Bombyx mandarina (Lepidoptera: Bombycidae), the presumed ancestor of B. mori, has long been a subject of study to illustrate the geographic relationships in connection with origin of B. mori. We report 97 mitochondrial genome (mitogenome) sequences of B. mandarina collected from Korea and Japan. Phylogenetic and population genetic analyses showed that all individuals of B. mandarina collected in Korean localities formed a strong group together with all individuals originated from northern China (mainly north of the Qinling-Huaihe line) and some of southern China. This group was placed as the sister group to B. mori strians suggesting that this group had been served as an immediate progenitor for B. mori.

Bumblebee, Bombus ardens ardens (Apidae: Hymenopera), is an important resource for pollination that is most widely distributed in Korea. This study utilized microsatellite markers for investigation of genetic diversity and geographic relevance of the B. a. ardens populations in Korea. Through Next Generation Sequencing analysis, we identified 10 microsatellite markers and genotyped for 107 individuals of B. ardens collected from 10 populations. At each locus the number of alleles ranged from 10 to 23; the observed and expected heterozygosities ranged from 0.8909 to 1.0000 and 0.6641 to 0.8422, respectively; and inbreeding coefficient(FIS) ranged from –0.5053 to –0.0891. Significant deviation from the Hardy–Weinberg equilibrium was not observed at any locus. Population structure analysis indicated that there are three genetic groups in Korea with each Jeongseon and Ulleung-do composed of different gene pool from the remaining other populations. Similarly, Principal coordinates analysis also showed the same pattern. FST and RST analyses showed that each Jeongseon and Ulleung-do population had a significant genetic distance from other populations. Considering these results, genetic isolation of Ulleung-do may be explained by “Oceanic island” status and Jeongseon, which showed the positive FIS (0.069) and genetic isolation may be caused by its location on the east side of Baekdudaegan and by on-going inbreeding with a small population size.

Most traditional genome sequencing projects involving infectious viruses include culturing and purification of the virus. This can present difficulties as an analysis of multiple populations from multiple locations may be required to acquire sufficient amount of high-quality DNA for sequence analysis. The electrophoretic method provides a strategy whereby the genomic DNA sequences of the Korean isolate of Pieris rapae granulovirus (PiraGV-K) were analyzed by purifying it from host DNA by pulsed-field gel electrophoresis, thus simplifying sampling and labor time. The genomic DNA of infected P. rapae was embedded in agarose plugs, digested with a restriction nuclease and methylase, and pulsed-field gel electrophoresis (PFGE) was used to separate PiraGV-K DNA from the DNA of P. rapae, followed by mapping of fosmid clones of the separated viral DNA. The double-stranded circular genome of PiraGV-K encodes 120 open reading frames (ORFs), covering 92% of the sequenced genome. BLAST and ORF arrangement showed the presence of 78 homologs to other genes in the database. The mean overall amino acid identity of PiraGV-K ORFs was highest with the Chinese isolate of PiraGV (~99%), followed up with Choristoneura occidentalis ORFs at 58%. PiraGV-K ORFs were grouped, according to function, into 10 genes involved in transcription, 11 involved in replication, 25 structural protein genes, and 15 auxiliary genes. Genes for Chitinase (ORF 10) and cathepsin (ORF11), involved in the liquefaction of the host, were found in the genome. The recovery of PiraGV-K DNA genome by pulse-field electrophoretic separation from host genomic DNA had several advantages, compared with its isolation from particles harvested as virions or inclusions from the P. rapae host. We have sequenced and analyzed the 108,658 bp PiraGV-K genome purified by the pulsed field electrophoretic method. The method appears to be applicable to the analysis of genomes of large viruses. The chitinase, identified by PiraGV-K genome sequence, was functionally characterized by quantitative PCR, Western blot analysis, immunohistochemistry and transmission electron microscopy.

