CB诱导熊本牡蛎三倍体及其存活率与倍化率的变化关系
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摘要
为诱导熊本牡蛎三倍体,研究了细胞松弛素B (CB)浓度、诱导起始时间、诱导持续时间等因素对卵裂率、D幼率、三倍体率的影响,并分析了幼虫、稚贝及成贝的存活率和三倍体率的变化特征。结果显示,CB浓度为0.5～0.6 mg/L,诱导起始时间为40%受精卵释放第一极体,诱导持续时间为20 min时可获得87%的三倍体率。卵裂率、D幼率、三倍体率的最大影响因素分别为CB浓度、诱导持续时间、诱导起始时间与诱导持续时间。三倍体率与卵裂率无显著负相关性,而与D幼率呈显著正相关。因此,减小CB浓度或诱导持续时间,可同时获得较高的三倍体率与幼虫产量。3～15日龄三倍体组与对照组的存活率分别由71.27%与96.09%降低至34.14%与58.80%,成贝期450日龄(9月)三倍体组与对照组的存活率分别为53.62%与44.67%。3～9日龄三倍体率从87%降低至77%,而90～450日龄三倍体率平均值为59.21%±4.99%,表明幼贝与成贝期三倍体率变化较小,三倍体率的维持与存活率无显著相关性。
In order to induce triploid using cytochalasin B(CB) in Crossostrea sikamea, three effecting factors,including CB concentration, initial time of induction, and duration time of induction, were optimized by the cleavage rate, D larvae rate, and triploidy rate. In addition, the variations of survival rate and triploidy rate were also compared for larvae and adults. The results suggested that the optimal method of triploid induction were 0.5 mg/L CB, 40% first polar body releasing for initial time of induction, and 20 min for duration time of induction. The triploidy rate of 87% was obtained by the optimal method. The first effecting factor for cleavage rate, D larvae rate, and triploidy rate was CB concentration, duration time of induction and initial time of induction, and duration time of induction, respectively. Furthermore, there was insignificant negative relationship between triploidy rate and cleavage rate. Whereas, the positive relationship was significant between triploidy rate and D larvae rate. As a result, high triploidy rate and high larvae production could be obtained by decreasing both CB concentration and duration time of induction. The survival rates were 71.27% and 96.09% at 3 days, and34.14% and 58.80% at 15 days for triploid and control groups, respectively. Comparatively, the survival rate were53.62% and 44.67% for adults at 450 days(September) in triploid and control groups, respectively. The triploidy rate declined from 87% to 77% during the period of 3 to 9 days. Whereas, the triploidy rate was 59.21%±4.99% on average from 90 to 450 days, suggesting that there was no correlativity between triploidy rate and survival rate,with a small variation of triploidy rate for adults. This study provides valuable data for further exploration into triploidy of C. sikamea.
In order to induce triploid using cytochalasin B(CB) in Crossostrea sikamea, three effecting factors,including CB concentration, initial time of induction, and duration time of induction, were optimized by the cleavage rate, D larvae rate, and triploidy rate. In addition, the variations of survival rate and triploidy rate were also compared for larvae and adults. The results suggested that the optimal method of triploid induction were 0.5 mg/L CB, 40% first polar body releasing for initial time of induction, and 20 min for duration time of induction. The triploidy rate of 87% was obtained by the optimal method. The first effecting factor for cleavage rate, D larvae rate, and triploidy rate was CB concentration, duration time of induction and initial time of induction, and duration time of induction, respectively. Furthermore, there was insignificant negative relationship between triploidy rate and cleavage rate. Whereas, the positive relationship was significant between triploidy rate and D larvae rate. As a result, high triploidy rate and high larvae production could be obtained by decreasing both CB concentration and duration time of induction. The survival rates were 71.27% and 96.09% at 3 days, and34.14% and 58.80% at 15 days for triploid and control groups, respectively. Comparatively, the survival rate were53.62% and 44.67% for adults at 450 days(September) in triploid and control groups, respectively. The triploidy rate declined from 87% to 77% during the period of 3 to 9 days. Whereas, the triploidy rate was 59.21%±4.99% on average from 90 to 450 days, suggesting that there was no correlativity between triploidy rate and survival rate,with a small variation of triploidy rate for adults. This study provides valuable data for further exploration into triploidy of C. sikamea.
