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1 2011 9, 18(6): Journal of Fishery Sciences of China 研究论文 DOI: /SP.J 柳学周 1, 宁鑫 1.2, 徐永江 1, 赵明 1, 李春广 1.2, 李军 1., ; 2., ; 3., 摘要 : (UV)(Pagrosomus major)(paralichthys lethostigma) UV 72 mj/cm 2,,, 40 s [(29±1.9)%], 100%, 0~2, 3 min 45 min,, (9.4±0.71)%,, (2n=48),, 64 h 59 min 55 h 49 min (P>0.05),,,, : ; ; ; ; 中图分类号 : S96 文献标志码 : A 文章编号 : (2011) (Paralichthys lethostigma), (Pleyronectiformes), (Bothidae), (Paralichthys),,, mm 230 mm [1],,, [2 3],, (Verasper moseri) [4] (Paralichthys olivaceus) [5 6] (Hippoglossus hippoglossus) [7] (Scophthalmus maximus) [8 9] (Cynoglossus semilaevis Günther) [10] [13], (Mugil cephalus) (Centropristis striata) [11 12] ; [13] (Lateolabrax japonicas),,,,,, 收稿日期 : ; 修订日期 : 基金项目 : 863 (2010AA10A402); ( ); (CARS-50); ( chg). 作者简介 : (1959 ),.. liuxz@ysfri.ac.cn

05),,,, : ; ; ; ; 中图分类号 : S96 文献标志码 : A 文章编号 : 1005 8737 (2011)06 1259 10 (Paralichthys lethostigma), (Pleyronectiformes), (Bothidae), (Paralichthys),,, 2 320 mm 230 mm [1],,, [2 3],,

2 , 3, (650±26) g, (33.5± 2.9) cm; (430±20) g, (26.3±3.1) cm , + +, : 12~26, 25~30, DO 6 mg/l, ph8.0~8.2, 500%~600%, 2 /, 2%~4% , GnRH(Syndel, Canada), 75 μg/,, 20~22 36 h 2 h 1 : MS-222 (150 mg/l),,,, 0.9% [14-15],,, s, 70% 0.2 ml Hank s 1..10, 9 cm 6, (Spectrolinker UVC(XL-1500)) 0 s 10 s 20 s 40 s 60 s 80 s 100 s 7 8 ( 10 ml), ml, 10 min L, (19±0.5) (, 401-H ), ph 8.0~8.2, 28~32,,, 36 h [14 17] 1.3 UV, UV 40 s, 72 mj/cm : 0 min 1 min 3 min 5 min 7 min 9 min( 0~2, 45 min),,, 36 h, 1.3.2, 7 : 0 min 15 min 30 min 46 min 60 min 75 min 90 min( 0~2, 3 min), , 30,, 20,, 260, DAPI Ploidy Analyzer(Paretec GmbH, Munster, germany) DNA [13] h, KC min,, 24 h 10 Giemsa (ph6.8 ) 30~35 min,, ; Olympus MS800,,, (2n=48) [18 20] L, (19±0.5), ph8.0~8.2, 28~32,,,, 30 min, (NIKON-MSZ800)

.10, 9 cm 6, (Spectrolinker UVC(XL-1500)) 0 s 10 s 20 s 40 s 60 s 80 s 100 s 7 8 ( 10 ml), 2 000 ml, 10 min 2 000 30 L, (19±0.5) (, 401-H ), ph 8.0~8.2, 28~32,,, 36 h [14 17] 1.

