三种香港牡蛎三倍体幼虫诱导方法的效果比较
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摘要
以卵裂率、D形幼虫发生率、三倍体率等为指标,比较了高盐、低温及咖啡因诱导香港牡蛎(Crassostrea hongkongensis)三倍体幼虫的效果,同时也分析了诱导因子强度(或浓度)、持续时间、受精卵密度及受精后开始处理时间对诱导效果的影响。结果表明,高盐诱导三倍体的最适条件组合是在卵子密度1.0×107个/L的情况下受精后15 min,用盐度40的海水持续处理10 min,此时卵裂率为(39.60±2.14)%, D形幼虫发生率为(31.46±1.06)%,三倍体率可以达到(59.53±5.90)%;低温诱导三倍体的最适条件组合是密度为2.0×10~7个/L的卵子在受精后15 min,用10℃海水持续处理10 min,此时卵裂率为(21.00±4.90)%, D形幼虫发生率为(12.68±1.21)%,三倍体率为(51.09±2.67)%;咖啡因诱导三倍体的最适条件组合是卵子密度为1.0×10~8个/L,用咖啡因终浓度2.0g/L的海水在受精后15min,持续处理20 min,卵裂率为(85.46±4.78)%, D形幼虫发生率为(71.79±3.92)%,三倍体率可以达到(56.36±2.07)%。最适诱导条件下,咖啡因的诱导效率指数0.405高于高盐处理0.187、低温处理0.065。高盐及低温处理方法诱导出的幼虫三倍体率随生长降低极快且存活率低于咖啡因诱导方法,说明3种方法中咖啡因可能更适合用于香港牡蛎三倍体幼虫的诱导。本研究为香港牡蛎三倍体育种提供了基础数据和实践经验。
We compared the effects of different salinities, different temperatures, and caffeine on triploid induction of Crassostrea hongkongensis in terms of cleavage rate, D larval rate and triploid rate. The effects of intensity or concentration, duration of treatment, egg density, and induction time were also studied. After the zygotes were fertilized at 31℃ and salinity of 15, optimum results were obtained 15 min post fertilization after hyperosmotic shock in the salinity of 40 for 10 min treatment, with a zygote density of 1.0×107 ind/L. Under these conditions,the cleavage rate was(39.60±2.14)%, D larval rate was(31.46±1.06)%, and triploid rate was(59.53±5.90)%. The induction time and duration of treatment obtained the same results as that of the high salinity treatment; however,the zygote density was 2.0×10~7 ind/L and zygotes were induced by 10℃ seawater, with these conditions optimum at low temperature. Here, the cleavage rate was(21.00±4.90)%, D larval rate was(12.68±1.21)%, and triploid rate was(51.09±2.67)%. Additionally, spawn was fertilized at 31℃ and a salinity of 15 and, 15 min after fertilization,2.0 g/L caffeine was added for a duration of 20 min at a zygote density of 1.0×10~8 ind/L, which were the optimum conditions of triploid induction by caffeine. Under these conditions, the cleavage rate was(85.46±4.78)%, D larval rate was(71.79±3.92)%, and triploid rate was(56.36±2.07)%. At the optimum conditions, the efficiency of triploid induction in caffeine(0.405) was higher than that of the high salinity(0.187) and low temperature(0.065)treatments. In addition, the triploid rate of larvae induced by the high salinity or low temperature was greatly decreased and the survival rates were lower than those noted for caffeine treatment. Therefore, caffeine was more applicable for triploid induction of C. hongkongensis than the other two methods, i.e., high salinity and low temperature. This study provides basic knowledge and practical evidence for triploid induction of C. hongkongensis.
