• ISSN 1000-0615
  • CN 31-1283/S
Volume 9 Issue 11
Nov.  2021
Article Contents
Turn off MathJax

Citation:

Continuous observation of early embryonic development of Hyriopsis cumingii under in vitro culture condition and analysis of biological zero point and effective accumulated temperature

  • Corresponding author: BAI Zhiyi, zybai@shou.edu.cn
  • Received Date: 2020-12-08
    Accepted Date: 2021-03-07
    Available Online: 2021-10-14
  • Hyriopsis cumingii is a unique freshwater breeding pearl mussel in our country. It has the reproductive biology characteristics that the fertilized eggs develop to the larvae in gills. The unique reproductive biology characteristics make the offspring produced by the mussels random in the process of artificial breeding and restrict the application of modern biological breeding techniques to mussels such as triploid induction, gynogenesis and gene editing. In this study, in order to do research on development of H. cumingii embryos and explore the in vitro culture of its early embryos, male and female gametes obtained by anatomy were used as materials for in vitro fertilization and in vitro culture with isoosmotic balance salt solution (BSS) suitable for freshwater mussels. And the morphological characteristics of each stage of embryo were observed with the required time for development recorded. Meanwhile, the embryos in its nurturing pouch of outer gill were continuously observed and the water temperatures were recorded day and night at the same time, then we calculated the embryonic developmental biological zero point and examined the reliability of the biological zero point. After that, effective accumulated temperature of each embryonic development stage was calculated according to the biological zero point confirmed. Results show that when the water temperature is (25 ± 1) °C, the anatomy of the sperms and eggs were mixed after diluting by BSS saline and fertilized eggs could develop to morula stage. The fertilized eggs developed to polar body emissions at 1.8 h, developed to 2-cell stage at 2.8 h, developed to 4-cell stage at 5.6 h, developed to 8-cell stage at 10.5 h, developed to 16-cell stage at 13.9 h, and developed to morula stage at 17.3 h after observing under optical microscope. And then embryos did not change, and some of embryos were deformed. The morphological characteristics of embryos at each stage under in vitro culture condition were similar to those in the gills. Meanwhile, when the average water temperature in the pond was 26.54 °C, 28.08 °C and 29.51 °C, it took respectively 10 d, 9 d and 8 d for embryos to develop into mature glochidium. And the biological zero point for embryonic development of H. cumingii was 14.81 °C which was confirmed reliable after examining. The effective accumulated temperature were 12.95 °C×d for embryos developing to cleavage stage, 25.99 °C×d for embryos developing to blastocyst stage, 42.27 °C×d for embryos developing to gastrulation stage, 69.21 °C×d for embryos developing to glochidium stage and 118.14 °C×d for embryos developing to cleavage stage mature mlochidium stage, respectively, according to the biological zero point calculated previously. The study tried to cultivate H. cumingii embryos under in vitro condition, which confirmed the feasibility to cultivate H. cumingii embryos under in vitro condition, and then we analyzed and calculated the biological zero point and effective accumulated temperature of early embryonic development. The results of this study can provide reference for the researches on artificial breeding and development of modern biological genetic breeding technology of H. cumingii.
  • 加载中
  • [1] 刘月英, 张文珍, 王跃先, 等. 中国经济动物志(淡水软体动物)[M]. 北京: 科学出版社, 1979: 68-132.Liu Y Y, Zhang W Z, Wang Y X, et al. Chinese economic animal data ( Freshwater mollusk)[M]. Beijing: Science Press, 1979: 68-132 (in Chinese).