3 6 : , 3 ± ( x ±SD) SPSS, P 0.05, P<0.05 [21] : (%)=( )/ 100% (%)= / 100% (%)= 36 h / 100% (%)= / 100% UV,, (Hertwig) ( 1) (69±3.5)%,,, 10 s (2.0±0.1)%,, 40 s, (29±1.9)%, 80 s 0 80 s (15±1.2)%,, UV 40 s, (VLX-3W) UV µw/cm 2, 72 mj/cm 2, (n=24) 2.2, 0~2, 3 min 45 min, (37.2± 5.1)%, (9.4±0.71)%, (8.3±2.5)% ( 1), (2n=48),,, 1 min 5 min 3 min (P>0.05), (P<0.05), 1 min 5 min (27.7±3.2)% (21.3±1.9)%( 2); 30 min 45 min (P>0.05), ;, 45 min [(37.2±5.1)%], 90 min (10.4±1.9)%, (1.8±0.21)%( 3) 2 Fig. 2 Effects of initial moment of cold shock on hatching rate and malformation rate of gynogenetic diploid larvae Fig. 1 1 Relationship among UV irradiation duration, fertilization rate and hatching rate 3 Fig. 3 Effects of cold shock duration on hatching rate and malformation rate of gynogenetic diploid larvae

05), 1 min 5 min (27.7±3.2)% (21.3±1.9)%( 2); 30 min 45 min (P>0.05), ;, 45 min [(37.2±5.1)%], 90 min (10.4±1.9)%, (1.8±0.21)%( 3) 2 Fig.

4 : DNA DNA ( 4),,,,,,, (P<0.05)( 2), 36 h n=24, 2n=48, [20] ( 5) 2.4 (19±0.5), ph8.0~8.2, 28~32, DO 6 mg/l, 64 h 59 min 55 h 49 min (49 h 45 min) (P<0.05) [21], 3, Tab.1 condition for cold shock D 45 min, T 0~2 TA 3 min T 0~2 表 1 漠斑牙鲆冷休克梯度实验结果 Results from cold shock duration experiment in gynogenesis induction of Paralichthys lethostigma x ±SD; % group hatching rate : TA ; D. Note: TA Treatment initial time after fertilization; D Duration time. abnormallity rate fertilization rate ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±1.9 4 Fig. 4 (a):; (b):. Patterns of ploidy analysis of haploid, normal diploid and gynogentic diploid larvae a: haploid; b: normal diploid and gynogentic diploid.

hatching rate : TA ; D. Note: TA Treatment initial time after fertilization; D Duration time. abnormallity rate fertilization rate 0 0.47±0.032 43.4±3.5 46.2±5.1 1 5.10±0.63 27.7±3.2 37.5±4.5 3 8.

5 6 : 1263 Tab.2 表 2 正常二倍体雌核发育二倍体和单倍体 1 日龄仔鱼的几个形态指标比较 Comparison of morphological index among haploid, gynogenetic diploid and normal fry of southern flounder n=12; x ±SD sample normal fry gynogentic diploid haploid /mm body length /mm body depth /mm eye diameter /mm yolk sac length 5.4± ± ± ± ± ± ± ± ± ± ± ± a b: ; c: ; d: ; e:; f:. Fig.5 Morphological characteristics and chromosome karyotype of haploid larvae, gynogentic diploid larvae, normal diploid larvae of Paralichthys lethostigma a and b: haploid larvae; c: normal diploid larvae; d: gynogentic diploid larvae; e: chromosome karyotype made from hapiloid embryo; f: chromosome karyotype made from gynogentic diploid larvae , ( 6B C D E G H), 32,,, ( 6-F3); ( 6-F1), : 2.4.2, ;,,, ( 6 I1 I2 I3) h 33 min ( 6 J1 J2 J3),, ( 6 K1 2);, ( 6 1 L2), 1, ( 6 K3);,,, ( 6 L3), (1 h 56 min)(1 h 21 min), 3 h 3 min: /5, ;,, ( 6 M1 ),

fry gynogentic diploid haploid /mm body length /mm body depth /mm eye diameter /mm yolk sac length 5.4±0.91 1.7±0.23 0.13±0.02 2.1±0.32 5.3±1.20 1.9±0.32 0.18±0.03 2.2±0.26 5.0±0.87 2.1±0.34 0.09±0.

