한수지 47(5), , 2014 Original Article Kor J Fish Aquat Sci 47(5), ,2014 서해안천수만일대양식조피볼락 (Sebastes schlegeli) 의상피선충감염현황 서한길 서정수 류민경 이은혜 권세련 1

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1 한수지 47(5), , 2014 Original Article Kor J Fish Aquat Sci 47(5), ,2014 서해안천수만일대양식조피볼락 (Sebastes schlegeli) 의상피선충감염현황 서한길 서정수 류민경 이은혜 권세련 1 강종순 2 노윤산 2 최혜승 정승희 한현자 * 국립수산과학원병리연구과, 1 선문대학교수산생명의학과, 2 충청남도수산관리소 A Nematode Infection in the pithelial Tissue of ultured Rockfish Sebastes schlegel in heonsu Bay, Western Korea Han-Gill Seo, Jung Soo Seo, Min-Kyung Ryu, un Hye Lee, Se Ryun Kwon 1, Jong Soon Kang 2, Yun-San No 2, Hye-Sung hoi, Sung Hee Jung and Hyun-Ja Han* Pathology Division, National Fisheries Research and Development Institute, Busan , Korea 1 Department of Aqualife Medicine, Sun Moon University, Asan , Korea 2 hungnam Fisheries Management Office, Boryoung , Korea We investigated a nematode infection in the epithelial tissue of rockfish Sebastes schlegeli cultured in heonsu Bay, western Korea, from May 2013 to April Nematodes infected the epithelial tissues of various external organs, including the fins, operculum, nares, mouth, and head. Over a 1-year period, the overall nematode infection rate in rockfish was 55% (n=89/163): 56.7% (n=17/30) in May 2013, 88% (n=29/33) in August 2013, 0% (n=0/30) in October 2013, 70% (n=21/30) in January 2014, and 55% (n=22/40) in April During this study, the only mass mortality of cultured rockfish in heonsu Bay was in August 2013, when we identified skin wounds on the rockfish caused by the nematodes escaping from their hosts. During this period, the accumulated mortality for 2 weeks was % in different farms. In addition, several pathogenic bacteria (Photobacterium damselae, Vibrio spp., and Streptococcus iniae) were isolated from the moribund rockfish; these were thought to be transmitted through the skin wounds caused by the nematodes. Key words: Rockfish, Seabastes schlegeli, Nematodes, heonsu Bay 서론 (Sebastes schlegeli), m,. 1990, , ,757, (KOSIS, 2013)..,,,. Microcotyle sebastis, Benedenia sp., Trichodina sp., Scutica, caligus (National Fisheries Research & Development Institute, 2012),. British olumbia (Sebastes spp.) Trichosomoidid Huffmanela spp., Huffmanela spp. Kor J Fish Aquat Sci 47(5) , October 2014 This is an Open Access article distributed under the terms of the reative ommons Attribution Non-ommercial Licens ( permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Received 9 June 2014; Revised 5 August 2014; Accepted 8 August 2014 *orresponding author: Tel: Fax: mail address: hjhan77@korea.kr opyright 2014 The Korean Society of Fisheries and Aquatic Science 603 pissn: , eissn:

2 604 서한길ㆍ서정수ㆍ류민경ㆍ이은혜ㆍ권세련ㆍ강종순ㆍ노윤산ㆍ최혜승ㆍ정승희ㆍ한현자 (Gonboy and Speare 2002, Moravec et al., 2005). (Nematode), 650.,. amallanoidea Ascaroidea, Ascaridoidoiea (ontracecum, Pseudoterranova, Anisakis), amallanoidea (amallanus, ulcullanus), Dracunculoidea (Philonema, Philometra), Spiruroidea (Metabronema, Ascarophis) (dward, 1996)..,.. 4, 5 (gusa, 2006; Park and Oh, 2009).,,, amallanus Philometra,.,,,, (dward, 1996) ( N, ),, ,,. 재료및방법 조피볼락양식현황조사및시료채취 , 8, 10, , (Fig. 1) West sea (Table 1), 2. 기생충검사... 세균검사 Taean-gun F Sabsido Anmyeondo Seosan-si heonsu-bay A Wonsando Hongseong-gun Boryeong-si Fig. 1. Map of sampling sites in Korea. A, Wonsando; B, Sodo;, Tangye; D, Gumae;, Daeyado; F, Dangamri. 1.5% Nal Brain Heart Infusion Agar (BHIA, Gibco, USA) 25., 16S rrna. genomic DNA PR High pure template preparation kit (Roche, Ger- D B

