한수지 50(6), 829-836, 2017 Note Korean J Fish Aquat Sci 50(6),829-836,2017 제주도사계연안어란의분자동정과격월별출현양상 한송헌 김맹진 김준상 1 송춘복 2 * 국립수산과학원서해수산연구소, 1 한국수산자원관리공단제주지사, 2 제주대학교해양과학대학 Molecular Identification and Bimonthly Abundance of Fish Eggs Collected in the Coastal Waters of Sagye, Jejudo Island Song-Hun Han, Maeng Jin Kim, Joon Sang Kim 1 and Choon Bok Song 2 * Fisheries Resources and Environment Division, National Fisheries Research and Development Institute, Incheon 22383, Korea 1 Korea Fisheries Resources Agency, Jeju Branch, Jeju 63005, Korea 2 College of Ocean Sciences, Jeju National University, Jeju 63243, Korea This study investigated the species composition and abundance of floating fish eggs to determine the timing and location of spawning of fish inhabiting the coastal waters of Sagye, Jejudo Island. Eggs were collected with a Bongo net bimonthly from May 2009 to February 2010. Identifications were based on nucleotide sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene. Eggs were determined to belong to 43 distinct taxa, 35 of which were identified to the species level. The assemblage spanned eight orders, 23 families, and 32 genera. The number of taxa collected varied from month to month, with 14 taxa (12 species) found in June 2009, 11 taxa (10 species) in October 2009, 10 taxa (nine species) each in August 2009 and February 2010, eight taxa (six species) in April 2009, and five taxa (four species) in December 2009. Five abundant species (Branchiostegus japonicus, Engraulis japonicus, Pseudolabrus sieboldi, Goniistius zonatus, and Halichoeres tenuispinis) together represented 52.8% of the total number of eggs collected during the study. Key words: Fish eggs, Identification, Molecular biology, Jejudo Island 서론,,,. DNA barcode, (Hebert et al., 2003)., mitochondrial DNA cytochrome c oxidase subunit I (COI) (species specific), (Ratnasingham and Hebert, 2007). COI (Lee and Kim, 2014a; Lee and Kim, 2014b; Lee and Kim, 2016)., PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism)., (Cocolin et al., 2000; Dooly et al., 2005) (Bivalvia) (Gastropoda) (Wang et al., 2006)., PCR-RFLP (pre-screening). COI PCR RFLP,,,, https://doi.org/10.5657/kfas.2017.0829 Korean J Fish Aquat Sci 50(6) 829-836, December 2017 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licens (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Received 19 October 2017; Revised 27 November 2017; Accepted 5 December 2017 *Corresponding author: Tel: +82. 64. 754. 3471 Fax: +82. 64. 756. 3493 E-mail address: cbsong@jejunu.ac.kr Copyright 2017 The Korean Society of Fisheries and Aquatic Science 829 pissn:0374-8111, eissn:2287-8815
