Cryopreservation and in vitro fertilization (IVF) protocols are important in genetic studies and applications to transgenic animals. Various studies about boar sperm cryopreservation have been studied for a long time. Those were about the use of extenders, the choice of sugars, the cooling and warming rates. The factors that influence the boar sperm are the dramatic changes in temperatures, osmotic and toxic stresses, and reactive oxygen species (ROS) generation. Among these factors, ROS generation is the main damage to DNA which is a principal genetic material and the most important for the practical applications. So we wondered whether ROS generation could be reduced. In previous study, monothioglycerol (MTG) was essential for the culture of embryo stem cells. Therefore we added MTG in the freezing extender based on lactose-egg yolk (LEY) with trehalose. For the assessment of the frozen-thawed sperm, we focused onmotility, membrane integrity and DNA damage. First, we used a computer-aided sperm analysis system for overall conditions of sperm such as motility and viability. Then we performed the sperm chromatin structure assay for DNA integrity and hypo-osmotic swelling test for membrane integrity. And our result showed the existence of MTG in the freezing extender caused less damage to DNA and higher motility in frozen-thawed boar sperm. Also we checked a relative antioxidant activity of MTG in modified Modena B extender. We concluded that this reagent can activate sperm mitochondria at MTG 0.2 μM, contribute to sperm motility and DNA integrity but there was no significant difference on membrane integrity. Also antioxidant activity of MTG in modified Modena B extender was proved.

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.

The bumblebee, Bombus ignitus (Hymenoptera: Apidae), is a valuable natural resource that is widely utilized for greenhouse pollination in South Korea. Understanding the magnitude of genetic diversity and geographic relationships is of fundamental importance for long term preservation and utilization. As a first step, we sequenced a partial COI gene of mitochondrial DNA (mtDNA) corresponding to the “DNA barcode” region and the complete internal transcribed spacer 2 (ITS2) of nuclear ribosomal DNA from 88 individuals collected in nine South Korean localities. The complete ITS2 sequences were longest among known insects, ranging in size from 2,034 bp ~ 2,052 bp, harboring two duplicated 112-bp long repeats. The 658-bp long mtDNA sequences provided only six haplotypes with a maximum sequence divergence of 0.61% (4 bp), whereas the ITS sequences provided 84 sequence types with a maximum sequence divergence of 1.02% (21 sites). The combination of the current COI data with those of published data suggest that the B. ignitus in South Korea and China are genetically a large group, but those in Japan can be roughly separated into another group. Overall, a very high per generation migration ratio, a very low level of genetic fixation, and no discernable hierarchical population were found to exist among the South Korean populations of B. ignitus, which suggests panmixia. This finding is consistent with our understanding of the dispersal capability of the species.

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.

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.

Worldwide studies on Apis cerana variation for biogeography and genetic diversity depended largely on a 86~93 bp-long mitochondrial non-coding region (internal spacer region) located between tRNALeu and COII (named as NC2), possibly due to higher variability among available markers. In order to incorporate the A. cerana occurring in South Korea into world extensive data, we also sequenced the NC2 from 118 A. cerana samples collected over nine Korean localities and 66 A. cerana samples over seven Asian localities, such as China, Vietnam, and Thailand. These data were combined with preexisting world data to scrutinize genetic relationships of A. cerana in South Korea to outside distributional range. Sequencing of 184 samples provided a total of ten haplotypes: five from Korea, six from China, one from Vietnam, and two from Thailand. Among them eight were new, whereas two were previously reported ones. Phylogenetic analysis of A. cerana NC2 haplotypes so far found including ours has confirmed the presence of four major groups of A. cerana (Asian mainland group, Sundaland group, Palawan group, and Luzon-Mindahnao group) and all haplotypes found in this study also were included in the Asian mainland group. In order to find further variable regions that can be used as sequence-based marker several mitochondrial non-coding regions and nuclear intron regions are in the middle of testing.

콩과(Fabaceae) 작물 해충인 팥나방(Matsumuraeses phaseoli)과 어리팥나방(M. falcana) (나비목: 잎말이나방과)은 형태적으로 매우 유사하여 종 구별이 힘든 것으로 알려져 있다. 본 연구에서는 PCR-SSP(PCR with Sequence Specific Primers) 방법으로 두 종을 빠르고 정확하게 구별할 수 있는 판별법을 찾고자 두 종의 미토콘드리아 시토크롬 옥시다제 I(mtCOI) DNA 부분영역(439 bp)의 염기서열을 해독하였다. 그리고 다른 나방 종의 mtCOI 염기서열과 함께 나열하여 비교한 후 팥나방과 어리팥나방에서 종 특이적으로 차이가 나는 단일 뉴클레오티드를 프라이머의 3ʹ말단으로 하는 염기서열 특이 프라이머 조합을 만들었다. PCR 산물들을 전기영동 한 결과, 어리팥나방은 245 bp, 팥나방은 409 bp와 245 bp의 특이적 밴드 패턴을 보여 두 종을 구별할 수 있었다.

