The Scarites aterrimus (Coleoptera: Carabidae), is one of the carabid beetles dwelling exclusively on coastal sandy dunes. Recent habitat deterioration has greatly concerned population declines in several species dwelling on the coastal sandy dunes. Asa first step to establish long-term conservation strategy, we investigated the nation-wide magnitude and nature of genetic diversity of the species. As a first step, we sequenced a portion of mitochondrial COI gene, corresponding to “DNA Barcode” region (658 bp) from a total of 24 S. aterrimus individuals collected over nine sandy dunes belonging to four Korean provinces. The sequence analysis evidenced moderate to low magnitude of sequence diversity compared with other insect species distributed in Korean peninsula (0.152% to 0.912%). The presence of closely related haplotypes and relatively high gene flow estimate collectively suggest that there had been no historical barriers that bolster genetic subdivision. Population decline was postulated on the basis of several missing haplotypes that are well found in the species with a large population size. This interpretation is consistent with field observation of small population size in the coastal sandy dune habitats. The highest genetic diversity estimates were found in the coastal sand dune population of Seogwipo, Jeju Island, justifying a prior attention to the population, in order to sustain overall genetic diversity of the species. Further scrutinized study might be required for further robust conclusion.

The leaf beetle, Chrysolina aurichalcea (Coleoptera: Chysomelidae), is a pest damaging plants of Compositae. In order to understand the genetic diversity and geographic variation of the species we sequenced a portion of mitochondrial COI gene (658 bp) and complete nuclear internal transcribed spacer 2 (ITS2) collected from seven Korean localities. A total of 18 haplotypes (BARCA01 ~ BARCA18), with the maximum sequence divergence of 3.04% (20 bp) were obtained from COI gene sequence, whereas 17 sequence types (ITS2CA01 ~ ITS2CA17), with the maximum sequence divergence of 2.013% (9 bp) were obtained from ITS2, indicating substantially larger sequence divergence in mitochondrial gene sequence. Phylogenetically, the mitochondrial DNA has shown several haplotypes formed independent groups with substantially high node support (≥ 90%), whereas no such grouping was evidenced for ITS2, indicating different behaviors of the two molecules. Such difference may reflect a diverse dynamics of the species such as biogeographic history, mating behaviors, and also possibly different mode of inheritance of the two molecules, but requires further scrutinized examination of the dataset. In terms of population genetic perspective, overall no population subdivision was detected from both molecules, except for locality 7 (Eocheong islet) from mitochondrial DNA. As more scrutinized analysis is performed, further fruitful inference on the geographic contour of the species might be available.

The seven-spotted lady beetle, Coccinella septempunctata (Coleoptera: Coccinellidae), known also as the seven-spot ladybird, is natural enemy for aphids and has a broad ecological range, living almost anywhere there are aphids for it to eat. In order to understand the genetic diversity and geographic variation of the species we sequenced a portion of mitochondrial COI gene (658 bp) and complete nuclear internal transcribed spacer 2 (ITS2) collected from nine Korean localities. A total of 21 haplotypes (CSCOI01 ~ CSCOI21), with the maximum sequence divergence of 4.56% (30 bp) were obtained from COI gene sequence (from 78 individuals), whereas 65 sequence types (CSITS201 ~ CSITS265), with the maximum sequence divergence of 2.06% (11 positions) were obtained from ITS2 (from 79 individuals), indicating substantially larger sequence divergence in mitochondrial gene sequence. Both COI gene and ITS2 shows the distribution pattern that only a few haplotypes or sequence types are widely distributed, whereas majority of them are highly restricted in one geographic location, even represented as a single individual. Unlikely the ITS2 sequence types the mitochondrial COI haplotypes evidenced the presence of two main phylogenetic groups, reciprocally monophyletic to each other. Geographically, these two groups are spread in all localities surveyed. Considering both COI gene and ITS2 sequence together, current our data may suggest the presence of ancestral polymorphism, rather than on-going speciation, but more scrutinized analysis will be performed soon. Due partially by the presence of both COI groups in all surveyed localities, the genetic diversity estimates of all localities are similar from the perspective of COI gene, but ITS data showed extremely lower genetic diversity of one islet locality, Anmyeon-do (locality 2; 0.002530 vs. 0.008054 ~ 0.012060). Analysis of gene flew estimates between localities indicates that most populations are highly interconnected to each other. However, one islet locality, Anmyeon-do (locality) has shown statistically significant distance from the remaining localities on the basis of only ITS2 data (FST = 0.19 ~ 0.34), requiring scrutinized phylogeographic inference on this population with expanded sampling. As more scrutinized analysis is performed, further fruitful inference on the geographic contour of the species might be available.

