Cytochrome c oxidase subunit 1과 RAPD 분석에 의한 한국 전복속의 계통 연구

서용배(Yong Bae Seo),강성철(Sung Chul Kang),최성석(Seong Seok Choi),이종규(Jong Kyu Lee),정태혁(Tae Hyug Jeong),임한규(Han Kyu Lim),김군도(Gun-Do Kim) 한국생명과학회 2016 생명과학회지 Vol.26 No.4

전복은 전복속(Haliotis)에 속하며 전 세계적으로 식품산업에서 중요한 복족류 연체동물이다. 우리나라에는 6개종; 북방전복(Haliotis discus hannai), 둥근전복(Haliotis discus discus), 왕전복(Haliotis madaka), 말전복(Haliotis gigantea), 오분자기(Haliotis diversicolor diversicolor), 마대오분자기(Haliotis diversicolor supertexta)가 보고되어 있다. 이 연구에서는 우리나라 해역에 서식하는 중 · 대형 전복과 4종인 북방전복, 둥근전복, 왕전복, 말전복의 유전학적 유연관계를 분석하기 위하여 미토콘드리아의 cytochrome c oxidase subunit I (COI) 유전자와 Random Amplified Polymorphic DNA (RAPD) 분석법을 실시 하였다. 본 연구의 결과 COI 유전자 분석과 RAPD 분석을 활용하면 4종의 전복 중 북방전복, 둥근전복, 왕전복을 한 그룹으로 나머지 한 그룹을 말전복으로 구분하는 종 분류는 명확히 구분할 수 있었다. 이러한 결과는 전복 교잡육종을 이용한 수출용 전복 신종자 개발에 있어 주요 대상종인 전복과 4종에 대한 유전적 근연 관계를 규정함으로써 향후 교잡육종 연구의 기초 자료를 제공할 수 있을 것으로 사료된다. Abalones are gastropod mollusks belonging to the genus Haliotis. Pacific abalones are regarded as a very important marine gastropod mollusk in Korea, Japan, China, and also in food industries around the world. In Korea, 6 species of abalone have been reported to occur along the coasts: Haliotis discus hannai, Haliotis discus discus, Haliotis madaka, Haliotis gigantea, Haliotis diversicolor supertexta, and Haliotis diversicolor diversicolor. This study was performed to discriminate the genetic variances by the partial sequences of the mitochondrial cytochrome c oxidase subunit I (COI) genes and random amplified polymorphic DNA (RAPD) analysis against four species of Pacific abalone (H. discus hannai, H. discus, H. madaka, H. gigantea). COI gene is reasonably well conserved and has been sequenced in various invertebrate taxa. The RAPD analysis technique is a relatively simple and low cost method that allows differentiation of taxa without the need to know their genomes. In this study, we investigated the genetic diversity, phylogenetic relationships within each species. The COI and RAPD analysis were able to distinguish between H. gigantea and the other three species. However, these analysis methods were inadequate to distinguish between H. discus and H. madaka. These results are believed to be able to provide a basis data for future hybrid breeding research by defining the genetically closely related four species of abalone, which is to develop new hybrid abalone for export using hybrid breeding.

수온 상승에 따른 북방전복, Haliotis discus hannai과 둥근전복, H. discus discus 체액의 생리학적 변화

민은영,이정식,김재원,전미애,강주찬,Min, Eun-Young,Lee, Jung Sick,Kim, Jae Won,Jeon, Mi Ae,Kang, Ju-Chan 한국패류학회 2015 The Korean Journal of Malacology Vol.31 No.1

