The spotted-wing drosophila (SWD), Drosophila suzukii (Diptera: Drosophilidae), originally distributed across a few Asian countries, including South Korea, has invaded North America and Europe, but is absent from Australia. In order to export the South Korean grape cultivar Campbell Early to Australia, its potential to serve as oviposition and development medium for SWD must first be determined. In this study, we determined the oviposition and development potential of SWD on Campbell Early, after elucidating the SWD life cycle and establishing an artificial diet-based mass-culturing system. An investigation of the life cycle under five temperature regimes (16, 19, 22, 25, and 28°C) showed that the durations of the egg, larval, and adult stages were shorten when temperature was increased from 16, 19, 22, 25, and 28°C, but pupal duration was shortest at 25°C and extended again at 28°C. A test of oviposition and development potential of SWD on Campbell Early grape clusters showed oviposition of 30.8 ± 6.8 eggs per cluster of injured grapes and 157.7 ± 16.2 eggs on a culture dish of artificial diet. However, in a similar experiment using uninjured grape clusters, only a single egg was deposited on the grape skin, which soon dried. In light of these results, newly harvested grapes left at vineyards during daily harvests are unlikely to serve as an oviposition and development medium for SWD, as long as the grapes remain uninjured.

Kentrochrysalis streckeri Staudinger, 1880 is distributed in Russia, Mongolia, China, and North Korea, whereas K. consimilis Rothschild & Jordan, 1903 has been known to occur South Korea and Japan. In the present study we found that the specimens from South Korea were K. streckeri, rather than K. consimilis based on morphology, DNA barcode, and nuclear elongation factor 1 alpha (EF-1α) sequences. The major morphological differences between K. streckeri and K. consimilis include the shape of wing pattern elements of fore- and hindwings and male and female genitalia. A DNA barcode analysis of the South Korean specimens and K. streckeri originated from Russia showed a maximum sequence divergence of only 0.659% (four bp), whereas the barcoding sequence of K. consimilis sequenced in this study and GenBank-registered sequence (JN678086), both of which originated from Japan showed the minimum sequence divergence of 2.965% (18 bp), indicating that the Korean specimens are, in fact, K. streckeri, instead of wrongly known K. consimilis. Phylogenetic analyses both by Bayesian Inference and maximum likelihood methods well supported the monophyly of South Korean specimens and Russian K. streckeri, excluding K. consimilis. The EF-1α-based sequence and phylogenetic analyses of the two species also supported the data from DNA barcode, indicating distribution of K. streckeri in South Korea, instead of K. consimilis.

The lycanid butterfly, Shijimiaeoides divina (Lepidoptera: Lycaenidae) is listed as the second-degree endangered wild animal in Korea from 2012. The 15,259-bp long complete mitochondrial genome of the species consisted of a typical set of genes (13 protein-coding genes, two rRNA genes and 22 tRNA genes) and one major non-coding A+T-rich region, with the typical arrangement found in majority of Lepidoptera. The 15,259-bp long S. divina mitogenome is well within the range found in Lycaenidae and has typical sets of 37 genes and a major non-coding A+T-rich region as 379 bp. As other lycanid butterflies S. divina COI also started with CGA. The gene arrangement of S. divina is identical to that of the Ditrysia in Lepidoptera that has the order trnM-trnI-trnQ (underline for inverted gene) between the A+T-rich region and ND2. Comparison of the skewness between the PCGs encoded in major and minor strand indicates a substantial difference between them in GC skewness (0.261 ~ 0.340 in minor strand vs. -0.081 ~ -0.115 in major strand). The 151-bp intergenic spacer sequence of the S. divina mitogenome is spread over 16 regions ranging in size from 1-53 bp. The longest one (53 bp) located between trnQ and ND2 shows substantially high sequence homology to neighboring ND2 may indicating the origination of the region by a partial duplication of the ND2 gene. One of the unusual features of the S. divina mitogenome is the presence of a trnK-like sequence that is encoded at the major strand of the genome in the A+T-rich region.

