中国科学院海洋研究所机构知识库

初孵仔鱼需摄入大量能量用于器官发育并为变态做准备，因此，在长期的进化过程中形成了与生存相关骨骼优先发育的特征。骨骼发育至关重要，骨骼发育起始于孵化后并与功能相适应，异常的骨骼影响仔稚幼鱼外部形态和功能需求。海水鱼类苗种培育过程中常发生骨骼畸形，导致运动困难、生长迟滞、甚至高的死亡率，并造成劳动力的浪费及较大的经济损失。大菱鲆（Scophthalmus maximus）隶属于鲽形目鲆科菱鲆属，自然分布于东北大西洋沿岸，生长迅速、适应低水温生活。鞍带石斑鱼（Epinephelus lanceol）隶属鲈形目鮨科石斑鱼属，为暖水性珊瑚礁鱼类，主要分布在东南亚与澳大利亚附近海域，我国南海诸岛和海南岛等海域，但数量稀少。大菱鲆和鞍带石斑鱼均是我国重要的海水养殖鱼类，在苗种培育过程中易产生骨骼畸形，尤其是鞍带石斑鱼，畸形率达96%。我们系统的分析了大菱鲆及鞍带石斑鱼仔稚幼鱼的生长特性、骨骼发育与畸形，结果如下：

1.大菱鲆仔稚幼鱼生长特性、骨骼发育与畸形分析

大菱鲆仔稚幼鱼养殖条件：初孵仔鱼培育水温维持在16±0.5℃直到孵化后4天（4dph），从5 dph到24 dph水温维持在21±0.5℃，从25 dph直到实验结束水温维持在23±0.5℃。绿水养殖、溶解氧量 7.0 – 8.5 mg L^–1、盐度 30 – 32、PH 7.7 – 8.0、持续曝气、全天光照。仔鱼孵化后 4 -19 dph投喂褶皱臂尾轮虫，13 -35 dph投喂卤虫无节幼体，25 -45dph投喂配合饲料。

变态前期仔鱼全长增加缓慢，变态高峰期稚鱼进入快速生长期。在变态前期和变态早期颅骨呈正异速生长，而脊柱为负异速生长，表现为与摄食和呼吸相关的器官优先发育；在变态高峰期和变态晚期颅骨呈负异速生长，而脊柱为近等速生长；变态后期颅骨和脊柱均为近等速生长。

大菱鲆与营养摄取和呼吸相关的颅骨元件优先发育，包括麦克尔氏软骨、舌骨棒、下舌骨和角鳃骨等；变态发育起始时颅骨元件基本形成，颌骨、齿骨、鳃盖骨优先骨化。颅骨变态发育伴随右侧眼睛移向左侧，部分骨骼呈现不对称发育，如右侧续骨和舌颌骨比左侧的短小，而齿骨、外翼骨和方骨等比左侧长且宽等。脊柱发育起始于仔鱼腹部前端脉弓的形成，随后神经弓出现在背部前端并依次向尾部形成，且伴随着颅骨的不对称发育，脊柱骨化顺序与发育顺序一致。胸鳍原基是仔鱼开口前存在的唯一鳍原基，随后是尾鳍、背鳍、臀鳍和腹鳍，但胸鳍是最后发育完全的鳍。

大菱鲆苗种培育过程中观察到8.15%骨骼畸形率，身体不协调畸形率最高为4.5%，其次是脊柱畸形约为2.3%，主要包括脊柱侧凸、脊柱前弯、脊柱后凸和椎骨融合；颅骨畸形约为0.5%，主要包括颌骨畸形和鳃畸形；畸形率最低的为鳍畸形约0.85%，主要包括背鳍畸形、臀鳍畸形和尾鳍畸形。脊柱畸形和颅骨畸形对仔稚幼鱼的生长发育及外观有显著影响，导致身体短小或扭曲。

骨骼发育相关基因BMP4、Sox9和Gpc4组织表达广泛，发育早期仔鱼中高表达与早期骨骼发育相关，随骨骼发育逐步完善，表达量逐渐降低并维持在一定范围内，参与其他信号通路维持机体正常生命活动。