    [2] Li J L, Wu X P, Bai Z Y. Freshwater pearl culture: success stories and modern trends[M]//Aquaculture in China. New York: John Wiley & Sons, 2018: 185-196.
    [3] 张根芳, 方爱萍, 李家乐. 淡水蚌类繁殖生物学研究进展[J]. 水产学报, 2005, 29(4): 560-564.Zhang G F, Fang A P, Li J L. A review in reproductive biology of freshwater mussel (Unionidae)[J]. Journal of Fisheries of China, 2005, 29(4): 560-564 (in Chinese).
    [4] 李阳春, 王昭萍, 马培振, 等. 低盐处理对长牡蛎胚胎发育及氧化应激、能量供应和渗透调节相关酶的影响[J]. 中国海洋大学学报, 2020, 50(10): 19-25.Li Y C, Wang Z P, Ma P Z, et al. Effect of hypotonic induction on embryonic development, energy metabolism, oxidation resistant and osmoregulation in Pacific oyster Crassostrea gigas[J]. Periodical of Ocean University of China, 2020, 50(10): 19-25 (in Chinese).
    [5] 沈永忱, 郑宝太, 刘吉明, 等. 中国蛤蜊的诱导产卵和胚胎发育[J]. 齐鲁渔业, 2005, 22(3): 2-3+7.Shen Y C, Zheng B T, Liu J M, et al. Induced spawning and embryo development of Mactra chinensis Philippi[J]. Shandong Fisheries, 2005, 22(3): 2-3+7 (in Chinese).
    [6] 梁玉波, 张福绥. 温度、盐度对栉孔扇贝(Chlamys farreri)胚胎和幼虫的影响[J]. 海洋与湖沼, 2008, 39(4): 334-340. doi: 10.3321/j.issn:0029-814X.2008.04.006Liang Y B, Zhang F S. Effects of temperature/salinity on development of embryos and larvae of scallop Chlamys farreri[J]. Oceanologia et Limnologia Sinica, 2008, 39(4): 334-340 (in Chinese). doi: 10.3321/j.issn:0029-814X.2008.04.006
    [7] 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. doi: 10.1016/0044-8486(81)90002-8
    [8] 孙秀鹏. 贝类三倍体育种研究进展[J]. 现代农业科技, 2017(4): 234-236. doi: 10.3969/j.issn.1007-5739.2017.04.130Sun X P. Research progress of shellfish triploid breeding[J]. Modern Agricultural Science and Technology, 2017(4): 234-236 (in Chinese). doi: 10.3969/j.issn.1007-5739.2017.04.130
    [9] Yu H, Li H J, Li Q, et al. Targeted gene disruption in Pacific oyster based on CRISPR/Cas9 ribonucleoprotein complexes[J]. Marine Biotechnology, 2019, 21(3): 301-309. doi: 10.1007/s10126-019-09885-y
    [10] 弭忠祥, 陈海军, 韩继卫, 等. 一种诱导三倍体三角帆蚌培育珍珠的方法: 中国, CN201010292013.2[P]. 2011-01-12.Mi Z X, Chen H J, Han J W, et al. A method for inducing triploid Hyriopsis cumingii to cultivate pearls: China: CN201010292013.2[P]. 2011-01-12.
    [11] Jirka K J, Neves R J. Reproductive biology of four species of freshwater mussels (Molluscs: Unionidae) in the New River, Virginia and West Virginia[J]. Journal of Freshwater Ecology, 1992, 7(1): 35-44. doi: 10.1080/02705060.1992.9664668
    [12] 陈修报, 杨健. 淡水蚌类发生与发育研究进展[J]. 中国水产科学, 2011, 18(4): 944-952.Chen X B, Yang J. Gametogenesis and development of freshwater bivalve molluscs: a review[J]. Journal of Fishery Sciences of China, 2011, 18(4): 944-952 (in Chinese).
    [13] Uthaiwan K, Pakkong P, Noparatnaraporn N, et al. Study of a suitable fish plasma for in vitro culture of glochidia Hyriopsis myersiana[J]. Aquaculture, 2002, 209(1-4): 197-208. doi: 10.1016/S0044-8486(01)00870-5
    [14] 马学艳, 闻海波, 金武, 等. 血浆种类和温度对褶纹冠蚌钩介幼虫非寄生变态发育及早期稚蚌生长的影响[J]. 农学学报, 2019, 9(2): 72-77. doi: 10.11923/j.issn.2095-4050.cjas18030014Ma X Y, Wen H B, Jin W, et al. Plasma type and temperature: effects on the non-parasitic metamorphosis of glochidia and growth of early juvenile mussel of Cristaria plicata[J]. Journal of Agriculture, 2019, 9(2): 72-77 (in Chinese). doi: 10.11923/j.issn.2095-4050.cjas18030014
    [15] 余颖, 洪一江, 邱齐骏, 等. 池蝶蚌胚胎发育与繁殖季节性腺的观察[J]. 动物学杂志, 2008, 43(3): 102-107. doi: 10.3969/j.issn.0250-3263.2008.03.017Yu Y, Hong Y J, Qiu Q J, et al. Embryonic development and breading-season gonad in Hyriopsis schlegeli[J]. Chinese Journal of Zoology, 2008, 43(3): 102-107 (in Chinese). doi: 10.3969/j.issn.0250-3263.2008.03.017
    [16] 李爽, 李耕, 潘玉洲, 等. 刺参生殖腺发育的生物学零度和有效积温研究[J]. 安徽农业科学, 2016, 44(1): 81-82. doi: 10.3969/j.issn.0517-6611.2016.01.027Li S, Li G, Pan Y Z, et al. Study on the biological zero point and effective accumulated temperature for the gonad development of sea cucumber[J]. Journal of Anhui Agricultural Sciences, 2016, 44(1): 81-82 (in Chinese). doi: 10.3969/j.issn.0517-6611.2016.01.027