The spotted-wing drosophila (SWD), Drosophila suzukii (Diptera: Drosophilidae), was originally observed in a few Asian countries, but is now found even in North America and Europe. Genetic information on geographic variation and relationship may broaden our understanding of origin and migration. As a first step, in this study, a portion of mitochondrial COI gene was sequenced to understand genetic relationship and diversity in Korea. Sequencing of 104 individuals provided 57 haplotypes, with the maximum sequence divergence of 1.5%, suggesting high haplotype diversity and moderate sequence divergence. Comparison to GenBankregistered D. suzukii haplotypes (possibly from Spain, Portugal, USA) has shown 100% sequence identity to most of the haplotypes found in this study, but two USA sequences were found to be independent haplotypes, with the sequence divergence ranging from 0.5% ~ 1.4% from our samples in the 553-bp comparison. Phylogenetically, no separable group was found, but, population genetically, the only Chinese population, Sandong, was significantly differentiated (p < 0.05) from all Korean populations, without sharing any haplotype. Among 28 pairwise comparisons of Korean populations only two comparisons showed a significant genetic differentiation, indicating that no population in Korea is completely isolated. Geographically, one haplotype (SWDBA06) was relatively widespread (five among nine localities) and a few haplotypes were found in more than one locality, but most haplotypes were restricted in a locality as a single individual. Overall, high rate of per generation female migration (Nm = 0.75 ~ infinite) and low level of geographic separation (FST=0~0.40) among localities were characteristic. Current data is limited mainly to Korean localities, thus, an expanded study may provide further scrutinized analysis for the fly.

A partial sequence of the mitochondrial cytochrome oxidase subunit I (COI) gene is widely used as a molecular marker for species identification in animals, also termed a DNA barcode. However, the presence of more than one sequence type in a single individual, also known as heteroplasmy, is one of the shortcomings of barcode identification. In this study, we examined the extent and divergence of COI heteroplasmy, including nuclear-encoded mitochondrial pseudogenes (NUMTs), at the genomic-DNA level from 13 insect species, including four individuals of orthopteran Anapodisma miramae. Furthermore, a long fragment of mitochondrial DNA (~13.5 kb) and cDNA from A. miramae were used as a template for COI PCR to compare the patterns of heteroplasmy between DNA sources and to investigate a possible way to avoid ambiguity in DNA barcoding. When multiple numbers of clones originated from genomic DNA were sequenced, heteroplasmy was prevalent in all species (3~16 heteroplasmic copies), with a varying degree of maximum sequence divergence (<1% in 7 species, <4% in 3 species, <6% in 2 species and 2.15-8.03% in four A. miramae individuals). In five species, NUMTs also were observed when genomic DNA was used as a template. Long fragment DNA also is a source of heteroplasmic amplification, but the divergent haplotypes and NUMTs obtained in the genomic DNA-based PCR were not detected in A. miramae. On the other hand, cDNA was heteroplasmy-free, without NUMTs when multiple numbers of clones were sequenced. Consistently, one dominant haplotype was always obtained from the genomic DNA-origin clones in all species and also from the long fragment- and cDNA-origin clones of A. miramae. Furthermore, the dominant haplotype was identical in sequence, regardless of the DNA source. Thus, one possible solution to avoid the barcoding problem in relationship to heteroplasmy could be the acquisition of multiple numbers of barcoding sequences to determine a dominant haplotype that can be assigned as barcoding sequence for a given species.

The sister relationship between Trichoptera and Lepidoptera has often been supported in a diverse study, but mitochondrial genomes (mitogenomes) based lepidopteran phylogenetic studies have never utilized Trichoptera as outgroup mainly due to unavailability. Therefore, the effect of alternatives that were previously used (e.g., Diptera, Hymenoptera, and Orthoptera) or Trichoptera as outgroups on the lepidopteran phylogeny remained unknown. In this study, we sequenced three complete mitogenomes of Trichoptera belonging to two suborders and characterized the genomic features of Trichoptera and tested the outgroup effect for lepidopteran phylogeny. The 15,208 ~ 15,285-bp long caddisfly mitogenomes harbor gene content typical of the animal mitogenomes. The orientation and gene order of the three species belonging to the suborder Integripalpia was identical to that of the most common type that has been hypothesized as ancestral for insects, but Cheumatopsyche brevilineata belonging to another suborder Annulipalpia has rearranged QIM, all encoded in forward direction between the A+T-rich region and ND2, instead of the ancestral IQM, with Q inverted. Further, the annulipalpian species had a typical start codon ATG, instead of CGA that are commonly found in other trichopteran species and majority of Lepidoptera. Phylogenetic analysis with different outgroups (Diptera, Hymenoptera, and Orthoptera, Coleoptera, and Trichoptera) and 115 lepidopteran mitogenomes has shown insensitivity either with Trichoptera, Diptera, or Coleoptera, but artificial grouping and lowered nodal support were found with Hymenoptera. The Trichoptera-based consensus topology were: (((((((Bombycoidea + Noctuoidea) + Pyraloidea) + Papilionoidea) + Cossoidea) + Tortricoidea) + Yponomeutoidea) + Hepialoidea).

