KOREAN JOURNAL OF ICHTHYOLOGY, Vol. 27, No. 1, 16-, March 15 ISSN: 1225-8598 (Print), 2288-3371 (Online) Received: February 2, 15 Revised: March 11, 15 Accepted: March 16, 15 붉바리 (Epinephelus akaara) 와 Brown-Marbled Grouper (E. fuscoguttatus) 종간잡종수정란의부화율 노충환 * 김근식 명정구 조재권 1 윤락진 2 임한규 3 방인철 4 한국해양과학기술원동해연구소, 1 국립수산과학원남서해수산연구소, 2 청솔수산, 3 목포대학교해양수산자원학과, 4 순천향대학교생명시스템학과 The Hatchability of Fertilized Eggs of Interspecific Hybrid between Red Spotted Grouper (Epinephelus akaara) and Brown-Marbled Grouper (E. fuscoguttatus) by Choong Hwan Noh*, Keun-Sik Kim, Jung Goo Myoung, Jae Kwon Cho 1, Nak Jin Yun 2, Han Gyu Lim 3 and In-Chul Bang 4 (East Sea Research Institute, Korea Institute of Ocean Science & Technology, Uljin 767-813, Korea; 1 South Sea Fisheries Research Institute, National Fisheries Research & Development Institute, Yeosu 556-823, Korea; 2 Cheong Sol Fisheries Co., Muan 534-841, Korea; 3 Department of Marine & Fisheries Resources, Mokpo National University, Mokpo 534-729, Korea; 4 Department of Life Science & Biotechnology, Soonchunhyang University, Asan 336-475, Korea) ABSTRACT The hatchability of the artificially induced hybrid between two groupers (family Serranidae), red spotted grouper (Epinephelus akaara) and brown-marbled grouper (E. fuscoguttatus) that lives in different habit environment was investigated. There was no difference in the required time of each developmental stages after fertilization between hybrid (red spotted grouper brown-marbled grouper ) and purebred (red spotted grouper ) and required 25.6 hours to hatch at incubated in 25 C, but a noticeable unequal cleavage in cell size was observed in hybrid eggs unlikely to purebred. The hatching rate of fertilized eggs of hybrid was generally low across the four incubate temperatures (22, 25, 28, 31 C) with highest 9.8% in 25 C. This study demonstrated the possibility of artificial hybridization between two groupers, red spotted grouper and brown-marbled grouper, thus preparing the groundwork on developmental characteristics, deformities of hatched larvae and early survival ability for further studies on aquaculture. Key words : Epinephelus akaara, Epinephelus fuscoguttatus, interspecific hybrid, embryonic development, hatchability 서 생물종은다른종과번식적격리를통해구분되어지고종특이성을유지하게되지만, 진화학적으로종간잡종 (interspecific hybrid) 을통하여새로운종의분화가가능하게된다 (Barton and Hewitt, 1989; Avise, 1). 