mass and isoelectric point of the protein estimated from the amino-acid sequence are 26.4 kDa and 4.92, respectively. The absence of any signal sequence for secretion in the N-terminus suggests that the protein exists in the cytoplasmic matrix. All-trans 3-hydroxyretinol is the major ligand of the Papilio RBP in dark-adapted eyes. Light illumination of the eyes increases the 11-cis isomer of the ligand and induces redistribution of the Papilio RBP from the proximal to the distal part of the photoreceptor layer. These results suggest that the Papilio RBP is involved in visual pigment turnover.     

A partitioned likelihood analysis of swallowtail butterfly phylogeny (Lepidoptera:Papilionidae)

Although it is widely agreed that data from multiple sources are necessary to confidently resolve phylogenetic relationships, procedures for accommodating and incorporating heterogeneity in such data remain underdeveloped. We explored the use of partitioned, model-based analyses of heterogeneous molecular data in the context of a phylogenetic study of swallowtail butterflies (Lepidoptera: Papilionidae). Despite substantial basic and applied study, phylogenetic relationships among the major lineages of this prominent group remain contentious. We sequenced 3.3 kb of mitochondrial and nuclear DNA (2.3 kb of cytochrome oxidase I and II and 1.0 kb of elongation factor-1 alpha, respectively) from 22 swallowtails, including representatives of Baroniinae, Parnassiinae, and Papilioninae, and from several moth and butterfly outgroups. Using parsimony, we encountered considerable difficulty in resolving the deepest splits among these taxa. We therefore chose two outgroups with undisputed relationships to each other and to Papilionidae and undertook detailed likelihood analyses of alternative topologies. Following from previous studies that have demonstrated substantial heterogeneity in the evolutionary dynamics among process partitions of these genes, we estimated evolutionary parameters separately for gene-based and codon-based partitions. These values were then used as the basis for examining the likelihoods of possible resolutions and rootings under several partitioned and unpartitioned likelihood models. Partitioned models gave markedly better fits to the data than did unpartitioned models and supported different topologies. However, the most likely topology varied from model to model. The most likely ingroup topology under the best-fitting, six-partition GTR + gamma model favors a paraphyletic Parnassiinae. However, when examining the likelihoods of alternative rootings of this tree relative to rootings of the classical hypothesis, two rootings of the latter emerge as most likely. Of these two, the most likely rooting is within the Papilioninae, although a rooting between Baronia and the remaining Papilionidae is only nonsignificantly less likely.     

Environmental factors affecting black/white coloration of the silken girdle in the swallowtail butterfly, Atrophaneura alcinous (Lepidoptera: Papilionidae)

The silken girdles of pupae of the swallowtail butterfly Atrophaneura alcinous show black and white color diphenism. Field observations revealed that all pupae observed on non-food plants and the leaves and stems of the larval food plant Aristolochia debilis were classified as a silken girdle of a black type, while a large portion of pupae pupating on the twigs and trunks of cherry trees in close proximity to A. debilis were classified as a silken girdle of a black type. Additionally, all pupae observed on the surfaces of artificial objects in areas where there are no surrounding plants or trees were classified as a silken girdle of a white type. We demonstrated the effect of day length and the texture, light, plant odor and humidity of pupation sites on the coloration of the silken girdle in A. alcinous. Regardless of long-day or short-day day length conditions, light conditions of constant light or dark, or the presence of a plant odor of A. debilis as environmental cues, all larvae placed at over 80% relative humidity (R.H.) developed into pupae with a silken girdle of a black type. However, all larvae developed into pupae with a silken girdle of a white type when R.H. was below 75%. Furthermore, when pupae with a silken girdle of a white type were transferred to conditions of 90% R.H. within 24 hr of pupation, the white color of the silken girdle changed into a black type within 24 hr of the transfer. The present data suggest that the induction of a black coloration of the silken girdle in A. alcinous requires a R.H. of approximately 80% or more as an environmental factor.     

Structure and metamorphic changes in the brain of the lemon butterfly Papilio demoleus L. (Lepidoptera: Papilionidae)

The morphology of the larval and adult brain of Papilio demoleus, and changes in the cell population and neuropile morphology during the pupal period have been described. The larval brain has more simple fibre areas than that of the adult. Dividing neuroblasts have been found which form the adult neurones. The larval brain contains the three neuromeres (proto-, deuto-, and tritocerebrum). The protocerebrum has well developed corpora pedunculata, a central body, a pons cerebralis and developing optic centres. The corpora ventralia are joined with each other by paired ventral commissures (single in adult). The deutocerebrum is simple and small, the antennal centres are small and simple (ef. adult). The glomerular tritocerebrum is posteroventral to the deutocerebrum, and fibres from the former travel to the crura cerebri. The cortex of the brain consists of four types of glial cells and of association cells, and large and medium sized motor neurones. The number of mitoses is greatest in the larval and prepupal stages; in the pupa it decreases gradually and in late stages it does not occur. Histolysis and pyknosis begin in the prepupa and decrease considerably in the late pupa. The entire neural lamella is broken down in the early pupa. Numerous haemocytes penetrate the laminae of the neural lambella and envelop the entire brain. In the adult, behind the well-developed central body is an ellipsoid body. The medulla interna is divided into two smaller lobes and the deutocerebral lobes are differentiated into cortical and medullary zones. Chiasmata between optic centres are also formed during the pupal period.     

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 Namino 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.     

Multiple matings increase the fecundity of the yellow swallowtail butterfly,Papilio xuthus L., in summer generations

Females of the yellow swallowtail butterfly, Papilio xuthus,were reared in the laboratory. They were divided into four groups held under different mating conditions: nonmating (virgin) and mated once, twice, and three times. The number of eggs in the ovaries was counted by dissection. Virgin females produced increasing numbers of mature eggs, up to about 30, in the week following emergence. When the female had mated once, the number of mature eggs was significantly higher than that of virgin females by the second day after emergence. However, the double- and triplemated females did not increase the number of eggs in each state further than the singlemated females. The double-mated females deposited significantly more eggs than the singlemated females in the laboratory. The triplemated females also deposited more eggs on the day after the third mating than the doublemated females. Thus, multiple matings increased the number of eggs deposited. The change in the hatchability and the morphology of the spermatophore in the bursa copulatrix suggested that the sperm from the last mating had precedence.