The mulberry white caterpillar, Rondotia menciana, belongs to the lepidopteran family Bombycidae, in which the domestic silkworm, Bombyx mori is included. In this study, we describe the complete mitochondrial genome (mitogenome) sequences of the species in terms of general genomic features and characteristic features found in the A+T-rich region. The 15,364-bp long genome consisted of a typical set of genes (13 protein-coding genes, two rRNA genes and 22 tRNA genes) and one major non-coding A+T-rich region, with the typical arrangement found in Lepidoptera. Twelve of the 13 PCGs start with typical ATN codons, except for the COI, which begins with CGA. Twelve of the 13 PCGs have complete stop codon, except for COII, which ends up with a single T. The 360-bp long A+T-rich region harbored the conserved sequence blocks that are typically found in lepidopteran insects. Additionally, the A+T-rich region of R. menciana contained one tRNAMet-like structure, which has a proper anticodon and secondary structure.

We newly sequenced mitogenomes of five skippers belonging to Lepidoptera to obtain further insight into characteristics of butterfly mitogenomes and performed phylogenetic reconstruction using all available gene sequences (PCGs, rRNAs, and tRNAs) from 85 species in 19 families in eight superfamilies. The general genomic features found in the butterflies also were found in the five skippers: a high A/T composition (79.3% - 80.9%), dominant usage of TAA stop codon, similar skewness pattern in various levels, consistently long intergenic spacer sequence between tRNAGln - ND2 (64-87 bp), the ATACTAA motif betweent RNASer(UCN) and ND1, and characteristic features of the A+T-rich region (the motif ATAGA, varying length of poly-T stretch, and poly-A stretch). The start codon for COI was CGA in four skippers as typical, but Lobocla bifasciatus evidently possessed canonical ATG as start codon. Phylogenetic analyses mainly yielded the consensus superfamilial relationships ((((((Bombycoidea + Noctuoidea + Geometroidea) + Pyraloidea) + Papilionoidea) + Tortricoidea) + Yponomeutoidea) + Hepialoidea) with a high support for most nodes, confirming the validity of Macroheterocera and its sister relationship to Pyraloidea. Within Rhopalocera the familial relationships (Papilionidae + (Hesperiidae + (Pieridae + ((Lycaenidae + Riodinidae) + Nymphalidae))) were strongly supported, confirming invalidity of the superfamily Hesperioidea. On the other hand, superfamilial relationships among Noctuoidea, Geometroidea, and Bombycoidea and the familial relationships among Saturniidae, Sphingidae, and Bombycidae were dubious, requiring further representative taxon sampling.

We report the complete mitochondrial genome (mitogenome) of Apodemia mormo, which belongs to the lepidopteran family Riodinidae. The 15,262-bp long complete genome is comprised of 13 protein-coding genes, two rRNA genes, 22 tRNA genes, and one major non-coding A+T-rich region, with the arrangement typically found in majority of Lepidoptera. The genes of A. mormo are interleaved with a total of 168 bp, which are spread over 16 regions and overlap in a total of 58 bp at eight locations. All tRNAs of the A. mormo mitogenome formed typical cloverleaf structure, except for tRNASer(AGN), which formed the truncated dihydrouridine arm. COI gene started with CGA, instead of canonical ATN as seen in other Lepidoptera. The 349-bp long A+T-rich region harbored the conserved sequence blocks, such as ATAGA motif, poly-T stretch, the conserved ATTTA sequence, and microsatellite A/T repeat that are typically found in Lepidoptera, but absent for tRNA-like pseudogene.