본 연구에서는 수온 증가에 따른 북방전복 (Haliotis discus hannai) 과 둥근전복 (H. discus discus) hemolymph의 생리 및 면역학적 변화를 관찰하기 위하여 위 두 종을 20, 22, 24, 26 및 $28^{\circ}C$ 수온에 각각 4 일간 노출시켰다. 노출 결과, hemolymph의 total-protein (TP), glucose, 칼슘 (Ca) 은 둥근전복이 북방전복보다 높은 값을 보였으나, 마그네슘 (Mg), alkaline phosphatase (ALP) 및 lysozyme은 두 종에서 유사한 값을 보였다. 수온 증가에 따른 hemolymph의 TP, glucose 및 마그네슘은 두 전복 모두에서 유의한 변화가 관찰되지 않았다. 그러나 칼슘과 면역관련 인자인 ALP 및 lysozyme은 둥근전복에서 유의한 차이가 없었으나, 북방전복에서는 $26^{\circ}C$ 또는 $28^{\circ}C$의 고수온에 노출되었을 때, 유의하게 높은 활성을 보였다. 한편 phenoloxidase (PO)는 북방전복에서 높은 값을 보였으며, 수온이 높을수록 증가하는 경향을 보였다. 결론적으로 북방전복은 둥근전복과 달리 여러 지표에서 유의한 변화가 관찰된 것으로 보아 북방전복이 둥근전복보다 고수온에 보다 민감한 것으로 사료된다. This study was conducted to investigate the effects of elevated water temperature (WT) on biochemical and immunological factors in the hemolymph of the abalones, Haliotis discus hannai and H. discus discus. The abalone were exposed to various WT; 20, 22, 24, 26 and $28^{\circ}C$ for 4 days. In the control and $20^{\circ}C$, total-protein (TP), glucose and calcium (Ca) in hemolymph of H. discus discus were higher than the values in H. discus hannai. The values of magnesium (Mg), alkaline phosphatase (ALP) and lysozyme in H. discus hannai were similar to the H. discus discus in the control. There were no significant alterations in TP, glucose and Mg levels of hemolymph in H. discus hannai and H. discus discus by WT increases. The values of Ca, ALP and lysozyme were increased in H. discus hannai exposed to the high temperature (26 and $28^{\circ}C$) compared to control, while the values in H. discus discus were not significant difference between the WT groups. The phenoloxidase (PO) activity was increased in hemolymph of H. discus hannai exposed to high temperature (${\geq}24^{\circ}C$) compared to the control (P < 0.05). These physiological and immunological parameters were significantly changed in H. discus hannai. However, these parameters in H. discus discus were barely altered at the high WT (P < 0.05). These results suggested that H. discus hannai is considered to be more sensitive than H. discus discus at the high WT.

고수온 스트레스에 대한 둥근전복, Haliotis discus discus (Reeve, 1846), 말전복, Haliotis gigantea의 항산화 및 면역학적 반응

민은영(EunYoung Min),김신후(Shin Hu Kim),이정식(Jung Sick Lee),강주찬(Ju-Chan Kang) 한국패류학회 2017 The Korean Journal of Malacology Vol.33 No.2

A study was conducted to evaluate the effects of elevated water temperature on antioxidant and immunological response in the abalones, Haliotis discus discus and Haliotis gigantea. The abalones were exposed to various water temperature; 20, 21, 22, 24, 26 and 28℃ for 96 hours. After thermal stress for 96 hours, in the control, superoxide dismutase (SOD) activities in hepatopancreas of H. gigantea were higher (1.3 times) than the values in H. discus discus. Although SOD activities showed no significant differences in H. discus discus, the values in H. gigantea were significantly increased in 24℃ or higher compared to the control after 24 hours exposure. Catalase (CAT) activities in hepatopancreas of H. discus discus were higher (1.4 times) than the values of H. gigantea in the control. After thermal stress (≥ 26℃), CAT activities significantly increased in both species compared to the control. The phenoloxidase (PO) activity of hemolymph was decreased in both species exposed to high temperature (≥ 26℃) compared to the control (P < 0.05). The values of lysozyme were significantly decreased in H. discus discus exposed to 28℃, and in H. gigantea to 26℃. In conclusion, in both species, there was a significant increase on antioxidant abilities (CAT) and a decrease on immunological parameters (PO and lysozyme) after thermal stress of 26℃ or higher. Also, antioxidant enzymes, SOD activities significantly increased in H. gigantea, but there was no significance in H. discus discus after thermal stress. These results suggested that H. gigantea is considered to be more sensitive than H. discus discus on thermal stress even though the similar warm current type.

수온 증가에 따른 북방전복, Haliotis discus hannai과 둥근전복, Haliotis discus discus의 체액의 생리학적 변화

