实验海洋生物学重点实验室知识库

鱼类养殖是我国海水养殖的支柱产业，工厂化养殖是主要的养殖模式之一，尤其是工厂化人工繁育和苗种生产的主要方式。迄今，工厂化海水鱼类苗种培育和养殖生产中的光照调控及应用均是经验使之，相关理论和技术研究甚少，尚未形成完善的光调控策略。光谱作为光环境的主要要素之一，对养殖鱼类的生长发育、生理状态和行为响应具有重要影响，鱼类在早期发育及生长过程中的光环境适应和需求尚未阐明。本研究以我国工厂化主养鱼种大菱鲆（Scophthalmus maximus）为研究对象，采用发光二极管（light emitting diode，LED）光谱调控技术，构建了红、橙、绿、蓝及全光谱等五种不同光谱环境，围绕大菱鲆不同发育阶段的感光系统发育特征及不同光谱环境下的生长发育、生理响应开展研究，旨在探究大菱鲆对光谱环境的适应性特征，深入了解鱼类养殖过程的光谱环境需求，为构建大菱鲆苗种培育和福利化养殖的适宜光环境提供理论指导。具体研究结果如下：

（1）光谱环境对大菱鲆胚胎发育的影响：

大菱鲆胚胎发育过程中，紫外视蛋白（short wavelength sensitive opsin, sws1）基因为母源性遗传基因，在大菱鲆胚胎中存在表达，可能介导了大菱鲆胚胎的紫外感知功能。孵化后35.5小时（hours post fertilization，hpf），大菱鲆胚胎进入体节期，视觉系统开始发育，眼原基（optic rudiment，OR）出现，同时，视觉系统外视紫质（exrh）开始在松果体上表达，古视蛋白（va-opsin）在下丘脑和后脑上表达，并开始介导大脑非视觉系统的感光功能，两种非视觉视蛋白表达量均随发育进行逐渐升高，自体节期至孵化期，va-opsin和exrh的表达水平分别增加了4604% ± 897%和534% ± 87%。42.7 hpf，出现晶体囊，此时，视网膜神经节细胞呈放射状紧密围绕晶体囊；62.8 hpf，胚胎发育至心跳期，视网膜神经节细胞和晶体囊之间出现空隙，开始出现零散的色素上皮细胞。光谱环境对大菱鲆胚胎发育具有显著影响，非视觉系统感知光信号后，会影响神经系统的发育和分化。在胚胎发育方面，蓝、绿光下大菱鲆胚胎较早发育至心跳期，62.8 hpf，蓝、绿光下具有心跳的大菱鲆胚胎比例分别达到100% ± 0%和71% ± 14%，显著高于其他光谱。但蓝、绿光并未加速胚胎的孵化速率，各光谱下大菱鲆的孵化时间和孵化率差异不显著，其中绿光下大菱鲆初孵仔鱼的畸形率最高，达39% ± 5%，其它组在6.7%至7.1%之间。高畸形率进一步造成了绿光下仔鱼死亡率升高，达38% ± 6%。转录组分析表明，光信号在被感光系统接收后，会影响大菱鲆神经系统发育和信号转导功能。并通过神经-内分泌系统调控胚胎生理状态。与全光谱相比，单色光下胚胎糖代谢、脂代谢等能量代谢过程显著加强。应激反应结果表明，红光、蓝光和绿光引起了大菱鲆初孵仔鱼的应激反应，表现为热休克蛋白70（heat shock protein 70，hsp70）表达量显著增加，同时，蓝光组初孵仔鱼组织蛋白酶D（cathepsin D，ctsd）、组织蛋白酶F（cathepsin F，ctsf）、过氧化氢酶（catalyase，cat）和金属硫蛋白（metallothionein，mt）的mRNA表达水平显著高于其他光谱组，且Cat活性显著低于其他光谱；红光下初孵仔鱼溶菌酶（lysozyme，lzm）的mRNA表达水平显著高于其他光谱。然而，Lzm酶活力并未出现显著差异。因此，为了提高初孵仔鱼免疫力，降低畸形率和死亡率，减少应激反应，应采用橙光或全光谱用于构建大菱鲆受精卵孵化的光谱环境。