시클리드과 (family Cichlidae) 와바리과 (family Serranidae) 어류에서종간잡종을통한종분화가오랜기간에걸쳐진행된분자 론 *Corresponding author: Choong Hwan Noh Tel: 82-54-7-5321, Fax.: 82-54-7-5349, E-mail: chnoh@kiost.ac 생물학적증거가발견되고있다 (Crapon De Caprona and Fritzch, 1983; Domeier, 1994; Palumbi, 1994; Kocher, 4). 어류에있어서종간잡종은진화학적연구뿐만아니라산업적측면에서도매우중요한역할을하며, 잡종강세 (hybrid vigor) 를통해상업적으로유용한형질을발현시킬수있다 (Bartley et al., 1). 바리과중능성어아과 (sub-family Epinephelinae) 는 15속 159종으로산업적가치가높은중요한분류군으로서 (Heemstra and Randall, 1993; Froese and Pauly, 14), 인위적인종간잡종이비교적수월하게이루어지는것으로알려져있다 (James et al., 1999; Herwerden et al., 2; Koh, 8, 16 http://www.fishkorea.or.kr
붉바리와 brown-marbled grouper 잡종의부화율 17 1). 현재까지바리과어류의종간잡종은자손집단의생존율, 성장률, 내병성그리고체색향상목적으로 brownmarbled grouper (Epinephelus fuscoguttatus) 대왕바리 (E. lanceolatus), brown-marbled grouper 꼬리큰점바리 (E. polyphekadion), 갈색둥근바리 (E. coioides) 대왕바리, 갈색둥근바리 brown-marbled grouper 등다양하게유도된바있다 (Tseng and Poon, 1983; James et al., 1999; Glamuzina et al., 1999, 1; Bartley et al., 1; Koh et al., 8, 1; Kiriyakit et al., 11). 그러나앞서의연구는동일환경에서식하는바리과어류의종간잡종에관한것으로서식환경이다른종간잡종에관한연구는아직까지없다. 붉바리 (E. akaara) 는우리나라를포함한서태평양해역의암반에주로서식하는온대성어종으로서서식수온이 11.7~26.3 C 이며, 성어의전장은최대 58 cm ( 체중 2.5 kg) 인것으로보고된바있다 (Froese and Pauly, 14). 이와달리 Brown-marbled grouper는인도태평양해역의암반이나산호초에서식하는열대및아열대성어종으로서서식수온이 21.4~33.1 C 이며, 성어는전장 11 cm ( 체중 11 kg) 까지성장하는대형종에속하는것으로알려져있다 (Chan et al., 1974; Heemstra and Randall, 1993; Froese and Pauly, 14). 본연구는온대에주로서식하는붉바리와열대및아열대지역에서식하는 brown-marbled grouper의종간잡종가능성을탐색하기위하여두종간교배를통해생산한수정란의부화율을조사하였다. 재료및방법 1. 친어와배우자생산붉바리친어는전라남도와제주도인근에서수집한후여수시거문도에위치한해상가두리에서 3년간양성한성숙개체를선별하여전남무안군소재청솔수산으로옮겨성숙및산란유도호르몬을주사하였다. 암컷친어는 LHRHa (Sigma-Aldrich, USA) μg/kg과축산용 HCG (Daihan Pharmaceutical Co., LTD., Korea) 1, IU/kg를혼합하여주사하였고, 수컷친어는축산용 HCG만을 5 IU/kg 주사하였다. 호르몬주사후 36시간에암컷 (3마리, 평균전장 33.5 ±1.8 cm, 평균체중 598.±124.1 g) 과수컷 (2마리, 평균전장 37.7±1.9 cm, 평균체중 872.±144.3 g) 친어의복부를압박하여채란과채정을실시하였다. Brown-marbled grouper 수컷친어는말레이시아사바주산다칸 (Sandakan, Sabah) 인근의해상가두리에서관리한개체중 7마리 ( 평균전장 71.6±3.9 cm, 평균체중 7.7±1.1 kg ) 를선별하여친어로사용하였으며, 호르몬주사없이유치도뇨관 (Sewoon medical Co., LTD., Korea) 을생식공에삽입하여채정하였다. 2. 정자동결보존 Brown-marbled grouper 정액은풀링 (pooling) 하여정자동결보존을실시하였다. 희석제로는인공정장 (artificial seminal plasma, ASP1) 을이용하였고, 동해방지제로는 DMSO와 glycerol을사용하였다. 정액과희석액의평형시간은 1분으로하였고, 정자동결보존은.5 ml 용량의정자보존용 straw를이용하여액체질소탱크에보관하였다. 