Rearing the pale grass blue Zizeeria maha (Lepidoptera,Lycaenidae): Toward the establishment of a lycaenid model system for butterfly physiology and genetics

Although some nymphalid butterflies have been intensively used to study mechanisms of the colour pattern formation on butterfly wings, lycaenid butterflies are equally attractive, having easily identifiable distinct spot patterns and highly diverse colour patterns among species. To establish a lycaenid model system for physiological and genetic experiments, we here describe a series of methods for rearing the Japanese pale grass blue Zizeeria maha (Kollar) (Lepidoptera, Lycaenidae) in a small laboratory space with an artificial diet for generations. Adult individuals readily mated and oviposited in a small cage with sufficient light, flowers, and host plants. Eggs were harvested in the cage, and larvae were successfully reared to normal adults with an artificial diet made from fresh leaves (AD‐F), although they were smaller than those reared with a natural diet. Feeding an artificial diet made from dried leaves (AD‐D) frequently produced adult individuals with aberrant wing colour patterns. Using our rearing methods, it is now possible to rear this species in a laboratory and to establish specific strains for physiological and genetic experiments on the wing colour pattern development, diversity, and evolution.     

Sampling efficiency of the pale grass blue butterfly Zizeeria maha (Lepidoptera: Lycaenidae): A versatile indicator species for environmental risk assessment in Japan

The pale grass blue butterfly, Zizeeria maha (Lepidoptera, Lycaenidae), has successfully been employed as an indicator species to evaluate acute biological changes in polluted human-living environments after the Fukushima nuclear accident. Here, we quantitatively examined the field sampling efficiency of the butterfly and its associated sexual sampling bias under different conditions, which may provide important information for environmental risk assessment. Sampling data were obtained in 2014 and 2015 from 87 localities across Japan. Across the localities, the mean capture rate was 17 individuals per hour per person, and males represented approximately 80% of the collected butterflies. No significant difference was detected in the capture rate under different weather conditions. Among the four habitats, the riverside showed a significantly higher capture rate than the city park. A shorter sampling time tended to yield a higher capture rate. Comparisons among prefectures and districts of collection localities revealed that the Kanto district had significantly higher percentages of collected males than 4 other districts. Fukushima prefecture also had high percentage, although not significant. A generalized linear mixed model indicated that the capture rate was significantly negatively affected by the agricultural village, the city park, the rainy weather, and latitude and positively by longitude. Together, the present study showed high sampling efficiency and versatility of this butterfly as an indicator for environmental risk assessment but simultaneously suggested preferable conditions for a field sampling design. This study also suggested a possibility that the Fukushima nuclear accident or other factors might have affected behavioral or population dynamics of the butterfly.     

The fragmented population structure of the Sardinian chalk hill blue butterfly (Lepidoptera, Lycaenidae)

The endemic Sardinian chalk hill blue butterfly, Polyommatus coridon gennargenti, is considered vulnerable to extinction because of its low genetic variation and restricted distribution. The species also has a fragmented distribution, which follows the patchy distribution pattern of its larval host-plant. A preliminary investigation of the population structure of P. coridon gennargenti was carried out on a small network of four local populations by means of capture–recapture methods. Estimated population sizes and movement rates among the four adjacent local populations suggest that this taxon has a metapopulation structure composed of loosely connected small local populations. Natural fragmentation, isolation, and traditional land use contribute to the vulnerability of P. coridon gennargenti to extinction. Low effective population sizes and restricted movement between habitat patches lead to inbreeding and an increased vulnerability to extinction of this island population.     

Clinal variation in populations of the common blue butterfly Polyommatus icarus rott. (Lepidoptera, Lycaenidae)

Geographical trends in variation of wing pattern characters of the common blue butterfly Polyommatus icarus Rott. have been established. Peripheral populations were shown to be more rich and diverse phenotypically than those of the central parts of the species range. Phenotypic diversity of the populations increased from the center to the periphery of the range. The range boundaries were characterized by strong fluctuations of frequencies of wing pattern phenotypes. Hybrid areas were found on the part of the range examined.     