    [17] 田斌, 王璐. 虾夷扇贝性腺发育的生物学零度与有效积温研究[J]. 中国水产, 2018(5): 93-96.Tian B, Wang L. The zero point temperature and the effective accumulative temperature for the biology of the gonad development of Patinopecten yessoensis[J]. China Fisheries, 2018(5): 93-96 (in Chinese).
    [18] 梁峻, 闫喜武, 李霞, 等. 菲律宾蛤仔性腺发育生物学零度的研究[J]. 海洋科学, 2007, 31(9): 67-72. doi: 10.3969/j.issn.1000-3096.2007.09.013Liang J, Yan X W, Li X, et al. Biological zero point of gonad development of Ruditapes philippinarum[J]. Marine Sciences, 2007, 31(9): 67-72 (in Chinese). doi: 10.3969/j.issn.1000-3096.2007.09.013
    [19] 白志毅, 李家乐, 杨光. 三角帆蚌钩介幼虫发育的生物学零度和有效积温初步研究[J]. 特产研究, 2008, 30(1): 12-14. doi: 10.3969/j.issn.1001-4721.2008.01.004Bai Z Y, Li J L, Yang G. Studies on the biological zero temperature and effective accumulated temperature of glochidia development of triangle mussel (Hyriopsis cumingii)[J]. Special Wild Economic Animal and Plant Research, 2008, 30(1): 12-14 (in Chinese). doi: 10.3969/j.issn.1001-4721.2008.01.004
    [20] 邬祥光. 昆虫生态学的常用数学分析方法[M]. 北京: 农业出版社, 1963: 232-233.Wu X G. Common mathematical analysis of insect ecology[M]. Beijing: Agriculture Press, 1963: 232-233 (in Chinese).
    [21] 闻海波, 顾若波, 华丹, 等. 三角帆蚌钩介幼虫体外培养及变态稚贝的形态变化[J]. 武汉大学学报(理学版), 2011, 57(1): 57-62.Wen H B, Gu R B, Hua D, et al. Culture of glochidia of Hyriopsis cumingii in artificial media and morphological variation of transformed juvenile[J]. Journal of Wuhan University (Natural Science Edition), 2011, 57(1): 57-62 (in Chinese).
    [22] Pynnönen K. Accumulation of 45Ca in the freshwater unionids Anodonta anatina and Unio tumidus, as influenced by water hardness, protons, and aluminum[J]. Journal of Experimental Zoology, 1991, 260(1): 18-27. doi: 10.1002/jez.1402600103
    [23] Wang N, Ingersoll C G, Ivey C D, et al. Sensitivity of early life stages of freshwater mussels (Unionidae) to acute and chronic toxicity of lead, cadmium, and zinc in water[J]. Environmental Toxicology and Chemistry, 2010, 29(9): 2053-2063.
    [24] 曹林泉, 秦政, 姜玉声, 等. 日本米虾胚胎发育及离体孵化[J]. 渔业科学进展, 2020, 41(1): 145-152.Cao L Q, Qin Z, Jiang Y S, et al. Embryonic development of Caridina japonica and in vitro incubation of its fertilized eggs[J]. Progress in Fishery Sciences, 2020, 41(1): 145-152 (in Chinese).
    [25] 肖友翔. 环境因子对日本海神蛤早期生长发育的影响[D]. 大连: 大连海洋大学, 2016.Xiao Y X. Effects of environmental factors on early growth and development of Panopea japonica[D]. Dalian: Dalian Ocean University, 2016 (in Chinese).
    [26] 何苗. pH、温度、饵料浓度对缢蛏钙化率及能量代谢的影响[D]. 上海: 上海海洋大学, 2017.He M. Studies on calcification and metabolism of Sinonovacula constricta[D]. Shanghai: Shanghai Ocean University, 2017 (in Chinese).
    [27] 王国强. 软体动物胚胎发育的温度效应研究进展[J]. 安徽农业科学, 2012, 40(28): 13830-13831. doi: 10.3969/j.issn.0517-6611.2012.28.057Wang G Q. Research progress in the effect of temperature on embryonic development of mollusk[J]. Journal of Anhui Agricultural Sciences, 2012, 40(28): 13830-13831 (in Chinese). doi: 10.3969/j.issn.0517-6611.2012.28.057
    [28] 林瑞才, 陈敏, 林笔水. 温度和盐度对海湾扇贝幼虫附着及变态的影响[J]. 台湾海峡, 1989, 8(1): 60-67.Lin R C, Chen M, Lin B S. Effects of temperature and salinity on attachment and metamorphosis of bay scallop larvae Argopecten irrdians (Lamarck)[J]. Journal of Oceanography in Taiwan Strait, 1989, 8(1): 60-67 (in Chinese).
    [29] 王宏, 白志毅, 李家乐, 等. 三角帆蚌胚胎在外鳃育儿囊内形态变化初步研究[J]. 上海水产大学学报, 2007, 16(3): 219-223.Wang H, Bai Z Y, Li J L, et al. A primary study on the morphological changes of embryo of Hyriopsis cumingii in nurturing pouch of outer gill[J]. Journal of Shanghai Fisheries University, 2007, 16(3): 219-223 (in Chinese).
    [30] 薛素燕, 毛玉泽, 方建光, 等. 中华原钩虾的胚胎发育及其与水温的关系[J]. 水产学报, 2016, 40(11): 1705-1712.Xue S Y, Mao Y Z, Fang J G, et al. The embryonic development of Eogammarus possjeticus and its relationship with variation in incubation temperature[J]. Journal of Fisheries of China, 2016, 40(11): 1705-1712 (in Chinese).
  • Relative Articles