6 , ( 6 M3) : 2.4.5, ( 6 N1 N2);,, ( 6 N3) 2/5,, ( 6 O1 O2);, ( 6 O3) 3/5,,,, ( 6 P1 P2 P3) 3, 8 h 58 min 6 h 3 min, : /5,,, 10~15 /min; 60~70 /min( 6 Q1 Q2),, (45~55 /min), ( 6 Q3) ,, ( 6 R1 R2);,,, 1 h 3 min( 6 R3) ( 6 S1 S2);, ( 6 S3) 2.5,,, (P<0.05); (P>0.05), (29.0±1.9)%, 表 3 漠斑牙鲆单倍体雌核发育二倍体及正常二倍体在孵化水温 (19±0.5) 的发育持续时间 Tab. 3 Embryonic development of haploid, gynogenetic diploid and normal diploid Paralichthys lethostigma at (19±0.5) development stage fertilized egg 2 2-cell stage 4 4-cell stage 8 8-cell stage cell stage cell stage cell stage cell stage morula stage high blastula stage low blastula stage late blastula female gynogenetic diploid haploid normal 0h00min 0h00min 0h00min 1h46min 1h35min 2h03min 2h21min 1h59min 2h19min 3h11min 2h45min 2h56min 4h07min 3h39min 3h56min 4h58min 4h02min 4h35min 5h58min 5h32min 5h43min 7h26min 6h56min 7h11min 8h21min 8h12min 8h38min 10h8min 9h21min 10h00min 12h15min 11h05min 11h58min 13h27min 12h21min 13h12min development stage early gastrula stage mid gastrula stage late gastrula stage neurala stage formation of optic vesicle formation of somite formation of olfactory plate formation of eye lens formation of optic capsule muscular contraction hatching larvae hatched out female gynogenetic diploid haploid normal 15h23min 15h25min 14h33min 19h6min 18h6min 17h11min 21h49min 20h58min 20h12min 27h56min 27h58min 26h21min 31h21min 32h03min 28h17min 33h49min 34h12min 30h02min 35h43min 35h52min 32h53min 42h48min 41h27min 39h10min 51h12min 48h17min 42h14min 57h29min 53h29min 43h49min 63h12min 53h57min 48h56min 64h59min 55h49min 49h45min

3 Embryonic development of haploid, gynogenetic diploid and normal diploid Paralichthys lethostigma at (19±0.

7 6 : (A H, I1 S1)(I2 S2)(F3, I3 S3), 32,,. A: ; B: 2 ; C: 4 ; D: 8 ; E: 16 ; F1: 32 ; F3: 32, ; G: 128 ; H: ; I1 I3:, ; J1 J3:, ; K1 K3:, ; L1 L3:, ; M1 M3: ; N1 N3:, ; O1 O3:, ; P1 P3:, ; Q1 Q3:, ; R1 R3: ; S1 S3:. Fig.6 Comparison of morphological characteristics of embryonic development of normal diploid(a H, I1 S1), gynogenetic diploid(i2 S2) and haploid(f3, I3 S3) Paralichthys lethostigma A: fertilized egg; B: 2 cell stage; C: 4 cell stage; D: 8 cell stage;e: 16 cell stage; F: 32 cell stage; F3: 32 cell stage, arrow showing flower teratocytes; G: 128 cell stage; H: morula stage; I1 I3: high blastula stage, arrow showing high blastocyst; J1 J3: early gastrula stage, arrow showing germ ring; K1 K3: mid gastrula stage, arrow showing dead cells; L1 L3: Telo-phase of gastrulastage; M1 and M3: neurula stage; N1 N3: formation of optic vesicle; O1 O3: Kuppefs vesicle stage; P1 P3: formation of eye lens; Q1 Q3: muscular contraction, arrow showing abnormal embryo; R1 and R3: Hatching out; S1 S3: newly-hatched larva.

