海洋生态与环境科学重点实验室知识库

仿刺参（Apostichopus japonicus），也称刺参，是我国重要的海水养殖物种之一。近年来，我国刺参养殖业蓬勃发展，现已成为海水养殖业中的支柱性产业。刺参养殖过程中存在生长差异较大的现象，这严重阻碍了刺参养殖效率的提高。为实现刺参高效养殖，亟需加深对刺参生长差异的理解，但不同生长速率刺参的行为、生理和分子调控特征有什么差异？影响个体生长差异的关键因素有哪些？针对这些问题，本研究利用缩时摄影技术、行为量化分析、16S rDNA测序技术、转录组学和代谢组学技术系统探究了不同生长速率刺参的行为和生理差异及分子调控特征，并研究了个性因素和社会互动因素对刺参个体生长差异的影响。主要研究结果如下：

1．不同生长速率刺参的行为及生理比较

摄食行为的差异是造成刺参个体生长差异的重要原因。利用同一家系刺参（体重为19.18±0.42 g）开展30天的养殖实验，刺参体重变异系数从起始的2.16%迅速增加到27.67%，表现出明显的个体生长差异。快速生长刺参和缓慢生长刺参的特定生长率分别为2.41±0.89%d-1和1.10±0.66%d-1。利用缩时摄影技术和行为分析软件，对刺参的运动行为和摄食行为进行定量分析，发现不同生长速率刺参的运动行为特征（运动距离和运动时间）没有显著差异。但缓慢生长刺参的摄食时间（133.5±150.91 min）和排粪量（0.53±0.44 g）均显著低于快速生长个体（摄食时间：283±168.98 min；排粪量：1.22±0.66 g），表明摄食行为显著减少，可能是导致其生长速率较低的重要原因。

刺参的生长差异可能与生理结构、能量代谢、消化能力以及肠道微生物的差异有关。通过比较分析发现，在生理结构层面，快速生长刺参体壁占比为66.60±2.79%，显著低于缓慢生长个体（71.14±4.25%），这意味着其具有更高的呼吸树和肠道占比，有利于食物的摄入、吸收和转化，进而促进生长。在能量代谢和消化能力层面，缓慢生长刺参的静息代谢率（21.60±4.33 μgO2g-1h-1）和胰蛋白酶活性（65.62±12.42 U/mg）显著降低，表明其能量代谢水平和消化能力显著下降，可能是对摄食行为减少的生理适应。此外，利用16S rDNA测序技术对不同生长速率刺参的肠道微生物进行分析，发现缓慢生长刺参的肠道微生物多样性下降，潜在的致病菌（Vibrionaceae和Burkholderiaceae）丰度升高，而快速生长刺参中相对丰度较高的细菌（Rhodobacteraceae、Rubritaleaceae和Pirellulaceae）大多与藻类多糖的降解有关。

2. 不同生长速率刺参的分子调控特征

通过对不同生长速率刺参的差异表达基因和差异代谢物的分析发现，缓慢生长刺参的生长受到抑制，并且遭受严重的氧化应激。利用RNA测序技术和超高效液相色谱-质谱技术（Ultra-Performance Liquid Chromatography-Mass Spectrometry, UPLC-MS），鉴定不同生长速率刺参中的差异表达基因和差异代谢物，并进行关联分析。RNA测序结果显示，与快速生长个体相比，在缓慢生长个体中分别发现了216和169个显著上调和显著下调的基因。对所有差异表达基因进行KEGG富集分析，发现富集到的通路包括“真核生物中的核糖体发生”、“剪接体”、“细胞凋亡”和“Hippo信号通路”等。差异代谢物鉴定结果显示，在正离子模式下，与快速生长刺参相比，缓慢生长刺参中显著上调的代谢物有29种，显著下调的代谢物有28种；在负离子模式下，缓慢生长刺参中显著上调的代谢物有8种，显著下调的代谢物有17种。通过KEGG富集分析发现，富集到的关键代谢通路有“牛磺酸和亚牛磺酸代谢”、“精氨酸生物合成”、“抗坏血酸和阿尔尿酸代谢”以及“氧化磷酸化”等。转录组和代谢组的关联分析表明，缓慢生长刺参中胶原蛋白编码基因显著下调，部分生长负调控因子和细胞凋亡诱导基因显著上调，糖异生和氧化应激代谢途径加剧，表明其生长受到抑制，且遭受严重的氧化应激，需消耗自身能量来维持机体正常运转。