Sequestration and metabolism of host-plant flavonoids by the Pale Grass Blue, Pseudozizeeria maha (Lepidoptera: Lycaenidae)

Flavonoids in the eggs, larvae, pupae and adult butterflies of Pseudozizeeria maha were identified and quantified. Isovitexin (apigenin 6- C -glucoside) was the dominant flavonoid detected in pupae and imagines, whereas saponarin (isovitexin 7- O -glucoside) was the dominant flavonoid in larvae. Saponarin, biotransformed from isovitexin by the larvae, was found to be reconverted into isovitexin at the pupal stages. The total amounts of accumulated flavonoids differed between sexes, particularly in adults where the bodies of females had a much higher flavonoid concentration than that of the males. These flavonoids were also detected in the eggs. These results indicate the possibility that flavonoids are utilized in not only wing coloration but also as a chemical defense compound against natural threats.     

Variation in number of copulations in Theclini butterfly females (Lycaenidae: Lepidoptera)

The number of spermatophores inseminated in the bursa copulatrix of females of 21 species of Theclini butterflies varied from 0 to 38, a maximum much larger than reported so far. The variation of spermatophore number observed was analyzed on a phylogenetic tree recently determined, together with variation of wing colors and male mating tactics. Monandrous species tended to be sexually dimorphic in wing color and associated with male territoriality.     

Impact of game bird release on the Adonis blue butterfly Polyommatus bellargus (Lepidoptera Lycaenidae) on chalk grassland

Polyommatus bellargus is a priority species of butterfly in the UK as a result of its scarcity and the rate of population decline over the last few years. In the UK, the species is associated with chalk grassland on hot, south-facing slopes suitable for the growth of the food plant Hippocrepis comosa. Shooting game birds is a popular pastime in the UK. Over 40 million game birds, principally Phasianus colchicus and Alectoris rufa, are bred and released into the countryside each year for shooting interests. There is a concern that the release of such a large number of non-native birds has an adverse effect on native wildlife. A study was carried out over a period of 3 years out to examine whether there was any evidence that A. rufa released into chalk grassland habitat negatively affects populations of P. bellargus. A comparison was made between sites where large numbers of A. rufa were released versus sites where no, or few, birds were released. The study involved the construction of exclosures in these sites to allow an examination of the number of butterflies emerging from H. comosa when the birds were excluded versus when the birds had free range across the area. Where birds were present the on-site vegetation was shorter than where they were absent indicating that the birds were definitely influencing habitat structure. However, the evidence that A. rufa was negatively influencing the number of adult butterflies emerging was not strong, although there was a largely non-significant tendency for higher butterfly emergence when the birds were excluded or absent.     

Sex-related differences in reaction norms in the butterfly Lycaena tityrus (Lepidoptera: Lycaenidae)

We investigate sexual differences in reaction norms in directly developing individuals of the copper butterfly Lycaena tityrus predicted from sexual selection theory. As recent studies on butterflies revealed a high degree of adaptive plasticity in growth and development, which may undermine the basic trade‐offs assumed in life‐history theory, we focus on effects of temperature, trying to drive growth rates to their physiological upper limit and thus disclosing otherwise potentially concealed responses. Development time strongly depended on temperature, leading in accordance with a central assumption in life‐history theory to a larger size at low temperatures, and vice versa. At all temperatures larval development time of males was significantly shorter compared to females, as was predicted by protandry theory. This was partially due to an invariably higher growth rate of males. However, sexes responded in different ways to developmental time constraints caused by increasing temperatures. Despite the shorter larval time of males, both sexes achieved similar body sizes at lower temperatures, because males avoided a reduction in weight due to plastic growth. At high temperatures, in contrast, males were forced to make a trade‐off in which they favoured early emergence over large size, leading to a dramatic weight loss. Weight of females, however, remained similar throughout showing no trade‐off. These different reaction norms reflect divergent selective pressures acting on males and females, which can be explained in relation to the reproductive system. The strong selection for early emergence in males is likely to be due to monandry, discrete non‐overlapping generations (as was already predicted by theory), and territoriality, because prior ownership of a territory seems to be a major advantage for successful reproduction. On the other hand, the preference of females for large body size was expected due to the close relationship between this trait and fecundity. Thus, our results highlight the extraordinary importance of the specific reproductive system, which can influence central life‐history traits in manifold ways.     