The seven-spotted lady beetle, Coccinella septempunctata (Coleoptera: Coccinellidae) has a broad ecological range, living almost anywhere there are aphids for feeding. In this study, we isolated and characterized a total of 10 microsatellite loci from the species. The loci were validated and characterized using 25 samples collected from five Korean localities. The number of alleles and heterozygosity observed at each locus ranged from 4 to 16 and from 0.37 to 0.89, respectively. None of the loci deviated significantly from Hardy-Weinberg equilibrium and there was no indication of significant linkage disequilibrium among pairs of loci. These microsatellite markers should be very valuable markers for population genetic studies of Coccinella septempunctata.

The dung beetle, Copris tripartitus, (Coleoptera: Scarabaeidae), is one of the insect species listed as endangered wildlife in Korea. In order to establish conservation strategy an investigation on nation-wide genetic magnitude and nature of genetic diversity would be required. In this study, we sequenced each partial sequences of mitochondrial COI (658 bp) and CytB (433 bop) genes and complete internal transcribed spacer 2 (ITS2) of the nuclear ribosomal DNA (411~420 bp) from 70 individuals of C. tripartitus collected from five Korean localities. The sequence divergence of mitochondrial genes was unexpectedly substantial as 5.0% (33 bp) for COI and 4.6% (20 bp) for CytB, whereas that of ITS2 was only 1.9% (6 bp), revealing much higher variability in mitochondrial DNA. In phylogenetic analysis, each 57 and 47 haplotypes obtained from COI and CytB gene sequences was subdivided into two groups (groups A and B), but the node supports for each group was not strong enough to consider each group as independent monophyletic groups in both genes. This interpretation was further supported by the same analysis with ITS2, showing no subdivision at all. These findings reinforce importance for the inference of phylogenetic result using both mitochondrial and nuclear encoded sequences. The C. tripartitus occurring in Korean peninsula was genetically well connected to each other and no obvious population genetic structure was detectable.

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.

Two complete mitochondrial genomes of the tobacco cutworm, Spodoptera litura (Lepidoptera: Noctuoidea) and the rice leaf roller, Cnaphalocrocis medinalis (Lepidoptera: Pyralidae), were sequenced. Each 15,388 bp and 15,368 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 other lepidopterans was also found both in S. litura and C. medinalis. The evolutionary rates among 13 protein-coding genes (PCGs) in Lepidoptera showed ATP8 the highest, whereas COI the lowest. The high A+T-content, which is characteristic of mitochondrial genome was well reflected in the two lepidopteran mitochondrial genomes: higher frequency of A/T-rich codons, severe A/T bias in 3rd codon position, and extremely high A/T content in the A+T-rich region. Because insect mitochondrial genomes harbor biased nucleotide and resultantly biased amino acid sequences, phylogenetic inference is often misled by them. Although each recoded and unrecoded datasets for nucleotide sequences and amino acid sequences of PCGs provided overall identical topology, regardless of recoded scheme, each nucleotide and amino acid dataset provided difference in the status of Macrolepidoptera, providing a monophyletic group by amino acid dataset, whereas non-monophyletic group by nucleotide dataset.