We compared the effects of different salinities, different temperatures, and caffeine on triploid induction of Crassostrea hongkongensis in terms of cleavage rate, D larval rate and triploid rate. The effects of intensity or concentration, duration of treatment, egg density, and induction time were also studied. After the zygotes were fertilized at 31℃ and salinity of 15, optimum results were obtained 15 min post fertilization after hyperosmotic shock in the salinity of 40 for 10 min treatment, with a zygote density of 1.0×107 ind/L. Under these conditions,the cleavage rate was(39.60±2.14)%, D larval rate was(31.46±1.06)%, and triploid rate was(59.53±5.90)%. The induction time and duration of treatment obtained the same results as that of the high salinity treatment; however,the zygote density was 2.0×10~7 ind/L and zygotes were induced by 10℃ seawater, with these conditions optimum at low temperature. Here, the cleavage rate was(21.00±4.90)%, D larval rate was(12.68±1.21)%, and triploid rate was(51.09±2.67)%. Additionally, spawn was fertilized at 31℃ and a salinity of 15 and, 15 min after fertilization,2.0 g/L caffeine was added for a duration of 20 min at a zygote density of 1.0×10~8 ind/L, which were the optimum conditions of triploid induction by caffeine. Under these conditions, the cleavage rate was(85.46±4.78)%, D larval rate was(71.79±3.92)%, and triploid rate was(56.36±2.07)%. At the optimum conditions, the efficiency of triploid induction in caffeine(0.405) was higher than that of the high salinity(0.187) and low temperature(0.065)treatments. In addition, the triploid rate of larvae induced by the high salinity or low temperature was greatly decreased and the survival rates were lower than those noted for caffeine treatment. Therefore, caffeine was more applicable for triploid induction of C. hongkongensis than the other two methods, i.e., high salinity and low temperature. This study provides basic knowledge and practical evidence for triploid induction of C. hongkongensis.
引文
[1]Bureau of Fisheries,Ministry of Agriculture.China Fishery Statistical Yearbook[M].Beijing:China Agriculture Press,2016.[农业部渔业渔政管理局.中国渔业统计年鉴[M].北京:中国农业出版社,2016.]
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[3]Stanley J,Allen S K,Hidu H.Polyploidy induced in the American oyster,Crassostrea virginica,with cytochalasin B[J].Aquaculture,1981,23(1-4):1-10.
[4]Jeung H D,Keshavmurthy S,Lim H J,et al.Quantification of reproductive effort of the triploid Pacific oyster,Crassostrea gigas raised in intertidal rack and bag oyster culture system off the west coast of Korea during spawning season[J].Aquaculture,2016,464:374-380.
[5]Downing S L,Allen S K.Induced triploidy in the Pacific oyster,Crassostrea gigas:Optimal treatments with cytochalasin B depend on temperature[J].Aquaculture,1987,61(1):1-15.
[6]Desrosiers R R,Gerard A,Peignon J M,et al.A novel method to produce triploids in bivalve molluscs by the use of6-dimethylaminopurine[J].Journal of Experimental Marine Biology and Ecology,1993,170(1):29-43.
[7]Fong P P,Kyozuka K,Duncan J,et al.The effect of salinity and temperature on spawning and fertilization in the zebra mussel Dreissena polymorpha(Pallas)from North America[J].Biological Bulletin,1995,189(3):320-329.
[8]Peachey B L,Allen S K.Evaluation of cytochalasin B and6-dimethylaminopurine for tetraploidy induction in the Eastern oyster,Crassostrea virginica[J].Aquaculture,2016,450:199-205.
[9]Qin Y P,Zhang Y H,Zhou Y L,et al.Comparative studies on triploidy induction using CB and 6-DMAP in Crassostrea hongkongensis[J].Journal of Fisheries of China,2017,41(2):250-257.[秦艳平,张跃环,周颖力,等.CB与6-DMAP诱导香港牡蛎三倍体的效果比较[J].水产学报,2017,41(2):250-257.]
[10]Yu R H,Wang Z P,Wang R C,et al.Comparative studies on triploidy induction using three chemicals in Pacific oyster[J].Journal of Ocean University of Qingdao,2000,30(4):590-592.[于瑞海,王昭萍,王如才,等.三种化学诱导剂诱导太平洋牡蛎三倍体的比较研究[J].青岛海洋大学报,2000,30(4):590-592.]
[11]Komaru A,Wada K T.Different processes of pronuclear events in pressure-treated and CB-treated zygotes at the second meiosis in scallop[J].Nippon Suisan Gakkaishi,1991,57(7):1219-1223.
[12]Kong J,Wang Z P,Yu R H,et al.Triploid induction in Pacific oyster(Crassostrea gigas)by hypotonic treatment and comparison with other induction methods[J].Journal of Fishery Sciences of China,2011,18(3):581-587.[孔静,王昭萍,于瑞海,等.低渗诱导太平洋牡蛎三倍体以及与其他诱导方法的比较[J].中国水产科学,2011,18(3):581-587.]