（2）大菱鲆感光器官发育及其对光谱环境的适应性特征

大菱鲆视觉系统发育表现出胚后发育的典型特征。孵化后第1天（days post hatching，dph），视网膜出现外核层、内核层、神经节细胞层的分化；2 dph出现色素上皮层和纯视锥结构的感光细胞层，此时，视锥细胞排列疏松。5 dph，可在组织学水平观察到视网膜的十层结构。18 dph，双锥细胞和视杆细胞开始出现，在随后的发育过程中视杆细胞和双锥细胞比例增加，单锥细胞比例降低，40 dph左右，感光细胞比例趋于稳定，双锥细胞占比达75% ± 4%，成为视网膜感光细胞的主体。蓝光加速了感光细胞的变化，对视觉系统发育具有显著促进作用。在生长方面，大菱鲆仔稚幼鱼生长的最适光谱具有阶段特异性。蓝光显著促进了仔稚幼鱼，尤其是附底后幼鱼的生长。此外，蓝光下大菱鲆变态发育起止时间为24-50 dph，变态起始时间提前且过程加快。在生理响应上，红、橙光可引起大菱鲆仔稚幼鱼的氧化应激反应，造成hsp70、抗氧化基因cat、gst、mt、先天性免疫基因ctsd、ctsf和lzm表达增加，但酶活力受到抑制，不利于仔稚幼鱼机体氧化-还原平衡的维持和免疫力的提高。为了促进大菱鲆仔稚幼鱼的生长和变态发育，减少应激反应，应在变态发育前采用全光谱，并在20 dph左右逐渐转换为蓝光，构建大菱鲆早期发育阶段的光谱环境。

（3）大菱鲆幼鱼对光谱环境的生长和生理响应

在生长方面，蓝光下幼鱼长速率最快，表现为更高的特定生长率 （specific growth rate，SGR）（1.62 ± 0.01）和更低的饲料转化率（feed conversion rate，FCR）（0.95 ± 0.02）。长波长的红、橙光可引起幼鱼的氧化应激反应，抑制幼鱼抗氧化能力和抗病能力，表现为hsp70、铜/锌-超氧化物歧化酶（copper/zinc superoxide dismutase，cu/zn-sod）、锰-超氧化物歧化酶（manganese superoxide dismutase，mn-sod）、cat、谷胱甘肽过氧化物酶（glutathione peroxidase，gsh-px）和lzm的表达量增加，但T-sod、Cat、Gsh-px和Lzm酶活性降低。但是，各组间蛋白羰基（PC）、丙二醛（MDA）含量不存在显著差异，可推断红、橙光并未造成大菱鲆幼鱼的氧化损伤。在代谢方面，大菱鲆幼鱼表现出对长波长光谱环境的适应。当应激反应发生时，幼鱼增加了皮质醇的合成和分泌，红、橙光下幼鱼血清皮质醇含量均达到60 ng/mL以上，显著高于绿、蓝和全光谱。同时，红、橙光下幼鱼ampk的表达量增加，使机体能量代谢向分解营养物质、产生能量的方向进行，促进了糖代谢和脂代谢过程，以维持红、橙光下的能量稳态。但能量消耗增加，会导致机体营养物质积累减少，进而造成生长速度降低。因此，建议采用蓝光优化大菱鲆幼鱼养殖系统的光谱环境。