6 Comparison of morphological characteristics of embryonic development of normal diploid(a H, I1 S1), gynogenetic diploid(i2 S2) and haploid(f3, I3 S3) Paralichthys lethostigma A: fertilized egg; B:

8 , ( 4) 表 4 Tab. 4 各组受精率孵化率和初孵仔鱼畸形率统计 Fertilization rate, hatching rate and malformation rate of newly hatched larvae in each group % trial normal diploid haploid gynogenetic diploid 3 fertilization rate hatching rate rate of abnormality ± ± ± ± ± ± ± ± ,, (Centropristis striata)(lateolabrax japonicus) (Mugil cephalus) [11-13] 3, 3, (Perciformes) (Pleuronectiformes),, [20, 27],,,,, :, UV 40 s, 72 mj/cm 2, [6,25-26], : (), [22],, [28],, (19±0.5), 3 min, (8.3±2.5)%, (9.4±0.71)%, 1 min 5 min 3 min (P>0.05), (P<0.05)( 1); [13] Adam [12] TA 4~5 min, Andrew [11] TA 1 min, (18±0.5),,, [6-13],,,,, 90 min (10.4±1.9)%, (1.8±0.21)% ( 1) Johnson [29],, (9.4±0.71)%, (16.13±5.12)% [13] (1.48±0.52)% [11] ;,,, [22, 31], ; [30], 3.2 (19±0.5), 64 h 59 min 55 h 49 min(49 h 45 min)

rate hatching rate rate of abnormality 76.5.4±7.6 69.0±3.5 1.2±0.9 63.3±4.1 29.0±1.9 100 37.2±5.1 9.4±0.71 8.3±2.5 3.

9 6 : 1267 (P<0.05) 32,,,, 3 h 3 min( 3),, (Paralichthys olivaceus) [6] (Cynoglossus semilaevis Günther) [10] (Paramisgurnus dabryanus) [23] (Scophthalmus maximus) [26], [33],, DNA,, [26] [34] (Hemicentrotus pulcherrimus),,, (DCB ),,,,,, [22, 35],,,, ;, 1 h 3 min 参考文献 : [1].[C]., 2004: [2]. [J]., 1986, 24(1): [3],,,. [J]., 1981, 8(1): [4],,,. [J]., 2009, 33(3): [5],,,, [J]., 2005, 35(6): [6],,,. [J]., 2010, 34(4): [7] Harald B T, Tillmann J B, Deborah J M R, et al. Gynogenesis and sex determination in Atlantic halibut (Hippoglossush ippoglossus)[j]. Aquaculture, 2006, 252: [8] Frances C P, Rosa M C, Castora G, et al. Induction of gynogenesis in the turbot (Scophthalmus maximus): Effects of UV irradiation on sperm motility, the Hertwig effect and viability during the first 6 months of age [J]. Aquaculture, 2004,238: [9] Xu Jian-He, You Feng, Sun Wei, et al. Induction of diploid gynogenesis in turbot (Scophthalmus maximus) with left-eyed flounder Paralichthys olivaceus sperm [J]. Aqua Internat, 2008, 16: [10],,,. [J]., 2009, 33(1): [11] Andrew J M, Ryan Murashige, Christopher A W, et al. Effective UV dose and pressure shock for induction of meiotic gynogenesis in southern flounder (Paralichthys lethostigma) using black sea bass (Centropristis striata) sperm [J]. Aquaculture, 2006, 259: [12] Adam L J, Godwin J, Daniels H V, et al. Induction of diploid gynogenesis in southern flounder with homologous and heterologous sperm [J]. Aquaculture, 2004, 237: [13],,,. [J]., 2010, 34(8): [14],,,.( ) ( ) [J]., 2007, 14(4): [15]. [J]., 2003, 9(3): [16],,,. [J]., 2006, 27(2): [17],,,. [J]., 2007, 14(2): [18]. [J]., 1987, 1: [19],,,. [J]., 2009, 30(2): [20],,. [J]., 2007, 28(4): [21],. [J]., 2008, 4(1): [22]. [M]. :, 1992: [23],. [J]., 1998, 25(5):

pulcherrimus),,, (DCB ),,,,,, [22, 35],,,, ;, 1 h 3 min 参考文献 : [1].[C]., 2004: 202 209. [2]. [J]., 1986, 24(1): 80 90. [3],,,. [J]., 1981, 8(1): 50 55. [4],,,. [J]., 2009, 33(3): 372 378.