2.鞍带石斑鱼仔稚幼鱼生长特性、骨骼发育与畸形分析

鞍带石斑鱼仔稚幼鱼养殖条件：绿水养殖、水温维持在29±0.5 ℃、溶解氧量7.0–8.5 mg L^–1、盐度30–32、PH 7.7–8.0、持续曝气、弱光。仔鱼孵化后3天开口，2-10dph投喂S型轮虫，6-27dph投喂L型轮虫，16-40dph投喂卤虫无节幼体，30-45dph投喂大卤虫。

在变态前期和变态早期仔鱼全长增加缓慢，在变态晚期和变态后期稚鱼进入快速生长期。颅骨及尾鳍在变态前期和变态早期呈正异速增长，而脊柱为负异速生长，表现出与摄食、呼吸及游泳相关的骨骼优先发育特征；在变态晚期及变态后期颅骨、脊柱及尾鳍复合物均呈近等速生长。

鞍带石斑鱼中与摄食和呼吸相关的颅骨元件优先发育，如麦克尔氏软骨、舌骨棒、下舌骨和角鳃骨等。骨化起始前，舌续骨、舌骨棒、颚方骨和前四对角鳃骨等部分退化。变态晚期，颌骨、齿骨和鳃盖骨等颅骨骨骼优先骨化。脊柱的发育起始于背部前端神经弓的形成并向尾部延伸，胸区部位神经弓形成后向头区和尾区延伸，脉弓形成起始于胸区并向头区和尾区延伸，脊柱的骨化从头区向尾区进行。胸鳍原基是仔鱼开口前存在的唯一鳍原基，背鳍和腹鳍发育优先于脊柱以及尾鳍和臀鳍的发育，经历背棘和腹棘发育的“起始-延伸-退化”过程。

鞍带石斑鱼苗种培育过程中畸形率高达96%，颅骨畸形率最高为82%，主要包括颌骨畸形和鳃骨畸形，其次为脊柱畸形为12%，主要包括脊柱前弯、脊柱后凸、脊柱侧凸和椎骨融合，畸形率最低的为鳍畸形约2%。颅骨畸形和脊柱畸形对仔稚鱼的生长发育及外观有显著影响，是变态前期和变态早期导致高死亡率的主要原因。此外，在40dph筛苗时20-30%的较大个体中存在残食现象，是变态后期导致成活率降低的主要原因。

The newly hatched larvae needs to uptake quantity of energy to meet the needs of development and the skeleton related to survival are preferentially developed. Skeletal development is important in fish fry breeding because of its influence on external morphology and functional exercise. Skeletal development in larval fish originates with the formation of cartilage. Skeletal deformity usually occurs in many cultured fishes and results in movement difficulty, growth retardation, even extremely high mortality, and leading to significant finical lose and labor force waste. Scophthalmus maximus (L. 1758) belongs to the order Pleuronectiformes, family Scophthalmidae, and category Scophthalmus, and it was suitable to inhabit in cold water and was natural distribution in the coast of Northeastern Atlantic. Epinephelus lanceolatus belongs to the order Perciformes, family Serranidae, and category Epinephelus, and mainly distributed in the Southeast Asia and Australia waters, the islands of South China Sea and Hainan Island. S. maximus and E. lanceolatus are important mariculture species in China with high growth rates and economic values. However, the incidence of skeletal deformities are ubiquitous, particularly the E. lanceolatus, the incidence of abnormalities could up to 96%. In the present study, we systematically investigated the growth characteristics, timing and progression of skeletal development, types and incidence of skeletal deformities during the breeding process of S. maximus and E. lanceolatus. The results are as follows:

1. Growth characteristics, timing and process of skeletal development, types and incidence of deformity in larva and juvenile S. maximus.

The cultivating temperature of newly hatched larvae maintained at 16±0.5 °C until 4 days post hatching (dph). The temperature was maintained at 21 °C from 5 dph to 24 dph and then the temperature was maintained at 23±0.5 °C until to the end of the experiment. The culture conditions were as follows: greenwater, dissolved oxygen 7.0–8.5 mg L^–1, salinity 30–32, pH 7.7–8.0, and constant aeration were provided. After hatching, the larvae were fed with Brachionus plicatilis from 4 dph to 19 dph, then the larvae were fed with Artemia sp. nauplii from 13 dph to 35 dph, and compounded diet was fed from 25 dph.