    [1] KUANG Yulan, LIANG Xufang, CAI Wenjing, HE Shan, XU Jing, GAO Junjie, WEI Junran. Primary cell culture of hypothalamus neurons in Siniperca chuatsi. Journal of fisheries of china, 2021, 45(2): 179-186.  doi: 10.11964/jfc.20200112136
    [2] LI Ziqi, CHENG Meiling, WU Yuping, ZHANG Jingjing, LI Zhentong, MA Wenhui, PANG Zunfang, ZHAI Jieming, TIAN Yongsheng. Early development of hybrids of Epinephelus lanceolatus(♀) × Epinephelus moara() and growth characteristics of reciprocal crosses. Journal of fisheries of china, 2020, 44(3): 436-446.  doi: 10.11964/jfc.20190311680
    [3] CHAO Qinghe, SHEN Fengfeng, CAI Zhenxi, ZHANG Junling. Expression pattern and localization analysis of cbx2 during embryonic and gonadal development in the medaka (Oryzias latipes). Journal of fisheries of china, 2021, 45(3): 357-364.  doi: 10.11964/jfc.20200112133
    [4] LIANG Keying, ZHANG Ziping, LIAO Jiaqian, ZOU Zhihua, WANG Yilei. Expression analysis of Sp-Sox14 gene during embryonic and gonadal development in Scylla paramamosain. Journal of fisheries of china, 2018, 42(2): 204-215.  doi: 10.11964/jfc.20170210712
    [5] MA Huan, CHU Jixing, WANG Liyan, LI Fuhua, XIANG Jianhai. Cloning and expression analysis of shrimp ovarian peritrophin-like gene during early embryonic development in Litopenaeus vannamei. Journal of fisheries of china, 2017, 41(5): 649-657.  doi: 10.11964/jfc.20160710479
    [6] CUI Wenyao, ZHOU Liqing, YANG Jihui, XIONG Jiafeng, LI Weiming, REN Jianfeng. Embryonic development and phylogenetic analysis of Puntius tetrazona. Journal of fisheries of china, 2020, 44(8): 1286-1295.  doi: 10.11964/jfc.20190411725
    [7] MAO Yuanyuan, ZHENG Rongquan, ZHANG Qipeng, MEI Yiyun, KONG Shenshen, LIU Zhifang. Transcriptome sequencing analysis on pearl color related genes in Hyriopsis cumingii. Journal of fisheries of china, 2018, 42(3): 322-333.  doi: 10.11964/jfc.20161110620
    [8] LI Xilei, LI Qingqing, REN Mingdong, BAI Zhiyi, LI Jiale. Cloning and tissue expression of a novel hcSRCR1 gene in differential inner-shell color pearl mussel Hyriopsis cumingii. Journal of fisheries of china, 2018, 42(11): 1719-1728.  doi: 10.11964/jfc.20171211107
    [9] XIA Zhonghui, LIU Xiaojun, LI Jiale. The role of Hyriopsis cumingii shell silk-like matrix protein gene silkmaxin in the shell and pearl biosmineralization. Journal of fisheries of china, 2019, 43(12): 2449-2458.  doi: 10.11964/jfc.20181111542
    [10] CHEN Ya, WANG Yayu, WANG Guiling, HE Fangshu, LI Jiale. Full-length cDNA cloning of M and F-type COⅡ genes and expression in different age freshwater mussel Hyriopsis cumingii. Journal of fisheries of china, 2018, 42(1): 29-38.  doi: 10.11964/jfc.20161210644
    [11] CHEN Xiajun, HAN Xuekai, BAI Zhiyi, LI Jiale. Detection of nacre colour-related SNPs and genetic mapping of HcTyp-1 gene in Hyriopsis cumingii. Journal of fisheries of china, 2019, 43(2): 467-473.  doi: 10.11964/jfc.20171111066
    [12] LIU Xiaojun, GUO Wei, JIN Can, BAI Zhiyi, LI Jiale. A novel shell matrix protein hic9 from Hyriopsis cumingiiinvolved in the pearl biomineralization. Journal of fisheries of china, 2019, 43(4): 782-789.  doi: 10.11964/jfc.20171111020
    [13] HAN Xuekai, CHEN Xiajun, BAI Zhiyi, LIU Xiaojun, LI Jiale. Detection of shell nacre colour-related SNP and gene mapping of HcTyr gene in Hyriopsis cumingii. Journal of fisheries of china, 2017, 41(7): 1044-1053.  doi: 10.11964/jfc.20170210717
    [14] CHEN Qiang, JIANG Xiamin, PENG Ruibing, HAN Qingxi, SHENG Peng, XUE Ruiping. Effects of four microalgae diets on growth, nutritional composition, pearl producing function, shell nacre color and trace element content of Hyriopsis cumingii. Journal of fisheries of china, 2021, 45(6): 899-909.  doi: 10.11964/jfc.20191212114
    [15] TANG Jinyu, WANG Yan, REN Gang, LI Youming. Effects of stocking density of the fishes fed formulated feed and EM product on phytoplankton community in a mussel-fish integrated system. Journal of fisheries of china, 2019, 43(6): 1438-1448.  doi: 10.11964/jfc.20180511289
  • 加载中
通讯作者: 陈斌, bchen63@163.com
  • 1. 