3. 수정및발생두종간잡종교배수정란은붉바리로부터채란한알에 1개월간동결보존중인 brown-marbled grouper 정액을사용하여건식법으로인공수정하여생산하였고, 대조구로서붉바리수정란은채란, 채정후즉시같은방법으로수정하여생산하였다. 수정란은멸균여과된해수로 2~3회세란후포장하여실험실로운반하였다. 수온별부화율조사는 22, 25, 28 그리고 31 C 가유지되는인큐베이터에설치한 5 L 용량의유리비이커각각에부상란을약 3,~5,개수용하여수행하였다. 발생기간중수질악화를방지하기위해마이크로필터로여과한후자외선살균한해수 ( 염분은 33~34 psu) 를이용하여 1일 1회환수하였으며, 각비이커에에어스톤을설치하여산소를공급하였다. 난발생은각발생단계에서매번 3개내외의수정란을만능투영기 (NIKON JP V-12B, Japan) 와입체해부현미경 (Leica MZ1 F, Taiwan) 으로관찰하였으며, 디지털카메라 (Leica DFC45 C, Taiwan) 로촬영하여기록하였다. 그리고각발생단계에전체알의약 5% 가도달한시점을기준으로발생단계별소요시간을구하였다. 결과및고찰본연구에이용한붉바리의성숙란은구형의투명한분리부성란이었고, 유구수는대부분 1개였다. 난의크기는 754~ 822 μm( 평균 798.9±16.9 μm, n=) 였으며, 유구의크기는 165.7~188.6 μm( 평균 173.7±5.8 μm, n=) 였다. 붉바리 brown-marbled grouper 잡종의수정률은부상란기준으로 76.7% 로서붉바리순종 (9.9%) 에비해낮았으며, 발생과정중상실기이전단계에서붉바리순종과는다르게난할이균등하게이루어지지않는기형현상이발생하였다 (Fig. 1B). 이러한현상은 brown-marbled grouper 대왕바리 잡종과 goldblotch grouper (E. costae) dusky
18 노충환 김근식 명정구 조재권 윤락진 임한규 방인철 Fig. 1. Egg development of hybrid (red spotted grouper brown-marbled grouper ). A, fertilized egg; B, 16-cell stage; C, morula; D, gastrula; E, embryo formation; F, optic vesicle appearance; G, tail beating; H, hatched larva. grouper (E. marginatus) 잡종에서도나타났으며, 비균등난할의원인과부화율에미치는영향에관해서는알려진바가없다 (Glamuzina et al., 1; Ch ng and Senoo, 8). 두종의자연산란기간동안부화수온은붉바리가 21 C ~26 C(6월 ~8월 ) 그리고 brown-marbled grouper는 28~31 C 인것으로알려져있다 (Sugama et al., 12). 이에따라수온을달리하여두종간잡종수정란의난발생과정을조사한결과는 Table 1에정리하였다. 낭배기 (gastrula) 까지소요시간은 22 C 실험구와 31 C 실험구간 3.8시간으로큰차이가나타났다. 배형성단계 (embryonic formation) 이후에서도수온에따른발생시간의차이는계속나타나부화까지소요시간은 25 C 실험구에서 25.6시간, 28 C 실험구에서 21.9시간그리고 31 C 실험구에서 18.1시간으로서수온에비례하여짧아졌다. 잡종의부화소요시간은 goldblotch grouper dusky grouper 잡종의경우순종에비해빨랐지만, 갈색둥근바리 대왕바리 잡종은순종과차이 Table 1. Required times of hybrid (red spotted grouper brownmarbled grouper ) to reach each of the embryonic developmental stages after fertilization in four different water temperature Embryonic Water temperature developmental stage 22 C 25 C 28 C 31 C 2~4 cells.78 h.73 h. h.58 h 16 cells 2.6 h 1. h 1.25 h 1.21 h Morula 2.5 h 2.27 h 1.87 h 1. h Gastrula 9.87 h 6.57 h 6.5 h 6.7 h Embryonic formation 14. h 1. h 8.92 h 8.25 h Optic vesicle appearance Dead 17.1 h 13.1 h 12.1 h 5% hatching Dead 25. h 21.92 h 18.1 h 가없는것으로보고된바있다 (Glamuzina et al., 1999; Kiriyaki et al., 11). 본연구에서붉바리 brown-marbled grouper 잡종의부화소요시간은수정란이모두죽은 22 C 를제외하고붉바리순종과차이가나타나지않았다.
