【作者】 佟雪红；

【导师】 李军；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生物学， 2010， 博士

【摘要】 大菱鲆Scophthalmus maximus自然分布于东北大西洋沿岸,具有生长快、肉质优良等特点,目前已成为我国北方重要的海水养殖种类,其工厂化繁育和养殖技术的建立,有效推动了我国海水鱼类养殖产业的发展。鱼类早期阶段的发育规律,是解释其生活史发生机理途径的重要基础,也是提高实践生产中鱼苗培育技术的重要理论依据。因此,本文借助于生理学、组织学和生物化学的方法,研究了大菱鲆早期发育过程中的胚胎发育特性、生长特性、消化生理和免疫特性及骨骼发生发育规律。结果如下:1.大菱鲆胚胎发育主要经历6个时期;在14°C时,胚体经108h即可孵化出膜。胚胎在多细胞期时形成卵黄合胞体层,在低囊胚期时形成囊胚腔。胚盘下包90%时,神经索和体节形成;胚盘下包完全时,脊索原基形成。在受精后59h35min,克氏囊出现;89 h时,克氏囊退化消失。孵化前,大菱鲆胚胎消化管的后端出现纤毛。初孵仔鱼鳍褶上形成特有的色素丛。2.大菱鲆消化系统的发育分为四个阶段。从孵化到仔鱼开口之前,消化道逐渐延长;4日龄时仔鱼开始摄食外源饵料,此时其消化道逐步分化为口咽腔、食管、胃、前中肠和后肠,具备了独立进行外源性摄食的能力。4日龄到16日龄,嗜伊红颗粒和核上空泡状结构出现在消化道中,显示了细胞内消化的开始;消化道粘膜皱褶数量逐步增加,肝脏和胰脏进一步发育。16日龄到35日龄,该阶段16日龄时胃腺出现,胃的功能逐渐完善;20日龄时幽门盲囊形成;肠道粘膜皱褶和纹状缘的丰度以及粘液细胞的数量都快速增加。在60日龄时,消化系统基本发育完成。3.大菱鲆免疫器官原基出现的先后顺序是头肾、脾脏和胸腺。初孵仔鱼,肾为一对简单的直肾管,出现未分化的干细胞。5日龄时,脾脏原基出现,其淋巴化速度较慢。13日龄时,胸腺原基出现,发育速度较快,分为外区和内区。在胸腺和头肾之间出现细胞迁移现象。免疫器官淋巴化的顺序是胸腺、头肾和脾脏。在免疫器官发育后期,脾脏和头肾中均发现了黑色素-巨噬细胞中心(MMCs),脾脏中较丰富,但在胸腺中尚未发现。4.消化酶发育变化表明:卵及胚胎中均未检测到淀粉酶的活性;碱性磷酸酶(AP)、胰蛋白酶、胃蛋白酶和亮氨酸-丙氨酸肽酶(Leu-ala)在胚胎发育早期和后期活性较高,中期较低,但胃蛋白酶活性在孵化时基本消失;亮氨酸氨基肽酶(LAP)在胚胎发育阶段规律不明显。胰蛋白酶在17日龄时达到最高值,然后急剧下降至31日龄并一直保持较低的活性。淀粉酶在4日龄时出现,39日龄之后活性较低。胃蛋白酶在17日龄时出现,在34日龄时快速升高至稳定的活性水平,表明了消化机制的转变。AP和LAP活性在23日龄前呈上升趋势,然后急剧下降至50日龄,之后逐渐增高。Leu-ala在23日龄前活性较低,23-39日龄时活性较高,然后活性下降。刷状缘膜酶的活性占相应酶总活性的比率从31日龄时的30%上升至38日龄时的81%,表明了肠道成熟和消化方式转变的过程。5.大菱鲆仔稚鱼在变态期特定生长率显著增加,在变态后期特定生长率下降。DNA、RNA及总蛋白的变化都带有发育阶段特异性。RNA: DNA比值在12日龄之前呈下降趋势,然后快速增加直至19日龄并在较高的水平上保持波动至35日龄,之后呈下降趋势。在变态后,RNA: DNA比值与生长呈明显正相关;但是在变态前和变态期,RNA: DNA比值并不是评价生长的有效指标。蛋白: DNA比值的变化趋势跟RNA: DNA比值相似。DNA含量和蛋白: DNA比值的变化表明:在变态前,大菱鲆仔鱼4至12日龄的生长以细胞增殖为主,然后以细胞增大为主;在变态早期和变态高峰期,大菱鲆的生长以细胞增大为主;之后以细胞增殖为主。6.骨骼发育结果表明,在5.1 mm体长(LS),脊柱的发生开始于最前端的脉弓;在16.9 mm LS时,椎体横突骨化完全,标志着脊柱的发育完成。在6.3 mm LS ,脊椎骨开始发生;11.0 mm LS时,脊椎骨的骨化全部可见。在5.1 mm LS,尾鳍发育开始;在20.6 mm LS ,尾鳍骨化可见。背鳍及臀鳍分别在5.8 mm LS,6.3 mm LS开始发育;并在20.6 mm LS时,两者的骨化全部可见。在23.5 mm LS时,胸鳍的骨化可见。在7.2 mm LS时,腹鳍开始发育;在19.8 mm LS时,腹鳍骨化可见。本实验中,共发现了24种骨骼畸形类型。在不同的发育期,畸形出现频率最高的分别是背鳍畸形(变态早期),脊椎骨融合(变态高峰期)和尾鳍畸形(变态后期和变态后)。更多还原