Fish farming is the pillar industry of mariculture in China. Industrial aquaculture is one of the main aquacultural modes, especially for industrial artificial breeding and aquacultural fry production. The light environmental regulation and application in aquaculture has been performed mainly according to experience for a long time. The researches on light regulation theory and technology are limited, and refining light regulation strategy has not been formed. As one of the main characteristics of light environment, spectrum has vital influences on the growth, development, physiological status and behavioral response in fish. The light environment adaptation and requirement of fish during early developmental stage have not been well elucidated. In this study, turbot (Scophthalmus maximus), an important economy fish species in China, was selected as the research material. Five different spectra (red, orange, green, blue and full spectrum) were constructed by using light emitting diode (light emitting diode, LED) spectral regulation technology. The development characteristics of photosensitive system, growth, development and physiological responses of turbot to spectral environment at different developmental stages were studied. The aim of the study is to explore the adaptive traits of turbot to spectral environment, thoroughly understand the spectral environmental requirements of fish in aquaculture, provide theoretical guidance for optimizing light environment of turbot cultural system and improving fish welfare. The specific results are as follows:

(1) Effect of light spectrum on embryonic development in turbot:

During the embryonic development of turbot, the short wavelength sensitive opsin (sws1) which was a maternal genetic gene and expressed in turbot embryos, might mediate the ultraviolet perception function of turbot embryos. At 35.5 hpf, turbot embryos developed into the somites stage, the visual system began to develop, with optic rudiment appearing. At the same time, exo-rhodopsin (exrh) beginning to express in the pineal gland, and vertebrate ancient-opsin (va-opsin) beginning to express in the hypothalamus and hindbrain, which mediated the photosensitive function of the brain non-visual system. The expression of the two non-visual opsins increases gradually with the development. From somites stage to hatching stage, the expression levels of va-opsin and exrh increased by 4604% ± 897% and 534% ± 87%, respectively. At 42.7 hpf, lens capsule appeared and was closely surrounded by retinal nerve cell layer which was radiately arranged. Since 62.8 hpf, when embryonic heartbeat started, a gap was formed between retinal nerve cells and lens capsule. During this period, scattered pigment epithelial cells began to appear. The spectral environment had significant effects on the embryonic development of turbot. Embryos exposed to blue and green light developed into heartbeat onset stage earlier, characterized by significantly higher number of embryos with heartbeat at 62.8 hpf, the percentage of which reached to 100% ± 0% and 71% ± 14%, respectively. However, blue and green light did not accelerate the hatching progress of embryos. There was no significant difference in the hatching time and hatching rate of turbot under different spectra. The malformation rate of newly hatched larvae exposed to green light was the highest, reaching 39% ± 5%, while that in other groups was between 6.7% and 7.1%. The high malformation rate further increased the mortality rate of larvae under green light, reaching to 38% ± 6%. Transcriptome analysis indicated that after received by photosensitive system, light signal could alert nervous system development and signal transduction function in turbot embryo. The physiological state was further regulated by the neuroendocrine system. Compared with the full spectrum, the energy metabolism of the embryo under monochromatic light was significantly enhanced, such as glucose metabolism and lipid metabolism. The results of stress response analysis indicated that red, blue and green light induced stress response in newly hatched turbot larvae characterized by significantly higher mRNA expression level of heat shock protein 70 (hsp70). Meanwhile, the expressions of cathepsin D (ctsd), cathepsin F (ctsf), catalase (cat) and metallothionein (mt) in blue light group were significantly higher than those under other spectra. The Cat activity in blue group was significantly lower than that in other groups. lysozyme (lzm) expression level of newly hatched larvae under red light was significantly higher. However, Lzm activity was not significantly higher in red light group. Therefore, in order to improve the immunity of newly hatched larvae, reduce the deformity rate, mortality rate, and avoid the stress response, orange or full spectrum should be used to optimize the spectral environment for turbot embryo.