붉바리와 brown-marbled grouper 잡종의부화율 19 22 C 25 C Cumulative % survival F M G OV TB H 28 C F M G OV TB H 31 C F M G OV TB H F M G OV TB H Embryonic developmental stage Red spotted grouper hybrid (red spotted grouper brown-marbled grouper ) Fig. 2. Cumulative survival rate of hybrid (red spotted grouper brown-marbled grouper ) and red spotted grouper fertilized eggs in the four different water temperatures. F, fertilization; M, morula; G, gastrula; OV, optical lens appearance; TB, tail beating; H, hatching. 발생과정중생존율은붉바리 brown-marbled grouper 잡종수정란이상실기 ~ 낭배기로이행하는단계에서급격히낮아졌으며, 붉바리순종역시동일한경향을보였다 (Fig. 2). 그리고모든수온에서잡종의부화율은붉바리순종에비해낮았다. 붉바리 x brown-marbled grouper 잡종수정란은 22 C 실험구에서안포형성기이후모두폐사하였고 ( 붉바리순종.1%), 25 C 실험구에서부화율이 9.8 % 로서가장높았으며 ( 붉바리순종 36.3%), 28 C 와 31 C 에서는부화율이각각 5.5% 와 2.% 로서수온이높을수록감소하면서붉바리순종 ( 각각 32.2% 와 13.5%) 과부화율의격차가커지는경향이나타났다. 이전의연구에서잡종과순종의수정률과부화율은 goldblotch grouper dusky grouper 잡종이각각 5.% 와 35.% 로서 goldblotch 순종 ( 각각 5.% 와 37.5%) 와차이가없었으며, 갈색둥근바리 대왕바리 잡종이각각 76.7% 와 51.3% 로서갈색둥근바리순종 ( 각각 86.% 와 48.%) 과역시차이가없었다 (Glamuzina et al., 1; Kiriyakit et al., 11). 그리고순종에관한보고는없지만다른잡종의수정률과부화율은 brownmarbled grouper 대왕바리 잡종이각각 86.8% 와 87.2% (Ch ng and Senoo, 8), 갈색둥근바리 대왕바리 잡종이각각 91.% 와 33.6% (Koh et al., 1), 갈색둥근바리 brown-marbled grouper 잡종이각각 93.9% 와 5.3% (Koh et al., 8) 였다. 본연구에서잡종과순종모두수정률에비해부화율이낮은것은발생중중층부상란이많이관찰되어난질에의한것으로판단된다. 그러나잡종의수정률과부화율이붉바리순종에비해낮게나타난것은종간교배에따른영향일수도있으며, 신선한정액을사용한붉바리순종과는달리동결보존된정액을사용한것에기인할수도있다 (Suquet et al., ; Kiriyaki et al., 11). 이전의연구에서바리과어류의종간잡종은동일서식지에분포하는종간에만이루어졌으며, 다른해양어류분류군에서도서식지가다른어종간잡종에관한보고는찾을수없었다. 본연구에서서식환경이다른붉바리와 brownmarbled grouper 잡종의수정란은적정수온에서부화가가능한것으로조사되었으며, 향후양질의알과신선한정액으로수정란을생산하여발생양상, 기형률및초기생존율등에관한면밀한연구가필요할것으로판단된다. 요약서식환경이다른바리과 (family Serranidae) 어류두종, 붉바리 (Epinephelus akaara) 와 brown-marbled grouper (E. fuscoguttatus) 의종간잡종수정란의부화율을조사하였다.