Lepidoptera is one of the largest insect orders, but the phylogenetic relationships within this order, have yet to be completely described. One of the unresolved relationships includes the monophyly of Papilionoidea in relationship with the monotypic superfamily Hesperioidea. We newly sequenced five hesperid mitochondrial genomes (mitogenomes), representing four subfamilies: Pyrginae (Daimio tethys and Lobocla bifasciatus), Coeliadinae (Choaspes benjaminii), and Hesperiinae (Potanthus flavus), and Heteropterinae (Carterocephalus silvicola). Along with these newly sequenced hesperid genomes phylogenetic analysis was conducted with all available lepidopteran mitogenomes including three reported species of Hesperiidae that consisted of ~70 species in ten lepidopteran superfamilies. The test for the effect of optimization schemes, such as exclusion and inclusion of third codon position of 13 PCGs, other genes (22 tRNAs and two rRNAs), and with and without partitions also was performed. Majority of datasets consistently placed the monophyletic Hesperiidae the sister to ((Pieridae + Lycaenidae) + Nymphalidae), placing another true butterfly family Papilionidae as the basal lineage of this group, presenting the relationships (Papilionidae + (Hesperiidae + ((Pieridae + Lycaenidae) + Nymphalidae))). Consistent to previous result, Pyraloidea was placed as the sister to ((Bombycoidea + Geometroidea) + Noctuoidea), placing the Macrolepidoptera as non-monophyletic group.

Gene arrangement in the mitochondrial genome (mitogenome) has been regarded as an important evolutionary event that is useful as a phylogenetic signal. The mountainous duskywing, Erynnis montanus, belongs to a lepidopteran family Hesperiidae. We sequenced 15,530-bp long complete mitogenome of the species. The genome has the typical gene content of animals (13 protein-coding genes, two rRNA genes, 22 tRNA genes, and one major non-coding A+T-rich region). Further, E. montanus mitogenome also contained a high A/T content in the whole genome (81.7%) and the CGA (arginine) as the start codon for the COI gene, as typical in lepidopteran mitogenome. However, unlike other lepidopteran species, including two sequenced skippers, the E. montanus mitogenome has a unique arrangement tRNASer-tRNAAsn, instead of the tRNAAsn-tRNASer found unanimously in other lepidopteran species, providing a new gene arrangement in Lepidoptera. Such rearrangement probably was likely caused by duplication of gene block tRNASer-tRNAAsn and subsequent random loss of tRNAAsn in the first copy and tRNASer in the second copy, resulting in the arrangement tRNASer-tRNAAsn. Considering current phylogenetic relationships among available lepidopteran groups in connection with lepidopteran gene arrangement the new gene arrangement found in E. montanus seems to be apomorphy, requiring cautious interpretation as a phylogenetic signal.

The phylogenetic relationships among the Nymphalidae (Lepidoptera: Papilionoidea) have been controversial in several perspective. The present study sequenced a total of ~ 3,500 bp from cytochrome oxidase subunit I (COI), 16S ribosomal RNA (16S rRNA), and elongation factor-1 alpha (EF-1α) in 80 nymphalid species belonging to seven subfamilies (Linmenitidinae, Heliconiinae, Nymphalinae, Apaturinae, Libytheinae, Satyrinae, and Danainae), along with those of six lycaenid species as outgroups. Phylogenetic analyses via Bayesian Inference (BI) and Maximum Likelihood (ML) algorithms concordantly supported the subfamilial relationships of (((((Linmenitidinae + Heliconiinae) + (Nymphalinae + Apaturinae)) + Libytheinae) + Satyrinae) + Danainae), with high nodal support for monophyletic subfamilies and tribes. This result is largely consistent with a previous study performed with a substantially large sequence information and morphological characters, except for the position of Libytheinae that has previously been placed as the sister to all reminder of Nymphalidae.

The complete mitochondrial genome sequence of the nerippe fritillary butterfly, Argynnis nerippe, which is listed as an endangered species in Korea, is described with an emphasis on the A+T-rich region. The 15,140-bp long circular molecule consisted of 13 protein-coding genes, two rRNA genes, 22 tRNA genes and one control region, known in insect as the A+T-rich region, as found in typical metazoans. The 329-bp long A+T-rich region located between srRNA and tRNAMet possessed the highest A/T content (95.7%) than any other region of the genome. Along with the several conserved sequences found typically in the lepidopteran insects the genome contained one tRNAMet-like and tRNALeu(UUR) -like sequence in the A+T-rich region.

