Complete nucleotide sequence and organization of the mitogenome of the silk moth Caligula boisduvalii (Lepidoptera: Saturniidae) and comparison with other lepidopteran insects

The 15,360-bp long complete mitogenome of Caligula boisduvalii possesses a gene arrangement and content identical to other completely sequenced lepidopteran mitogenomes, but different from the common arrangement found in most insect order, as the result of the movement of tRNA(Met) to a position 5'-upstream of tRNA Ile. The 330-bp A+T-rich region is apparently capable of forming a stem-and-loop structure, which harbors the conserved flanking sequences at both ends. Dissimilar to what has been seen in other sequenced lepidopteran insects, the initiation codon for C. boisduvalii COI appears to be TTG, which is a rare, but apparently possible initiation codon. The ATP8, ATP6, ND4L, and ND6 genes, which neighbor another PCG at their 3' end, all harbored potential sequences for the formation of a hairpin structure. This is suggestive of the importance of such structures for the precise cleavage of the mRNA of mature PCGs. Phylogenetic analyses of available sequenced species of Bombycoidea, Pyraloidea, and Tortricidea supported the morphology-based current hypothesis that Bombycoidea and Pyraloidea are monophyletic (Obtectomera). As previously suggested, Bombycidae (Bombyx mori and B. mandarina) and Saturniidae (Antheraea pernyi and C. boisduvalii) formed a reciprocal monophyletic group.     

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 complete mitogenome of Bombyx mori strain Dazao (Lepidoptera: Bombycidae) and comparison with other lepidopteran insects

The complete mitochondrial genome (mitogenome) of Bombyx mori strain Dazao (Lepidoptera: Bombycidae) was determined to be 15,653 bp, including 13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes and a A + T-rich region. It has the typical gene organization and order of mitogenomes from lepidopteran insects. The AT skew of this mitogenome was slightly positive and the nucleotide composition was also biased toward A + T nucleotides (81.31%). All PCGs were initiated by ATN codons, except for cytochrome c oxidase subunit 1 (cox1) gene which was initiated by CGA. The cox1 and cox2 genes had incomplete stop codons consisting of just a T. All the tRNA genes displayed a typical clover-leaf structure of mitochondrial tRNA. The A + T-rich region of the mitogenome was 495 bp in length and consisted of several features common to the lepidopteras. Phylogenetic analysis showed that the B. mori Dazao was close to Bombycidae.     

The complete mitogenome of Apocheima cinerarius (Lepidoptera: Geometridae: Ennominae) and comparison with that of other lepidopteran insects

The complete mitochondrial genome (mitogenome) of a female flightless geometrid moth Apocheima cinerarius was found to be 15,722 bp in length, containing 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and a control region. The A + T content of the complete mitogenome is 80.83%. The AT skew value ([A − T] / [A + T]) is 0.027. The 13 PCGs of the mitogenome start with typical ATN codons, except for cox1 with the start codon CGA. All the tRNA genes have typical cloverleaf secondary structures, except for trnSer(AGN). The secondary structures of rrnL and rrnS were predicted. Six structural domains including conserved regions (IV, V) and variable regions (I, II, III, VI) were identified in the secondary structure of rrnL. The secondary structure of rrnS consists of 3 structural domains. The control region of A. cinerarius begins with conserved motifs of “ATAGA” + 19-bp poly T. It also contains a microsatellite-like (TA)₂₆, a stem-and-loop structure, and a poly-A stretch. Phylogenetic analysis showed that Geometroidea is more closely related to Bombycoidea than to Noctuoidea. A. cinerarius is more closely related to Biston panterinaria than to Phthonandria atrilineata, which is in accordance with the conventional morphology-based classification.     

The demonstration of natural hybridization between two swallowtail species Parnassius nomion and Parnassius bremeri (Lepidoptera, Papilionidae) using RAPD-PCR technique]

Genetic evidence for interspecific hybridization between Parnassius nomion and Parnassius bremeri in nature is presented. To demonstrate hybridization between these species, RAPD analysis was used. By testing 25 decamer primers, three and two diagnostic markers were revealed for P. nomion and P. bremeri, respectively. Out of 28 animals examined, 4 were shown to be interspecific hybrids. According to the distribution of diagnostic markers, the interspecific hybrids were intermediate with regard to the parental species. Ecological and biological characteristics of two swallowtail species that promote their hybridization in nature are discussed.     

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.     

