Isolation of novel microsatellite markers for the clouded Apollo (P. mnemosyne Linnaeus, 1758; Lepidoptera,Papilionidae)

Five novel polymorphic microsatellite loci were isolated and characterized using an enriched genomic DNA library for Parnassius mnemosyne, a European butterfly of conservation concern, and a valuable model for the study of metapopulation dynamics. Allele numbers ranged from 4 to 12 and observed and expected heterozygosities from 0.17 to 0.74 and from 0.26 to 0.835, respectively. Two samples from geographically close populations were analyzed, demonstrating that the new markers can be successfully employed to investigate fine-scale population structure.     

The Effect of Metals on the Mortality of Parnassius Apollo Larvae (Lepidoptera: Papilionidae)

The population sizes and range of Parnassius apollo started to decline in Finland in the 1930s and resulted in complete extinction in many parts of its former range 30 years later. It has been suggested that metal pollution is one potentially very important reason for the decline. Recently, a recovery of P. apollo and widening of its range has started in the southwestern Finland, simultaneously with a decline in metal fallout from air. Metal analyses were performed with larval and host plant samples collected from an area where P. apollo is recolonising its former range (the archipelago between the island of Kemiö and the Hankoniemi peninsula). In the host plants (Sedum telephium), the level of Fe was significantly lower and the levels of Cd and Zn suggestively lower in the current range (n = 35) of P. apollo than in the former range (n = 12). Analyses on larvae of P. apollo (n = 29) showed lower levels of Cd, Fe and Mn than in the host plant leaves. The excretion of these metals, as well as Zn, via the faeces was effective. The level of the Cd-antagonist Cu was considerably higher in caterpillars than in the food. When caterpillars were fed with metal-polluted food from Helsinki city (4.35ppm Cd/dwt; n = 5) they died, whereas control individuals fed with food from the archipelago (2.49ppm Cd/dwt; n = 5) survived. These results indicate that metals may play an important role in the large-scale decline and partial recovery of P. apollo in the western Europe.     

Genetic structure of French populations of the mountain butterfly Parnassius mnemosyne L. (Lepidoptera: Papilionidae)

Geographical variation in allozyme frequencies among 24 populations of the butterfly Parnassius mnemosyne in southern France was studied by enzyme electrophoresis. Clustering procedures were used to define the geographical organization of genetic variation and an analysis of genetic differentiation was performed to test the degree of potential gene flow between the colonies using the methodologies derived from F-statistics and the private alleles model. Further, an approach using reduced space ordinations (factorial analysis of correspondences and discriminant analysis), was used to allow the visualization of genetically intermediate populations and to determine the rôles of individual alleles at various levels of geographical structure. In P. mnemosyne , the effect of both isolation by distance and geographical barriers upon gene flow is complex. Some barriers appear to be very efficient, but others, at least as strong geographically, do not act in such a way. Multivariate analyses underlined the progressive transition between some groups while in other instances, a sharp cut was observed without obvious topographical barriers. Ecogeographical data and historical events (such as colonization processes) should probably be taken into account in order to explain the geographical variation of genetic structure.     

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 complete mitochondrial genome of the endangered Apollo butterfly,Parnassius apollo (Lepidoptera: Papilionidae) and its comparison to other Papilionidae species

The Apollo butterfly, Parnassius apollo is a representative species of the butterfly subfamily Parnassiinae. This charming species is one of the most endangered butterfly species in the world. In this study, we sequenced its complete mitochondrial genome (mitogenome), with the aim of accumulating genetic information for further studies of population genetics and mitogenome evolution in the Papilionidae. The 15,404-bp long mitogenome harbors a typical set of 37 genes and is the largest butterfly mitogenome determined, except for Papilio maraho (16,094 bp). Like many other sequenced lepidopteran species, one tRNA^Trp-like and one tRNA^Leu(UUR)-like sequences were detected in the AT-rich region. A total of 164 bp of non-coding sequences are dispersed in 14 regions throughout the genome. The longest intergenic spacer (68 bp) is located between tRNA^Ser(AGN) and tRNA^Glu, and is the largest spacer at this location among Papilionidae species. This spacer may have resulted from an 8-fold repetition of a TTTCTTCT motif or a 4-fold repetition of a CTTTATTT motif.     

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.     

