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.     

Genetic variation and Wolbachia infection status of the lycaenid butterfly Zizina emelina (Lepidoptera,Lycaenidae) from Korean populations

The lycaenid butterfly Zizina emelina emelina (de l’Orza) (previously Zizina otis emelina; Lepidoptera, Lycaenidae) has populations distributed across Japan and Korea. Zizina emelina is listed on Japan’s Red List as an endangered species, owing to the loss of its principal plant food and habitat. Researchers previously investigated the genetic diversity and structure in Japanese populations by using mitochondrial and nuclear DNA analyses alongside their Wolbachia infection status to consider the conservation strategy. In this study, we investigated the genetic variation and Wolbachia infection status of Korean populations and compared our findings with these traits in the Japanese populations. Four novel mitochondrial haplotypes were found in the Korean populations, whereas nuclear DNA analysis revealed one new haplotype and another that matched the one reported previously in Japan. The two known strains of Wolbachia in the Japanese populations were also found in the Korean ones. Mitochondrial DNA analysis demonstrated that no gene flow had occurred between the Japanese and Korean populations. The fact that they share the same nuclear haplotype and Wolbachia strains, however, indicates a low level of population differentiation between the two.     

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.     

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.     

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.     

Evolution of Euphilotes (Lepidoptera: Lycaenidae) by seasonal and host shifts

The lycaenid butterfly genus Euphilotes , consisting of five species and several dozen subspecies, is confined to western North America. Each subspecies utilizes one or a few species of Eriogonum (Polygonaceae) as larval hosts; larvae feed entirely on pollen and developing seeds. Euphilotes populations are (with few exceptions) univoltine and fly in synchrony with the initial (c. 2 weeks) annual flowering periods of their hosts. Euphilotes evolution coincides with shifts to new hosts, frequently with different bloom periods. Often both inter- and intraspecific populations occur in sympatry. Although interspecific populations may fly in synchrony and utilize the same hosts, sympatric intraspecific populations use different hosts and generally fly allochronically. Analyses of Euphilotes populations using biochemical, morphological, and life history characters, do not support either coevolution or sequential evolution with Eriogonum but are more consistent with opportunistic adaptation to new hosts having different bloom periods.     

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.     

Effects of Argentine ants (Hymenoptera: Formicidae) on myrmecophilous lycaenid butterfly,Narathura bazalus (Lepidoptera: Lycaenidae), in western Japan

Effects of the invasive Argentine ant, Linepithema humile (Mayr), on a myrmecophilous butterfly, Narathura bazalus (Hewiston), were investigated in the field in western Japan. Larvae of N. bazalus were attended by workers of Argentine ants in invaded parks and of ten native and one cosmopolitan ant species in uninvaded parks. The abundance of eggs and larvae were not significantly different between invaded and uninvaded parks. Pupal weight and parasitized ratio by tachinid flies were also not different between the two types of parks. These results indicate that the role of Argentine ants for the butterfly might be almost equivalent to the native ants.     

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.     

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.     

Phylogeography of silver‐studded blue,Plebejus subsolanus (Lepidoptera: Lycaenidae), in the central mountainous regions of Japan

The silver‐studded blue, Plebejus subsolanus, is widely distributed in the Russian Altai mountains, northeastern China, the Korean Peninsula, and the Japanese archipelago. In Japan, the species is distributed across wide elevation ranges from the lowlands of Hokkaido to the subalpine zone of Honshu. Current subspecies classification in Japan is as follows: ssp. iburiensis, occurring in lowland grasslands in Hokkaido; ssp. yaginus in lower mountain grasslands in Honshu; and ssp. yarigadakeanus in higher mountain grasslands in Honshu. The habitat of this species has been markedly reduced due to recent habitat destruction and land‐use changes. Here, we undertook phylogeographic analyses of two subspecies, ssp. yaginus and yarigadakeanus in the central mountainous regions of Japan, based on two mitochondrial gene sequences, in order to collect information for establishing effective conservation strategies. From 57 samples from the four mountain ranges, we obtained a haplotype network comprised of 12 haplotypes. Because of the haplotype network topology, the geographic distribution of haplotypes and the correspondence of haplotype divergence to subspecies taxonomy, we provisionally divided the haplotypes into three haplogroups: YR1 and YR2, which comprised ssp. yarigadakeanus, and YG, which comprised ssp. yaginus. Mitochondrial DNA genetic differentiation generally agreed with morphological subspecies classification. The haplotype network suggested that ssp. yarigadakeanus populations had multiple origins, and the subspecies character of “bright blue of the male's wings” was assumed to have evolved independently in each subalpine meadow. We found that P. subsolanus was genetically differentiated depending upon the elevation at each mountain region, suggesting that each haplogroup should be a conservation unit.     