3. 个性因素对刺参个体生长差异的影响

刺参中存在个性（行为差异的一致性），部分个性行为特征（运动距离）和静息代谢率具有较好的可重复性，个体之间行为和生理差异可能是造成生长差异的重要原因。通过刺参隔离养殖实验，发现刺参个体之间的摄食量、食物转化率和特定生长率存在较大差异，差异可达3.47、6.42和5.64倍。生长速率与食物摄入量和食物转化率均呈正相关关系。通过对刺参静息代谢率和个性行为特征的重复测量，发现刺参的静息代谢率的可重复性较好，而个性行为特征中只有运动距离具有较好的可重复性，运动时间比、潜伏时间、摄食时间均不具备可重复性。此外，相关性分析表明，刺参生长速率与能量代谢存在正相关关系，与应激反应存在负相关关系，但个性行为特征与刺参生长速率、静息代谢率和皮质醇均不存在相关性。

4. 社会互动因素对刺参个体生长差异的影响

在社会互动因素的研究中发现，影响刺参个体生长差异的主要原因是对食物的竞争，采取适当的食物投喂方式能够有效减少个体的生长差异。在食物充足且分布比较均匀的条件下，隔离养殖和群体养殖刺参的生长速率没有明显差异。此外，不同于以往的研究结果，本研究中群体养殖的刺参没有表现出更大的生长差异。通过比较隔离养殖刺参和群体养殖刺参的行为指标和能量代谢指标，发现它们的运动行为、摄食行为和能量代谢均没有显著差异。利用16S rDNA测序技术对隔离养殖和群体养殖刺参的肠道微生物进行分析，发现它们的肠道微生物组成在门和科水平上基本相似，均发现了与藻类多糖降解相关的细菌，但在隔离养殖刺参中发现潜在致病菌（Shewanella）的丰度有所增加，这表明社会接触和互动可能对维持肠道微生物的稳态与健康具有一定影响。

综上，本研究系统探究了刺参个体生长差异的行为、生理和分子调控特征，阐明了个性因素与刺参生长和生理之间的关系，探明了群体养殖条件下影响刺参个体生长差异的关键因素，并提出了在养殖环境中解释刺参个体生长差异的理论模型。研究结果为理解刺参个体生长差异现象提供了新认知，并为提高刺参养殖效率提供了理论依据，有利于刺参养殖产业的持续健康发展。

Apostichopus japonicus is one of the most important mariculture species.In recent years, the cultivation of A. japonicus has developed vigorously in China and has become the pillar industry of mariculture. There was a great growth difference in the culture of A. japonicus, which seriously hindered the improvement of the cultivation efficiency of A. japonicus. In order to achieve efficient culture, the understanding of growth differences needs to be improved. However, what are the differences in behavioral, physiological and molecular regulatory characteristics of A. japonicus at different growth rates? What are the key factors affecting individual growth differences? Aiming at the questions above, this study systematically investigated the behavioral, physiological and molecular regulatory mechanisms of individual growth difference of A. japonicus by using time-lapse photography technology, behavioral quantification software, 16S rDNA sequencing technology transcriptomic technology, and metabolomics technology. The effects of personality factors and social interaction factors on individual growth difference of A. japonicus were also investigated. The main study results are as follows:

1. Comparison of behavior and physiology of A. japonicus with different growth rates

The difference in feeding behavior was an important cause of individual growth differences in A. japonicus. Using the same family of A. japonicus (weighed 19.18±0.42 g). A. japonicus showed significant individual growth differences during the 30-day experimental period, with the coefficient of variation of body weight increases rapidly from 2.16% to 27.67%. The specific growth rates were 2.41±0.89%d-1 and 1.10±0.66%d-1 respectively. Using time-lapse photography and behavioral analysis software, we quantified the locomotor and feeding behavior of A. japonicus and found that there were no significant differences in the locomotor behavior characteristics (total distance and movement time) of A. japonicus with different growth rates. However, the feeding time (133.5±150.91 min) and fecal discharge (0.53±0.44 g) of slow-growing A. japonicus were significantly lower than those of fast-growing individuals (feeding time: 283±168.98 min; fecal discharge: 1.22±0.66 g), indicating that its feeding behavior was significantly reduced, which might be related to their lower growth rate.

There were significant differences in physiological structure, energy metabolism, digestive capacity, and intestinal microorganisms among different growth rates of A. japonicus. At the level of the physiological structure, fast-growing A. japonicus had a lower body wall percentage, which meant that it had a higher percentage of body luminal fluid and intestinal tract, which facilitated the intake, absorption, and transformation of food, and thus promoted growth. At the level of energy metabolism and digestive capacity, the resting metabolic rate (21.60±4.33μgO2g-1h-1) and trypsin activity (65.62±12.42 U/mg) of slow-growing A. japonicus were significantly lower, indicating a significant decrease in its energy metabolism level and digestive capacity, which may be a physiological adaptation to the reduced feeding behavior. In addition, analysis of gut microorganisms in A. japonicus with different growth rates using 16S rDNA sequencing revealed a decrease in gut microbial diversity and an increase in the abundance of potentially pathogenic bacteria (Vibrionaceae and Burkholderiaceae) in slow-growing A. japonicus, while the relatively high abundance of bacteria in fast-growing A. japonicus ( Rhodobacteraceae, Rubritaleaceae, and Pirellulaceae) were mostly associated with the degradation of algal polysaccharides. Decreased energy metabolism and digestibility as well as imbalance of gut microbes may be associated with lower growth rates.