We newly sequenced mitochondrial genomes of Spodoptera litura and Cnaphalocrocis medinalis (Lepidoptera) to obtain further insight into mitochondrial genome evolution and investigated the influence of optimal strategies on phylogenetic reconstruction of Lepidoptera. Estimation of p-distances of each mitochondrial gene for available taxonomic levels has shown the highest value in ND6, whereas the lowest values in COI and COII at the nucleotide level, suggesting different utility of each gene for different hierarchical group when individual genes are utilized for phylogenetic analysis. Phylogenetic analyses mainly yielded the relationships (((((Bombycoidea + Geometroidea) + Noctuoidea) + Pyraloidea) + Papilionoidea) + Tortricoidea), evidencing the polyphyly of Macrolepidoptera. The tests of optimality strategies, such as exclusion of third codon positions, inclusion of rRNA and tRNA genes, data partitioning, RY recoding approach, and recoding nucleotides into amino acids suggested that the majority of the strategies did not substantially alter phylogenetic topologies or nodal supports, except for some familial relationship only in the amino acid dataset.

Glyphodes quadrimaculalis (Lepidoptera: Crambidae) feed on a root tuber of Cynanchum wilfordii (Asclepiadaceae) that is one of the most famous traditional medicines in Korea. The genus Glyphodes includes ~130 species distributed worldwide, so the complete mitochondrial genome (mitogenome) would be helpful for bio-identification, biogeographic studies, and multigene-based phylogeny. The 15,255-bp long G. quadrimaculalis genome comprises 37 typical genes and one large non-coding region, with the typical arrangement found in Lepidoptera. Of the 13 protein coding genes (PCGs), 12 begin with typical start codons found in insect mitochondrial PCGs, but the COI gene starts with atypical CGA. One of the noteworthy features of the genome includes the presence of a 51-bp long non-coding space sequence located between tRNAGln and ND2 that reveals high sequence homology (71.4%) to the neighboring ND2 gene, indicating the origin of the region by partial duplication of the ND2 gene.

We present the nearly complete mitogenome sequences of the garden chafer, Polyphylla laticollis manchurica, which is listed as an endangered species in Korea. The P. l. manchurica mitogenome, which includes unfinished whole A+T-rich region and a partial srRNA was 14,473-bp long, possessing typical sets of genes (13 PCGs, 22 tRNA genes, and 2 rRNA genes). Gene arrangement of the P. l. manchurica mitogenome was identical to the common one found in the majority of insects. The 5 bp-long motif sequence (TAGTA) that has been suggested to be the possible binding site for the transcription termination peptide for the major-strand was also found in the P. l. manchurica mitogenome between tRNASer(UCN) and ND1. As has been previously determined, the high A/T content was unanimously observed in P. l. manchurica in terms of A/T bias in the third codon position (73.5%) compared with the first (66.4%) and second codon position (66.2%), and a high frequency of A/T-containing codons (a total of 28.22% for TTA, ATT, TTT, and ATA). The PCGs encoded in major-strands are slightly T-skewed, whereas those of the minor-strand are A-skewed, indicating strand asymmetry in nucleotide composition in the Coleoptera including P. l. manchurica.

Hipparchia autonoe belongs to the family Nymphalidae (Lepidoptera) and is designated as an endangered insect and national monument in Korea. It only inhabits a very restricted area on Mt. Halla but is widely distributed in several Asian countries including Mongolia. A previous study conducted to understand the genetic relationship between Mt. Halla and Mongolian H. autonoe for conservation purposes suffered from a limited number of samples. Therefore, we sequenced the DNA barcode region of an additional 36 H. autonoe individuals, combined them with previous data from 19 individuals, and performed phylogenetic and population genetic analyses. Furthermore, the internal transcribed spacer 2 (ITS2) region was also sequenced from the 36 samples as a nuclear DNA marker. The existence of independent haplotypes, sequence types, and significant FST estimates (p < 0.05) between Mt. Halla and Mongolian populations indicated hampered gene flow between the populations. Nevertheless, an absence of a reciprocal monophyletic group in Mt. Halla and Mongolian populations by cytochrome oxidase subunit I gene- and ITS2-based phylogeny suggests that the genetic isolation of the Mt. Halla population from the Mongolian populations seemed not large enough to consider them independent genetic entities.