민은영,이정식,김재원,전미애,강주찬 한국패류학회 2015 The Korean Journal of Malacology Vol.31 No.1

This study was conducted to investigate the effects of elevated water temperature (WT) on biochemical and immunological factors in the hemolymph of the abalones, Haliotis discus hannai and Haliotis discus discus. The abalone were exposed to various WT; 20, 22, 24, 26 and 28℃ for 4 days. In the control, 20℃, total-protein (TP), glucose and calcium (Ca) in hemolymph of H. discus discus were higher than the values in H. discus hannai. The values of manesium (Mg), alkaline phosphatase (ALP) and lysozyme in H. discus hannai were similar to the H. discus discus in the control. There were no significant alterations in TP, glucose, and Mg levels of hemolymph in H. discus hannai and H. discus discus by WT increases. The values of Ca, ALP, and lysozyme were increased in H. discus hannai exposed to the high temperature (26 and 28℃) compared to control, while the values in H. discus discus were not significant difference between the WT groups. The phenoloxidase (PO) activity was increased in hemolymph of H. discus hannai exposed to high temperature (≥ 24℃) compared to the control (P < 0.05). These physiological and immunological parameters were significantly changed in H. discus hannai. However, these parameters in H. discus discus were barely altered at the high WT (P < 0.05). These results suggested that H. discus hannai is considered to be more sensitive than H.D. discus at the high WT.

수온 증가에 따른 말전복, Haliotis gigantea과 둥근전복, Haliotis discus discus (Reeve, 1846) hemolymph의 생리학적 변화

민은영,김신후,황인기,김경욱,박보미,이정식,강주찬,Min, Eun-Young,Kim, Shin-Hu,Hwang, In-Ki,Kim, Kyeong-Wook,Park, Bo-Mi,Lee, Jung Sick,Kang, Ju-Chan 한국패류학회 2015 The Korean Journal of Malacology Vol.31 No.3

This study was conducted to examine the effects of alterations in water temperature (WT) on biochemical and immunological factors in the hemolymph of the abalones, Haliotis gigantea and H. discus discus. The abalone were exposed to various WT; 18, 20, 22, 24, 26 and $28^{\circ}C$ for 96 hours. In biochemical factors, total-protein (TP), glucose, magnesium (Mg), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were not significant changes in hemolymph of H. gigantea and H. discus discus. But calcium was significantly increased by high WT (${\geq}24^{\circ}C$). In immunological factor, The phenoloxidase (PO) activity was decreased in hemolymph of H. gigantea and H. discus discus exposed to high temperature (${\geq}22^{\circ}C$) compared to the control (P < 0.05). Whereas alkaline phosphatase (ALP) was not significantly changed. These results suggested that high temperature adversely affects the immunity of H. gigantea and H. discus discus. 본 연구에서는 수온 증가에 따른 말전복 (Haliotis gigantea) 과 둥근전복 (H. discus discus) 헤모림프의 생리 및 면역학적 변화를 관찰하기 위하여 위 두 전복을 20, 22, 24, 26 및 $28^{\circ}C$ 수온에 각각 4 일간 노출시켰다. 노출 결과, 헤모림프의 totalo-protein (TP), glucose, calcium (Ca) 은 둥근전복 (H. discus discus)이 말전복 (H. gigantea) 보다 높은 값을 보였으나, magnesium (Mg), alkaline phosphatase (ALP) 두 전복에서 유사한 값을 보였다. 그리고 면역인자인 PO에서는 둥근전복 (H. discus discus)이 더 높게 나타났다. 수온 증가에 따른 헤모림프의 TP, glucose, Mg, AST, ALT 및 ALP는 유의적인 변화가 나타나지 않았다. 하지만 헤모림프의 Ca 농도는 둥근전복 (H. discus discus)에서 $24^{\circ}C$ 이상, 말전복 (H. gigantea)에서는 $26^{\circ}C$ 이상의 수온에서 유의적 증가를 보였다. 그리고 면역인자인 phenoloxidase (PO) 는 둥근전복 (H. discus discus)은 $22^{\circ}C$ 이상 말전복 (H. gigantea) 에서는 $26^{\circ}C$ 이상의 수온에서 유의적 감소를 나타내었다. 결론적으로 말전복 (H. gigantea)은 $24^{\circ}C$ 이상에서 혈액학적 변화를 보였고, $26^{\circ}C$ 이상에서 면역학적 변화를 보였다. 둥근전복 (H. discus discus)은 $26^{\circ}C$ 이상에서 혈액학적 변화를 보였고, $22^{\circ}C$ 이상에서 면역학적 변화를 나타내었다.