Attacin is a well-studied glycine-rich antibacterial protein in insect immune response, which has limitary antibacterial effect to some Gram-negative bacteria. A cDNA encoding the attacin gene was screened and isolated from the immunized larvae of the swallowtail butterfly, Papilio xuthus. The complete P. xuthus attacin cDNA is 949 nucleotides encoding a 250 amino acid precursor that contains a putative 18-residue signal peptide, a common 42-residue propeptide sequence and a presumed 190-residue mature protein with a theoretical mass of 19904.01 and a pI of 9.13. The putative mature protein of P. xuthus attacin showed 48%~52% and 24%~30% identity in amino acid sequences with that of lepidopteran and dipteran insects, respectively. The attacin transcript was induced at significant level after injection with bacterial lipopolysaccharide (LPS). Recombinant attacin was highly expressed in E. coli BL21 (DE3) cells by fusing with an N-terminal S-tag/thrombin cleavage site configuration protein to avoid the cell death during induction. The expressed fusion protein was purified by Ni-NTA immobilized metal affinity chromatography (IMAC). After desalting and cleavage with thrombin, the recombinant attacin was released and showed considerably antibacterial activity against Gram-negative bacteria, E. coli ML 35. Our results proved that this protein family with a potent antibacterial activity may play a role in the immune response of butterflies.

We analyzed the chirp sound and behavior of Teleogryllus emma with observation system, which was consisted of computer, ccd-camera and microphone. Computational methods of wavelet transformation and Self-Organizing Maps (SOM) were utilized to characterized the chirp sound of insect species for automatic counting in this study. Wavelets were initially applied to feature extraction of the chirp sound. Wavelet coefficients were accordingly calculated based on the basis function (e.g., Morlet). The obtained coefficients were subsequently provided to count number of chirps in each song. Sound structure of insect specimens consisted with long chirp and short chirp and the patterns of song were grouped by frequency of long chirp and short chirp. The song patterns of insect specimens were divided by Self-Organizing Map (SOM) that was used number of chirp as input data. Application of computational methods to automatic detection of chirp sound was further discussed for obtaining objective assessment in behavior science.

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).

The bumblebee, Bombus ignitus (Hymenoptera: Apidae), is a valuable natural resource that is one of the most notably utilized for greenhouse pollination in Korea. In order to understand the nature of genetic relationships, gene flow, and population structure of the species we sequenced a partial COI gene of mitochondrial DNA (mtDNA) corresponding to “animal barcode” region and the complete internal transcribed spacer 2 (ITS2) of the nuclear ribosomal DNA (nrDNA) collected from Korean localities. Although the 658-bp long mtDNA sequence provided only six haplotypes with the maximum sequence divergence of 0.61% (4 bp), the ITS sequences provided 84 sequence types with the maximum sequence divergence of 1.02% (21 sites), confirming better applicability of the ITS sequences to the study of intraspecific variation. The complete ITS2 sequences of B. ignitus were shown to be longest among known insects, ranging in size from 2,034 bp ~ 2,052 bp, harboring two duplicated repeats. Overall, a very high per generation migration ratio, a very low level of genetic fixation, and no discernable hierarchical population/ population group were noted to exist among populations of B. ignitus on the basis of both molecules, thus suggesting that the B. ignitus populations on the Korean peninsula are panmictic, which is consistent with our understanding of the dispersal capability of the species