(2) Development of photosensitive organs and adaptation traits of turbot to spectral environment:

The visual system development of turbot showed the typical characteristics of postembryonic development. On 1 day post hatching (dph), the retina had three layers, including outer nuclear layer, inner nuclear layer and ganglion cell layer. On 2 dph, the pigment epithelium layer and photoreceptor cell layer which was formed by pure cone cell began to appear. At this time, the cone cells were loosely arranged. On 5 dph, the structure of ten layers of retina could be observed at the histological level. On 18 dph, double cones and rod cells began to appear. During the subsequent development process, the proportion of rod cells and double cone cells increased while the proportion of single cone cells decreased. On about 40 dph, the proportion of photoreceptor cells tended to be stable. The proportion of double cones reached 75 ± 4% and became the main body of retinal photoreceptor cells. Blue light accelerates the changes of photoreceptor cells and promotes the development of visual system. In terms of growth, the optimal spectrum of turbot larvae and juveniles was developmental stage specific. Blue light significantly promoted the growth of larvae and juveniles, especially after the migration to water bottom. In addition, the metamorphosis of larvae turbot exposed to blue spectrum began at 24 dph and lasted for 26 days, which was significantly faster than other groups. In terms of physiological response, red and orange light could cause oxidative stress in larvae and juvenile turbot, resulting in increased expression of hsp70, antioxidant genes (cat, gst, mt), and innate immune genes (ctsd, ctsf, lzm). However, the corresponding enzyme activities were not significantly increased, indicating that long wavelength was not conducive to maintaining redox balance and improving immunity of turbot larvae and juveniles. In order to promote the growth and metamorphosis of turbot, reduce stress response, full spectrum should be used prior to metamorphosis and blue light should be used since 20 dph in turbot aquacultural system.

(3) Growth and physiological responses of juvenile turbot to spectral environments:

Juveniles under blue light had the highest growth rate, characterized by higher specific growth rate (SGR) (1.62 ± 0.01) and lower feed conversion rate (FCR) (0.95 ± 0.02). Long wavelength (red and orange spectra) could induce oxidative stress and inhibit the antioxidant and disease resistance of juvenile fish. The expression of hsp70, copper/zinc superoxide dismutase (cu/zn-sod), manganese superoxide dismutase，(mn-sod), cat、glutathione peroxidase (gsh-px) and lzm increased, but the activities of T-sod, Cat, Gsh-px and Lzm decreased. However, there were no significant differences in protein carbonyl (PC) and malondialdehyde (MDA) content, which suggested that red and orange light did not cause oxidative damage in juvenile turbot. In terms of metabolism, juvenile turbot showed adaptation to the long wavelength spectral environment. When the stress reaction occurred, the synthesis and secretion of cortisol were increased. The serum cortisol content of juveniles under red and orange light reached above 60 ng/mL, which was significantly higher than that of green, blue and full spectrum. At the same time, the expression of adenosine monophosphate-activated protein kinase (ampk) genes in red and orange group was also increased to promote nutrients decomposition and energy generation. Following this, glucose and lipid metabolism were promoted so as to maintain the energy homeostasis under red and orange spectra. However, the increase of energy consumption will lead to a decrease in the accumulation of nutrients, resulting in growth retardation. Therefore, blue light was suggested for optimizing the spectral environment of juvenile turbot aquacultural system.