Analysis of ovipositional environment using Quantification Theory Type I: the case of the butterfly, Luehdorfia puziloi inexpecta (Papilionidae)

The habitat of Luehdorfia puziloi inexpecta was analyzed by using Quantification Theory Type I, a method for quantifying qualitative data. The investigation area was divided into 92 small sections and was surveyed for its environmental characteristics such as type of trees, tree trunk girth, condition of undergrowth, geography, aspect of slope, and amount of food plants. Environmental factors suitable for oviposition were analyzed using the area density of eggs in each section as the external criterion. The results showed the order of importance of environmental factors to be: aspect of slope > amount of food plants > tree type = tree trunk girth > condition of undergrowth > geography. A slope facing northeast, rich in food plants, with deciduous trees of trunk girth larger than 40 cm, with undergrowth below 30 cm and along a ridgeline was the most preferable habitat for the butterfly.     

基于线粒体基因组控制区序列的中国冰清绢蝶遗传分化和谱系生物地理研究

【目的】分析中国冰清绢蝶Parnassius glacialis遗传多样性和遗传分化情况及其系统发生关系,推测其起源及分歧时间,并探讨其历史生物地理分布格局的成因。【方法】以2008-2016年采集于中国13个地区的冰清绢蝶的325头及绢蝶属其他11个种的11头个体样品,对其线粒体基因组的AT富集区(控制区)序列进行PCR扩增和序列测定,采用MEGA 6.0, Dna SP 5.1和Arlequin 3.5等软件分析其遗传多样性和遗传分化情况;以其他近缘绢蝶种类作为外类群,采用PhyML3.0, MrBayes 3.2和BEAST V1.8.3等软件重建冰清绢蝶的系统发生树,并利用宽松分子钟和校准方法推测中国冰清绢蝶的起源和分歧时间;同时结合现今冰清绢蝶的地理分布特点和第四纪以来的地球环境事件,分析其扩散路径,探讨其历史生物地理分布格局及成因。【结果】冰清绢蝶AT富集区序列长度介于487～495 bp之间,平均为491 bp,其长度的差异主要体现在Poly(T)或Poly(A)的数目差异,A+T平均含量为95.76%。供试冰清绢蝶13个地理居群325头个体中,共检测出基于线粒体基因组AT富集区序列的239个单倍型,单倍型多样性Hd为0.9971;核苷酸多态性指数π值为0.02948, Theta (per site)Eta值为0.06594。系统发生分析和分子定年显示,冰清绢蝶和白绢蝶P.stubbendorfii为近缘姊妹种,二者在距今7.52百万年(Ma)的中新世晚期开始分化。冰清绢蝶的祖先可能起源于我国西南地区(现今横断山脉-喜马拉雅附近的青藏高原东北缘)距今1.53 Ma的更新世时期,先由起源地先期扩散到达小陇山、秦岭一带;后来,由于第四纪的冰期-间冰期轮回事件和扩散路径的不同而分化出2大支系,但两个支系之间存在少量的单倍型相互混杂现象;随后,2大支系继续向低海拔、低纬度地区扩散,即通过伏牛山脉向东北方向扩散到嵩山、泰山、昆嵛山一带,经大别山系向东南方向扩散到琅琊山、紫金山和天目山一带。【结论】中国13个居群的冰清绢蝶没有形成明显的地理分化格局,其遗传变异主要来自居群内部,居群间的遗传分化现象尚不明显;冰清绢蝶起源于更新世时期的较高海拔地区,由于第四纪的冰期-间冰期轮回事件以及扩散路径的不同而形成2大支系并向中国东部和南部低海拔和低纬度地区扩散。     

Effects of meadow suitability on female behaviour in the alpine butterfly Parnassius smintheus

Abstract  1. Many butterfly populations persist in networks of naturally fragmented habitat patches. Movement and reproductive decisions made by adult females are critical to the persistence of these populations because colonisation of extinct habitat patches in the network requires emigration of fecund adult females from their natal meadow and their subsequent establishment in the extinct patch.
2. Movement and oviposition behaviours of mated Parnassius smintheus females released in suitable meadows (a good- and a poor-quality meadow) and an unsuitable meadow were compared, to determine whether adult females consider meadow suitability for their offspring despite frequent oviposition events off the larval host plant.
3. Bootstrap and correlated random walk analyses of female step lengths and turn angles demonstrated that females flew more randomly in the unsuitable meadow than in the suitable meadows. Although females tended to turn the sharpest angle between landing sites in the good-quality meadow, and fly the smallest distance between landing sites and displace the smallest distance from the release site in the suitable meadows, no significant differences were detected in turn angle, step length, and dispersal rates between suitable and unsuitable meadows.
4. Results from female flight observations and a caged oviposition study suggest that females lay significantly more eggs in suitable habitat than in unsuitable habitat despite not ovipositing on the host plant, and support the above findings.
5. Movement and oviposition behaviours of adult female P. smintheus promote their retention within meadows that can support their offspring.     