Early comers occupy popular territories in a lycaenid butterfly,Chrysozephyrus smaragdinus (Lepidoptera: Lycaenidae)

Territorial activities of a lycaenid butterfly, Chrysozephyrus smaragdinus, were observed throughout its adult season to investigate the territorial dynamics. The territories occupied earlier in the season were abandoned later than later‐occupied territories, indicating that the site preference of male C. smaragdinus was largely maintained during the adult season. Three indexes of territory quality based on animal preference, occupation duration (OD), initial occupation date (IOD) and the number of contests (NOC), were highly correlated. Therefore, these preference‐based indexes are consistent and reliable, although they are indirect estimates of benefits obtained from territories. Males arriving at territory sites early in the season were more likely to hold territories of better quality when NOC was used as the index of territory quality. The positive relationship between territory quality and male arrival date is congruent with the result of a previous study showing that early comers tend to hold a territory whereas late comers do not. However, male arrival date was not related to territory quality when OD or IOD was used as an index of territory quality. One possible reason for this apparent discrepancy is that the coefficient of variation of NOC is larger than those of OD and IOD, and therefore NOC could evaluate territory quality more quantitatively than the other two indexes. To date, studies of territoriality in butterflies have not considered the quality of territories. More studies considering the quality of territories will provide new insights into butterfly territoriality.     

World travellers: phylogeny and biogeography of the butterfly genus Leptotes (Lepidoptera: Lycaenidae)

Leptotina butterflies (Lycaenidae, Polyommatiinae) are found mostly in tropical and subtropical areas around the globe, marginally penetrating into temperate regions. Here, we investigated phylogenetic and biogeographical relationships of most representatives of the subtribe, using both likelihood and Bayesian approaches. We also estimated the timing of their diversification. And lastly, we studied phylogeographic patterns of the most widespread species, Leptotes pirithous. DNA sequences from two mitochondrial (COI, COII) and two nuclear genes (wingless, Ef1α) were analysed for 13 species of the genus Leptotes Scudder and one species of the genus Cyclyrius Butler. Both genera together form a monophyletic clade, and Cyclyrius is rooted deep inside Leptotes. Therefore, we designate Cyclyrius to be a junior synonym of Leptotes. According to our study, the genus Leptotes originated between the late Eocene and early Oligocene (35–31 Ma). During the Miocene it dispersed to the rest of the southern hemisphere, with further speciation events within the Indo‐Australian region, and separate radiations in the Americas and the Afrotropics. Leptotes webbianus from the Canary Islands turned out to be sister to the American clade from which it split c. 12 Ma. Leptotes pirithous originated in Madagascar c. 4 Ma and invaded the whole of Africa and southern Europe, including numerous surrounding islands. Populations of L. pirithous from Mauritius and Madagascar turned out to represent a distinct species (Leptotes durrelli sp.n. ) and the same applies to the Australasian populations of Leptotes plinius (Leptotes lybas stat. rev. ). This published work has been registered in ZooBank, http://zoobank.org/urn:lsid:zoobank.org:pub:20308930‐988B‐4327‐A35F‐CC983D46263B .     

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。     

Geographical distribution of morphological abnormalities and wing color pattern modifications of the pale grass blue butterfly in northeastern Japan

The pale grass blue butterfly Zizeeria maha has been used as an environmental indicator to evaluate the biological impacts of the Fukushima nuclear accident. A high morphological abnormality rate (AR) of this butterfly was detected in 2011 from radioactively contaminated areas at 37–38°N. However, the geographical AR distribution has not been documented for the entirety of northeastern Japan. Additionally, the geographical distribution of the wing color pattern modification rate (MR) of temperature‐shock type remains undocumented. Here, we collected adult butterflies from many localities in northeastern Japan in 2014 and examined the local AR and MR. Both AR and MR were generally low throughout the 44 local populations surveyed. Latitudinal AR and MR distributions indicated a gap zone at approximately 39°N. The mean AR and the mean MR of the populations south of the gap zone were low (AR = 3.0%, MR = 1.1%), whereas those of the northern populations were relatively high (AR = 10.6%, MR =10.3%). Logistic regression analyses revealed that abnormalities and modifications were associated with temperature‐related variables. We conclude that abnormalities and modifications are generally rare, but that their rates are higher in the northern populations than in the southern ones. These results, along with evidence from other studies, strongly suggest that the high AR detected in 2011 from contaminated areas was induced by anthropogenic radioactive mutagens. This study presents a basic dataset of the current wildlife state of Z. maha in northeastern Japan, which facilitates a future use of this butterfly species as an environmental indicator.     