Tumor cells express altered metabolic activities often linked to mitochondrial dysfunction. Such mitochondrial defects can inhibit oxidative phosphorylation, change the cellular redox status (NAD+/NADH), increase production of reactive oxygen species (ROS), and cause DNA damage that further supports tumorigenesis and a metastatic phenotype1,2. Mitochondrial Complex I (NADH dehydrogenase) is a major site of ROS production in mitochondria and regulator of the NAD+/NADH ratio. This study is focused on mitochondrial complex I as a possible modulator of tumorigenesis and progression in breast cancer. We used NADH dehydrogenase from yeast, called NDI1, to augment complexI activity in metastatic human breast cancer cells. We followed NDI1 functionality and impact on tumor cell behavior in vitro and tumor progression in vivo. Augmentation of NADH dehydrogenase activity through NDI1 resulted in an enhanced NAD+/NADH ratio and slight inhibition of ROS production. Importantly, NDI1 expression inhibited metastasis and tumor growth in the mammary fad pad of immune deficient mice, as seen by non-invasive bioluminescence imaging and histology. The mechanisms involve NDI1-induced inhibition of the AKT/mTOR survival pathway and autophagy stimulation. Knock-down of ATG5 partially reversed the anti-metastatic effect of NDI1, demonstrating that enhancement of autophagy is responsible for NDI1-mediated inhibition of breast cancer spreading. The results indicate that mitochondrial complex I activity can drastically impact tumorigenesis and metastasis in breast cancer, and that augmentation of complex I function through NDI1 can inhibit tumor formation and cancer progression through NAD+/NADH ratio modulation.

Three tortricid pests, Grapholta dimorpha (Komai), G. molesta (Busck), and Carposina sasakii (Matsumura) are known as internal apple feeders in Korea. For identify young larvae which occurring serious damage in fruits, the molecular maker was developed from their mitochondrial DNA (mtDNA) sequences. To develop of PCR-RFLP marker, ND4 locus was digested with Swa Ⅰ. ND4-Swa Ⅰ digests showed two bands (396, 292 bp), one band (700 bp), and three bands (408, 178, and 103 bp) of G. dimorpha, G. molesta, and C. sasakii, respectively. Species-specific diagnostic PCR primers were developed in the ND4 locus and gave species-specific PCR products. Finally, these markers were applied to diagnose larvae infesting apples and showed species-specific fruit damage patterns, in which most feeders of G. dimorpha, G. molesta, and C. sasakii showed major feedings in apex, surface, and core of apple fruits, respectively.

The ant species, Vollenhovia emeryi, is distributed in Far East. The species can be divided into two major groups by their wing morphology of reproductives: short-winged and long-winged. A nationwide survey of the species was conducted for analyzing the mitochondrial haplotype diversity and genetic population structure. We collected 91 samples from 40 locations. A total of the 1239 bp partial COI (cytochrome C oxidase 1) region was used for the analyses. We found the total of 21 haplotypes. The mitochondrial haplotypes may correspond to the wing morphology. The genetic population structure examined potential geographic barriers of gene flow such as distance, mountains, rivers and plains which are non-mountain areas to prevent dispersal through mountain range. The result implied that no barriers considered in this study affected differently gene flow. Therefore, the behavioral characteristics of the ant may be the causal constraint of its genetic exchange.

Recent studies indicate that reactive oxygen species (ROS) are critically involved in persistent pain primarily through spinal mechanisms, and that mitochondria are the main source of ROS in the spinal dorsal horn. To investigate whether mitochondrial ROS can induce changes in mem¬brane excitability on spinal substantia gelatonosa (SG) neurons, we examined the effects of mitochondrial electron transport complex (ETC) substrates and inhibitors on the membrane potential of SG neurons in spinal slices. Application of ETC inhibitors, rotenone or antimycin A, resulted in a slowly developing and slight membrane depolarization in SG neurons. Also, application of both malate, a complex I substrate, and succinate, a complex II substrate, caused reversible membrane depolarization and enhanced firing activity. Changes in membrane potential after malate exposure were more prominent than succinate exposure. When slices were pretreated with ROS scavengers such as phenyl-N-tert-buthylnitrone (PBN), catalase and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), malate-induced depolarization was significantly decreased. Intracellular calcium above 100 µM increased malate-induced depolarization, witch was suppressed by cyclosporin A, a mitochondrial permeability transition (MPT) inhibitor. These results suggest that enhanced production of spinal mitochondrial ROS can induce nociception through central sensitization.