수온 증가에 따른 말전복, Nordotis gigantea과 둥근전복, Haliotis discus discus (Reeve, 1846) hemolymph의 생리학적 변화

민은영,김신후,황인기,김경욱,박보미,이정식,강주찬 한국패류학회 2015 The Korean Journal of Malacology Vol.31 No.3

This study was conducted to examine the effects of alterations in water temperature (WT) on biochemical and immunological factors in the hemolymph of the abalones, Nordotis gigantea and Haliotis discus discus. The abalone were exposed to various WT; 18, 20, 22, 24, 26 and 28℃ for 96 hours. In biochemical factors, total-protein (TP), glucose, magnesium (Mg), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were not significant changes in hemolymph of N. gigantea and H. discus. But calcium was significantly increased by high WT (≥ 24℃). In immunological factor, The phenoloxidase (PO) activity was decreased in hemolymph of N. gigantea and H.D. hannai exposed to high temperature (≥ 22℃) compared to the control (P < 0.05). Whereas alkaline phosphatase (ALP) was not significantly changed. These results suggested that high temperature adversely affects the immunity of N. gigantea and H.D. hannai.

감귤박 및 감태추출물의 사료첨가제 급여에 따른 둥근전복 (Haliotis discus discus)의 성장 및 생리적 변화

좌민석 ( Min Seok Jwa ),여인규 ( In Kyu Yeo ) 한국어병학회 2014 한국어병학회지 Vol.28 No.1

Here, we report the physiological changes and growth in disk abalone, Haliotis discus discus, in relation to dietary supplementation with citrus pomace (CP) 6%, Ecklonia cava residue (ECR) 6%, and CP + ECR (3% + 3%). The composition and nutrient content, survival rate and growth rate were measured 0, 4, 8 and 12 weeks after feeding the supplemented diets of CP and/or ECR. Moreover, the experiment of low salinity stress (25psu) for environmental resistance was examined for a period of 48 hours after feeding the supplemented diets for 12 weeks. The activities of superoxide dismutase (SOD), catalase (CAT), lysozymes, respiratory burst, and phenoloxidase were measured. The moisture content and crude protein condition of the body were increased with the addition of ECR only (P<0.05). We observed higher levels of survival in the experimental group compared with the control group. Moreover, the growth disk abalone that were fed a diet containing ECR was higher compared with the control group. However, the growth of abalone fed a diet containing CP was similar to the control group. With a rearing condition of low salinity stress, survival rate and lysozyme activity were increased in the ECR group compared with the control group. Dietary ECR reduced the level of CAT activity to approximately 30% of the control, however the level of CAT activity in the ECR group was similar to the start level of the previous stress. These results suggest that dietary ECR gives rise to an enhanced immunity in disk abalone, as a result of the decrease in CAT and lysozyme activity in particular. Accordingly, the growth and survival rate were increased by feeding an ECR-supplemented diet in the rearing of disk abalone, Haliotis discus discus.

감귤박 및 감태추출물의 사료첨가제 급여에 따른 둥근전복 (Haliotis discus discus)의 성장 및 생리적 변화

좌민석,여인규,Jwa, Min-Seok,Yeo, In-Kyu 한국어병학회 2015 한국어병학회지 Vol.28 No.1

Here, we report the physiological changes and growth in disk abalone, Haliotis discus discus, in relation to dietary supplementation with citrus pomace (CP) 6%, Ecklonia cava residue (ECR) 6%, and CP + ECR (3% + 3%). The composition and nutrient content, survival rate and growth rate were measured 0, 4, 8 and 12 weeks after feeding the supplemented diets of CP and/or ECR. Moreover, the experiment of low salinity stress (25psu) for environmental resistance was examined for a period of 48 hours after feeding the supplemented diets for 12 weeks. The activities of superoxide dismutase (SOD), catalase (CAT), lysozymes, respiratory burst, and phenoloxidase were measured. The moisture content and crude protein condition of the body were increased with the addition of ECR only (P<0.05). We observed higher levels of survival in the experimental group compared with the control group. Moreover, the growth disk abalone that were fed a diet containing ECR was higher compared with the control group. However, the growth of abalone fed a diet containing CP was similar to the control group. With a rearing condition of low salinity stress, survival rate and lysozyme activity were increased in the ECR group compared with the control group. Dietary ECR reduced the level of CAT activity to approximately 30% of the control, however the level of CAT activity in the ECR group was similar to the start level of the previous stress. These results suggest that dietary ECR gives rise to an enhanced immunity in disk abalone, as a result of the decrease in CAT and lysozyme activity in particular. Accordingly, the growth and survival rate were increased by feeding an ECR-supplemented diet in the rearing of disk abalone, Haliotis discus discus.