[13]Yu R H,Wang Z P,Wang R C,et al.Induced triploid in pacific oyster using caffeine combined with thermal shock[J].Journal of Ocean University of Qingdao,2001,31(4):518-522.[于瑞海,王昭萍,王如才,等.利用咖啡因和热休克诱导太平洋牡蛎三倍体[J].青岛海洋大学学报,2001,31(4):518-522.]
[14]Zhang Q Z,Qiu M Y,Wu X Z,et al.Heat pretreatment induces thermotolerance in the Jinjiang oyster[J].Ecological Science,2005,24(1):35-37.[张其中,邱马银,吴信忠,等.热休克诱导近江牡蛎对高温的耐受性[J].生态科学,2005,24(1):35-37.]
[15]Qin Y P,Zhang Y H,Ma H T,et al.Comparison of the biochemical composition and nutritional quality between diploid and triploid Hong Kong oysters,Crassostrea hongkongensis[J].Frontiers in Physiology,2018,9:1674.
[16]Lin Q,Wu J S,Zeng Z N,et al.Studies on induction of triploid and technology of culture in Crassostrea gigas[J].Journal of Oceanography in Taiwan Strait,2000,19(4):479-483.[林琪,吴建绍,曾志南,等.长牡蛎三倍体的诱导与培育[J].台湾海峡,2000,19(4):479-483.]
[17]Zhang G F,Chang Y Q,Song J,et al.Comprehensive comparison between triploid oysters induced with CB and6-DMAP[J].Journal of Fisheries of China,2000,24(4):325-328.[张国范,常亚青,宋坚,等.不同方法制备的三倍体长牡蛎养殖效果的比较[J].水产学报,2000,24(4):325-328.]
[18]Zheng X D,Wang R C,Wang Z P,et al.Studies on karyotypes of diploid and triploid of Crassostrea gigas[J].Journal of Fishery Sciences of China,2000,7(2):96-97.[郑小东,王如才,王昭萍,等.太平洋牡蛎二倍体与三倍体的核型研究[J].中国水产科学,2000,7(2):96-97.]
[19]Qin Y P,Xiao S,Ma H T,et al.Effects of salinity and temperature on the timing of germinal vesicle breakdown and polar body release in diploid and triploid Hong Kong oysters,Crassostrea hongkongensis,in relation to tetraploid induction[J].Aquaculture Research,2018,49(11):3647-3657.
[20]Yu R H,Wang R C,Wang Z P,et al.A study on the effect of spawn density on triploid induction of Pacific oyster[J].Transactions of Oceanology and Limnology,2002(1):57-60.[于瑞海,王如才,王昭萍,等.不同卵密度对太平洋牡蛎三倍体诱导效果影响的研究[J].海洋湖沼通报,2002(1):57-60.]
[21]Tan S H A,Teh C P,Chang G O,et al.Tetraploid induction in tropical oysters,Crassostrea belcheri(Sowerby)and Crassostrea iredalei(Faustino)[J].Aquaculture Research,2017,48(4):1406-1412.
[22]Yamamoto S,Sugawara Y,et al.Induced triploidy in the mussel,Mytilus edulis,by temperature shock[J].Aquaculture,1988,72(1-2):21-29.
[23]Allen S K,Bushek D.Large-scale production of triploid oyster,Crassostrea virginica(Gmelin),using“stripped”gametes[J].Aquaculture,1992,103(3-4):241-251.
[24]Yang H P,Guo X M.Polyploid induction by heat shock-induced meiosis and mitosis inhibition in the dwarf surfclam,Mulinia lateralis Say[J].Aquaculture,2006,252(2-4):171-182.
[25]Yu R H,Wang Z P,Wang R C,et al.Comparison of economic efficiency of triploidy induction in Pacific oysters Crassostrea gigas by two methods[J].Transactions of Oceanology and Limnology,2003(1):57-61.[于瑞海,王昭萍,王如才,等.两种方法诱导太平洋牡蛎三倍体在生产上的应用效果[J].海洋湖沼通报,2003(1):57-61.]
[26]Zhang Y H,Li J,Qin Y P,et al.A comparative study of the survival,growth and gonad development of the diploid and triploid Hong Kong oyster,Crassostrea hongkongensis(Lam&Morton 2003)[J].Aquaculture Research,2017,48(5):2453-2462.