    沈阳化工大学材料科学与工程学院 沈阳 110142

  1. 本站搜索
  2. 百度学术搜索
  3. 万方数据库搜索
  4. CNKI搜索

Figures(1) / Tables(3)

Article views(364) PDF downloads(6) Cited by()

Related
Proportional views

Continuous observation of early embryonic development of Hyriopsis cumingii under in vitro culture condition and analysis of biological zero point and effective accumulated temperature

    Corresponding author: BAI Zhiyi, zybai@shou.edu.cn
  • 1. Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture and Rural Affairs, Shanghai Ocean University, Shanghai 201306, China
  • 2. Shanghai Collaborative Innovation Center for Cultivating Elite Breeds and Green-culture of Aquaculture Animals, Shanghai Ocean University, Shanghai    201306, China
  • 3. Zhejiang Wuyi Weimin Aquaculture Co. Ltd., Jinhua    321200, China

Abstract: Hyriopsis cumingii is a unique freshwater breeding pearl mussel in our country. It has the reproductive biology characteristics that the fertilized eggs develop to the larvae in gills. The unique reproductive biology characteristics make the offspring produced by the mussels random in the process of artificial breeding and restrict the application of modern biological breeding techniques to mussels such as triploid induction, gynogenesis and gene editing. In this study, in order to do research on development of H. cumingii embryos and explore the in vitro culture of its early embryos, male and female gametes obtained by anatomy were used as materials for in vitro fertilization and in vitro culture with isoosmotic balance salt solution (BSS) suitable for freshwater mussels. And the morphological characteristics of each stage of embryo were observed with the required time for development recorded. Meanwhile, the embryos in its nurturing pouch of outer gill were continuously observed and the water temperatures were recorded day and night at the same time, then we calculated the embryonic developmental biological zero point and examined the reliability of the biological zero point. After that, effective accumulated temperature of each embryonic development stage was calculated according to the biological zero point confirmed. Results show that when the water temperature is (25 ± 1) °C, the anatomy of the sperms and eggs were mixed after diluting by BSS saline and fertilized eggs could develop to morula stage. The fertilized eggs developed to polar body emissions at 1.8 h, developed to 2-cell stage at 2.8 h, developed to 4-cell stage at 5.6 h, developed to 8-cell stage at 10.5 h, developed to 16-cell stage at 13.9 h, and developed to morula stage at 17.3 h after observing under optical microscope. And then embryos did not change, and some of embryos were deformed. The morphological characteristics of embryos at each stage under in vitro culture condition were similar to those in the gills. Meanwhile, when the average water temperature in the pond was 26.54 °C, 28.08 °C and 29.51 °C, it took respectively 10 d, 9 d and 8 d for embryos to develop into mature glochidium. And the biological zero point for embryonic development of H. cumingii was 14.81 °C which was confirmed reliable after examining. The effective accumulated temperature were 12.95 °C×d for embryos developing to cleavage stage, 25.99 °C×d for embryos developing to blastocyst stage, 42.27 °C×d for embryos developing to gastrulation stage, 69.21 °C×d for embryos developing to glochidium stage and 118.14 °C×d for embryos developing to cleavage stage mature mlochidium stage, respectively, according to the biological zero point calculated previously. The study tried to cultivate H. cumingii embryos under in vitro condition, which confirmed the feasibility to cultivate H. cumingii embryos under in vitro condition, and then we analyzed and calculated the biological zero point and effective accumulated temperature of early embryonic development. The results of this study can provide reference for the researches on artificial breeding and development of modern biological genetic breeding technology of H. cumingii.