引文
[1]Guo X M,DeBrosse G A,Allen S K Jr.All-triploid Pacific oysters(Crassostrea gigas Thunberg)produced by mating tetraploids and diploids[J].Aquaculture,1996,142(3-4):149-161.
[2]Nell J A,Perkins B.Studies on triploid oysters in Australia:farming potential of all-triploid Pacific oysters,Crassostrea gigas Thunberg,in Port Stephens,New South Wales,Australia[J].Aquaculture Research,2005,36(6):530-536.
[3]Piferrer F,Beaumont A,Falguière J C,et al.Polyploid fish and shellfish:production,biology and applications to aquaculture for performance improvement and genetic containment[J].Aquaculture,2009,293(3-4):125-156.
[4]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.
[5]Stanley J G,Allen S K Jr,Hidu H.Polyploidy induced in the American oyster,Crassostrea virginica,with cytochalasin B[J].Aquaculture,1981,23(1-4):1-10.
[6]Allen S K Jr,Downing S L.Performance of triploid Pacific oyster,Crassostrea gigas Thunberg.I.Survival,growth,glycogen content,and sexual maturation in yearlings[J].Journal of Experimental Marine Biology and Ecology,1986,102(2-3):197-208.
[7]Nell J A,Hand R E,Goard L J,et al.Studies on triploid oysters in Australia:evaluation of cytochalasin B and 6-dimethylaminopurine for triploidy induction in Sydney rock oysters Saccostrea commercialis(Iredale and Roughley)[J].Aquaculture Research,1996,27(9):689-698.
[8]Gendreau S,Grizel H.Induced triploidy and tetraploidy in the European flat oyster,Ostrea edulis L[J].Aquaculture,1990,90(3-4):229-238.
[9]曾志南,陈木,林琪,等.僧帽牡蛎三倍体的研究[J].海洋通报,1994,13(6):34-40.Zeng Z N,Chen M,Lin Q,et al.Triploidization of oyster Ostrea cucullata with ctyochalasin B and temperature shock[J].Marine Science Bulletin,1994,13(6):34-40(in Chinese).
[10]杨春玲,陈晓汉,李咏梅,等.应用6-DMAP人工诱导近江牡蛎三倍体的初步研究[J].西南农业学报,2008,21(3):825-828.Yang C L,Chen X H,Li Y M,et al.Artificial induction of triploidy in Crassostrea rivularis with 6-DMAP[J].Southwest China Journal of Agricultural Sciences,2008,21(3):825-828(in Chinese).
[11]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.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(in Chinese).
[12]Wang H Y,Qian L M,Wang A M,et al.Occurrence and distribution of Crassostrea sikamea(Amemiya 1928)in China[J].Journal of Shellfish Research,2013,32(2):439-446.
[13]Camara M D,Davis J P,Sekino M,et al.The Kumamoto oyster Crassostrea sikamea is neither rare nor threatened by hybridization in the northern Ariake sea,Japan[J].Journal of Shellfish Research,2008,27(2):313-322.
[14]Hong J S,Sekino M,Sato S.Molecular species diagnosis confirmed the occurrence of Kumamoto oyster Crassostrea sikamea in Korean waters[J].Fisheries Science,2012,78(2):259-267.
[15]Hedgecock D,Banks M A,Mcgoldrick D k.The status of the Kumamoto oyster Crassostrea sikamea(Amemiya1928)in US commercial brood stocks[J].Journal of Shellfish Research,1993,12(2):215-221.