In an effort to gain a better understanding of the nature of the population genetic structure of the pest insect, Spodoptera litura (Lepidoptera: Noctuidae), tobacco cutworms were collected from six Korean and five Chinese localities and their mitochondrial A+T-rich region and nuclear internal transcribed spacer 2 (ITS2) regions were cloned and sequenced. A total of 106 A+T-rich region haplotypes and 92 ITS2 sequence types were obtained from 158 individuals. Compared to the preliminary mitochondrial COI gene sequence data that provided all identical sequence types in all samples, the A+T-rich region and nuclear ITS2 are highly variable. Overall, a low level of genetic fixation in the A+T-rich region (FST = 0 ~ 0.02965) and nuclear ITS2 (FST = 0 ~ 0.34491), and no discernable isolated population was noted to exist among most S. litura populations. The presence of a majority of within-population variations, rather than variations among populations or between China and Korea, indicates that the S. litura populations are composed of heterogeneous individuals. The fixation index at hierarchical level of among regions shows no significance, suggesting that the S. litura populations in both Korea and China are profoundly interrelated with one another. This finding is in accord with the current knowledge that S. litura has sufficient flight capacity for dispersal.

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.

A four-year-old sexually intact male dog and a seven-year-old sexually intact female dog presenting with heavy tick infestation were tentatively diagnosed with tick paralysis. Both dogs died due to respiratory failure. A number of ticks were observed in the head, ears, neck, trunk, and limbs of each dog. In these cases, a presumptive diagnosis of tick paralysis could be based on the presence of a species of tick known to cause paralysis combined with the absence of any other disease previously. Most ticks were identified as Haemaphysalis longicornis according to morphological features. Tick paralysis in dogs has rarely been reported in Korea.

In order to understand the nature of population genetic structure of the notorious pest insect, Spodoptera litura (Lepidoptera: Noctuidae), a total of 159 individuals of the tobacco cutworm collected from six Korean and five Chinese localities were cloned and sequenced their mitochondrial A+T-rich region. A total of 90 haplotypes ranged 324 bp ~ 372 bp in length and 0.30% ~ 4.85% in sequence divergence. Compared to the preliminary mitochondrial COI gene sequence data that provided nearly identical sequences in all samples, the A+T-rich region provided substantially high sequence variation, validating the applicability of this molecule to the study of intraspecific variation. Overall, a very high per generation migration ratio (Nm = 5.91 ~ infinite), a very low level of genetic fixation (FST = 0 ~ 0.077796), and no discernable isolated population were noted to exist among populations of S. litura, although some significant genetic differentiations were found between several pairs of populations. These results collectively suggest that the S. litura populations in both Korea and China are very well interrelated to each other. The structural analysis revealed that the S. litura A+T-rich region harbored two stretches of the [TA(A)]n sequence, the poly-A stretch, and a stem-and-loop structure that are well found in other lepidopteran A+T-rich region. Furthermore, the region harbors the 13 ~ 19-bp long T stretch and 5-bp long motif that have been suggested to function as a possible recognition site for the initiation of replication of the minor strand of mtDNA also were found.

The bumblebee, Bombus ignitus (Hymenoptera: Apidae), is a valuable natural resource that is one of the most notably utilized for greenhouse pollination in Korea. In order to understand the nature of genetic relationships, gene flow, and population structure of the species we sequenced a partial COI gene of mitochondrial DNA (mtDNA) corresponding to “animal barcode” region and the complete internal transcribed spacer 2 (ITS2) of the nuclear ribosomal DNA (nrDNA) with the individuals collected from Korean localities. Although the 658-bp long mtDNA sequence provided only six haplotypes with the maximum sequence divergence of 0.456% (3 bp, from 91 individuals), but the ITS sequences provided 33 sequence types with the maximum sequence divergence of 0.78% (16 bp, from 35 individuals), confirming better applicability of the ITS sequences to the study of intraspecific variation. The ITS2 sequences of B. ignitus were shown to be the longest among known insects, ranging in size from 2,034 bp ~ 2,045 bp and harbored two duplicated repeats, indicating unusual structure of B. ignitus ITS2 sequences compared with other insect ITS sequences. Overall, a very high per generation migration ratio, a very low level of genetic fixation, and no discernable hierarchical population/population group were noted to exist among populations of B. ignitus on the basis of both molecules, thus suggesting that the B. ignitus populations on the Korean peninsula are panmictic, which is consistent with our understanding of the dispersal capability.