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.     

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.     

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.     

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.     

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.     

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.     

Pattern of sperm storage and migration in the reproductive tract of the swallowtail butterfly Papilio xuthus: cryptic female choice after second mating

Females of the swallowtail butterfly Papilio xuthus L. (Lepidoptera: Papilionidae) mate multiply during their life span and use the spermatophores transferred to increase their longevity as well as fecundity. Sperm from different males may be stored in the sperm storage organs (bursa copulatrix and spermatheca). To clarify the pattern of sperm storage and migration in the reproductive tract, mated females are dissected after various intervals subsequent to the first mating, and the type and activity of sperm in the spermatheca are observed. When virgin females are mated with virgin males, the females store sperm in the spermatheca for more than 10 days. Sperm displacement is found in females that are remated 7 days after the first mating. Immediately after remating, these females flush out the sperm of the first male from the spermatheca before sperm migration of the second male has started. However, females receiving a small spermatophore at the second mating show little sperm displacement, and the sperm derived from the small spermatophore might not be able to enter the spermatheca. Females appear to use spermatophore size to monitor male quality.     

Identification of amine receptors from a swallowtail butterfly, Papilio xuthus L.: cloning and mRNA localization in foreleg chemosensory organ for recognition of host plants

The swallowtail butterfly, Papilio xuthus L., feeds exclusively on members of the plant family, Rutaceae. Female butterflies lay eggs in response to specific chemicals contained in their host plants. They perceive a variety of polar compounds as oviposition stimulants through the tarsal chemosensilla of the foreleg by drumming upon the leaf surface. Some biogenic amine analogs have been characterized as oviposition stimulants. We have cloned three amine receptors, serotonin, tyramine, and dopamine, from cDNA derived from foreleg tarsus of P. xuthus, and determined structures of both cDNA and genomic genes. The phenylethylamine (tyramine and dopamine) receptors were expressed preferentially in brain and chemosensory organs. Moreover, we observed the localized expression of dopamine receptors at the base of tarsal chemosensilla by in situ hybridization. These results suggest that amine receptors in tarsal chemosensilla have a functional role in chemoreception for host plant recognition.     

Characterization of a new insect cell line that is derived from the neonate larvae of Papilio xuthus (Lepidoptera: Papilionidae) and its susceptibility to AcNPV

The cell line RIRI-PX1 was established from neonate larval tissues of Papilio xuthus by performing primary cultures in the modified Grace medium that was supplemented with 20% fetal bovine serum (FBS). The cell line primarily consisted of spindle-shaped and spherical cells which attached themselves to the flask. The population-doubling times (PDTs) at the 50th and 60th passage were 42.5 h and 42.1 h respectively. The average chromosome numbers of RIRI-PX1 cell line from passage 5 to passage 50 ranged from 103 to 199. It was confirmed that RIRI-PX1 cell line was derived from P. xuthus by comparing the mitochondrial cytochrome c oxidase subunit I gene (COI) of RIRI-PX1 cells and P. xuthus eggs. This cell line was susceptible to the Autographa californica nucleopolyhedrovirus (AcNPV) and produced high yield of polyhedral occlusion bodies (43.9 OBs/cell) after 10 days of infection by AcNPV. The virus titer of AcNPV infected RIRI-PX1 cells was 3.25 × 10⁷ TCID₅₀/ml. We concluded that the RIRI-PX1 cell line is established from the neonate larvae tissues successfully and the cells of the cell line are sensitive to AcNPV.     

Identification of cytochrome P450 and glutathione-S-transferase genes preferentially expressed in chemosensory organs of the swallowtail butterfly, Papilio xuthus L

The swallowtail butterfly, Papilio xuthus L., feeds exclusively on members of the plant family, Rutaceae. Female butterflies lay eggs in response to specific chemicals contained in their host plants. They perceive a variety of polar compounds as oviposition stimulants through the tarsal chemosensilla of the foreleg by drumming upon the leaf surface. We undertook an expressed sequence tag (EST) analysis to identify the chemosensory-related genes that are expressed in chemosensilla on the tarsus of P. xuthus. Several genes that showed similarity with biotransformation enzymes were identified from the ESTs. Among them, a cytochrome P450 and a glutathione-S-transferase (GST) were preferentially expressed in the chemosensory organs. We have determined the structure of both cDNA and genomic sequences encoding these enzymes and designated the P450 as CYP341A2, a novel member of CYP341A subfamily, and the GST as GST-pxcs1, respectively. We observed a localized expression of CYP341A2 at the base of tarsal chemosensilla by in situ hybridization. These results suggest that these degrading enzymes play a role in the chemosensory reception for host plant recognition.     