[16]张跃环,秦艳平,张扬,等.熊本牡蛎多嵴和无嵴品系F1生长性状的群体选育[J].中国水产科学,2016,23(4):882-889.Zhang Y H,Qin Y P,Zhang Y,et al.Population selection for growth in two strains of the Kumamoto oyster Crassostrea sikamea[J].Journal of Fishery Sciences of China,2016,23(4):882-889(in Chinese).
[17]张跃环,秦艳平,张扬,等.熊本牡蛎(Crassostrea sikamea)多嵴和无嵴品系子二代生长性状的选择效应[J].海洋与湖沼,2016,47(3):581-586.Zhang Y H,Qin Y P,Zhang Y,et al.Selective effect of the second generation for fast growth in two strains of the Kumamoto oyster Crassostrea sikamea[J].Oceanologia et Limnologia Sinica,2016,47(3):581-586(in Chinese).
[18]张跃环,武祥伟,秦艳平,等.熊本牡蛎多嵴和无嵴品系F3生长性状的连续选择效应[J].中国水产科学,2017,24(6):1161-1167.Zhang Y H,Wu X W,Qin Y P,et al.Sustained response to selection of growth traits to the third generation for two strains of Kumamoto oyster Crassostrea sikamea[J].Journal of Fishery Sciences of China,2017,24(6):1161-1167(in Chinese).
[19]张跃环,秦艳平,武祥伟,等.熊本牡蛎无嵴和多嵴品系生产性状比较[J].水产学报,2016,40(12):1883-1888.Zhang Y H,Qin Y P,Wu X W,et al.Evaluation of phenotypic traits of two strains of the Kumamoto oyster Crassostrea sikamea[J].Journal of Fishery Sciences of China,2016,40(12):1883-1888(in Chinese).
[20]Zhang Y H,Li J,Zhang Y,et al.Performance evaluation of reciprocal hybrids derived from the two brackish oysters,Crassostrea hongkongensis and Crassostrea sikamea in southern China[J].Aquaculture,2017,473:310-316.
[21]苏家齐.葡萄牙牡蛎与熊本牡蛎、僧帽牡蛎的种间杂交研究[D].青岛:中国海洋大学,2015.Su J Q.Hybridization between Crassostrea Angulata and C.Sikamea,Saccostrea Cucullata[D].Qingdao:Ocean University of China,2015(in Chinese).
[22]Xu F,Li L,Wang J F,et al.Use of high-resolution melting analysis for detecting hybrids between the oysters Crassostrea sikamea and C.angulata reveals bidirectional gametic compatibility[J].Journal of Molluscan Studies,2014,80(4):435-443.
[23]Yan L L,Su J Q,Wang Z P,et al.Growth performance and biochemical composition of the oysters Crassostrea sikamea,Crassostrea angulata and their hybrids in southern China[J].Aquaculture Research,2018,49(2):1020-1028.
[24]Eudeline B,Allen S K Jr,Guo X M.Optimization of tetraploid induction in Pacific oysters,Crassostrea gigas,using first polar body as a natural indicator[J].Aquaculture,2000,187(1-2):73-84.
[25]Schatten G,Schatten H.Effects of motility inhibitors during sea urchin fertilization:microfilament inhibitors prevent sperm incorporation and restructuring of fertilized egg cortex,whereas microtubule inhibitors prevent pronuclear migrations[J].Experimental Cell Research,1981,135(2):311-330.
[26]田传远,梁英,王如才,等.6-DMAP诱导太平洋牡蛎三倍体:诱导因素对孵化率和D幼畸形率的影响[J].中国水产科学,2000,7(1):110-112.Tian C Y,Liang Y,Wang R C,et al.Triploid Crassostrea gigas induced with 6-DMAP:the influences of 3factors on hatching rate and D-shaped larvae abnormality rate[J].Journal of Fishery Sciences of China,2000,7(1):110-112(in Chinese).
[27]Desrosiers R R,Gérard A,Peignon J M,et al.A novel method to produce triploids in bivalve molluscs by the use of 6-dimethylaminopurine[J].Journal of Experimental Marine Biology and Ecology,1993,170(1):29-43.