The adaptive significance of alpine melanism in the butterfly Parnassius phoebus F. (Lepidoptera: Papilionidae)

Summary The adaptive significance of alpine melanism, the tendancy for insects to become darker with increased elevation and latitude, was investigated using the butterfly Parnassius phoebus. The effects on temperature dependent activity of five components of overall wing melanism, as well as size, were examined. The components of wing melanism examined were the transparency of the basal hindwing and distal fore-wing areas, the width of the black patch in the basal hind-wing area and the proportion of black to white scales in that area, and the proportion of the distal fore-wing covered by predominantly black scaling.The body temperature of dead specimens was correlated with air temperature, solar radiation, the width of the black patch at the base of the wings, and the proportion of black to white scales at the base of the wings. The minimum air temperatures and solar radiation levels required for initiation of flight did not vary with wing melanism of P. phoebus, in contrast to the results found for Colias butterflies by Roland (1982). However, under environmental conditions suitable for flight initiation, males with a higher proportion of black to white scales in the basal area of the hind-wing, and wider basal black patches, spent a greater proportion of time in flight at low air temperatures and low insolation. Increased basal wing melanism was also associated with increased movement of males within a population. In contrast, melanism in the distal area of the wings has no effect on activities which are dependant on body temperature. The amount of time spent feeding did not vary with differences in wing melanism. I suggest that in dorsal basking, slow-flying butterflies (Parnassius) basal wing color affects body temperature primarily during flight (rather than while basking), such that butterflies with darker wing bases cool down less rapidly because they absorb more solar radiation during flight.     

Twenty new microsatellite loci for population structure and parentage studies of Parnassius apollo nevadensis (Lepidoptera; Papilionidae)

We characterized 20 new microsatellite loci isolated from Parnassius apollo nevadensis. The 20 loci displayed 2–12 alleles and expected heterozygosities varied from 0.22 to 0.89; the markers were amplified in nine multiplex reactions. The combined non-exclusion probabilities of this set are 0.001084 and 0.000005 for the first and second parents, respectively. This set of markers will thus enable other studies on reproductive strategies and population genetics in this endangered and previously understudied species. We show, for the first time, evidence of multiple paternity and that females may successfully copulate with more than one male despite the presence of mating plugs. Future studies on this mating behavior and population genetics of the species using the 20 loci here presented will be very important to define its conservation status and management measures.     

柳紫闪蛱蝶线粒体基因组全序列及与相关蛱蝶类的比较分析(英文)

该文对柳紫闪蛱蝶Apaturailia(鳞翅目:蛱蝶科)的线粒体基因组全序列进行了测定,同时结合其它已知蛱蝶类的相应序列进行了比较分析。结果显示:柳紫闪蛱蝶的线粒体基因组(GenBankaccessionno.:JF437925)是一个15242bp的环状DNA分子,包含13个蛋白质编码基因、2个rRNA基因和22个tRNA基因。13个蛋白编码基因中,除了COI基因的起始密码子是CGA外,其余12个蛋白编码基因都具有标准的ATN起始密码子;柳紫闪蛱蝶与其它已测的10种蛱蝶在基因定位和排列顺序方面几乎相同,只是在非编码序列上存在细微的差异,其核苷酸的构成及密码子使用频率都处于鳞翅目昆虫的范围之内。22个的tRNA基因中,除了tRNASer(AGN)缺少DHU臂,其余的tRNA基因都显示为典型的三叶草结构。基因组共存在9处基因间重叠区(总长度为33bp)以及12个基因间隔区(总长为155bp,最长间隔是49bp,最短的是1bp)。在ND6和Cytb间的间隔区中还发现有(TA)23似微卫星结构。与其他蛱蝶类相似,403bp的AT富集区包含有ATAGA,ATTTA二个保守模块(一个21bp的poly-T,一个10bp的poly-A),以及二个似微卫星的重复结构((TA)10和(TA)7)。     

Complete nucleotide sequence and organization of the mitochondrial genome of eri-silkworm,Samia cynthia ricini (Lepidoptera: Saturniidae)

Samia cynthia ricini is a commercial silk-producing insect that is now reared year-round in Korea, with the expectation of being utilized for diverse purposes. In this report, we present the complete mitochondrial genome (mitogenome) of S. c. ricini. The 15,384-bp long S. cynthia ricini mitogenome was amplified into 26 short fragments using three long overlapping fragments using primers designed from reported lepidopteran mitogenome sequences. The genome comprises 37 genes (13 protein-coding genes, two rRNA genes, and 22 tRNA genes), and one large non-coding region termed the A + T-rich region. The A/T content of the third codon position was 91.7%, which was 18.8% and 21.6% higher than those of first and second codon positions, respectively. The high A/T content in the genome is reflected in codon usage, accounting for 39.5% of A/T-composed codons (TTA, ATT, TTT, and ATA). Unlike a previous report on the start codon for the COI gene, the S. c. ricini COI gene commences with a typical ATT codon. A total of 221 bp of non-coding sequences are dispersed in 17 regions, ranging in size from 1 to 54 bp, which comprise 1.4% of the total genome. One of the non-coding sequence located between tRNA^Gln and ND2 (54 bp) has 77% sequence homology with the 5′-sequence of the neighboring ND2 gene, suggesting partial duplication of the sequence during evolution. The 361-bp long A + T-rich region contains an 18 bp-long poly-T stretch, ATAGA motif, ATTTA element, microsatellite-like A/T sequence, poly-A stretch and one tRNA-like sequence, as typically found in Lepidoptera including Bombycoidea.     