  • 三角帆蚌(Hyriopsis cumingii)属于软体动物门(Mollusca)双壳纲(Bivalve)真瓣鳃目(Eulamellibranchia)蚌科(Unionidae)帆蚌属(Hyriopsis)[1],是我国特有的淡水珍珠养殖品种之一[2]。淡水蚌类具有受精卵在雌蚌鳃腔内进行胚胎发育的繁殖生物学特性[3],目前,三角帆蚌人工繁育通常利用该特性,直接采集怀成熟钩介幼虫的雌蚌进行采苗、寄苗。这种苗种繁育方法中,三角帆蚌自受精卵发育至钩介幼虫属于自然采苗,无法进行人工干预,因此制约了多倍体诱导、雌核发育、基因编辑等现代生物育种技术的应用。海水双壳贝类多为体外受精,迄今为止,长牡蛎(Crassostrea gigas)[4]、中国蛤蜊(Mactra chinensis)[5]和栉孔扇贝(Chlamys farreri)[6] 等典型海水双壳贝类受精卵体外培养技术已非常成熟,现代生物技术也已在海水双壳贝类的研究中广泛应用,早在20世纪80年代已开展多倍体育种工作[7],其中多倍体牡蛎已商业化生产[8],近年来基因编辑技术也已在牡蛎上开展[9]。由于淡水双壳类受精卵体外培养仍未获得成功,后续的研究受到较大限制,仅有少数关于三角帆蚌培育多倍体的研究,且研究成功率不高[10]

    淡水蚌类胚胎在雌性体内被称为“育儿囊”[11]的鳃腔内进行发育,有研究表明,鳃“育儿囊”与外界水体环境在一定程度上是相同的,但同时具有很高的封闭性[12],因此,“育儿囊”与外界环境存在一定的差异。目前,关于淡水蚌类幼体体外培养的研究主要集中在成熟钩介幼虫寄生变态发育阶段[13-14],余颖等[15]通过自制平衡盐溶液培养池蝶蚌 (H. schlegeii) 早期胚胎取得一定进展。温度是影响无脊椎动物的重要环境因素之一[16],研究生长发育尤其是胚胎发育与生物学零度和积温的关系,不仅可以深入认识生物生长发育特征,而且可为该物种的人工育苗技术提供理论支持,目前,关于海水双壳类繁殖及其发育生物学零度和有效积温的研究已开展[17-19],但是对于三角帆蚌胚胎发育早期阶段生物学零度和有效积温的研究尚未见报道。

    本实验研究根据三角帆蚌体液渗透压配制等渗无机盐溶液,在该溶液中对解剖获得的三角帆蚌成熟配子进行人工体外受精和受精卵体外培养,结合三角帆蚌“育儿囊”中胚胎发育观察,阐明不同阶段胚胎发育特征和发育时间,计算三角帆蚌胚胎发育不同阶段的生物学零度和有效积温,以期补充三角帆蚌繁殖生物学资料,为突破三角帆蚌受精卵体外培养技术奠定理论基础。

1.   材料与方法
  • 实验用三角帆蚌取自上海海洋大学与浙江省武义伟民水产养殖有限公司合作的良种繁育基地。选用亲蚌为3+冬龄(经历了3个冬季),壳长为(135.32±7.97) mm,体质量为(255.73±43.36) g。雌雄鉴别后,对雌雄亲蚌隔离暂养促熟,至性腺成熟,用人工解剖法获得三角帆蚌雌雄配子。

    实验配制的平衡盐溶液 (BSS) 培养液参考余颖等[15]的配方,即每升BSS溶液含有0.503 g NaCl、0.051 g KCl、0.235 g Na2SO4、0.350 g CaCl2、0.090 g MgSO4·7H2O、0.750 g NaHCO3、0.014 g NaH2PO4及0.030 g 葡萄糖。

  • 成熟卵子准备,镜检挑选出梨形或卵圆形且细胞质丰富的成熟卵子,用上述BSS培养液稀释10倍,经60目(孔径约300 μm)的筛绢网筛去组织块等杂质;成熟精子准备,镜检判断雄蚌精子活力,筛选精子活力强的雄蚌,吸取精子,并用BSS培养液稀释10倍,用200目(孔径约75 μm)的筛绢网筛去组织块等杂质。将配子稀释液按1∶1的比例混合受精后移入直径为6 cm的培养皿中,每只培养皿中放入100粒受精卵。取样时池塘平均水温为25 °C,因此,将培养皿置于水浴中,控制培养皿内水温为(25±1) °C。卵裂期前每隔0.5 h,卵裂期后每隔2 h,将含有精卵混合液的培养皿置于解剖镜下,观察在人工培养环境下胚胎发育各时期的特征,用相机拍照,并记录相应的发育时间,实验设置5个重复。

  • 在三角帆蚌繁殖期,将钩介幼虫成熟的三角帆蚌取出,阴干1~2 h后,置于水体中促使其将成熟钩介幼虫排空,将亲蚌按雌雄比为1∶1的比例放入池塘网箱中使雌蚌自然受精怀卵,每隔24 h用开壳器略微打开蚌壳,用注射器刺破外鳃吸取适量胚胎,涂抹在载玻片上,置于生物显微镜下观察胚胎发育特征,直至观察到发育成熟的钩介幼虫,并用相机拍照记录胚胎发育各时期形态特征,三角帆蚌胚胎发育各时期特征参照陈修报等[12]对淡水蚌类胚胎发育分期的描述。