[28]田传远,梁英,王如才,等.6-DMAP诱导太平洋牡蛎三倍体-5.孵化率和D幼畸形率与三倍体诱导率的关系[J].青岛海洋大学学报,1998,28(3):421-425.Tian C Y,Liang Y,Wang R C,et al.Triploid Pacific oyster(Crassostrea gigas)induced by 6-DMAP:5.The relationship between embryo hatching rate and D-shaped larval deformity rate and triploidy[J].Journal of Ocean University of Qingdao,1998,28(3):421-425(in Chinese).
[29]田传远,王如才,梁英,等.6-DMAP诱导太平洋牡蛎三倍体:抑制受精卵第二极体释放[J].中国水产科学,1999,6(2):1-4.Tian C Y,Wang R C,Liang Y,et al.Triploidy of Crassostrea gigas induced with 6-DMA P:by blocking the second polar body of the zygotes[J].Journal of Fishery Sciences of China,1999,6(2):1-4(in Chinese).
[30]Cox E S,Smith M S R,Nell J A,et al.Studies on triploid oysters in Australia.VI.Gonad development in diploid and triploid Sydney rock oysters Saccostrea commercialis(Iredale and Roughley)[J].Journal of Experimental Marine Biology and Ecology,1996,197(1):101-120.
[31]He M X,Lin Y G,Shen Q,et al.Production of tetraploid pearl oyster(Pinctada martensii Dunker)by inhibiting the first polar body in eggs from triploids[J].Journal of Shellfish Research,2000,19(1):147-151.
[32]Hand R E,Nell J A,Maguire G B.Studies on triploid oysters in Australia.X.Growth and mortality of diploid and triploid Sydney rock oysters Saccostrea commercialis(Iredale and Roughley)[J].Journal of Shellfish Research,1998,17(4):1115-1127.
[33]Cotter E,Malham S K,O′Keeffe S,et al.Summer mortality of the Pacific oyster,Crassostrea gigas,in the Irish Sea:the influence of growth,biochemistry and gametogenesis[J].Aquaculture,2010,303(1-4):8-21.
[34]Allen S K.Triploids for field study?The good,the bad,and the ugly[J].Journal of Shellfish Research,1993,12(1):125.
[2]Nell J A,Perkins B.Studies on triploid oysters in Australia:farming potential of all-triploid Pacific oysters,Crassostrea gigas Thunberg,in Port Stephens,New South Wales,Australia[J].Aquaculture Research,2005,36(6):530-536.
[3]Piferrer F,Beaumont A,Falguière J C,et al.Polyploid fish and shellfish:production,biology and applications to aquaculture for performance improvement and genetic containment[J].Aquaculture,2009,293(3-4):125-156.
[4]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.
[5]Stanley J G,Allen S K Jr,Hidu H.Polyploidy induced in the American oyster,Crassostrea virginica,with cytochalasin B[J].Aquaculture,1981,23(1-4):1-10.
[6]Allen S K Jr,Downing S L.Performance of triploid Pacific oyster,Crassostrea gigas Thunberg.I.Survival,growth,glycogen content,and sexual maturation in yearlings[J].Journal of Experimental Marine Biology and Ecology,1986,102(2-3):197-208.
[7]Nell J A,Hand R E,Goard L J,et al.Studies on triploid oysters in Australia:evaluation of cytochalasin B and 6-dimethylaminopurine for triploidy induction in Sydney rock oysters Saccostrea commercialis(Iredale and Roughley)[J].Aquaculture Research,1996,27(9):689-698.
[8]Gendreau S,Grizel H.Induced triploidy and tetraploidy in the European flat oyster,Ostrea edulis L[J].Aquaculture,1990,90(3-4):229-238.
[9]曾志南,陈木,林琪,等.僧帽牡蛎三倍体的研究[J].海洋通报,1994,13(6):34-40.Zeng Z N,Chen M,Lin Q,et al.Triploidization of oyster Ostrea cucullata with ctyochalasin B and temperature shock[J].Marine Science Bulletin,1994,13(6):34-40(in Chinese).