노충환 김근식 명정구 조재권 윤락진 임한규 방인철 잡종 ( 붉바리 brown marbled grouper ) 의수정후각발생단계까지소요시간은붉바리순종과차이가없었으며, 부화소요시간은 25 C 에서 25.6시간이었다. 잡종은붉바리순종과는달리비균등난할이분명하게관찰되었다. 잡종의부화율은네수온 (22, 25, 28, 31 C) 에서대체적으로낮았으며, 25 C 에서 9.8% 로가장높았다. 이상의연구에서서식환경이서로다른바리과두종간잡종이생산가능한것으로조사되었으며, 향후산업적활용성을검증하기위하여발생양상, 기형률및초기생존율등에관한면밀한연구가필요할것으로판단된다. 사 본논문은농림축산식품부, 해양수산부, 농촌진흥청, 산림청 Golden Seed 프로젝트사업 (2134-4-2-SB53) 과한국해양과학기술원 (PE-991) 의지원에의해수행되었습니다. 사 인용문헌 Avise, J.C. 1. Cytonuclear genetic signatures of hybridization phenomena: rationale, utility and empirical examples from fishes and other aquatic animals. Reviews in Fish Biology and Fisheries, 1: 253-263. Bartley, D.M., K. Rana and A.J. Immink. 1. The use of inter-specific hybrids in aquaculture and fisheries. Reviews in Fish Biology and Fisheries, 1: 325-337. Chan, W., D. Carlsson and N. Loharkan. 1974. Serranidae. In: Fischer, W. and P. J. P. Whitehead (eds.), FAO species identification sheets for fishery purposes. Eastern Indian Ocean (Fishing Area 57) and Western Central Pacific (Fishing Area 71). Volume 4. FAO, Rome. Ch ng, C.L and S. Senoo. 8. Eggs and larval development of a new hybrid grouper, tiger grouper Epinephelus fuscoguttatus giant grouper E. lanceolatus. Aquaculture Science, 56: 55-512. Crapon De Caprona, M.D. and B. Fritzsch. 1983. Interspecific fertile hybrids of Haplochromine Cichlidae (Teleostei) and their possible importance for speciation. Netherlands Journal of Zoology, 34: 53-538. Domier, M.L. 1994. Speciation in the Serranid fish Hypoplectrus. Bulletin of Marine Science, 54: 13-141. Froese, R. and D. Pauly. 14. FishBase. World wide web electronic publication. www.fishbase.org (version 2/14). Glamuzina, B., N. Glavic, B. Skaramuca, V. Kožul and P. Tutman. 1. Early development of the hybrid Epinephelus costae E. marginatus. Aquaculture, 198: 55-61. Glamuzina, B., V. Kožul, P. Tutman and B. Skaramuca. 1999. Hybridization of Mediterranean groupers: Epinephelus marginatus E. aeneus and early development. Aquaculture Research, 3: 626-628. Heemstra, P.C. and J.E. Randall. 1993. FAO species catalogue. Vol. 16. Groupers of the world (family Serranidae, subfamily Epinephelinae). An annotated and illustrated catalogue of the grouper, rockcod, hind, coral grouper and lyretail species known to date. FAO Fishieries Synopsis, 125: p. 382. Herwerden, L., C.R. Davies and J.H. Choat. 2. Phylogenetic and evolutionary perspectives of the Indo-Pacific grouper Plectvopomus species on the Great Barrier Reef, Australia. Journal of Fish Biology, : 1591-1596. James, C.M., S.A. Al-Thobaiti, B.M. Rasem and M.H. Carlos. 1999. Potential of grouper hybrid (Epinephelus fuscoguttatus E. polyphekadion) for aquaculture. Naga, The ICLARM Quarterly, 22: 19-23. Kiriyakit, A., W.G. Gallardo and N. Bart. 11. Successful hybridization of groupers (Epinephelus coioides Epinephleus lanceolatus) using cryopreserved sperm. Aquaculture, 3: 16-112. Kocher, T.D. 4. Adaptive evolution and explosive speciation: the Cichlid fish model. Nature Reviews Genetics, 5: 288-298. Koh, I.C.C., S.R.M. Shaleh and S. Senoo. 8. Egg and larval development of a new hybrid orange-spotted gruper Epinephelus coioides tiger grouper E. fuscoguttatus. Aquaculture Science, 56: 441-451. Koh, I.C.C., S.R.M. Shaleh, N. Akazawa, Y. Oota and S. Senoo. 1. Egg and larval development of a new hybrid orangespotted grouper Epinephelus coioides giant grouper E. lanceolatus. Aquaculture Science, 58: 1-1. Palumbi, S.R. 1994. Genetic divergence, reproductive isolation and marine speciation. Annual Reviews, 25: 547-572. Suquet, M., C. Dreanno, C. Fauvel, J. Cosson and R. Billard.. Cryopreservation of sperm in marine fish. Aquaculture Research, 31: 231-243. Tseng, W.Y. and C.T. Poon. 1983. Hybridization of Epinephelus species. Aquaculture, 34: 177-182.