830 한송헌ㆍ김맹진ㆍ김준상ㆍ송춘복. 재료및방법 시료채집 (33 13 N, 126 19 E) 2009 4 2010 2 (Fig. 1). ( 0.6 m, 2 m, 333 m) 2-3 knots 10, (HYDRO-BIOS flowmeter) 1,000 m 3 (ind./1,000 m 3 ). 70% ethanol. (YSI, USA). Total DNA 추출 DNA 70% ethanol 1.5 ml eppendorf tube micropipet tip. tissue lysis buffer (Bioneer Inc., Korea) 50 μl 0.2 μg/μl proteinase K (Bioneer Inc., Korea) 4 μl 60 1. binding buffer (Bioneer Inc., Korea) 50 L 60 10, 100% isopropanol (Sigma, USA) 25 μl, DNA binding column (Bioneer Inc., Korea). Accuprep Genomic DNA Extraction Kit (Bioneer Inc., Korea). DNA Unicam UV/VIS Spectrometer (He ios, Unicam Ltd., UK). PCR 증폭 PCR COI trna forward primer (Asn_F, 5'-YGATCCYRCAAAHWCTTAGT- TAACAGCTAA-3') reverse primer (Ser_R, 5'-GGGGTTC- DAYTCCYYCCTTTCT-3'). PCR volume 40 μl, 10 mm Tris-HCl (ph 8.3) 50 mm KCl, 2 mm MgCl 2, 0.2 mm dntp mix, 0.5 mm of each primer, 50 ng template DNA solution, 1 U Ex Taq DNA polymerase (Takara, Japan) TaKaRa PCR Thermal Cycler Dice (TP600, Takara Bio Inc.). PCR denaturing step 94 1 1, 94 30, primer annealing 54 1, primer extension 72 1 50 34, 72 7 extension. PCR 1X TAE buffer 0.5 mg/ L ethidium bromide 0.8% agarose (LE agarose, Cambrex co.) gel. PCR AccuPrep Gel Pruification Kit (Bioneer Inc., Korea). 제한효소처리 Korea 120 50 129 10 East Sea Fig. 1. A map showing a sampling site located at the coastal waters of Sagye, Jejudo Island. COI HinfI (Bioneer Inc., Korea) Hpy188I (NEB Inc., UK). HinfI 50 mm Tris-HCl (ph 7.6) 10 mm MgCl 2, 100 mm NaCl, 1 mm DTT, 10 ng PCR, 1 U HinfI volume 15 μl. Hpy188I volume 15 μl, 20 mm Tris-acetate (ph 7.9) 50 mm potassium acetate, 10 mm Mg acetate, 1 mm DTT, 10 ng PCR, 1 U Hpy188I. 37 2. 1X TAE buffer 0.5 μg/μl ethidium bromide 2% agarose (LE agarose, Cambrex co.) gel band. band, PCR. 염기서열결정및종동정 41 40 33 20 Sampling location 126 00 Jejudo Island 126 30 127 00 33 45 33 15 PCR-RFLP PCR Big Dye Terminator v3.1 Cycle Sequencing Kits ABI 3730xl DNA Analyzer (Applied Biosystems, USA). COI ( 1,551 bp) DNAssist program 2.2 (Patterton and Graves, 2000). (species identification)
제주도사계연안어란의분자동정과격월별출현양상 831 GenBank database (species), (genus), (family) (order). Kimura-2-parameter distance model (Kimura, 1980), MEGA6 (Tamura et al., 2013) Neighbor- Joining (Saitou and Nei, 1987). 군집구조분석및환경요인과의상관관계 (Shannon and Weaver, 1963), (Pielou, 1966), (McNaughton, 1967), SPSS (Ver 14.0 SPSS Inc.) (Pearson correlation coefficient Pearson s r),. 결과 수온과염분 Fig. 2. 14.9-28.2, 19.2. 2010 2, 2009 8. 31.2 psu (2009 8 ) 34.3 psu (2009 12 ), 33.0 psu. (r=-0.916, P<0.01). 종동정과출현현황, 301 275 COI PCR (91.4%). PCR RFLP,., Table 1. 43, 35 (8 23 32 ) (81.8%). 8 2 (family), 1 (order), 5. (Order) (Perciformes) 14 24 ( 1 ), (Pleuronectiformes) 2 3 ( 1 ), (Aulopiformes) 2 Temperature ( ) 30 25 20 15 10 5 Apr. Jun. Aug. Oct. Dec. Feb. Fig. 2. Bimonthly variation of surface water temperature ( ) and salinity (psu) at the sampling location. 3. (Cupeiformes), (Ophidiiformes), (Gadiformes), (Scorpaeniformes), (Tetraodontiformes). (Family), (Labridae) 7, (Sparidae) 3. (Synodontidae), (Moridae), (Malacanthidae), (Cheilodactylidae), (Callionymidae) 2, 1. 출현종및출현량 Month Temp. Sal. (ind./1,000 m 3 ) Fig. 3 Table 1. 5,590, 2009 8 2,344, 2009 10 1,693, 2010 2 675, 2009 6 406, 12 254, 4 219 8 10. 2009 6 14 (12 ), 2009 10 11 (10 ), 2009 8 2010 2 10 (9 ), 2009 4 12 8 (6 ) 5 (4 ). (Branchiostegus japonicus) 2009 8 912, 16.3%. (Engraulis japonicus) 2009 4, 8 2010 2 668 (11.9%), (Pseudolabrus eoethinus) 2009 10 12 631 (11.3%), (Goniistius zonatus) 2009 10 388 (6.9%), (Halichoeres tenuispinis) 2009 10 12 359 (6.4%), 5 52.8%. 35 34 33 32 31 30 Salinity (psu)