The species status of Oeneis urda (Eversmann) and O. mongolica (Oberthür) has been argued based on morphological characters. Reexamination of their major morphological characters has shown a slight differentiation in the two species. Sequences of three mitochondrial genes (COI, ND6, and ND1) and one nuclear region (internal transcribed spacer 2, ITS2) from two O. urda populations (Yangyang and Mt. Hanla) and one O. mongolica population (Uljin) were performed for phylogenetic and population genetic inferences. Sharing of identical sequences in the ND6 gene and ITS2, minimal sequence divergence in the COI and ND1 genes, and phylogenetically undividable sequence types in all mitochondrial genes and ITS2 suggest genetic continuity between the two species. Nevertheless, significant FST estimates (p < 0.05) were found for the COI gene in comparisons between Yangyang (O. urda) and Uljin (O. mongolica), between Yangyang (O. urda) and Mt. Hanla (O. urda), and between Uljin (O. mongolica) and Mt. Hanla (O. urda) populations. These FST estimates, along with other gene-based analyses collectively suggest isolation of the two species at some point in the past, but incomplete separation between the two species on the mainland (Yangyang and Uljin) and biogeographically forced isolation of the O. urda population on Mt. Hanla collectively appear to complicate species status of these two species that were once further clearly separated.

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.

The larch hawk moth, Sphinx morio, belongs to the lepidopteran family Sphingidae that has long been studied as a family of model insects in a diverse field. In this study, we describe the complete mitochondrial genome (mitogenome) sequences of the species in terms of general genomic features and characteristic short repetitive sequences found in the A+T-rich region. The 15,299-bp long genome consisted of a typical set of genes (13 protein-coding genes, two rRNA genes and 22 tRNA genes) and one major non-coding A+T-rich region, with the typical arrangement found in Lepidoptera. The 316-bp long A+T-rich region located between srRNA and tRNAMet harbored the conserved sequence blocks that are typically found in lepidopteran insects. Additionally, the A+T-rich region of S. morio contained three characteristic repeat sequences that are rarely found in Lepidoptera: two identical 12-bp repeat, three identical 5-bp long tandem repeat, and six nearly identical 5~6 bp long repeat sequences.

In the present study, the 17,694-bp long complete mitochondrial genome (mitogenome) of the dwarf honey bee, Apis florea (Hymenoptera: Apidae), is described with an emphasis on the noteworthy triplicated tRNAser(AGN) region and an extraordinary long A+T-rich region with repeat regions. The gene arrangement of A. florea mitogenome is identical to that of A. mellifera, but has triplicated tRNASer(AGN), each of which contains the precedent 44 bp-long and following another 64 bp-long repeats plus one complete first repeat abutting to tRNAMet. A total of 1,610-bp long two repeat regions in 1,987 bp-long A+T-rich region is composed of nearly identical 141 ~ 219-bp long five tandem repeats and 50 ~ 52-bp long 12 tandem repeats that are encompassed by three non-repeat sequences. One of the common interpretations for such repeat sequence is slipped-strand mispairing and unequal crossing-over events during DNA replication.

Previously, several levels of phylogenetic relationships in an insect order Odonata have been estimated using morphological and molecular markers. For the molecular phylogeny rRNA sequences were mainly, but other markers were not frequently employed. In this study, we sequenced both two mitochondrial genes (COI and 16S rRNA) and nuclear genes (28S rRNA and elongation factor-1α), composed of ~4,002 bp from 71 species of Odonata, occurring mostly in South Korea. These concatenated sequences were utilized to test the previous phylogenetic hypotheses of Odonata via Bayesian Inference (BI) and Maximum Likelihood (ML) algorithms, along with the data partition option available in BI method. Each families and superfamilies represented by multiple taxa consistently supported monophylies with the highest nodal supports in both Anisoptera and Zygoptera. A close relationship of Anisozygoptera to Anisoptera represented by a single species was obvious. On the other hand, familial relationships within each suborder of Anisoptera and Zygoptera have shown two compelling topologies. The topology obtained by BI method with partitioning of the four genes showed an unresolved relationship among Gomphidae, Aeshnidae, and the suborder Anisozygoptera in Anisoptera clade, presenting the relationships ((((Libellulidae + Corduliidae) + Macromiidae) + (Gomphidae + Aeshnidae + Anisozygoptera)) + (((Coenagrionidae + Platycnemdidae) + Calopterygidae) + Lestidae)). Another topology obtained by both BI and ML methods without partitioning, on the other hand, placed Anisozygoptera the basal lineage of Anisoptera, but Lestidae in Zygoptera was placed as the sister to Anisoptera + Anisozygoptera, presenting the relationships (((((((Libellulidae + Corduliidae) + Macromiidae) + Aeshnidae) + Gomphidae) + Anisozygoptera) + Lestidae) + ((Coenagrionidae + Platycnemdidae) + Calopterygidae)). Topological test to find out better supported tree turned out a slight higher support for the former topology, but the monophyly of Zygoptera with the inclusion of Lestidae was supported only poorly (BPP = 0.68) in the former topology.