The total length of larva increased slowly during the pre-metamorphosis stage, during the metamorphosis and post-metamorphosis stages, the juvenile enter into a rapid growth period. During the pre- and early-metamorphosis stage, the development of cranium presented as positive allometric growth, while the vertebral column was negative allometric. These results shows that skeleton related to food ingestion and respiration were preferentially developed. In climax- and late-metamorphosis period, cranium was negative allometric, and the vertebral column was nearly isometric growth. The cranium and vertebral were both nearly isometric growth when the juvenile enter into the late-metamorphosis stage.

The cranium elements relevant to food ingestion and inspiration developed preferentially, such as the Michael's cartilage, hyoid bar, hypohyal and ceratobranchial. The cranium skeletal elements were basically formed before the larva enter into early-metamorphosis stage, then the jaw, dentary and opercle ossified preferentially. Asymmetries gradually presented in some elements of cranium, for example, the sympletic and hyomandibular on the right side became shorter or smaller than the one on the left, the dentary, ectopterygoid and quadrated became wider or longer than the one on the left. And the eye on the right side moved to the left side. Vertebral elements were not observed until the formation of haemal and neural arches in the midriff and anterodorsal regions, respectively. Subsequently, it formed backwards and the ossification of spinal following a similarly pattern. Only the pectoral fin was observed before first feeding, followed by the caudal fin, dorsal fin, anal fin and pelvic fin, however, the pectoral fin was the last fully developed fin.

8.15% incidence of deformities were observed during the the breeding process, uncoordinated appearance possed the hightest incidence 4.5%, followed by spinal deformities, its incidence was 2.3% and primarily presented as vertebra fusion, scoliosis, lordosis, and kyphosis, the incidence of cranium deformities was 0.5% and mainly included jaw and operculum deformities, and fin deformities occurred at the lowest incidence 0.85% and mainly included caudal fin, dorsal and anal fins deformities. Spinal and cranium deformities could significantly affected the external morphology and growth.

BMP4, Sox9 and Gpc4 participate in a wide range of biological processes, and expressions of them maintain at a certain level in different tissues of adult fish. BMP4, Sox9 and Gpc4 had a higher expression level at the early development stage and gradually decreased, this pattern of gene expression corresponding to their founctions during the process of development.

2. Growth characteristics, timing and process of skeletal development and types and incidence of deformity in larva and juvenile E. lanceolatus.

The cultivating conditions were as follows: greenwater, the temperature maintained at 29±0.5 °C, dissolved oxygen 7.0–8.5 mg L^–1, salinity 30–32, pH 7.7–8.0, weak laser and constant aeration were provided. After hatching, from 2 dph to 10 dph the larvae were fed with S rotifers, from 6 dph to 27 dph the larvae were fed with L rotifers, from 16 dph to 40 dph the larvae were fed with Artemia sp. nauplii, and from 30 dph to 45 dph the larvae were fed with Artemia sp.

The total length of larva increased slowly during the pre- and early-metamorphosis stage, and the juvenile enter into a rapid growth period during the late- and post-metamorphosis stages. During the pre- and early-metamorphosis stage the development of cranium and caudal fin presented as positive allometric, while the vertebral column was negative allometric. In late- and post-metamorphosis period, the cranium, vertebral column and caudal fin complex were nearly isometric growth.

Cranium elements relevant to food ingestion and inspiration were preferentially developed, such as the Michael's cartilage, hyoid bar, hypohyal and ceratobranchial. After the cranium elements fully formed, some skeletal elements undergone a degradation processes, such as hyosympletic, hyoid bar, palatoquadrate and ceratobranchial. The ossification of cranium initiated during late-metamorphosis period, and the maxillary, premaxillary, dentary and opercle were ossified preferentially. The vertebral elements development initiated with the formation of neural arch at the anterior of the spinal, then the neural arch at the center part of spinal was formed accompanied with the formation of haemal arch in the midriff regions, and the haemal arch was formed forwards and backwards. Only the pectoral fin was observed before first feeding, followed by dorsal and pelvic fin. The dorsal and pelvic spine appeared at the time enter into early-metamorphosis stage, it lengthened rapidly and undergone a degenerative process.