832 한송헌ㆍ김맹진ㆍ김준상ㆍ송춘복 Table 1. Species occurrence and number of individuals of fish eggs (ind./1,000 m 3 ) collected bimonthly in the coastal waters of Sagye, Jejudo Island from April 2009 to February 2010 Order Family Species 2009 2010 Total no. Apr Jun Aug Oct Dec Feb of eggs (%) Cupeiformes Engraulidae Engraulis japonicus 6-646 - - 16 668(11.9) Aulopiformes Aulopodidae Aulopus japonicus - - - 35 - - 35(0.6) Synodontidae Saurida wanieso - 5 - - - - 5(0.1) Trachinocephalus myops - - - 212 - - 212(3.8) Ophidiiformes Ophidiidae Hoplobrotula armata - - 31 35 - - 66(1.2) Gadiformes Moridae Lotella phycis - - - - 14 27 41(0.7) Moridae sp. - - - - - 83 83(1.5) Scorpaeniformes Platycephalidae Platycephalus indicus - 5 - - - - 5(0.1) Perciformes Moronidae Lateolabrax japonicus - - - - - 83 83(1.5) Serranidae Caprodon schlegelii 65 - - - - 164 229(4.1) Callanthiidae Callanthias japonicus 98 65 - - 27 83 274(4.9) Malacanthidae Branchiostegus albus - - - - - 27 27(0.5) Branchiostegus japonicus - - 912 - - - 912(16.3) Sparidae Acanthopagrus schlegeli - 54 - - - - 54(1.0) Evynnis japonica - - - 35 - - 35(0.6) Sparus sarba 6 - - - - - 6(0.1) Mullidae Upeneus japonicas - 16 - - - - 16(0.3) Kyphosidae Girella punctate - 43 - - - - 43(0.8) Oplegnathidae Oplegnathus fasciatus - 76 - - - - 76(1.4) Cheilodactylidae Goniistius quadricornis - - - 106 - - 106(1.9) Goniistius zonatus - - - 388 - - 388(6.9) Labridae Halichoeres tenuispinis - 76 283 - - - 359(6.4) Pseudolabrus eoethinus - - - 494 137-631(11.3) Pseudolabrus sieboldi - - - 247 69-316(5.7) Semicossyphus reticulatus - 16 - - - - 16(0.3) Stethojulis terina - - 189 - - - 189(3.4) Suezichthys gracilis - 11 - - - - 11(0.2) Xyrichtys dea - - 31 71 - - 102(1.8) Pinguipedidae Pinguipedidae sp. - 5 - - - - 5(0.1) Callionymidae Neosynchiropus ijimae - 5 - - - - 5(0.1) Repomucenus virgis - - - - - - 27(0.5) Trichiuridae Trichiurus japonicus - - 31 35 - - 66(1.2) Scombridae Auxis rochei - - 63 - - - 63(1.1) Pleuronectiformes Paralichthyidae Paralichthys olivaceus 26 22 - - - - 159(2.8) Pseudorhombus pentophthalmus 6 - - - - - 6(0.1) Pleuronectidae Eopsetta grigorjewi - - - - - - 55(1.0) Pleuronectiformes sp. - 5 - - - - 5(0.1) Tetraodontiformes Diodontidae Diodon holocanthus - - 94 - - - 94(1.7) Unidentified sp. A 6 - - - - - 6(0.1) Unidentified sp. B 6 - - - - - 6(0.1) Unidentified sp. C - - 63 - - - 63(1.1) Unidentified sp. D - - - 35 - - 35(0.6) Unidentified sp. E - - - - 7 7 7(0.1) Total no. of eggs (%) 219 (3.9) 406 (7.3) 2,343 1,693 (41.9) (30.3) 254 (4.5) 254 (4.5) 5,590 (100.0) Number of taxa examined 8 14 10 11 5 5 43 Number of species identified 6 12 14 10 4 4 35 -, non-appearance.