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

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

Highly‐enhanced larval growth during the cold season mediated by the basking behavior of the butterfly Parnassius citrinarius (Lepidoptera: Papilionidae)

Some insect species are thought to grow quickly, even in low temperatures under natural conditions, presumably by conducting basking behaviors to use sunlight. However, whether basking behavior in fact enhances developmental speed and shortens the larval period in the field has not been determined. Moreover, few studies have examined whether basking is behavioral thermoregulation or simply the result of highly‐heterogeneous heat environments in the field. To examine these issues, we conducted field observations and laboratory experiments using larvae of Parnassius citrinarius Motschulsky, which mature within a short period after the thaw in early spring. First, body temperatures of larvae were measured under sunny and cloudy conditions. Second, larval preference for warmer locations was examined. Finally, we compared the developmental speed of larvae when they basked under field conditions and when did not bask in laboratory conditions under different air temperature regimes. Under sunny conditions, larval body temperature was substantially higher than either the temperature of the host plant or the air temperature, and was equivalent to the temperature of dead leaves, which the larvae used as basking sites. In contrast, no such tendency was observed under cloudy conditions. Larvae exhibited an exclusive preference for warmer locations. Moreover, in the field, despite the low ambient temperature, larvae grew much faster than those reared in the laboratory. These results imply that the basking behavior of P. citrinarius larvae is active thermoregulation to maintain high body temperatures in the cold season.     

Host plant-based territoriality in the white peacock butterfly,Anartia jatrophae (Lepidoptera: Nymphalidae)

Mate locating behavior of Anartia jatrophaewas studied near Ochopee, Collier County, Florida. Males were individually marked, and focal animal samples were used to determine activity, residency, and interactions with other butterflies. We recaptured more than half of 53 marked males within the study area on subsequent days. Marked males restricted non-feeding activity to roughly circular areas typically less than 15 m in diameter. Males showed considerable site fidelity and chased both conspecifics and a variety of other insects. Typically, encountered individuals flew away, and the resident peacock male returned to perch near his previous perch. Encounters with conspecific males averaged 38 sec, three times longer than with other insects. Such site fidelity combined with aggressive interactions indicates territorial behavior. When male density more than doubled, site fidelity decreased, resident turnover increased, and areas that had not been defended at lower density were defended. Without exception, defended sites contained the larval host, Bacopa monnieri.In butterfly mating systems, defense of host plants appears to be much less common than defense of natural features such as landmarks.     

Phylogenetic relationships of the New World Troidini swallowtails (Lepidoptera: Papilionidae) based on COI, COII, and EF-1α genes

A phylogeny of the Neotropical members of the Tribe Troidini (Lepidoptera: Papilionidae) was obtained with sequences of three protein-coding genes: two mitochondrial (COI and COII), and one nuclear (EF-1α). Parsimony and Bayesian analyses of 33 taxa resulted in very similar trees regardless of method used with the 27 troidines always forming a monophyletic clade. Within Troidini, the genus Battus is sister group to the remaining troidines, followed by a clade formed by the Paleotropical taxa (here represented by three exemplars). The genus Euryades is the next branch, and sister group of Parides. The genus Parides is monophyletic, and is divided into four main groups by Maximum Parsimony analysis, with the most basal group composed of tailed species restricted to SE Brazil. Character optimization of ecological and morphological traits over the phylogeny proposed for troidines indicated that the use of several species of Aristolochia is ancestral over the use of few or a single host-plant. For the other three characters, the ancestral states were the absence of long tails, forest as the primary habitat and oviposition solitary or in loose group of several eggs.     

Visual feedback in the control of pheromone-mediated flight ofHeliothis virescens males (Lepidoptera: Noctuidae)

MaleHeliothis virescens (F.) (Lepidoptera: Noctuidae) were made to fly into a uniformly white and translucent tube within a large wind tunnel while responding to sex pheromone. Different visual patterns placed within the tube greatly affected the ability of the male moths to maintain upwind progress or remain oriented to the wind while in contact with the plume. Over 89% of males attempting to fly through a blank tube, lacking visual patterns, became disoriented, the males gaining or losing altitude and repeatedly hitting the sides of the tube. Patterns of 20–40 dots placed on the sides of the tube at or slightly above plume level resulted in high levels of sustained upwind flight (47–74%) relative to patterns placed directly below (30–40%), directly above (35%), or slightly below the level of the flight path (26–44%). Optimal upwind progression in pheromone-responding males occurred when image motion could be resolved both transversely (T), orthogonally to the longitudinal axis of the body relative to the horizontal plane of the environment, and longitudinally (L), along the body axis. Even very sparse patterns (single rows of dots) could elicit high levels of sustained upwind flight (53–63%) when positioned within the tube such that the males' movements would create both L and T image motion. However, successful negotiation of the tube was also unexpectedly facilitated by patterns apparently providing no horizontal transverse component for flying males but providing longitudinal flow while centering the moth in the plume through a symmetrical left-right input (4–40%).     