酢浆灰蝶生物学特性研究

通过野外观察、室内饲养等方法研究了酢浆灰蝶Pseudozizeeria maha(Kollar)的生活史及生物学特性。在南京地区,该蝶幼虫以酢浆草科Oxalidaceae的黄花酢浆草(Oxalis corniculataL.)为食,1年5代,世代交替发生。酢浆灰蝶在10月末以蛹在枯枝落叶或土壤表层浅洞中越冬,越冬代成虫次年5月始见、中旬为高峰期,5月中旬始见第1代卵及幼虫,6—10月间各月各虫态均同时发生,期间每月中下旬为成虫活动高峰期。幼虫共4龄,野外2～4龄幼虫具迁移、避热与适蚁行为。在室内饲养条件下,卵期为3～4d,幼虫期为22～26d,蛹期为6～7d。     

An integumental anatomy for the butterfly Glaucopsyche lygdamus (Lepidoptera:Lycaenidae):a morphological terminology and homology

An integumental anatomy for the lycaenid butterfly Glaucopsyche lygdamus is presented. Comparisons with other lepidopteran taxa are made to rectify the homology of parts and contrast anatomical divergences within the Lycaenidae. A general terminology based on Snodgrass is given, to replace many of the specialized and often synonymous terms restricted to the Lepidoptera. Many common anatomical svnonyms are also given. Several reinterpretations of the anatomy and homology of various integumental regions are discussed. A previously unreported cuticular anomaly on abdominal tergum 2 of male Polyommatinae (Downey's area) is described. The following new or newly combined terms are used:postgenal-occipital area, postgenal-occipital protuberance, dorsal temporal sulci, postantennal projections, pronotal projection, infraepisternal-basisternal plate, paracoxal-marginopleural sulci, dorsal epimeral sulci, ventral epimeral sulci, secondary coxal sulci, ventral subcostal-radial process, lateral secondary sclerite and Downey's area.     

A butterfly with olive green eyes discovered in the United States and the Neotropics (Lepidoptera,Lycaenidae, Eumaeini)

We describe Ministrymon janevicroy Glassberg, sp. n., from the United States (Texas). Its wing pattern closely resembles that of the widespread and well-known lycaenid, Ministrymon azia (Hewitson). The new species is distinguished by the structure of its male and female genitalia, by the patterning of the ground color on the basal half of the ventral hindwing surface, and by the color of its eyes. Adults of Ministrymon janevicroy in nature have olive green eyes in contrast to the dark brown/black eyes of Ministrymon azia. Ministrymon janevicroy occurs in dry deciduous forest and scrub from the United States (Texas) to Costa Rica (Guanacaste) with disjunct populations on Curaçao and Isla Margarita (Venezuela). In contrast, Ministrymon azia occurs from the United States to southern Brazil and Chile in both dry and wet lowland habitats. Nomenclaturally, we remove the name Electrostrymon grumus K. Johnson & Kroenlein, 1993, from the synonymy of Ministrymon azia (where it had been listed as a synonym of Ministrymon hernandezi Schwartz & K. Johnson, 1992). We accord priority to Angulopis hernandezi K. Johnson & Kroenlein, 1993 over Electrostrymon grumus K. Johnson & Kroenlein, 1993, syn. n., which currently is placed in Ziegleria K. Johnson, 1993. The English name Vicroy’s Ministreak is proposed for Ministrymon janevicroy. We update biological records of dispersal and caterpillar food plants, previously attributed to Ministrymon azia, in light of the new taxonomy.     