제주도사계연안어란의분자동정과격월별출현양상 833 Apr, 2009 Jun, 2009 Aug, 2009 33 45 219 406 2,344 33 15 Oct, 2009 Dec, 2009 Feb, 2009 33 45 1,693 254 675 33 15 126 00 126 00 127 00 126 00 126 00 127 00 126 00 126 00 127 00 1-250 250-500 500-1,000 1,000-2,000 >2,000 (ind./1,000 m) Fig. 3. Bimonthly abundance of fish eggs collected at the sampling location of the coastal waters of Sagye, Jejudo Island. Values in parentheses indicate the number of species. 2009 4 (Callanthias japonicus) 4 98 (44.7%), 6 (Oplegnathus fasciatus) 76, 6 18.7%. 8 912 (38.9%). 10 12, 494 (29.2%) 137 (53.9%). 2010 2 (Caprodon schlegelii) 164 (24.3%). 생태지수의변동,, Fig. 4. 1.18-2.21 2009 6 2.21, 2009 12 1.18. 0.71-0.91, 2010 2 0.91, 2009 6 0.82, 10 0.81, 12 0.74, 8 0.73, 4 0.71. 0.38-0.81, 2009 12 0.81, 4 0.74. 2009 8 0.66, 10 0.52, 2010 2 0.41, 6 0.38. 환경요인과의상관관계 Diversity Evenness Dominant 2.5 2 1.5 1 0.5 0 1 0.8 0.6 0.4 0.2 0 1 0.8 0.6 0.4 0.2 0 1.47 0.71 0.74 2.21 0.82 0.37 1.68 0.73 0.66 0.52 0.81 0.41 Apr Jun Aug Oct Dec Feb Month 1.95 0.81 1.18 0.74 2.09 0.91 Fig. 4. Diversity, evenness and dominance indices of fish eggs collected during this study.
834 한송헌ㆍ김맹진ㆍ김준상ㆍ송춘복 Table 2. Comparison between present and previous studies on the mean abundance and number of identified species of fish eggs Sampling area Sampling period (Sampling times, no. of location) Mean abundance of fish eggs No. of species (No. of taxa) Reference Yeongil Bay Feb-Dec, 2001 (4, 8) 573.8 7 (9) Han et al., 2003 Saemangeum Jan-Aug, 2003 (5, 9-12) 610.2 4 (8) Cha et al., 2004 Uljin Jan-Oct, 2002 (4, 5) 298.7 5 (7) Han and Kim, 2007 Jejudo Island Aug, 2006-Jul, 2007 (12, 4) 506.0 34 (43) Han et al., 2015 Sagyeoi Apr, 2009-Feb, 2010 (6, 1) 931.7 35 (43) Present study Pearson s r. 0.897 (P<0.05), -0.910 (P<0.05)., 0.209-0.428 (P>0.05). 주요어종의출현시기및수온범위 (Table 1) (Fig. 2), 2009 4, 6, 12, 2010 2 4, 14.9-18.4., 2009 4, 8, 2010 2 3, (14.9-28.2 ), 8. (Paralichthys olivaceus) 2009 4, 6, 2010 2 14.0-16.2, 2010 2. (Family) (29.1%) (7 ), 2009 6 2009 12 16.4-28.2, 2009 10 12 16.4-20.7. 2009 8 (28.2 ) 1, 2010 2 (14.9 ). (Lateolabrax japonicus), (Acanthopagrus schlegeli), (Girella punctata),, (Trichiurus japonicus), (Eopsetta grigorjewi)., 2010 2 14.9,, 2009 6 18.4, 2009 8 10 28.2-20.7, 2010 2 14.9. 고찰 PCR. COI 94.7% cytochrome b (cyt b) PCR 68.3% (Han et al., 2015). cyt b trna (glutamic acid trna 5' threonine proline trna 3' ) trna (Inoue et al., 2001) trna Thr. Ivanova et al. (2007) COI PCR 96.8%. COI Ivanova et al. (2007) 650 bp, Lee and Kim (2014a) 510 bp, Lee and Kim (2016) 515 bp. PCR-RFLP band prescreening 2-3 1,550 bp COI.. (Table 2)., 932, 574 (Han et al., 2003), 610 (Cha et al., 2004), 299 (Han and Kim, 2007), 506 (Han et al., 2015). (Kim et al., 2014) (kg/1,000 m 2 ) CPUE (Catch Per Unit Effort),,,
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