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

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

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     

Hormonal control of the orange coloration of diapause pupae in the swallowtail butterfly, Papilio xuthus L. (Lepidoptera: Papilionidae)

Diapause pupae of Papilio xuthus show color polymorphism, represented by diapause-green, orange, and brownish-orange types that are each associated with specific pupation sites. We investigated the role of the site of pupation on the induction of the development of orange types (or brownish-orange types), and the endocrine mechanism underlying the control of color polymorphism in short-day pupae. All short-day larvae of the wandering stage developed into orange or brownish-orange type pupae when they were placed in rough-surfaced containers after gut-purge. Utilizing a pharate pupal ligation between the thorax and abdomen, the endocrine mechanism underlying the control of color polymorphism was shown to involve a head-thorax factor (Orange-Pupa-Inducing Factor: OPIF) that induced orange types in short-day pupae. OPIF was bioassayed using the ligated abdomens of short-day pharate pupae. OPIF was extractable with 2% NaCl solution from 5th-instar larval ganglia complexes following the mesothoracic complex (TG(2,3)-AG(1-7)), but it could not be extracted with either acetone or 80% ethanol solution. OPIF may not exist in the brains of day-0 pupae or in brain-subesophageal ganglion and prothoracic ganglion complexes of 5th-instar larvae. The short-day pharate pupae responded to OPIF in a dose-dependent manner.     

Effect of male and female multiple mating on the fecundity, fertility, and longevity of diamondback moth, Plutella xylostella (L.)

Abstract:  The effect of diamondback moth (DBM), Plutella xylostella (Lep., Plutellidae) male and female multiple mating on fecundity, fertility, and longevity was studied. Males could mate for five times with virgin females during scotophase. The successful copulation rates, fecundity of female, and longevity of both females and males decreased when male mating times increased, whereas copulation duration increased. Correlation coefficient between copulation duration and male mating times was significant ( r  = 0.7358, P = 0.0001, spearman rank-order correlation). There were linear relationships between mating history of males and longevities of males and females, and regression relationships between them were significant. Mated females had similar daily reproductive pattern, which laid the most eggs on the first day after mating in spite of their mates' mating history. Virgin females laid some infertile eggs before they died. Most of the females mated once during their lifespan but 19.9% of females mated twice when one female kept with one male during scotophase. There were no significant differences in the fecundity, fertility and longevity between the single- and twice-mated females. Correlation coefficient between copulation duration and female mating times was not significant ( r  = 0.0860, P = 0.8575). Results suggested that DBM females may be monandrous. Multiple mating did not increase male or female mating fitness.     

Host influence on mating behavior and spermatophore reception correlated with reproductive output and longevity of femaleEarias insulana (Boisduval) (Lepidoptera: Noctuidae)

When mating and oviposition behavior ofEarias insulana were studied in the absence or presence of host or by providing only host odor, in comparison to other treatments host absence caused a delayed and reduced reproductive activity with occasional impairment of egg viability. Presence of host or its odor resulted in a short premating period and a significant increase in mating frequency, number of spermatophores transferred, number of eggs oviposited, and proportion of eggs fertilized. Compared to host odor, host accessibility had a larger impact on insect behavior, indicating the importance of host cantact. Under conditions of host accessibility, there existed a significant and positive correlation between the number of spermatophores received by a female and both the number of eggs she oviposited and her longevity. This indicated a role for male-derived nutrients in the reproduction ofE. insulana.     

Effects of mating rate and delayed mating on the fecundity of Adoxophyes orana

Females of Adoxophyes orana F. v. R. (Lepidoptera: Tortricidae) could mate after one day, or after up to 7 days, after eclosion. It was recorded how many eggs per female were laid, had been fertilized and ultimately hatched. Mating rate was assessed by counting the spermatophores in a female.Egg production did not depend on mating rate, but proportion fertilization of eggs from females with three or more spermatophores was reduced. Delayed mating promoted longevity and changed the pattern of oviposition in time. No correlation was found between the proportion hatching of fertilized eggs and any of the other variables in the experiments.The data were introduced into a population model to compute the relation between fecundity and probability to mate.