Ecological significance of puddling as a behavioural phenomenon in the life history of butterfly Papilio polytes Linn. (Lepidoptera: Papilionidae)

Puddling in butterflies is an essential physiological phenomenon, by which they gain mineral nutrients required for their reproductive success and flight. The preference given to the puddling by butterflies has been analysed by two hypotheses: butterflies puddle when there is a scarcity of nutritional resource in one hypothesis and competition with other males to increase their probability of mating in the other. In the present investigation, an attempt has been made to verify these hypotheses through experimental evidences in the butterfly Papilio polytes, in which only males are known to puddle. Our investigation revealed the significance of puddling as an alternative source of nutrition in the absence of nectar. However, increase in the number of males in a given vicinity with constant number of females enhances the puddling activity in males. This shows the importance of puddling as a distinct competitive behaviour by which male improves their chances of mating. Additionally, in both cases the puddling is drastically regulated by the environmental temperature. Males prefer to puddle at a temperature ranging from 26 °C to 28 °C, which is considered as optimum for puddling activity. Further, the fecundity and longevity of imago are completely unaffected by puddling activity. All these results are in good agreement with the existing hypotheses and show the behavioural significance of puddling in butterflies.     

中国绮夜蛾属Acontia Ochsenheimer分类学修订（鳞翅目：夜蛾科：绮夜蛾亚科）

首次报道尺蛾科泽尺蛾属Zamarada Moore, 1887和超泽尺蛾Z. excisa Hampson, 1891在我国分布,给出了形态描述和特征图。     

Complete nucleotide sequence and organization of the mitogenome of the red-spotted apollo butterfly, Parnassius bremeri (Lepidoptera: Papilionidae) and comparison with other lepidopteran insects

The 15,389-bp long complete mitogenome of the endangered red-spotted apollo butterfly, Parnassius bremeri (Lepidoptera: Papilionidae) was determined in this study. The start codon for the COI gene in insects has been extensively discussed, and has long remained a matter of some controversy. Herein, we propose that the CGA (arginine) sequence functions as the start codon for the COI gene in lepidopteran insects, on the basis of complete mitogenome sequences of lepidopteran insects, including P. bremeri, as well as additional sequences of the COI start region from a diverse taxonomic range of lepidopteran species (a total of 53 species from 15 families). In our extensive search for a tRNA-like structure in the A+T-rich region, one tRNA^Trp-like sequence and one tRNA^Leu (UUR)-like sequence were detected in the P. bremeri A+T-rich region, and one or more tRNA-like structures were detected in the A+T-rich region of the majority of other sequenced lepidopteran insects, thereby indicating that such features occur frequently in the lepidopteran mitogenomes. Phylogenetic analysis using the concatenated 13 amino acid sequences and nucleotide sequences of PCGs of the four macrolepidopteran superfamilies together with the Tortricoidea and Pyraloidea resulted in the successful recovery of a monophyly of Papilionoidea and a monophyly of Bombycoidea. However, the Geometroidea were unexpectedly identified as a sister group of the Bombycoidea, rather than the Papilionoidea.     

Sensory cell degeneration in the ontogeny of the chemosensitive sensilla in the labial palp-pit organ of the butterfly,Pieris rapae L. (Insecta,Lepidoptera)

Summary The ontogeny of the chemoreceptive sensilla in the labial palp-pit organ was studied in Pieris rapae by examining twelve successive stages between pupation and emergence of the imago, which takes a period of 160 h under the experimental conditions. Mitoses occur until 20 h after pupation. They lead to anlagen of sensilla, 91% of which are comprised of three sensory cells. However, two sensory cells degenerate in each sensillum during a period of 28 h. The same process occurs in anlagen with four sensory cells resulting in bicellular sensilla. Axons grow out only after the number of sensory cells has been reduced. Further consecutive steps in sensory cell differentiation are: (a) outgrowth of dendritic outer segment and dendrite sheath; (b) outgrowth of trichogen process and change in structure of elongating dendrite sheath; (c) deposition of cuticle and pore tubules in the pegs; (d) retraction of trichogen process; (e) increase in diameter of dendritic outer segment accompanied by increase of microtubule number and appearance of regularly spaced electron-dense bodies at tubular doublets; (f) branching of dendritic outer segment; and (g) transformation of the dendritic branches into curled lamellae and partial destruction of the dendrite sheath. The unique process of sensory cell degeneration is interpreted as an event that revokes a step towards a possible functional improvement of the labial palp-pit organ during further evolutionSupported by the Deutsche Forschungsgemeinschaft (SFB 4/G1)     

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.