The effect of ant association on the population genetics of the Australian butterfly Jalmenus evagoras (Lepidoptera: Lycaenidae)

Populations of the myrmecophilous lycaenid Jalmenus evagoras Donovan were assessed for genetic structure at three hierarchical spatial scales: sites, geographically-defined subpopulations, and subpopulations defined by species of mutualistic ant-associate. Estimates of Wright's FST generated from multilocus electrophoretic data revealed low, though significant, amounts of genetic structure. Most structure was observed at the level of geographic subpopulations, suggesting that adult butterflies do not exhibit preferential mating and oviposition along the lines of ant associate. The genetic structure data, together with estimates of Nei's genetic distance (D) for pairwise site and subpopulation comparisons, suggest that J. evagoras populations are spatially and temporally dynamic. These patterns are considered in the context of extinction and recolonization models. The extreme patchiness of J. evagoras populations stems from the stringent requirements of both host plant and host ant, contributing to an extinction/ recolonization process. We discuss the key parameters influencing genetic cohesion versus differentiation under an extinction/recolonization regime, including mode of butterfly dispersal, site turnover rate, and the effects of host dispersal and phenology. This system provides a model of population-level consequences of certain mutualistic interactions as well as of a class of patterns arising from an extinction/recolonization process.     

Wolbachia infection associated with all-female broods in Hypolimnas bolina (Lepidoptera: Nymphalidae): evidence for horizontal transmission of a butterfly male killer

Inherited bacteria that kill male hosts during embryogenesis infect a wide range of insect species. In order to ascertain if there are patterns to host infection, with particular male killing bacteria specialising on particular taxa, we investigated the male killing trait in the butterfly Hypolimnas bolina. All-female broods were first reported in this species in the 1920s. Investigation of this system in the Fiji Islands revealed the causal agent of sex ratio distortion in H. bolina to be a male killing Wolbachia bacterium. This bacterium is identical in wsp and ftsZ sequence to a male killer in the butterfly Acraea encedon in Tanzania, suggesting it has moved between host species, yet retained its phenotype. The prevalence of the Wolbachia was calculated for three different island groups of Fiji, and found to vary significantly across the country. Antibiotics failed to cure either the male killing trait or the Wolbachia infection. The implications of these results are discussed.     

Effects of temperature on the development and survival of an endangered butterfly, Shijimiaeoides divinus barine (Leech) (Lepidoptera: Lycaenidae)

The effects of temperature on the development and survival of Shijimiaeoides divinus barine were examined in the laboratory in 2008. The eggs and larvae were reared at temperatures of 15, 17.5, 20, 25, 30 and 35°C with a long-day photoperiod of 16 h light : 8 h dark (16L : 8D). The highest hatchability of eggs was 88.0% at 20°C, but hatchability at high temperatures of 30 and 35°C was 30 and 0%, respectively. The lowest and highest survival rates from the first to third instar were 18.8% at 15°C and 76.9% at 20°C. Few deaths were observed after the fourth instar. The shortest developmental periods of the eggs and larvae were 4.0 and 15.8 days at 30°C, and the durations of the egg and larval stages increased significantly as the temperature decreased. The developmental zero and thermal constants were 9.6°C and 82.6 degree–days for the egg stage, and 10.7°C and 306.8 degree–days for the larval stage. The developmental period of the natural population of S. divinus barine in Azumino City, Nagano Prefecture was calculated using the developmental zero, thermal constants and Azumino City temperature data.     

Molecular and morphological divergence in the butterfly genus Lycaeides (Lepidoptera: Lycaenidae) in North America: evidence of recent speciation

Male genital morphology, allozyme allele frequencies and mtDNA sequence variation were surveyed in the butterfly species Lycaeides idas and L. melissa from across much of their range in North America. Despite clear differences in male genital morphology, wing colour patterns and habitat characteristics, genetic variation was not taxonomically or geographically structured and the species were not identifiable by either genetic data set. Genetic distances (Nei's D=0.002–0.078, calculated from allozyme data) between all populations of both species were within the range commonly observed for conspecific populations of other butterflies. The most frequent mtDNA haplotype was present in individuals of both species in populations from southern California to Wisconsin. We conclude that speciation has probably happened recently and the lack of genetic differentiation between the species is the product of either (1) recent or ongoing gene flow at neutral loci, and/or (2) an insufficiency of time for lineage sorting. The evolution of male genital morphology, wing colour patterns and ecological characteristics has proceeded more rapidly than allozyme or mtDNA evolution.     