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Effets du nombre d'accouplements et du retard de la date du premier accouplement sur la fécondité de Adoxophyes orana

Résumé Des femelles de A. orana F. v. R. (Lep., Tortricid.) ont eu la possibilité de s'accoupler le lendemain de leur émergence. Nous avons dénombré l'effectif d'oeufs pondus par chaque femelle, et calculé leur taux de fertilisation et d'éclosion. Les femelles ont été disséquées et les spermatophores comptés pour déterminer le nombre de copulations. La même procédure a été suivie avec d'autres femelles qui ont pu copuler librement jusquà leur mort, (leur longévité a été notée), mais avec une date du premier accouplement pouvant être retardée jusqu'à 6 jours après l'émergence.La production d'oeufs ne dépend pas du nombre de copulations, mais le taux de fertilisation des femelles avec 3 ou 4 spermatophores a été plus faible. Le retard de la date du premier accouplement augmente la longévité, mais réduit légérement le taux de fertilisation. La production d'oeufs était à peu près proportionnelle à la durée de la vie décomptée à partir du moment où la copulation était possible. Aucune corrélation n'a été mise en évidence entre la taux d'éclosion des oeufs fertilisés et les variables de l'habitat.Pour évaluer ces résultats et obtenir plus de connaissances sur les conséquences des copulations retardées, par exemple provenant de procédures d'interruption des copulations, les données ont été introduites successivement dans un modèle de populations pour établir la relations entre fécondité et probabilité d'accouplement.

     

Control mechanisms of larval diapause and quiescence in Trogus mactator, an endoparasite of papilionids

First instar larvae of Trogus mactator Tosquinet (Hymenoptera: Ichneumonidae) developed to second instar immediately after pupation of the host, Papilio xuthus L., under a short photoperiod (L10: D14) as well as under a long photoperiod (L15: D9). The host went into diapause as pupa under short photoperiods, and the parasite at the third instar in the host pupa. They responded independently to short photoperiods. The critical photoperiods for diapause induction both in the parasite and the host were between 12 and 13 h, but there was a slight disparity between them. Diapause of neither the parasite nor the parasitized host was terminated under short photoperiods. Diapause of the parasite in a diapausing host pupa was completed when it was transferred to an incubator having a long photoperiod. This suggests that the exposure to a long photoperiod breaks diapause in the parasite irrespective of physiological conditions of the host. The period from the change in photoperiod to time of parasite emergence shortened as the change was made later. The parasite remaining in a host pupa for 60 days completed diapause even under a short photoperiod when it was implanted into a nondiapausing host pupa, or when 20-hydroxyecdysone was injected into the parasitized and diapausing host pupa. These results indicate that the physiological development for diapause termination in T. mactator seems to complete 60 days after host pupation and endogenous ecdysteroids are somehow involved in termination of diapause in the parasite.

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Résumé Que la photopériode soit brève (L10/D14) ou longue (L15/D9), le premier stade de T. mactator Tosquinet (Hym. Ichneumonidae) donne un second stade immédiatement après la nymphose de son hôte, Papilio xuthus L.,. L'hôte entre en diapause au stade nymphal sous photopériodes courtest et le parasite au troisiéme stade larvaire dans la nymphe de l'hôte. Ils réagissent indépendamment aux courtes photopériodes. Les photopériodes critiques pour l'induction de la diapause, tant de l'hôte que du parasite, se situent entre 12 et 13 h, mais il y a une légère différence. Ni la diapause de l'hôte, ni celle du parasite ne se terminent sous de courtes photopériodes. Le transfert dans une étuve ayant une longue photopériode rompt la diapause du parasite dans un hôte diapausant. Ceci laisse supposer que l'exposition à une longue photopériode rompt la diapause du parasite quel que soit l'état physiologique de l'hôte. Le délai entre le moment du changement de la photopériode et la date d'émergence du parasite, est d'autant plus raccourci que le changement est tardif. Les parasites restés dans une nymphe hôte pendant 60 jours, achèvent leur diapause même avec une photopériode courte quand ils sont transplantés dans un hôte sans diapause, ou quand de la 20-hydroxyecdysone a été injectée dans la nymphe hôte parasitée et diapausante. Ces résultats montrent que le développement physiologique nécessaire à la fin de la diapause semble achevé 60 jours après la nymphose de l'hôte et que les ecdystéroïdes endogènes semblent impliqués d'une manière ou d'une autre dans la fin de la diapause du parasite.