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.     

丝带凤蝶线粒体基因组全序列及其系统学意义(英文)

锯凤蝶类与凤蝶科其他类群的系统发生关系及其分类学地位一直存在争议。本研究采用PCR和long PCR技术测定了属于锯凤蝶类的丝带凤蝶Sericinus montelus线粒体基因组全序列; 结合已有的其他凤蝶科物种的相应序列数据, 基于13个蛋白质编码基因重建了凤蝶科主要类群的系统发生树, 探讨了它们之间的系统发生关系。基因组分析结果表明： 丝带凤蝶线粒体基因组全长15 242 bp, 包括13个编码蛋白基因(ATP6, ATP8, COⅠ-Ⅲ, ND1-6, ND4L和Cytb)、 22个tRNA基因、 16S和12S rRNA基因以及非编码的控制区; 基因组A, T, G和C含量分别为40.1%, 40.8%, 7.4%和11.7%, 表现出明显的AT偏倚。所有的蛋白质编码基因都使用标准的起始密码子(ATN); 除ND4 和 ND4L基因使用单个的T作为终止密码子外, 其余蛋白编码基因都使用了标准的终止密码子(TAA)。除丝氨酸 tRNA的二氢尿苷突环缺失外, 所有tRNA基因都形成典型的三叶草型结构。基因组中共存在12个大小介于2~65 bp之间的基因间隔区以及15个大小介于1~8 bp之间的基因重叠区, 其中, 存在于COⅡ和tRNA^Lys之间的24 bp的间隔区在其他鳞翅目昆虫中未曾见到。以邻接法和最大简约法并基于13个蛋白质编码基因序列对凤蝶科进行了系统发生分析。结果显示, 丝带凤蝶和中华虎凤蝶Luehdorfia chinensis先构成一个支系, 再和冰清绢蝶Parnassius bremeri构成姊妹群; 表明锯凤蝶类应作为族级分类单元归于凤蝶科下的绢蝶亚科。     

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.     

Egg cluster size variation in relation to the larval food abundance inLuehdorfia puziloi (Lepidoptera: Papilionidae)

Mean egg cluster size of Luehdorfia puziloi yessoensis varied among habitats. The mean egg cluster size tended to be large when abundance of the larval food leaves expressed as the fresh weight of leaves per unit area at a given habitat was high. Since this variation was observed among closely located study plots (butterflies can easily move between study plots), the egg cluster size variation among habitats is likely to be a result of flexible response by females to varying food abundance for larvae.     

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

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

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.     

A new Heraclides swallowtail (Lepidoptera,Papilionidae) from North America is recognized by the pattern on its neck

Heraclides rumiko Shiraiwa & Grishin, sp. n. is described from southwestern United States, Mexico, and Central America (type locality: USA, Texas, Duval County). It is closely allied to Heraclides cresphontes (Cramer, 1777) and the two species are sympatric in central Texas. The new species is diagnosed by male genitalia and exhibits a nearly 3% difference from Heraclides cresphontes in the COI DNA barcode sequence of mitochondrial DNA. The two Heraclides species can usually be told apart by the shape and size of yellow spots on the neck, by the wing shape, and the details of wing patterns. “Western Giant Swallowtail” is proposed as the English name for Heraclides rumiko. To stabilize nomenclature, neotype for Papilio cresphontes Cramer, 1777, an eastern United States species, is designated from Brooklyn, New York, USA; and lectotype for Papilio thoas Linnaeus, 1771 is designated from Suriname. We sequenced DNA barcodes and ID tags of nearly 400 Papilionini specimens completing coverage of all Heraclides species. Comparative analyses of DNA barcodes, genitalia, and facies suggest that Heraclides oviedo (Gundlach, 1866), reinstated status, is a species-level taxon rather than a subspecies of Heraclides thoas (Linnaeus, 1771); and Heraclides pallas (G. Gray, [1853]), reinstated status, with its subspecies Heraclides Papilio bajaensis (J. Brown & Faulkner, 1992), comb. n., and Heraclides anchicayaensis Constantino, Le Crom & Salazar, 2002, stat. n., are not conspecific with Heraclides astyalus (Godart, 1819).     

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.     

Diapause pupal color diphenism induced by temperature and humidity conditions in Byasa alcinous (Lepidoptera: Papilionidae)

We investigated whether diapause pupae of Byasa alcinous exhibit pupal color diphenism (or polyphenism) similar to the diapause pupal color polyphenism shown by Papilio xuthus. All diapause pupae of B. alcinous observed in the field during winter showed pupal coloration of a dark-brown type. When larvae were reared and allowed to reach pupation under short-day conditions at 18 °C under a 60 ± 5% relative humidity, diapause pupae exhibited pupal color types of brown (33%), light-brown (25%), yellowish-brown (21%), diapause light-yellow (14%) and diapause yellow (7%). When mature larvae reared at 18 °C were transferred and allowed to reach pupation at 10 °C and 25 °C under a 60 ± 5% relative humidity after a gut purge, the developmental ratio of brown and light-brown, yellowish-brown, and diapause light-yellow and diapause yellow types was 91.2, 8.8 and 0.0% at 10 °C, and 12.2, 48.8 and 39.0% at 25 °C, respectively. On the other hand, when mature larvae reared at 18 °C were transferred and allowed to reach pupation at 10 °C, 18 °C and 25 °C under an over 90% relative humidity after a gut purge, the developmental ratio of brown and light-brown, yellowish-brown, and diapause light-yellow and diapause yellow types was 79.8, 16.9 and 3.3% at 10 °C, 14.5, 26.9 and 58.6% at 18 °C, and 8.3, 21.2 and 70.5% at 25 °C, respectively. These results indicate that diapause pupae of brown types are induced by lower temperature and humidity conditions, whereas yellow types are induced by higher temperature and humidity conditions. The findings of this study show that diapause pupae of B. alcinous exhibit pupal color diphenism comprising brown and diapause yellow types, and suggest that temperature and humidity experienced after a gut purge are the main factors that affect the diapause pupal coloration of B. alcinous as environmental cues.     