Two duplicated chicken-type lysozyme genes in disc abalone Haliotis discus discus: Molecular aspects in relevance to structure, genomic organization, mRNA expression and bacteriolytic function

Umasuthan, N.,Bathige, S.D.N.K.,Kasthuri, S.R.,Wan, Q.,Whang, I.,Lee, J. Academic Press 2013 FISH AND SHELLFISH IMMUNOLOGY Vol.35 No.2

Lysozymes are crucial antibacterial proteins that are associated with catalytic cleavage of peptidoglycan and subsequent bacteriolysis. The present study describes the identification of two lysozyme genes from disc abalone Haliotis discus discus and their characterization at sequence-, genomic-, transcriptional- and functional-levels. Two cDNAs and BAC clones bearing lysozyme genes were isolated from abalone transcriptome and BAC genomic libraries, respectively and sequences were determined. Corresponding deduced amino acid sequences harbored a chicken-type lysozyme (LysC) family profile and exhibited conserved characteristics of LysC family members including active residues (Glu and Asp) and GS(S/T)DYGIFQINS motif suggested that they are LysC counterparts in disc abalone and designated as abLysC1 and abLysC2. While abLysC1 represented the homolog recently reported in Ezo abalone [1], abLysC2 shared significant identity with LysC homologs. Unlike other vertebrate LysCs, coding sequence of abLysCs were distributed within five exons interrupted by four introns. Both abLysCs revealed a broader mRNA distribution with highest levels in mantle (abLysC1) and hepatopancreas (abLysC2) suggesting their likely main role in defense and digestion, respectively. Investigation of temporal transcriptional profiles post-LPS and -pathogen challenges revealed induced-responses of abLysCs in gills and hemocytes. The in vitro muramidase activity of purified recombinant (r) abLysCs proteins was evaluated, and findings indicated that they are active in acidic pH range (3.5-6.5) and over a broad temperature range (20-60 <SUP>o</SUP>C) and influenced by ionic strength. When the antibacterial spectra of (r)abLysCs were examined, they displayed differential activities against both Gram positive and Gram negative strains providing evidence for their involvement in bacteriolytic function in abalone physiology.

Three novel C1q domain containing proteins from the disk abalone Haliotis discus discus: Genomic organization and analysis of the transcriptional changes in response to bacterial pathogens

Bathige, S.D.N.K.,Umasuthan, N.,Jayasinghe, J.D.H.E.,Godahewa, G.I.,Park, H.C.,Lee, J. Academic Press 2016 FISH AND SHELLFISH IMMUNOLOGY Vol.56 No.-

<P>The globular C1q (gC1q) domain containing proteins, commonly referred as C1q domain containing (C1qDC) proteins, are an essential family of proteins involved in various innate immune responses. In this study, three novel C1qDC proteins were identified from the disk abalone (Haliotis discus discus) transcriptome database and designated as AbC1qDC1, AbC1qDC2, and AbC1qDC3. The cDNA sequences of AbC1qDC1, AbC1qDC2, and AbC1qD0 consisted of 807, 1305, and 660 bp open reading frames (ORFs) encoding 269, 435, and 220 amino acids (aa), respectively. Putative signal peptides and the N-terminal gC1q domain were identified in all three AbC1qDC proteins. An additional predicted motif region, known as the coiled coil region (CCR), was identified next to the signal sequence of AbC1qDC2. The genomic organization of the AbC1qDCs was determined using a bacterial artificial chromosome (BAC) library. It was found that the CDS of AbC1qDC1 was distributed among three exons, while the CDSs of AbC1qDC2 and AbC1qDC3 were distributed between two exons. Sequence analysis indicated that the AbC1qDC proteins shared <40% identity with other counterparts from different species. According to the neighbor joining phylogenetic tree, the proteins were grouped within an invertebrate group with high evolutionary distances, which suggests that they are new members of the C1qDC family. Higher expression of AbC1qDC1 and AbC1qDC2 was detected in hepatopancreas, muscle, and mantle tissues compare to the other tissues analyzed, using reverse transcription, followed by quantitative real-time PCR (qPCR) using SYBR Green, whereas AbC1qDC3 was predominantly expressed in gill tissues, followed by muscles and the hepatopancreas. The temporal expression of AbC1qDC transcripts in gills after bacterial (Vibrio parahaemolyticus and Listeria monocytogenes) and lipopolysaccharide stimulation indicated that AbC1qDCs can be strongly induced by both Gram-negative and Gram-positive bacterial species with different response profiles. The results of this study suggest that AbC1qDCs are involved in immune responses against invading bacterial pathogens. (C) 2016 Published by Elsevier Ltd.</P>