Environmental induction of larval diapause and life-history consequences of post-diapause development in the Large Copper butterfly,Lycaena dispar (Lepidoptera: Lycaenidae)

Many insects in temperate zones withstand the adverse conditions of winter through entering diapause and the two most important environmental stimuli that induce diapause are photoperiod and ambient temperature. The Large Copper butterfly, Lycaena dispar Haworth (Lepidoptera: Lycaenidae), is a Palearctic butterfly that hibernates as larvae. Since this butterfly is a near threatened species in some regions, there has been a growing need for a standardized protocol for mass rearing of this butterfly based on the adequate knowledge of its ecology. In the present study, we first identified that L. dispar larvae were sensitive to the photoperiodic induction of diapause during their first larval instar. We then investigated to what extent the diapause-inducing effects of photoperiod could be modified by ambient temperatures in L. dispar larvae by exposing them to the range of day-lengths (L:D 14:10, 12:12, 10:14 and 8:16) at three different temperatures (15, 20 and 25 °C). All larvae were induced to enter diapause at low ambient temperature (15 °C) regardless of photoperiod, whereas most of them (86 %) exhibited direct development when temperature was high (25 °C). The photoperiodic induction of diapause was evident when day-length was shorter than 14 h at intermediate temperature (20 °C). Pre-diapause development was prolonged at low temperatures. Finally, we found that post-diapause development of L. dispar larvae was determined by both the chilling temperature experienced by diapausing larvae and the duration of larval diapause. Adult emergence was enhanced when larvae were chilled at 8 °C and when they had been under the state of diapause for 20 days before they were treated to terminate diapause.     

A quantified ethogram for oviposition behavior and oviposition preference in the hemipterophagous butterfly Spalgis epius (Westwood) (Lepidoptera: Lycaenidae)

In this study, we examined the oviposition behavior and preference of Spalgis epius, a potential predator of mealybug crop pests. An ethogram of oviposition behavior was constructed based on observations made in an oviposition cage. Ovipositional behavioral acts were catalogued and separated into two behavioral repertoires: searching and egg laying. Gravid females of S. epius oviposited similar numbers of eggs on three mealybug species. Females preferred eggs and adults to nymphs of mealybugs for oviposition. Among three species of mealybugs attended by ants, females laid fewer eggs in the mealybug mass attended by Oecophylla smaragdina than on mealybugs attended by Tapinoma melanocephalum and Camponotus variegatus. Females preferred mealybug masses already containing conspecific eggs to mealybug masses containing conspecific larvae or Cryptolaemus montrouzieri larvae for egg deposition. Gravid females laid larger numbers of eggs under bright sunlight than in diffused sunlight or shade. The results of this study showed that S. epius can effectively attack any species of mealybugs, avoid intra- and interspecific competition, and co-exist with some species of ants attending mealybugs. With the knowledge of these behaviors, this predator can be effectively utilized as a major biological control agent of mealybugs.     

Host-ant trail following by myrmecophilous larvae of Liphyrinae (Lepidoptera,Lycaenidae)

In this study we report a case of ant-trail following by lycaenid caterpillars. Euliphyra mirifica and E. leucyana caterpillars are involved in a commensal association with the weaver ant Oecophylla longinoda. The host nests are made with leaves which over the course of time dry out or are broken open by storms, forcing the ants to migrate and build a new nest elsewhere. Euliphyra caterpillars are stimulated by recruitment behaviour which triggers the migration of their host. They then follow the host trails leading to the new nesting site. Laboratory experiments showed that these caterpillars are able to follow host trails under varied conditions: (1) fresh trails actually used by workers, (2) fresh trails in the absence of workers, (3) heterocolonial, 2-month-old trails, and (4) fresh trails washed with water (to simulate the effect of tropical rains). They can also bridge trail gaps of more than 1 cm. Under natural conditions, the trails are frequently situated along thin twigs. The forward progress of the ants in such a situation is not impeded by the presence of large Euliphyra larvae. Workers just climb over the caterpillars, even on larger trails where there is enough room to pass alongside them. This suggests that an allomone is secreted on the dorsal part of the caterpillars. When crawling along heterocolonial trails, the caterpillars are not attacked, even if about 21% of the workers from the new colony spread their mandibles when encountering them. They are then adopted and are admitted to the nest of the new host colony of O. longinoda.