Multiple factors correlating with wing malformations in the population of Parnassius apollo (Lepidoptera: Papilionidae) restituted from a low number of individuals: A mini review

The Apollo butterfly, Parnassius apollo (Linnaeus), was common in Europe over 100 years ago, but currently it is considered as near threatened. Different conservation programs have promoted the persistence of this species;however, it is still endangered. An example of such programs was the action devoted to reestablish the Apollo butterfly population in Pieniny National Park (Poland) from only 20-30 individuals which had survived till the last decade of the 20th century. This reintroduction has been successful;however, unexpected developmental problems appeared. Butterflies with deformed or reduced wings became frequent in the population living in the natural habitat, and particularly among those reared under seminatural conditions (in the same environment, but fenced by a net). Until recently, reasons for these malformations remained unknown. However, reports published during last months indicated that there are genetic, biochemical, and microbiological factors contributing to this phenomenon. In the malformed individuals, lesions in the wingless gene and dysfunctions of laccase 1 and 2 were found to be significantly more frequent than in normal insects. A large fraction of butterflies with deformed or reduced wings was devoid of the prokaryotic symbiont Wolbachia, which was present in most normal individuals. Moreover, Yersinia pseudotuberculosis (Pfeiffer) Smith and Thai, and Serratia sp., bacteria pathogenic to insects, were detected in the biological material from both normal and malformed butterflies from this population. These findings are summarized and discussed in this review, in the light of conservation of insects and restitution of their populations from a low number of individuals.     

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.     

Isolation and characterization of polymorphic microsatellites in Parnassius apollo and Euphydryas aurinia (Lepidoptera)

Microsatellite loci were developed from Parnassius apollo and Euphydryas aurinia, two endangered Palaearctic butterfly species. Respectively, six and five polymorphic loci were characterized from an enriched partial genomic library. Genetic diversity range from three to 25 alleles for the first species and from seven to 21 for the second. Although the presence of null alleles is suspected, these polymorphic loci are likely to provide important information on the fine scale genetic structure among populations of these 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.     

A partitioned likelihood analysis of swallowtail butterfly phylogeny (Lepidoptera:Papilionidae)

Although it is widely agreed that data from multiple sources are necessary to confidently resolve phylogenetic relationships, procedures for accommodating and incorporating heterogeneity in such data remain underdeveloped. We explored the use of partitioned, model-based analyses of heterogeneous molecular data in the context of a phylogenetic study of swallowtail butterflies (Lepidoptera: Papilionidae). Despite substantial basic and applied study, phylogenetic relationships among the major lineages of this prominent group remain contentious. We sequenced 3.3 kb of mitochondrial and nuclear DNA (2.3 kb of cytochrome oxidase I and II and 1.0 kb of elongation factor-1 alpha, respectively) from 22 swallowtails, including representatives of Baroniinae, Parnassiinae, and Papilioninae, and from several moth and butterfly outgroups. Using parsimony, we encountered considerable difficulty in resolving the deepest splits among these taxa. We therefore chose two outgroups with undisputed relationships to each other and to Papilionidae and undertook detailed likelihood analyses of alternative topologies. Following from previous studies that have demonstrated substantial heterogeneity in the evolutionary dynamics among process partitions of these genes, we estimated evolutionary parameters separately for gene-based and codon-based partitions. These values were then used as the basis for examining the likelihoods of possible resolutions and rootings under several partitioned and unpartitioned likelihood models. Partitioned models gave markedly better fits to the data than did unpartitioned models and supported different topologies. However, the most likely topology varied from model to model. The most likely ingroup topology under the best-fitting, six-partition GTR + gamma model favors a paraphyletic Parnassiinae. However, when examining the likelihoods of alternative rootings of this tree relative to rootings of the classical hypothesis, two rootings of the latter emerge as most likely. Of these two, the most likely rooting is within the Papilioninae, although a rooting between Baronia and the remaining Papilionidae is only nonsignificantly less likely.     

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。     

Feeding preferences of the Apollo butterfly (Parnassius apollo ssp. frankenbergeri) larvae inhabiting the Pieniny Mts (southern Poland)

Parnassius apollo (Lepidoptera, Papilionidae) is considered to be typical stenophagous species. Its European forms fall generally into 'telephiophagous' or 'albophagous' trophic groups. According to some authors, 'telephiophagous' P. apollo ssp. frankenbergeri SLABY, inhabiting the Pieniny Mts, has a rather broad spectrum of food-plants. We aimed at defining its feeding preferences for successful breeding in a semi-natural colony on more than one commercially-available Sedum species. Larval development (L5) and performance were assessed in three experimental groups fed on different plant species selected in a preliminary test. Apollo larvae appeared to be quite specific in the plant choice and developed poorly in the absence of Sedum telephium. Possible reasons of this are discussed.