第1章  引言... 1

1.1  自然海水光谱成分特征... 1

1.2  鱼类的光感受器... 2

1.2.1  视觉光感受器... 2

1.2.2  非视觉光感受器... 5

1.2.3  鱼类的光信号转导系统... 5

1.3  硬骨鱼类感光系统对光环境的适应性特征... 7

1.4  鱼类养殖光照调控研究现状... 8

1.4.1  光照强度对养殖鱼类的影响... 9

1.4.2  光周期对养殖鱼类的影响... 9

1.4.3  光谱环境对养殖鱼类的影响... 10

1.5  Light emitting diode（LED）技术在水产养殖中的应用... 11

1.6  大菱鲆的生物学特征... 12

1.7  研究目的与意义... 13

第2章  光谱环境对大菱鲆胚胎发育的影响... 15

2.1  前言... 15

2.2  材料与方法... 16

2.2.1  实验设备与日常管理... 16

2.2.2  实验材料... 18

2.2.3  样品采集... 18

2.2.4  实验仪器... 19

2.2.5  实验试剂... 19

2.2.6  组织学检测... 20

2.2.7  视蛋白与非视觉视蛋白表达检测... 21

2.2.8  胚胎va-opsin和exrh表达位置检测... 23

2.2.9  大菱鲆抗氧化系统及先天性免疫系统基因表达和酶活力测定... 26

2.2.10  有参转录组测序分析... 27

2.2.11  数据分析... 28

2.3  研究结果... 28

2.3.1  大菱鲆胚胎视觉器官发育及视蛋白表达... 28

2.3.2  大菱鲆非视觉系统的发育... 31

2.3.3  不同光谱下胚胎发育和孵化差异... 33

2.3.4  初孵仔鱼畸形率、死亡率... 35

2.3.5  不同光谱下初孵仔鱼抗氧化应激分析... 37

2.3.6  不同光谱下初孵仔鱼先天性免疫系统分析... 39

2.3.7  转录组组装结果... 41

2.3.8  差异基因表达分析... 42

2.3.9  基因注释与富集分析... 45

2.3.10  共同差异基因的GO富集分析... 50

2.3.11  转录组的荧光定量验证... 51

2.4  讨论... 53

2.5  小结... 58

第3章  大菱鲆感光器官发育及其对光谱环境的适应性特征... 59

3.1  前言... 59

3.2  材料与方法... 61

3.2.1  实验设备与日常管理... 61

3.2.2  实验材料... 62

3.2.3  样品采集... 62

3.2.4  实验仪器... 63

3.2.5  实验试剂... 63

3.2.6  组织学检测... 63

3.2.7  大菱鲆仔稚幼鱼氧化应激与先天性免疫相关基因表达... 63

3.2.8  酶活力检测... 64

3.2.9  数据分析... 64

3.3  研究结果... 64

3.3.1  大菱鲆仔稚幼鱼视觉器官发育结构特征... 64

3.3.2  不同光谱下大菱鲆仔稚幼鱼生长发育差异... 70

3.3.3  不同光谱下大菱鲆仔稚幼鱼抗氧化应激分析... 72

3.3.4  不同光谱下大菱鲆仔稚幼鱼先天性免疫分析... 76

3.4  讨论... 78

3.5  小结... 82

第4章  大菱鲆幼鱼对光谱环境的生长与生理响应... 83

4.1  前言... 83

4.2  材料与方法... 85

4.2.1  实验设备与日常管理... 85

4.2.2  实验材料... 85

4.2.3  排氨率检测... 85

4.2.4  样品采集... 86

4.2.5  实验仪器... 86

4.2.6  实验试剂... 86

4.2.7  大菱鲆幼鱼代谢与抗氧化相关基因表达检测... 86

4.2.8  大菱鲆幼鱼抗氧化相关酶活力与氧化损伤参数PC、MDA检测... 88

4.2.9  大菱鲆幼鱼皮质醇和代谢底物检测... 88

4.2.10  数据分析... 88

4.3  研究结果... 88

4.3.1  不同光谱下大菱鲆幼鱼生长的差异... 88

4.3.2  不同光谱下大菱鲆幼鱼应激反应分析... 89

4.3.3  不同光谱下大菱鲆幼鱼先天性免疫差异分析... 94

4.3.4  不同光谱下大菱鲆幼鱼血清皮质醇含量与能量代谢差异... 95

4.4  讨论... 102

4.5  小结... 104

第5章  结论与展望... 105

5.1  主要结论... 105

5.2  展望... 106

参考文献... 107

附  录... 129

致  谢... 133

作者简历及攻读学位期间发表的学术论文与研究成果    135