10 [24] Brown D B. Chromatin decondensation and DNA synthesis in human sperm activated in vitro by using Xenopus laevis egg exacts[j]. J Exp Zool, 1987, 242: [25] Yamakawa T, Matsuda H, Tsujigado A, et al. Optimum dose of UV irradiation for induction of gynogenesis in flounder Paralichthys olivaceus using red sea bream sperm [J]. Bull Fish Res Ins,1987, 2: [26],,,. [J]., 2010, 40(7): [27],. [J]., 1991, (3): [28],. [J]. :, 1991, 8(2): [29] Johnson M, Heath J W. Family induction methodology and interaction effects on the performance of diploid and triploid chinook salmon Oncorhynchus shawytscha[j]. Aquaculture, 2004, 234: [30]. [J]., 1988, 6: [31],,,. [J]., 2006, 13(2): [32],,. [J]., 2008, 23(3): [33],,,. [J]., 2007, 16(5): [34],,. [J]., 2008, 23(1): 1 7. [35] Pandian T J, Koteeswaran R. Ploidy induction and sex control in fish[j]. Hydrobiologia, 1998, 384: Induction of diploid gynogenesis in southern flounder (Paralichthys lethostigma) with heterologous sperm of Pagrosomus major LIU Xuezhou 1, NING Xin 1,2, XU Yongjiang 1, ZHAO Ming 1, LI Chunguang 1.2, LI Jun 3 1. Key Laboratory for Sustainable Utilization of Marine Fisheries Resource, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao , China; 2. College of Fisheries and Life Science, Shanghai Ocean University, Shanghai , China; 3. Institute of Oceanology, Chinese Academy of Sciences, Qingdao , China Abstract: Female southern flounder (Paralichthys lethostigma) grow faster than males. Therefore, farming all-female progenies could maximize profits from cultured fish. In the present study, diploid gynogenesis was induced by activating flounder egg development with UV-irradiated (72 mj/cm 2 ) Pagrosomus major sperm for 3 min in sea water, and then subjecting the eggs to a cold-shock (0 2 ) treatment for 45 min. Cold shock was used to prevent extrusion of the second polar body. In the control treatments, one or more of these steps was omitted to separately assess the effectiveness of UV irradiation and cold shock. When the initial treatment was applied 3 min after hatching (treatment duration, 45 min), the fertilization rate of eggs activated by UV-irradiated heterologous sperm was (37.2 ± 5.1)%, the malformation rate of newly hatched larvae was (8.3 ± 2.5)%, and the hatching rate was up to (9.4 ± 0.71)%. Flow cytometry and chromosome karyotyping were used to examine the ploidy level of larvae experienced to the cold-shock treatment. All of the gynogenetic larvae were diploid (2n = 48), and no haploid or aneuploid phenomena were detected. The duration of hatching of haploid embryos and gynogenetic embryos was significantly different from that of control normal embryos. There were no significant differences in the morphological index between gynogenetic diploids and normal larvae (P>0.05). All of the haploid larvae (100%) showed abnormal external morphology (P<0.05) while normal diploid larvae and gynogenetic diploid larvae showed normal morphologies. In summary, these methods resulted in successful diploid gynogenesis in southern flounder using heterologous sperm. This technique could serve as a useful tool to produce monosex progenies of southern flounder. Key words: Paralichthys lethostigma; heterologous sperm; gynogenesis; cold shock; embryonic development

Bull Fish Res Ins,1987, 2:51 53. [26],,,. [J]., 2010, 40(7): 36 42. [27],. [J]., 1991, (3): 64 67. [28],. [J]. :, 1991, 8(2): 51 55. [29] Johnson M, Heath J W.

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