96% incidence of deformities were observed during the breeding process of E. lanceolatus. The cranium deformities possessed the highest incidence 82% and primarily including jaw and branchial deformities. Followed by vertebral column deformities 12% and mainly including loidosis, kyphosis, scoliosis and vertebra fusion. The fin deformity had the lowest incidence 2%. Cranium and vertebral column anomalies could significantly affected the external morphology and growth, and even high mortality during the pre- and early-metamorphosis stage. In addition, a significant cannibalism were observed in about 20%-30% bigger individuals during the first fry separation, and it maybe another reason leading to lower survival rate during post-metamorphosis stage.

目 录

第一章 绪论. 1

1.1鱼类早期生长发育特性. 1

1.2鱼类早期发育过程中骨骼发育研究进展. 1

1.3鱼类骨骼畸形研究进展. 7

1.4骨骼发育相关基因研究进展. 10

第二章 大菱鲆仔稚幼鱼异速生长特性、骨骼发育与畸形研究. 14

2.1研究背景：. 14

2.2材料与方法. 14

2.2.1大菱鲆仔稚幼鱼培育. 14

2.2.2大菱鲆仔稚幼鱼样本及成鱼样本采集. 15

2.2.3实验试剂与仪器. 15

2.2.4大菱鲆仔稚幼鱼样本骨骼染色. 16

2.2.5大菱鲆仔稚幼鱼骨骼染色样本观察和生长数据采集. 17

2.2.6大菱鲆骨骼发育相关基因BMP4、Sox9、GPC4的表达分析. 18

2.3实验结果. 21

2.3.1大菱鲆仔稚幼鱼早期发育阶段异速生长特性研究. 21

2.3.2大菱鲆仔稚幼鱼早期骨骼发育时序研究. 23

2.3.3大菱鲆仔稚幼鱼椎骨数量性状统计分析. 32

2.3.4大菱鲆骨骼畸形类型与畸形率统计. 34

2.3.5 大菱鲆骨骼发育相关基因BMP4、Sox9和GPC4的生物信息学及表达分析  41

2.4讨论. 57

2.4.1大菱鲆仔稚幼鱼颅骨、脊柱和尾鳍的异速生长特性. 57

2.4.2大菱鲆仔稚幼鱼颅骨、脊柱和鳍的骨骼发育时序. 58

2.4.3大菱鲆仔稚幼鱼椎骨的数量性状. 60

2.4.4大菱鲆仔稚幼鱼颅骨、脊柱和鳍的骨骼畸形. 60

2.4.5大菱鲆骨骼发育相关基因BMP4、Sox9和Gpc4的生物信息学及表达分析  61

第三章 鞍带石斑鱼仔稚幼鱼生长特性、骨骼发育与畸形研究. 63

3.1研究背景. 63

3.2材料与方法. 64

3.2.1鞍带石斑鱼仔稚幼鱼培育. 64

3.2.2鞍带石斑鱼仔稚幼鱼样本采集. 64

3.2.3鞍带石斑鱼仔稚幼鱼样本骨骼染色. 64

3.2.4鞍带石斑鱼仔稚幼鱼骨骼染色样本观察和生长数据采集. 64

3.3实验结果. 65

3.3.1鞍带石斑鱼仔稚幼鱼早期异速生长特性研究. 65

3.3.2鞍带石斑鱼仔稚幼鱼早期骨骼发育时序研究. 67

3.3.3鞍带石斑鱼仔稚幼鱼骨骼畸形类型与畸形率统计及同类残食现象. 78

3.4讨论. 84

3.4.1鞍带石斑鱼仔稚幼鱼颅骨、脊柱和尾鳍的异速生长特性. 84

3.4.2鞍带石斑鱼仔稚幼鱼颅骨、脊柱和尾鳍的骨骼发育时序. 85

3.4.3鞍带石斑鱼仔稚幼鱼颅骨、脊柱和尾鳍的骨骼畸形及同类残食现象. 86

第四章 总结与展望. 89

4.1总结. 89

4.1.1大菱鲆仔稚幼鱼生长特性、骨骼发育与畸形. 89

4.1.2鞍带石斑鱼仔稚幼鱼生长特性、骨骼发育与畸形. 90

4.2展望. 91

参考文献. 92

致 谢. 103

作者简历及攻读学位期间发表的学术论文与研究成果. 104