Structure of the food web including the endangered lycaenid butterfly Shijimiaeoides divinus asonis (Lepidoptera: Lycaenidae)

The structure of the food web including the endangered lycaenid butterfly Shijimiaeoides divinus asonis (Matsumura) was analyzed to identify species contributing most to maintaining the equilibrium of the food web. Twenty‐seven species belonging to 17 families fed on Sophora flavescens Aiton, the host‐plant of S. divinus asonis: 15 species were leaf and stem feeders, seven (including S. divinus asonis) fed on flower buds, four were flower feeders and one fed on the seeds of So. flavescens. Of these 27 species, four were omnivores. The natural enemies of S. divinus asonis comprised six insect species, 11 spider species and one entomopathogenic fungus species, including six new predator records. The linkage density, total number of trophic links, connectance, average chain length and predator–prey ratio were 1.617, 97, 0.0548, 2.267 and 0.694, respectively. Exclusion of any of the 15 species with four or more trophic links reduced the connectance of the food web. These 15 species included facultative mutualistic attendant ants and predators of S. divinus asonis, herbivores to So. flavescens, an omnivore feeding on S. divinus asonis and So. flavescens, and prey insects. Therefore, future studies should monitor these 15 species.     

Incongruence between morphological and molecular markers in the butterfly genus Zizina (Lepidoptera: Lycaenidae) in New Zealand

The butterfly genus Zizina in New Zealand has a complex taxonomic history due to the presence of morphological intermediates between the two species, the endemic Z. oxleyi and the introduced Z. labradus, in a putative hybrid zone on the east coast of the South Island of New Zealand. This makes species identification in the field problematic, particularly as the presence of hybrids has not been confirmed. We address this uncertainty through morphological and molecular analyses. Specimens were collected from a range of locations in New Zealand, as well as from Australia, and measurements were made of male genitalia and ventral wing coloration. Two mitochondrial genes (COI, ND5) and three nuclear gene fragments (28S, ITS2 and wingless) were also sequenced for a selection of individuals, and the presence of Wolbachia species in genomic DNA was tested. The two species were separable in morphological space, although there was some overlap, and the contact zone appeared to be around Kaikoura on the east coast of the South Island. Furthermore, specimens from the putative hybrid zone could be classified as Z. oxleyi using morphological characters individually, but not when these were used in a principal component analysis. Molecular analysis showed that there was a mean sequence divergence of 2.0% between two clades for COI, and 4.1% for ND5, but suggested that the contact zone between them was in the north‐west of the South Island. However, there was only a single clade for each of the three nuclear markers. It is thought that this incongruence between morphological and molecular markers is indicative of hybridization which is more extensive than previously thought. However, the possibility that recent speciation has occurred or is occurring is not ruled out.     

The pale grass blue butterfly in ex-evacuation zones 5.5 years after the Fukushima nuclear accident: Contributions of initial high-dose exposure to transgenerational effects

Biological impacts of the Fukushima nuclear accident have been reported in various organisms but have not been evaluated sufficiently. Here, we collected the pale grass blue butterfly, Zizeeria maha, in September 2016, 5.5 years after the accident, from ex-evacuation zones in Fukushima Prefecture to evaluate the remaining biological effects of the accident. Although the morphological abnormality rate was not high, it was significantly positively correlated with the initial ¹³¹I and ¹³⁷Cs deposition levels (as of March 2011). The capture rate was negatively correlated with the initial ¹³⁷Cs deposition level at the borderline of significance. Neither the abnormality rate nor the capture rate was correlated with the ground radiation dose (as of September 2016). Generalized linear model (GLM) analysis also indicated contributions of the initial ¹³¹I and ¹³⁷Cs deposition levels but not the ground radiation dose. These results suggest that the butterflies present in the ex-evacuation zones in 2016 were still affected, although not severely, mainly because of the initial high-dose exposure to radioiodine, radiocesium, and other radionuclides. We conclude that transgenerational effects of radioactive contamination have successively manifested in the 5.5 years since the accident, possibly through genetic mutations, although potential contributions of other modes of damage cannot be ignored.