3 서해안조피볼락의선충감염현황 605 Table 1. Diagnosis results of this study Sampling Date (year.month) Sampling Site Fish Size (Avg.) Tested fish Length (cm) Weight (g) No. (n) Mortality rate for 2 weeks (%) Major sings Nematodes sp. infection rate (%) Microcotyle sebastis infection rate (%) Bacterial infection rate (%) NBI blast result of 16S rrna sequencing (% homology) A Skin ulcer A B Hole of go through abdomen, Skin and fin injury Snout, skin and fin injury Anal and operculum hemorrhage Photobacterium sp (99%). Aeromonas salmonicida(99%) Photobacterium damselae (98%) Vibrio sp.(99%) Skin and fin injury Vibrio sp (99%) D Snout, skin and fin injury Vibrio sp. (99%) D Skin and fin injury, Operculum, eyes hemorrahge Streptococcus iniae (99%) Skin and fin injury Vibrio sp. (99%) Skin and fin injury Vibrio sp. (99%) F Skin and fin injury, xophthalmos Lactococcus garviae (99%) yes hemorrahge, Operculum and abdominal wall hemorrhage Streptococcus iniae (99%) A D * No fish virus was detected with RSIV, VHSV, VNNV, MBV and HRV. RSIV, red seabream iridovirus; VHSV, viral hemorrhagic septicaemia virus; VNNV, viral nervous necrosis virus; MBV, marine birnavirus; HRV, hirame rhabdovirus.

4 606 서한길ㆍ서정수ㆍ류민경ㆍ이은혜ㆍ권세련ㆍ강종순ㆍ노윤산ㆍ최혜승ㆍ정승희ㆍ한현자 many) x Taq (Takara, Japan). 16S rrna Table 2 PR, PR band, Gel SV kit (GeneAll, Korea) Topo TA cloning (Invitrogen, USA) cloning. Genetyx Ver. 8.0 (SD Software Development, Japan), NBI BLAST program. 바이러스검사 High pure PR template preparation kit (Roche, Germany) DNA, asy-spin Total RNA xtraction Kit (Intron, Korea) RNA SuperScript TM Reverse Transcriptase (Invitrogen, USA) cdna. PR x Taq (TaKaRa, Japan), primer set PR Table 2., PR. (Red sea bream irido virus; RSIV), (Viral hemorrhagic septicaemia virus; VHSV), (Viral nervous necrosis virus; VNNV), (Marine birna virus; MBV), (Hirame rhabdo virus; HRV) polymerase chain reaction (PR ) (Table 1). 1.5% Agarose gel (Bioneer, Korea) UV band. 결과및고찰, (Fig. 2A, 2B, 2). (Fig. 2F),,,,.. genomic DNA 18S rrna (1,483 bp), NBI BLAST program. Philometroides carassii 97% (Data not shown),, Genbank, (A-F) (Fig. 1) Table , , , , ,. Table 2. Oligonucleotide primers used in PR amplification Gene Nucleotide sequence of primer PR condition Size (bp) RSIV VHSV VNNV MBV HRV Forward 5 -TAAAATTGGTAT-3 94 (30 s)-58 (1 min)-72 (1 min) Reverse 5 -GAAAAATTTAT-3 30 cycle Forward 5 -ATGGAAGGAGGAATTGTGAAGG-3 95 (30 sec)-55 (30 s)-72 (45 sec) Reverse 5 -GGGTGAAGTGTGAGTT-3 30 cycle Forward 5 -GTGTAGTATGTGTGT-3 95 (40 s)-50 (40 s)-72 (40 sec) Reverse 5 -GAGTAAAGGGTGAAGA-3 25 cycle Forward 5 -GAAGAAGGTAGAAAT-3 94 (1 min)-55 (1 min)-72 (1 min) Reverse 5 -GTAGTTGGTTTTGAT-3 30 cycle Forward 5 -TGAGGTAGAGAAGAAA-3 94 (30 s)-58 (1 min)-72 (1 min) Reverse 5 -AATGATGGGAAAAGAG-3 30 cycle Forward 5 -TGAGAGGATAAATT-3 95 (30 s)-49 (30 s)-72 (90 s) 16S rrna 1,404 Reverse 5 -GTTATTGTTAGATT-3 35 cycle RSIV, red seabream iridovirus; VHSV, viral hemorrhagic septicaemia virus; VNNV, viral nervous necrosis virus; MBV, marine birnavirus; HRV, hirame rhabdovirus

5 서해안조피볼락의선충감염현황 607 A B D F Fig. 2. Rockfish Sebastes schlegel infected by Nematodes. A, Nematodes infected rockfish; B, Pectoral fin;, Head; D, Oral cavity;, Base of pectoral fin; F, xtracted Nematodes % (F ), (Table 1),.

