近江牡蛎受精过程、胚胎及早期幼虫发育特征

Developmental characteristics during fertilization process,embryonic and early larval stages of Crassostrea ariakensis

为了解近江牡蛎(Crassostrea ariakensis)从受精到早期幼虫发育阶段的发育特性,在水温22~24℃、盐度20~22的实验室环境中对采自山东东营丁字湾海域的近江牡蛎进行人工授精,利用普通光学显微镜和荧光显微镜对近江牡蛎受精、早期胚胎和幼虫发育过程中的形态和核行为变化进行观察,并对这一过程中牡蛎的壳长变化进行了统计分析。结果表明,近江牡蛎成熟的未受精卵呈梨形,卵径为50μm左右,核相处于第一次成熟分裂中期。受精卵在受精后15~20min、25~30min,先后排出第一、第二极体,完成第一次和第二次成熟分裂,在受精后30min左右先后形成雄原核和雌原核;雌、雄原核各自形成染色体组,在卵子中央发生联合,染色体排列于赤道板上,形成第一次有丝分裂的中期分裂相;受精后45min左右,受精卵进行第一次卵裂,形成2个体积不等的卵裂球;受精75min后,受精卵开始第二次卵裂,90min后形成4个卵裂球,进入4细胞时期;6h后受精卵发育至桑葚期,8~10h进入囊胚期,12~15h后发育至担轮幼虫,27~30h左右发育成D形幼虫,5d左右进入壳顶幼虫时期,17d后幼虫陆续开始出现眼点,22d后发育为附着幼虫,而后在30 d左右变态为附着稚贝。研究中还发现了少数多精入卵现象,该过程会影响受精卵的正常分裂,造成胚胎发育畸形乃至死亡。研究结果可为近江牡蛎的苗种繁育、遗传育种等研究提供参考资料。

Crassostrea ariakensis is an important aquaculture oyster species in China.It has high nutritional value,and is also the main component of oyster reef,playing significant ecological functions of filtering water pollutants and maintaining ecological diversity.In recent years,the natural resources of oyster in coastal areas of China,especially in the northern coastal areas,have been declining due to changes in marine environment,overfishing by humans,limited supply of artificial seedlings and other factors.Large-scale breeding of artificial seedlings can provide support and guarantee to supplement natural seedlings and increase resources,and is also a necessary prerequisite for germplasm improvement.Fertilization and early embryo development is an important research content in genetic breeding of economic aquatic animals.The characteristics of fertilization and embryo development can provide important references for artificial breeding and cell engineering breeding.Fluorescent microscope observation and ordinary optic microscope observation have the advantages of simplicity,clarity and intuition,and have been applied in related researches on changes in nuclear behavior during fertilization and early embryo development in various economic shellfishes.Important data have been accumulated in research on fertilization mechanism,artificial seedlings and cross-breeding practices.However,research on fertilization and early development of embryos and larvae of C.ariakensis has only been briefly observed and described by other methods in a few literatures,and no in-depth systematic research has been reported.In this study,cytological changes during fertilization and early embryo development of C.ariakensis were observed by optic and fluorescent microscopy.The results showed that mature unfertilized eggs were pear-shaped with a diameter of about 50μm and the nuclear phase was in the middle of the first maturation division.Artificial insemination was carried out in a laboratory environment with water temperature from 22℃to 24℃and salinity from 20 to 22.The first and second polar bodies were discharged successively to complete the first and second mature division from 15 min to 20 min and from 25 min to 30 min after fertilization.About 30 min after fertilization,male and female pronuclei formed.Female and male pronuclei formed their own chromosomes and united in the center of the ovum.Chromosomes were arranged on the equatorial plate of the spindle to form the metaphase of the first mitosis.About 45 min after fertilization,the zygote cleaved for the first time,forming two blastomes of different sizes;the zygote started the second cleavage at 75 min.About 90 min after fertilization,zygote entered four-cell stage.About 6 h after fertilization it entered morula,from 8 h to 10 h it entered blastocyst stage,from 12 h to 15 h it developed into tricyclic larva stage.At 27 h it developed into D-shaped larva;it developed into apical larva 5 d later.In addition,polyspermy was found in this study,which would affect the normal division of fertilized eggs,resulting in abnormal embryonic development and even death.The formation mechanism and reasons were analyzed and discussed.In this paper,the nuclear phase and morphological changes during fertilization,embryo and early larval development of C.ariakensis were observed and studied to provide reference materials for research on seed breeding and genetic breeding of C.ariakensis.