  • 在连续观察“育儿囊”内胚胎发育情况的同时,记录亲蚌开始怀卵至钩介幼虫成熟所用时间,同时每日早晚2次测量记录水温,取平均值作为当日观测水温。实验分别在3个水温不同的时期(a:5月6—15日、b:6月2—10日、c:7月5—12日)进行,每次检查3只蚌的成熟度。

    运用K=t(TC)公式,t为发育所需时间(d),T为观测水温(°C),C为生物学零度(°C),K为有效积温 (°C×d),借助直线回归法[20],计算胚胎发育的生物学零度及有效积温,计算公式:

    式中,V=1/tn为数据组数。根据计算得出的有效积温运用公式C=TK/t对生物学零度进行验证,并用计算出的生物学零度计算胚胎发育至各时期的有效积温。

  • 运用Excel 2016及SPSS 19.0软件进行数据分析,方差分析采用One-Way ANOVA方法,多重比较采用最小显著差数法(LSD法)。

2.   结果
  • 水温(25±1) °C条件下,将解剖获得的三角帆蚌精卵经过BSS溶液稀释过滤后,进行体外人工授精,并在BSS溶液进行体外培养,结果显示,在BSS培养环境下,胚胎最长存活至桑葚期,受精卵发育至第二极体排放、二细胞期、四细胞期、八细胞期、十六细胞期和桑葚期的平均时间分别为(1.8±0.3)、(2.8±0.3)、(5.6±0.2)、(10.5±0.5)、(13.9±0.4)和(17.3±0.4) h。以蒸馏水为培养液的对照组中,未见受精卵发育。

  • 三角帆蚌胚胎发育主要分为受精卵、卵裂期、囊胚期、原肠期、膜内钩介幼虫期和成熟钩介幼虫期。通过三角帆蚌受精卵体外培养,对受精卵卵裂期发育的形态特征进行更细致地观察。结果显示,卵子受精后,受精卵在光镜下呈现深褐色,卵膜在培养皿内由于渗透压变化与卵细胞之间产生间隙,大小约为200~300 μm;发育至1.8 h,受精卵动物极出现一个小凸起,为第二极体(图版-1);随后,胚胎以相近速度经历了二细胞期、四细胞期、八细胞期、十六细胞期和桑葚期(图版-2~6),三角帆蚌受精卵体外培养发育至桑葚期所需时间与鳃“育儿囊”内发育时间基本一致,此后人工培养条件下所有胚胎停止发育。本研究鳃“育儿囊”内胚胎发育情况也较之前研究基本一致。囊胚期分裂球大小基本相同(图版-7),原肠期阴影区域颜色变浅,并形成闭壳肌、内幼虫足丝等结构(图版-8);膜内钩介幼虫两边半壳呈半透明(图版-9),成熟钩介幼虫贝壳孔中央和周围有无定形的白色点状物分布,且出现外幼虫足丝结构(图版-10)。

    Figure 图版.  Morphological characteristics of H. cumingii at different embryonic developmental stages

  • 三角帆蚌胚胎发育生物学零度的统计结果显示,三角帆蚌“育儿囊”内胚胎发育至成熟钩介幼虫的3个组别发育时间分别为10、9和8 d,对应的平均观测水温分别为(26.54±0.53)、(28.08±0.14)和(29.51±0.16) °C (表1)。对观察数据整理并求和,代入计算公式求得,C=14.81 °C;K=118.15 °C×d。

    组别
    group
    时间/d
    t
    平均温度/°C
    T
    发育速率/(1/d)
    V
    VTV2
    a 10 26.54±0.53 0.100 2.650 0.010
    b 9 28.08±0.14 0.111 3.122 0.012
    c 8 29.51±0.16 0.125 3.650 0.016
    84.13 0.336 9.422 0.037

    Table 1.  Statistical data of biological zero point and effective accumulated temperature in embryonic development of H. cumingii

    对各组统计结果代入K=118.15 °C×d对实验结果进行检验,计算分析得出的C值分别为 (14.73±0.53) 、(14.96±0.14) 和(14.74±0.16) °C,与公式计算得出的C值差异均不显著(P>0.05)。因此,可认为将生物学零度C值定为14.81 °C是适宜的(表2)。

    项目
    items
    abc
    时间/d t 10 9 8
    生物学零度/°C
    biological zero point
    14.73±0.53a 14.96±0.14a 14.74±0.16a
    注:同一行的相同字母表示差异不显著(P>0.05)
    Notes:the same letters in the same line represent no significant differences (P>0.05)

    Table 2.  Test for biological zero point and effective accumulative temperature from embryonic development to mature larvae of H. cumingii