[10]杨春玲,陈晓汉,李咏梅,等.应用6-DMAP人工诱导近江牡蛎三倍体的初步研究[J].西南农业学报,2008,21(3):825-828.Yang C L,Chen X H,Li Y M,et al.Artificial induction of triploidy in Crassostrea rivularis with 6-DMAP[J].Southwest China Journal of Agricultural Sciences,2008,21(3):825-828(in Chinese).
[11]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.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(in Chinese).
[12]Wang H Y,Qian L M,Wang A M,et al.Occurrence and distribution of Crassostrea sikamea(Amemiya 1928)in China[J].Journal of Shellfish Research,2013,32(2):439-446.
[13]Camara M D,Davis J P,Sekino M,et al.The Kumamoto oyster Crassostrea sikamea is neither rare nor threatened by hybridization in the northern Ariake sea,Japan[J].Journal of Shellfish Research,2008,27(2):313-322.
[14]Hong J S,Sekino M,Sato S.Molecular species diagnosis confirmed the occurrence of Kumamoto oyster Crassostrea sikamea in Korean waters[J].Fisheries Science,2012,78(2):259-267.
[15]Hedgecock D,Banks M A,Mcgoldrick D k.The status of the Kumamoto oyster Crassostrea sikamea(Amemiya1928)in US commercial brood stocks[J].Journal of Shellfish Research,1993,12(2):215-221.
[16]张跃环,秦艳平,张扬,等.熊本牡蛎多嵴和无嵴品系F1生长性状的群体选育[J].中国水产科学,2016,23(4):882-889.Zhang Y H,Qin Y P,Zhang Y,et al.Population selection for growth in two strains of the Kumamoto oyster Crassostrea sikamea[J].Journal of Fishery Sciences of China,2016,23(4):882-889(in Chinese).
[17]张跃环,秦艳平,张扬,等.熊本牡蛎(Crassostrea sikamea)多嵴和无嵴品系子二代生长性状的选择效应[J].海洋与湖沼,2016,47(3):581-586.Zhang Y H,Qin Y P,Zhang Y,et al.Selective effect of the second generation for fast growth in two strains of the Kumamoto oyster Crassostrea sikamea[J].Oceanologia et Limnologia Sinica,2016,47(3):581-586(in Chinese).
[18]张跃环,武祥伟,秦艳平,等.熊本牡蛎多嵴和无嵴品系F3生长性状的连续选择效应[J].中国水产科学,2017,24(6):1161-1167.Zhang Y H,Wu X W,Qin Y P,et al.Sustained response to selection of growth traits to the third generation for two strains of Kumamoto oyster Crassostrea sikamea[J].Journal of Fishery Sciences of China,2017,24(6):1161-1167(in Chinese).
[19]张跃环,秦艳平,武祥伟,等.熊本牡蛎无嵴和多嵴品系生产性状比较[J].水产学报,2016,40(12):1883-1888.Zhang Y H,Qin Y P,Wu X W,et al.Evaluation of phenotypic traits of two strains of the Kumamoto oyster Crassostrea sikamea[J].Journal of Fishery Sciences of China,2016,40(12):1883-1888(in Chinese).
[20]Zhang Y H,Li J,Zhang Y,et al.Performance evaluation of reciprocal hybrids derived from the two brackish oysters,Crassostrea hongkongensis and Crassostrea sikamea in southern China[J].Aquaculture,2017,473:310-316.
[21]苏家齐.葡萄牙牡蛎与熊本牡蛎、僧帽牡蛎的种间杂交研究[D].青岛:中国海洋大学,2015.Su J Q.Hybridization between Crassostrea Angulata and C.Sikamea,Saccostrea Cucullata[D].Qingdao:Ocean University of China,2015(in Chinese).