6 608 서한길ㆍ서정수ㆍ류민경ㆍ이은혜ㆍ권세련ㆍ강종순ㆍ노윤산ㆍ최혜승ㆍ정승희ㆍ한현자 A B D F Fig. 3. Rockfish Sebastes schlegel infected by Nematodes and pathogenic bacteria. A, Died rockfish in cultured cage; B, Fin and operculum injury;, Hole in fin; D, Hole in abdomen;, Lost of the caudal fin; F, Skin hemorrhage % (n=17/30), % (n=29/33), % (n=0/30), % (n=21/30) %

7 서해안조피볼락의선충감염현황 609 (n=22/40) (Fig. 3A),. (Fig. 3B, 3), (Fig. 3D), (Fig. 3) 2 (Fig. 3F)., (Table 1). 16S rrna Photobacterium damselae, Vibrio spp. Streptococcus iniae, (Table 1). (Vendrell et al., 2006; Won et al., 2008; Jung et al., 2012),., , , mm, mg, mm, 37 8 mg. (t-test), (, P=0.43;, P=0.22)., (Park and Oh, 2009).,. 2. Philometroides cyprini 6-7,.,, (gusa, 2006; Park and Oh, 2009) , , (copepod) (Wang, 2002). 8-10,. 5 m,.,.,..,.,. 사사 ( ; RP AQ-072). References dward JN Diagnosis and Treatment, Fish Disease. Blackwell, Iowa state university Press, USA, gusa S Parasites and Infectious Diseases of Fish and Shellfish. Life Science Inc. Seoul, Korea, Gonboy GA and Speare DJ Dermal Nematodosis in commercially captured rockfish (Sebasters spp.) from coastal British olumbia, anada. J omp Pathol 127, Jung SH, hoi HS, Do JW, Kim MS, Kwon MG, Seo JS, Hwang JY, Kim SR, ho YR, Kim JD, Park MA, Jee BY, ho MY

8 610 서한길ㆍ서정수ㆍ류민경ㆍ이은혜ㆍ권세련ㆍ강종순ㆍ노윤산ㆍ최혜승ㆍ정승희ㆍ한현자 and Kim JW Monitoring of bacteria and parasites in cultured olive flounder, black rockfish, red sea bream and shrimp during summer period in Korea from 2007 to J Fish Pathol 25, jfp KOSIS. Korean Statistical Information Service. Retrived from kosis.kr. on June Moravec F, onboy GA and Speare DJ A new trichosomoidid from the skin of Sebastes spp. (Pisces) from British olumbia, anada. J parasitol 91, org/ /g National Fisheries Research and Development Institute Field Guidebook of Fish Diseases, Bioscience Inc., National Fisheries Research and Development Institute, Busan, Korea, Park SW and Oh MJ Pathology of Aquatic Life. Bioscience Inc., Seoul, Korea, Vendrell D, Balcázar JL, Ruiz-Zarzuela I, Blas I, Gironés O and Múzquiz JL Lactococcus garvieae in fish: A review. omp Immunol Microbiol Infect Dis 29, dx.doi.org/ /j.cimid Wang GT Seasonal dynamics and distribution of Philometroides fulvidraconi (Philometridae) in the bullhead catfish, Pseudobagrus fulvidraco (Richardson). J Fish Dis 25, Won KM and Park SI Pathogenicity of Vibrio harveyi to cultured marine fishes in Korea. Aquaculture 285,

732 서한길ㆍ서정수ㆍ류민경ㆍ이은혜ㆍ정승희ㆍ한현자 (Moravec and Harrison, 1989; Moravec and Wang, 2002; Wijová et al., 2006).,,,., Philometridae., (Edward, 1996). Seo et al.

732 서한길ㆍ서정수ㆍ류민경ㆍ이은혜ㆍ정승희ㆍ한현자 (Moravec and Harrison, 1989; Moravec and Wang, 2002; Wijová et al., 2006).,,,., Philometridae., (Edward, 1996). Seo et al. 한수지 48(5), 731-738, 2015 Original Article Korean J Fish Aquat Sci 48(5),731-738,2015 조피볼락 (Sebastes schlegeli) 선충 (Nematode: Philometridae) 에대한분자생물학적동정및 PCR 검출법개발 서한길 서정수 류민경 이은혜 정승희 한현자 * 국립수산과학원병리연구과