    按有效积温公式计算得出三角帆蚌胚胎发育至各时期的有效积温。三角帆蚌胚胎发育至卵裂期、囊胚期、原肠期、膜内钩介幼虫期和成熟钩介幼虫的有效积温分别为(12.95±1.80)、(25.99±3.33)、(42.27±4.88)、(69.21±3.49)和(118.14±1.15) °C×d (表3)。

    胚胎发育时期     
    developmental      
    stage     
    有效积温/(°C×d)
    effective accumulated
    temperature
    卵裂期 cleavage stage 12.95±1.80
    囊胚期 blastocyst stage 25.99±3.33
    原肠期 gastrulation stage 42.27±4.88
    膜内钩介幼虫 glochidium stage 69.21±3.49
    成熟钩介幼虫期 mature mlochidium stage 118.14±1.15

    Table 3.  Effective accumulated temperature for each stage embryos of H. cumingii

3.   讨论
  • 大多数海水双壳类的胚胎发育都是在体外进行,而淡水贝类具有在鳃腔进行胚胎发育的繁殖生物学特性[3],发育场所具有一定的封闭性[12],导致其体外培养存在一定的难度。目前,关于淡水蚌类人工体外受精和胚胎的离体培养方面的报道比较少见,余颖等[15]通过自制的平衡盐溶液对池蝶蚌“育儿囊”内获得的早期胚胎进行了一定时间的体外培养,闻海波等[21]和马学艳等[14]分别对三角帆蚌和褶纹冠蚌(Cristaria plicata)的成熟钩介幼虫进行了体外培养,都取得了一定的成果。本研究使用了与三角帆蚌等渗的平衡盐溶液进行人工体外受精及胚胎体外培养,胚胎发育至桑葚期,这与对池蝶蚌胚胎进行体外培养得到的结果相似,说明只要保证渗透压平衡,三角帆蚌胚胎在不添加外源营养物质的情况下可以依靠卵黄所提供的能量物质正常发育。胚胎发育至桑葚期后停止发育,可能是由于胚胎发育进入到后续阶段,需要消耗的能量增加,受精卵内本身的卵黄物质不足以支撑胚胎的后续发育。淡水蚌类鳃腔内部的鳃丝和鳃瓣有许多血管,母体通过“育儿囊”对胚胎提供养分[22]。当胚胎处于鳃腔中发育时,母体会为幼体输送胚胎发育所需的各种养分[23],促使胚胎进一步发育。因此,在后续对培养液进行改良时,应适当添加营养物质来保证胚胎发育所需的能量需求。此外,在实验中发现在胚胎发育期间许多胚胎出现解体和破损的现象,这是由于胚胎发育过程中有大量的有害微生物附着于胚胎表面,使得胚胎被这些生物直接进食[24],从而影响胚胎发育。因此,特异性的抗生素也是在后续进行胚胎体外培养中不可缺少的一部分,具体种类和剂量有待进一步研究。

  • 温度是重要的环境因子,对变温动物双壳贝类更为重要,温度不仅影响其生长和代谢[25-26],更对胚胎发育起决定作用。当其他环境因子适宜时,在一定范围内,温度越高胚胎发育时间越短,如西施舌(Coelomactra antiquate)在 21、24、27 °C适宜温度条件下,受精卵发育至D型幼虫所需时间分别为32、22、7 h[27]。海湾扇贝(Argopecten irradias)在22~25 °C条件下20 h后即发育成D型幼虫,但是在13 °C条件下需65 h才能进入D型幼虫期[28]。本实验在平均水温为26.5、28.1和29.2 °C下三角帆蚌胚胎发育至成熟钩介幼虫的时间分别为10、9、8 d,短于王宏等[29]在水温为20~23 °C时的结果,说明随着温度的升高,三角帆蚌受精卵发育至钩介幼虫的时间缩短。有机体必须在温度达到生物学零度以上才能繁殖和发育[30],因此,掌握贝类胚胎发育生物学零度和各发育阶段的有效积温是突破贝类人工繁育技术的重要基础。本研究通过直线回归法分析得出三角帆蚌早期胚胎发育至成熟钩介幼虫的生物学零度为14.81 °C,与白志毅等[19]对三角帆蚌钩介幼虫发育的生物学零度和有效积温相比较,其衔接阶段差距较大,原因可能为三角帆蚌繁殖生物学特性有关,三角帆蚌胚胎在鳃“育儿囊”中发育,而钩介幼虫需要寄生在鱼体上,两个阶段的发育环境存在明显差异。另外,在胚胎发育这种形态特征变化较多的阶段,其发育过程易受光照、水质等其他环境因素的影响,也会存在较大的统计误差,因为有效积温法则仅考虑了温度这一影响因子,忽略了其他生态因子的作用。总体上,本实验统计的生物学零度经过检验得出,相对可靠,研究结果可以初步预报三角帆蚌发育至各阶段的时间,对推进三角帆蚌的人工繁育技术发展具有重大意义。

Reference (30)

Catalog

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return