【Abstract】 Turbot Scophthalmus maximus distributes mainly in northeast Atlantic, and is characterized by rapid growth and good meat quality. At present, turbot is an important commercial species in northern China. The foundation of its breeding and aquaculture technology pushes the development of marine fishes aquaculture in China. The early development of fish is important base for explanation of its life history. In this study, the traits of embryonic development, growth, digestive physiology, immunity and skeletal development of turbot in early life stages were analyzed. The results are as follows:1. Embryonic development of turbot was divided into six main stages. Hatching occurs 108 hours post-fertilisation (hpf) in 14°C. Yolk syncytial layer and blastocoel were formed at multiple cell stage and low stage, respectively. Neural rod and somite were formed at 90%-epiboly stage, and notochord primordium was formed at complete epiboly stage. Kupffer’s vesicle appeared at 59h35min hpf and degenerated at 89 hpf. The posterior digestive tract of embryo was ciliated. The specific pigment cluster appeared in fin fold of newly hatched larva.2. The development of digestive system had four stages in turbot. The first one was from hatching to mouth opening. Digestive tube developed rapidly at that period. The larvae started to open mouth and feeding on 4 DAH. At that day, the digestive tube was differentiated into buccopharynx, esophagus, stomach, anterior and posterior intestine. The larval digestive system was morphologically ready to digest external food at this time. The second stage was from 4 DAH to 16 DAH. During this period, the acidophilic granules and vascular structure appeared in digestive tube, indicating the start of pinocytosis ingestion. The number of mucosal folding in intestine increased gradually, and liver and panereas developed further. The third stage was from 16 DAH to 35 DAH. Gastric glands appeared at 16 DAH and pyloric caecum appeared at 20 DAH. Mucosal folding in intestine, brush border and goblet cells grew rapidly. At 60 DAH, the digestive system completed development.3. The sequence of immune organ anlages appearance were head kidney, spleen and thymus. At hatching, kidney was a paired of pronephric tubules and primordial stem cells were observed. Spleen anlage was present at 5 DAH and it developed slowly. Progenitor thymus appeared at 13 DAH and grew quickly. An outer zone and an inner zone in the thymus were observed. Cell migration occurred between thymus and head kidney. The first functional lymphoid organ was thymus followed by head kidney and spleen. During the posterior developmental period of the immune organs, the melano-macrophage centers (MMCs) were found in spleen and head kidney, but not in thymus. The abundance in spleen was higher.4. Amylase was not found in turbot eggs and embryo. Activities of alkaline phosphatase (AP), trypsin, pepsin and leucine-alanine peptidase (Leu-ala) were high in early and late embryonic developmental stages, but relatively low during middle stages. However, pepsin activity disappeared at hatching. Leucine aminopeptidase N (LAP) activity showed no obvious changes during different embryonic developmental stages. Trypsin sharply increased to the climax at 17 DAH. Then abrupt decrease was observed until 31 DAH followed by a relatively stable level until end of this experiment. Amylase was determined at 4 DAH and reached the maximum value at 19 DAH and then declined sharply to 39 DAH and remained at a low level. Pepsin was firstly detected at 17 DAH and increased to 34 DAH, and then remained at a stable level, indicating the transition of digestive mechanism. AP and LAP activities markedly increased to 23 DAH, decreased abruptly to 50 DAH and increased gradually to 60 DAH. Leu-ala specific activity was low before 23 DAH. After that, this enzyme reached the plateau until 39 DAH, followed by a decline to 60 DAH. The activities of the BBM bound enzymes increased from 30% of the total activities at 31 DAH to approximately 81% at 38 DAH, indicating the maturation of intestinal tract and the transition of digestion mode. 5. A significant increase of specific growth rate during metamorphosis and retarded growth rate during post-metamorphic phase were observed. Ontogenetic patterns of DNA, RNA and protein parameters all showed developmental stages-specific traits. RNA:DNA ratio decreased up to 12 DAH, then increased rapidly till 19 DAH and fluctuated until 35 DAH followed by a decline to the end. RNA:DNA ratio was positively correlated with growth rate of turbot juveniles during post-metamorphic phase, while this ratio was not a sensitive indicator of growth during pre-metamorphic phase and metamorphosis. Protein:DNA ratio showed a similar tendency to RNA:DNA ratio. Changes of DNA content and protein:DNA ratio revealed that growth of turbot performed mainly by hyperplasia from 4 DAH to 12 DAH and hypertrophy until 21 DAH during pre-metamorphic larval phase. Growth was dominantly hypertrophical from early- to mid-metamorphosing phase and hyperplastic thereafter.6. Skeletal development of turbot showed that vertebral ontogeny started with the formation of anterior haemal arches at 5.1 mm standard length (LS), and was completed by the full ossification of parapophyses at 16.9 mm LS. Vertebral centra started to develop at 6.3 mm LS and ossification in all centra was visible at 11.0 mm LS . The caudal fin appeared at 5.1 mm LS and ossification was visible at 20.6 mm LS. The onset of dorsal and anal fins elements appeared at 5.8 mm LS and 6.3 mm LS respectively. Ossifications of both dorsal fin and anal fin were visible at 20.6 mm LS. The pectorals were present before first feeding, their ossifications were completed at 23.5 mm LS . Pelvic fins began forming at 7.2 mm LS and calcification on the whole structure was visible at 19.8 mm LS. In turbot, 24 types of skeletal abnormalities were observed. As for each developmental stage, the most common were dorsal fin abnormalities during early-metamorphic period, vertebral fusion during climax metamorphosis, and caudal fin abnormality during both late-metamorphic period and post-metamorphic period.更多还原