The phylogenetic relationships among the Nymphalidae (Lepidoptera: Papilionoidea) have been controversial. The present study sequenced approximately 1,099 bp from cytochrome oxidase subunit I (COI), 1,336 ~ 1,551 bp from 16S ribosomal RNA (16S rRNA), and 1,066 bp from elongation factor-1 alpha (EF-1α) in 80 species belonging to seven subfamilies (Linmenitidinae, Heliconiinae, Nymphalinae, Apaturinae, Libytheinae, Satyrinae, and Danainae) of Nymphalidae, along with those of six lycaenid species as outgroups. The average base compositions for the three genes (COI, 16S rRNA, and EF-1α) are as follows: A (30.6%, 38.8%, and 25.8%), G (14.7, 5.2%, and 23.6%), T (39.8%, 45.2%, and 23.4%), and C (14.9%, 10.8%, and 27.3%). This result shows the A/T bias in the mitochondrial genes, but not for the nuclear EF-1α. Between the two mitochondrial genes, the 16S rRNA gene evidenced a significantly higher A/T content than was detected in the COI gene. These sequences were subjected to phylogenetic reconstruction via Bayesian Inference (BI) and Maximum Likelihood (ML) algorithms. Both analyses concordantly supported the subfamilial relationships of (((((Linmenitidinae + Heliconiinae) + (Nymphalinae + Apaturinae)) + Libytheinae) + Satyrinae) + Danainae), along with highly supported monophyletics of tribes within subfamilies. This result is largely consistent with a previous study performed with a large sequence information and morphological characters, except for the position of Libytheinae, which was suggested to be the basal lineage of Nymphalidae.

The phylogenetic relationships among true butterfly families (superfamily Papilionoidea) have been a matter of substantial controversy, and that debate has led to several competing hypotheses. Two of the most compelling of those hypotheses involve the relationships of (Nymphalidae + Lycaenidae) + (Pieridae + Papilionidae) and (((Nymphalidae + Lycaenidae) + Pieridae) + Papilionidae). In this study, approximately 3,500 nucleotide sequences from cytochrome oxidase subunit I (COI), 16S ribosomal RNA (16S rRNA), and elongation factor-1 alpha (EF-1α) were sequenced from 83 species belonging to four true butterfly families, along with those of eight outgroup species belonging to the skipper family (superfamily Hesperioidea). These sequences were subjected to phylogenetic reconstruction via Bayesian Inference (BI), Maximum Likelihood (ML), and Maximum Parsimony (MP) algorithms. All phylogenetic analyses among the four true butterfly families strongly indicated a sister relationship between the Nymphalidae and Lycaenidae on one hand, and relatively strongly indicated a sister relationship between the Pieridae and Papilionidae on another hand, thus supporting the hypothesis: (Nymphalidae + Lycaenidae) + (Pieridae + Papilionidae).

Two complete mitochondrial genomes (mitogenomes) of the endangered, lycaenid butterflies, Spindasis takanonis and Protantigius superans (Lepidoptera: Lycaenidae), were sequenced. Each 15,349 bp and 15,248 bp-long genome contained both the lepidopteran specific gene arrangement that differ from the most common arrangement of insects by the movement of tRNAMet to a position 5’-upstream of tRNAIle. Neither of the species have typical COI start codon. Instead, the CGA (arginine) sequence that is commonly present in all other lepidopterans was also found in both lycaenids. The possible binding site for the transcription termination peptide, TACTA sequence, also was well retained in both species. The high A+T-content, which is a characteristic of insect mitogenomes was well reflected in the genomes in the form of higher frequency of codons with A/T nucleotides, severe A/T bias in 3rd codon position, and extremely high A/T content in the A+T-rich region. The 19 bp-long poly-T stretch and the downstream conserved motif ATAG, which were suggested previously to function as a structural signal for minor-strand mtDNA replication, was also well conserved in the A+T-rich region of both lycaenids. Phylogenetic analysis among lepidopteran superfamilies supported the relationships of either (((((Bombycoidea + Geometroidea) + Noctuoidea) + Papilionoidea) + Pyraloidea) + Tortricoidea) by concatenated amino acid sequence or (((((Bombycoidea + Geometroidea) + Noctuoidea) + Pyraloidea) + Papilionoidea) + Tortricoidea) by concatenated nucleotide sequences of 1st and 2nd codon positions of 13 protein-coding genes, two rRNA genes, and 22 tRNA genes, revealing fluctuating positions of Papilionoidea and Pyraloidea between the two data sets.