[27]Eudeline B,Allen S K,Guo X M,et al.Delayed meiosis and polar body release in eggs of triploid Pacific oysters,Crassostrea gigas,in relation to tetraploid production[J].Journal of Experimental Marine Biology and Ecology,2000,248(2):151-161.
[28]Gerard A,Naciri Y,Peignon J M,et al.Optimization of triploid induction by the use of 6-DMAP for the oyster Crassostrea gigas(Thunberg)[J].Aquaculture Research,2012,25(7):709-719.
[29]Nell J A.Farming triploid oysters[J].Aquaculture,2002,210(1-4):69-88.
[2]Wang Q Y.Cell engineering applied to breeding of aquaculture organisms in seawater[M].Beijing:China Ocean Press,2007:187-222.[王清印.海水养殖生物的细胞生物工程育种[M].北京:海洋出版社,2007:187-222.]
[3]Stanley J,Allen S K,Hidu H.Polyploidy induced in the American oyster,Crassostrea virginica,with cytochalasin B[J].Aquaculture,1981,23(1-4):1-10.
[4]Jeung H D,Keshavmurthy S,Lim H J,et al.Quantification of reproductive effort of the triploid Pacific oyster,Crassostrea gigas raised in intertidal rack and bag oyster culture system off the west coast of Korea during spawning season[J].Aquaculture,2016,464:374-380.
[5]Downing S L,Allen S K.Induced triploidy in the Pacific oyster,Crassostrea gigas:Optimal treatments with cytochalasin B depend on temperature[J].Aquaculture,1987,61(1):1-15.
[6]Desrosiers R R,Gerard A,Peignon J M,et al.A novel method to produce triploids in bivalve molluscs by the use of6-dimethylaminopurine[J].Journal of Experimental Marine Biology and Ecology,1993,170(1):29-43.
[7]Fong P P,Kyozuka K,Duncan J,et al.The effect of salinity and temperature on spawning and fertilization in the zebra mussel Dreissena polymorpha(Pallas)from North America[J].Biological Bulletin,1995,189(3):320-329.
[8]Peachey B L,Allen S K.Evaluation of cytochalasin B and6-dimethylaminopurine for tetraploidy induction in the Eastern oyster,Crassostrea virginica[J].Aquaculture,2016,450:199-205.
[9]Qin Y P,Zhang Y H,Zhou Y L,et al.Comparative studies on triploidy induction using CB and 6-DMAP in Crassostrea hongkongensis[J].Journal of Fisheries of China,2017,41(2):250-257.[秦艳平,张跃环,周颖力,等.CB与6-DMAP诱导香港牡蛎三倍体的效果比较[J].水产学报,2017,41(2):250-257.]
[10]Yu R H,Wang Z P,Wang R C,et al.Comparative studies on triploidy induction using three chemicals in Pacific oyster[J].Journal of Ocean University of Qingdao,2000,30(4):590-592.[于瑞海,王昭萍,王如才,等.三种化学诱导剂诱导太平洋牡蛎三倍体的比较研究[J].青岛海洋大学报,2000,30(4):590-592.]
[11]Komaru A,Wada K T.Different processes of pronuclear events in pressure-treated and CB-treated zygotes at the second meiosis in scallop[J].Nippon Suisan Gakkaishi,1991,57(7):1219-1223.
[12]Kong J,Wang Z P,Yu R H,et al.Triploid induction in Pacific oyster(Crassostrea gigas)by hypotonic treatment and comparison with other induction methods[J].Journal of Fishery Sciences of China,2011,18(3):581-587.[孔静,王昭萍,于瑞海,等.低渗诱导太平洋牡蛎三倍体以及与其他诱导方法的比较[J].中国水产科学,2011,18(3):581-587.]
[13]Yu R H,Wang Z P,Wang R C,et al.Induced triploid in pacific oyster using caffeine combined with thermal shock[J].Journal of Ocean University of Qingdao,2001,31(4):518-522.[于瑞海,王昭萍,王如才,等.利用咖啡因和热休克诱导太平洋牡蛎三倍体[J].青岛海洋大学学报,2001,31(4):518-522.]
[14]Zhang Q Z,Qiu M Y,Wu X Z,et al.Heat pretreatment induces thermotolerance in the Jinjiang oyster[J].Ecological Science,2005,24(1):35-37.[张其中,邱马银,吴信忠,等.热休克诱导近江牡蛎对高温的耐受性[J].生态科学,2005,24(1):35-37.]