[22]Xu F,Li L,Wang J F,et al.Use of high-resolution melting analysis for detecting hybrids between the oysters Crassostrea sikamea and C.angulata reveals bidirectional gametic compatibility[J].Journal of Molluscan Studies,2014,80(4):435-443.
[23]Yan L L,Su J Q,Wang Z P,et al.Growth performance and biochemical composition of the oysters Crassostrea sikamea,Crassostrea angulata and their hybrids in southern China[J].Aquaculture Research,2018,49(2):1020-1028.
[24]Eudeline B,Allen S K Jr,Guo X M.Optimization of tetraploid induction in Pacific oysters,Crassostrea gigas,using first polar body as a natural indicator[J].Aquaculture,2000,187(1-2):73-84.
[25]Schatten G,Schatten H.Effects of motility inhibitors during sea urchin fertilization:microfilament inhibitors prevent sperm incorporation and restructuring of fertilized egg cortex,whereas microtubule inhibitors prevent pronuclear migrations[J].Experimental Cell Research,1981,135(2):311-330.
[26]田传远,梁英,王如才,等.6-DMAP诱导太平洋牡蛎三倍体:诱导因素对孵化率和D幼畸形率的影响[J].中国水产科学,2000,7(1):110-112.Tian C Y,Liang Y,Wang R C,et al.Triploid Crassostrea gigas induced with 6-DMAP:the influences of 3factors on hatching rate and D-shaped larvae abnormality rate[J].Journal of Fishery Sciences of China,2000,7(1):110-112(in Chinese).
[27]Desrosiers R R,Gérard A,Peignon J M,et al.A novel method to produce triploids in bivalve molluscs by the use of 6-dimethylaminopurine[J].Journal of Experimental Marine Biology and Ecology,1993,170(1):29-43.
[28]田传远,梁英,王如才,等.6-DMAP诱导太平洋牡蛎三倍体-5.孵化率和D幼畸形率与三倍体诱导率的关系[J].青岛海洋大学学报,1998,28(3):421-425.Tian C Y,Liang Y,Wang R C,et al.Triploid Pacific oyster(Crassostrea gigas)induced by 6-DMAP:5.The relationship between embryo hatching rate and D-shaped larval deformity rate and triploidy[J].Journal of Ocean University of Qingdao,1998,28(3):421-425(in Chinese).
[29]田传远,王如才,梁英,等.6-DMAP诱导太平洋牡蛎三倍体:抑制受精卵第二极体释放[J].中国水产科学,1999,6(2):1-4.Tian C Y,Wang R C,Liang Y,et al.Triploidy of Crassostrea gigas induced with 6-DMA P:by blocking the second polar body of the zygotes[J].Journal of Fishery Sciences of China,1999,6(2):1-4(in Chinese).
[30]Cox E S,Smith M S R,Nell J A,et al.Studies on triploid oysters in Australia.VI.Gonad development in diploid and triploid Sydney rock oysters Saccostrea commercialis(Iredale and Roughley)[J].Journal of Experimental Marine Biology and Ecology,1996,197(1):101-120.
[31]He M X,Lin Y G,Shen Q,et al.Production of tetraploid pearl oyster(Pinctada martensii Dunker)by inhibiting the first polar body in eggs from triploids[J].Journal of Shellfish Research,2000,19(1):147-151.
[32]Hand R E,Nell J A,Maguire G B.Studies on triploid oysters in Australia.X.Growth and mortality of diploid and triploid Sydney rock oysters Saccostrea commercialis(Iredale and Roughley)[J].Journal of Shellfish Research,1998,17(4):1115-1127.
[33]Cotter E,Malham S K,O′Keeffe S,et al.Summer mortality of the Pacific oyster,Crassostrea gigas,in the Irish Sea:the influence of growth,biochemistry and gametogenesis[J].Aquaculture,2010,303(1-4):8-21.
[34]Allen S K.Triploids for field study?The good,the bad,and the ugly[J].Journal of Shellfish Research,1993,12(1):125.