The two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), is one of the most important pest species devastating many horticultural, ornamental crops and fruit trees. Difficulty in managing this mite is largely attributed to its ability to develop resistance to many acaricides. Development of 3,700 folds resistance to etoxazole was found in the population of T. urticae collected from rose greenhouses in Buyeo, Chungnam Province in August 2000. This population has been selected for eleven years with etoxazole (over 500 times), and increased over 5,000,000 fold in resistance as compared with susceptible strain (S). Etoxazole-resistant strain was shown to be maternally inherited. The objective of this study was to determine whether resistance of T. urticae to etoxazole was linked with point mutations in the mitochondrial gene. DNA sequencing of cytochrome c oxidase subunit I (COX1), COX2, COX3, cytochrome b (CYTB), NADH dehydrogenase subunit 1 (ND1), ND2, ND3, ND4, ND5, and ND6 were analyzed by comparing two isogenic etoxazole-susceptible (EtoS) and etoxazole-resistant (EtoR) strains. As a result, all genes revealed no point mutations between the two strains.

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.

The complete mitogenome (20,456 bp) of Challia fletcheri (Dermaptera: Pygidicranidae) as the first dermapteran insect is the longest among sequenced insects. The genome contained typical gene sets, but harbored the largest TRU among Exopterygota and Palaeoptera. The AT- and GC-skewness showed more Ts and Gs encoded on the major strand, whereas more As and Cs on the minor strand, presenting a reversal to the general pattern found in most insect mitogenomes. This pattern was explained in terms of inversion of replication origin. The gene arrangement of C. fletcheri genome is unique in insects and differs from the ancestral type found in insects by a series of gene translocations and/or inversions. We hypothesize that the markedly different gene arrangement is probably due to some unique organism-level properties, which allow relaxed selection against mitochondrial gene rearrangement. All phylogenetic analyses consistently placed Orthoptera as the sister to the group composed of a monophyletic Isoptera + Mantodea + Blattodea and a monophyletic Grylloblattodea + Mantophasmatodea + Phasmatodea, and placed Dermaptera as the sister to Plecoptera, leaving them as the most basal lineage of Polyneoptera.

The Samia cynthia ricini (Lepidoptera: Saturniidae) is a commercial silk-producing insect belonging to an insect family Saturniidae in Bombycoidea. The species that has presumably been originated in India, is distributed in India, China, and Japan. Unlikely domestic silkworm the prime host plant for the species is a castor-oil plant (Ricinus communis in Euphorbiaceae). Recently, the eri-silkworm also is reared in Korea and is expected to be utilized for a diverse purpose. In this report, we present the complete mitochondrial genome of the species with the emphasis of a few major characteristics. The 15,384-bp long S. cynthia ricini (Lepidoptera: Saturniidae) mitochondrial genome was amplified into three long overlapping fragments (from COI ~ ND4, ND5 ~ lrRNA, and lrRNA ~ COI) and subsequent several short fragments using the long fragments as temperate. The primers for both long and short fragments were designed solely for lepidopteran genomes, without any species-specific primers. As a usual the genome is composed of 37 genes: 13 protein-coding genes (PCGs), two rRNA genes, and 22 tRNA genes, and one large non-coding region termed the A+T-rich region. Arrangement of the genome is identical to those of other lepidopteran mitochondrial genome, but this differs from the common arrangement found in a diverse insect order, by the movement of tRNAMet to a position 5’- up stream of tRNAIle. Unlikely previous report on the start codon for COI gene in Lepidoptera S. cynthia ricini COI gene starts with typical ATT codon located between tRNATyr and the beginning region of COI gene. The 22 tRNAs that are interspersed throughout the mitogenome ranged in length from 62 to 71 bp. All tRNAs but tRNASer(AGN) were shown to be folded into the expected cloverleaf secondary structures. More detailed structural and phylogenetic analyses among Bombycidae and Saturniidae in connection with other families in the Bombycoidea will be performed soon