[15]Qin Y P,Zhang Y H,Ma H T,et al.Comparison of the biochemical composition and nutritional quality between diploid and triploid Hong Kong oysters,Crassostrea hongkongensis[J].Frontiers in Physiology,2018,9:1674.
[16]Lin Q,Wu J S,Zeng Z N,et al.Studies on induction of triploid and technology of culture in Crassostrea gigas[J].Journal of Oceanography in Taiwan Strait,2000,19(4):479-483.[林琪,吴建绍,曾志南,等.长牡蛎三倍体的诱导与培育[J].台湾海峡,2000,19(4):479-483.]
[17]Zhang G F,Chang Y Q,Song J,et al.Comprehensive comparison between triploid oysters induced with CB and6-DMAP[J].Journal of Fisheries of China,2000,24(4):325-328.[张国范,常亚青,宋坚,等.不同方法制备的三倍体长牡蛎养殖效果的比较[J].水产学报,2000,24(4):325-328.]
[18]Zheng X D,Wang R C,Wang Z P,et al.Studies on karyotypes of diploid and triploid of Crassostrea gigas[J].Journal of Fishery Sciences of China,2000,7(2):96-97.[郑小东,王如才,王昭萍,等.太平洋牡蛎二倍体与三倍体的核型研究[J].中国水产科学,2000,7(2):96-97.]
[19]Qin Y P,Xiao S,Ma H T,et al.Effects of salinity and temperature on the timing of germinal vesicle breakdown and polar body release in diploid and triploid Hong Kong oysters,Crassostrea hongkongensis,in relation to tetraploid induction[J].Aquaculture Research,2018,49(11):3647-3657.
[20]Yu R H,Wang R C,Wang Z P,et al.A study on the effect of spawn density on triploid induction of Pacific oyster[J].Transactions of Oceanology and Limnology,2002(1):57-60.[于瑞海,王如才,王昭萍,等.不同卵密度对太平洋牡蛎三倍体诱导效果影响的研究[J].海洋湖沼通报,2002(1):57-60.]
[21]Tan S H A,Teh C P,Chang G O,et al.Tetraploid induction in tropical oysters,Crassostrea belcheri(Sowerby)and Crassostrea iredalei(Faustino)[J].Aquaculture Research,2017,48(4):1406-1412.
[22]Yamamoto S,Sugawara Y,et al.Induced triploidy in the mussel,Mytilus edulis,by temperature shock[J].Aquaculture,1988,72(1-2):21-29.
[23]Allen S K,Bushek D.Large-scale production of triploid oyster,Crassostrea virginica(Gmelin),using“stripped”gametes[J].Aquaculture,1992,103(3-4):241-251.
[24]Yang H P,Guo X M.Polyploid induction by heat shock-induced meiosis and mitosis inhibition in the dwarf surfclam,Mulinia lateralis Say[J].Aquaculture,2006,252(2-4):171-182.
[25]Yu R H,Wang Z P,Wang R C,et al.Comparison of economic efficiency of triploidy induction in Pacific oysters Crassostrea gigas by two methods[J].Transactions of Oceanology and Limnology,2003(1):57-61.[于瑞海,王昭萍,王如才,等.两种方法诱导太平洋牡蛎三倍体在生产上的应用效果[J].海洋湖沼通报,2003(1):57-61.]
[26]Zhang Y H,Li J,Qin Y P,et al.A comparative study of the survival,growth and gonad development of the diploid and triploid Hong Kong oyster,Crassostrea hongkongensis(Lam&Morton 2003)[J].Aquaculture Research,2017,48(5):2453-2462.
[27]Eudeline B,Allen S K,Guo X M,et al.Delayed meiosis and polar body release in eggs of triploid Pacific oysters,Crassostrea gigas,in relation to tetraploid production[J].Journal of Experimental Marine Biology and Ecology,2000,248(2):151-161.
[28]Gerard A,Naciri Y,Peignon J M,et al.Optimization of triploid induction by the use of 6-DMAP for the oyster Crassostrea gigas(Thunberg)[J].Aquaculture Research,2012,25(7):709-719.
[29]Nell J A.Farming triploid oysters[J].Aquaculture,2002,210(1-4):69-88.