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.     

Latitudinal variation of morphological characteristics in the swallowtail Sericinus montelus Gray, 1798 (Lepidoptera: Papilionidae)

Patterns of latitudinal variation in the phenotype or genotype of an organism may provide evidence for natural selection. In this study, we investigated seven populations of swallowtail Sericinus montelus Gray, 1798 (Lepidoptera: Papilionidae), a non‐migratory species, to explore the latitudinal variation of morphological characteristics in adults. The results showed that body size and the development of dark pigmentation on wings in this species responded strongly to latitude. The body size of both male and female adult of S. montelus was negatively correlated with latitude. These findings provided solid evidence to support the converse Bergmann's rule. We considered that the observed variation in morphological characteristics was most likely mediated by the seasonal length and thermoperiod to adapt to different latitudinal environment (e.g. shortened developmental time of immature stages for smaller body size at higher latitude). Moreover, the tendency towards progressively darker colour patterns (only in adult males) at increasingly low latitudes was consistent with Gloger's rule. We suggested that the observed colour variation was most likely associated with thermoregulation. Slight variation in the morphology of the W‐shaped stripe on the forewing of adult females was also found, and we presumed that the functions of sexual preferences, mimicry and thermoregulation might be involved.     

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.     

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.     

Geographic variation in photoperiodic diapause induction and diapause intensity in Sericinus montelus (Lepidoptera: Papilionidae)

Abstract Due to the risk of extinction and ornamental value of the swallowtail butterfly, Sericinus montelus Gray (Lepidoptera: Papilionidae) in China, knowledge about local adaptations is important for the conservation and economical utilization of the species. In the present study, photoperiodic diapause induction and diapause intensity of S. montelus populations from Jiamusi (46°37′N), Beijing (40°15′N), Zibo (36°48′N), Fangxian (32°36′N), Wuhan (30°33′N) and Huaihua (27°33′N) were characterized at 25°C. Logistic regression analysis revealed a significant population × hours of light interaction, confirming that photoperiodic responses varied among populations. The critical photoperiod was positively correlated with latitude and increased toward the north at a rate of about 1 h for each 6.67 degrees of latitude. Survival analyses indicated that survival time of diapausing pupae before adult eclosion differed significantly among populations at 25°C and 16 : 8 L : D h. The mean duration of pupal diapause was also positively correlated with latitude. Our study reveals geographic variation in the critical photoperiod for diapause induction and in diapause intensity of S. montelus. These results provide useful information for our general understanding about seasonal adaptation in insects and may also be used to predict how geographic populations respond to climate warming.     

The role of latitudinal,genetic and temperature variation in the induction of diapause of Papilio glaucus (Lepidoptera: Papilionidae)

A key adaptation in insects for dealing with variable environmental conditions is the ability to diapause. The tiger swallowtail butterflies, Papilio glaucus and P. canadensis are ideal species to explore the genetic causes and population genetic consequences of diapause because divergence in this trait is believed to be a salient factor in maintaining a hybrid zone between these species. Yet little is known about the factors that influence diapause induction in this system. Here we explored how spatial (latitudinal), environmental (temperature) and genetic (hybridization) factors affect diapause induction in this system. Specifically, a series of growth chamber experiments using wild caught individuals from across the eastern United States were performed to: (1) evaluate how critical photoperiod varies with latitude, (2) isolate the stage in which induction occurs, (3) test whether changes in temperature affected rates of diapause induction, and (4) explore how the incidence of diapause is affected in hybrid offspring. We find that induction occurs in the larval stage, is not sensitive to a relatively broad range of temperatures, appears to have a complex genetic basis (i.e., is not simply a dominant trait following a Mendelian inheritance pattern) and that the critical photoperiod increases by 0.4 h with each increasing degree in latitude. This work deepens our understanding of how spatial, environmental and genetic variation influences a key seasonal adaptation (diapause induction) in a well‐developed ecological model system and will make possible future studies that explore how climatic variation affects the population dynamics and genetics of this system.     

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 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.     

Diapause induction in the oblique-banded leafroller Choristoneura rosaceana (Lepidoptera: Tortricidae): Role of photoperiod and temperature

Induction of diapause in the larval stage of the oblique-banded leafroller, Choristoneura rosaceana (Harris), was found to be dependent on both photoperiod and temperature. At constant temperatures of 24, 20 and 16°C, short photoperiods induced diapause. The critical photoperiod was between 14–15 h of light per day at 20 and 16°C. At 14 h light: 10 h dark, all larvae expressed diapause. Temperature had a modifying effect, and slightly shifted the larval response to diapause-inducing photoperiods. High constant temperatures of 28°C and above induced diapause in some individuals (< 20%), while fluctuating temperatures of 32 and 16°C in a 12-h cycle resulted in 67% diapause induction, suggesting that diapause could also be induced by fluctuating temperatures, particularly if the higher temperature exceeds 25°C.The first- and the second-instar larvae were the only two stages sensitive to diapause induction. Exposure of adult, egg and third, fourth, and fifth-larval instars to diapause-inducing conditions did not produce diapause. Although diapause was induced in the first or the second instars, it was always expressed in the third or fourth instar.