The leaf beetle, Chrysolina aurichalcea (Coleoptera: Chysomelidae), is a pest damaging plants of Compositae. In order to understand the genetic diversity and geographic variation we sequenced a portion of mitochondrial COI gene (658 bp) and complete nuclear internal transcribed spacer 2 (ITS2) of the species collected from seven Korean localities. A total of 17 haplotypes (CACOI01 ~ CACOI17), with the maximum sequence divergence of 3.04% (20 bp) were obtained from COI gene sequence, whereas 16 sequence types (ITS2CA01 ~ ITS2CA16), with the maximum sequence divergence of 2.013% (9 bp) were obtained from ITS2, indicating substantially larger sequence divergence in COI gene sequence. Phylogenetically, the COI gene provided two haplotype groups with a high nodal support (≥ 87%), whereas ITS2 provided one sequence type group with a high nodal support (≥ 92%). The result of COI gene may suggest the presence of historical biogeographic barriers that bolster genetic subdivision in the species. Different grouping pattern between COI gene and ITS2 sequences were interpreted in terms of recent dispersal, reflected in the ITS2 sequence. Finally, finding of unique haplotypes and sequence types only from Beakryeng-Islet population was interpreted as an intact remnant of ancient polymorphism. As more samples are analyzed using further hyper-variable marker, further fruitful inference on the geographic contour of the species might be available.

The black-veined white, Aporia crataegi (Lepidoptera: Papilionoidea), is nearly extinct in South Korea, although substantial numbers of dried specimens are available. One of the common practices for such species is to launch re-introduction program after proper amount of genetic information are analyzed from donor and donee populations. In this study, we sequenced complete mitochondrial genome (mitogenome) of A. crataegi to design species-specific primers for subsequent population works and to further understand the mitogenome evolution in lepodiopteran Papilionoidea. The 15,140-bp long A. crataegi mitogenome that has typical sets of 37 genes is smallest among true butterfly species with overall slightly smaller size in genes and regions throughout the genome. Arrangement of the genome is identical to those of other lepidopteran mitogenomes, in which tRNA cluster located between the A+T-rich region and ND2 gene is translocated into tRNAMet, tRNAIle, and tRNAGln from ancestral arrangement, tRNAIle, and tRNAGln, tRNAMet. The A/T content of the genome at 81.3% is the highest in Pieridae, but lower than that of lycaenid species (81.7% ~ 82.7%) The high A/T content in the genome is also reflected in codon usage, accounting for 41.69% of A/T-composed codons (TTA, ATT, TTT, and ATA). Unlikely the diversified or modified usage of anticodon for tRNASer(AGN) the species of Pieridae including A. crataegi all unanimously have GCT that has been hypothesized as ancestral for Lepidoptera. A total of 111 bp of non-coding sequences are dispersed in 13 regions, ranging in size from 1–49 bp. Among them relatively longer ones (≥ 16 bp) all have relatively higher sequence identity to other regions of the genome, suggesting partial duplication of the sequences during A. crataegi evolution. As has been reported in some species of Lepidoptera, the A. crataegi A+T-region also has typically found conserved sequences (e.g., poly-T stretch, ATAGA motif, ATTTA element, microsatellite-like A/T sequence, and poly-A stretch) and one tRNA-like sequence, and this feature was commonly found in true butterfly species.