2. Molecular regulation characteristics of A. japonicus with different growth rates

Analysis of differentially expressed genes and differential metabolites in A. japonicus with different growth rates revealed that slow-growing A. japonicus was inhibited in growth and suffered from oxidative stress. RNA sequencing and Ultra-Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS) were used to identify and correlate differentially expressed genes and differential metabolites in A. japonicus with different growth rates. The RNA sequencing results showed that 216 and 169 significantly up-regulated and significantly down-regulated genes were identified in slow-growing individuals compared to fast-growing individuals, respectively. KEGG enrichment analysis of all differentially expressed genes revealed that the enriched pathways included "ribosome genesis in eukaryotes", "spliceosome", "apoptosis" and "Hippo signaling pathway", etc. The results of differential metabolite identification showed that 29 metabolites were significantly up-regulated, and 28 metabolites were significantly down-regulated in slow-growing A. japonicus compared with fast-growing A. japonicus in positive ionization mode, and 8 metabolites were significantly up-regulated, and 17 metabolites were significantly down-regulated in slow-growing A. japonicus in negative ionization mode. The KEGG enrichment analysis revealed that the key metabolic pathways were "taurine and subtaurine metabolism", "arginine biosynthesis", "ascorbic acid and alanuric acid metabolism" and "oxidative phosphorylation". "and "oxidative phosphorylation", etc. The correlation analysis of transcriptome and metabolome showed that collagen-encoding genes were significantly down-regulated, some negative growth regulators and apoptosis-inducing genes were significantly up-regulated, and gluconeogenesis and oxidative stress metabolic pathways were intentified in slow-growing A. japonicus, indicating that its growth was severely inhibited and it suffered from severe oxidative stress and needed to consume its own energy to maintain the normal functioning of the organism.

3. Effects of personality factors on individual growth differences of A. japonicus

A. japonicus possessed personality (consistency of behavioral differences), and some personality behavior characteristics (movement distance) and resting metabolic rate had good repeatability. Differences in feeding behavior and energy conversion between individuals may be important reasons for differences in growth. Through the isolation culture experiment, it was found that there were great differences in food intake, food conversion rate and specific growth rate among individuals, with the differences being 3.47, 6.42 and 5.64 times. The growth rate was positively correlated with food intake and food conversion. Through repeated measurement of resting metabolic rate and personality behavior characteristics, it was found that the resting metabolic rate has good repeatability, while only the movement distance has good repeatability in personality behavior characteristics, and the movement time ratio, latent time, feeding time all have no repeatability, which may be related to the adaptation to the test environment. In addition, the growth rate of A. japonicus was positively correlated with energy metabolism and negatively correlated with stress response. However, the growth rate, resting metabolic rate and cortisol of A. japonicus were not correlated with their personality behavior characteristics.

4. Effects of social interaction factors on individual growth differences of A. japonicus

The main reason affecting individual growth differences of A. japonicus was competition for food and adopting appropriate food feeding methods could effectively reduce individual growth differences. Under the condition of sufficient and evenly distributed food, there was no significant difference in the growth rate between isolated and group-farmed individuals. In addition, different from the results of previous studies, the group-farmed A. japonicus in this study did not show greater growth differences. By comparing the behavioral indicators and energy metabolism indicators of isolated cultured and group cultured A. japonicus, it was found that their locomotor behavior, feeding behavior and energy metabolism were not significantly different. Analysis of the gut microbes of isolated and group-cultured A. japonicus using 16S rDNA sequencing revealed that their gut microbial composition was largely similar at the phylum and family levels, and bacteria associated with algal polysaccharide degradation were found in both, but an increased abundance of potentially pathogenic bacteria (Shewanella) was found in isolated cultured A. japonicus, suggesting that social contact and interaction may have an effect on maintaining gut microbial homeostasis and health.

In conclusion, this study systematically investigated the behavioral, physiological and molecular regulatory mechanisms of individual growth differences in A. japonicus. The relationship between personality factors and the growth and physiology of A. japonicus was clarified. The key factor affecting the individual growth difference of A. japonicus cultured in group was identified. And a theoretical model was proposed to explain the individual growth differences of A. japonicus in the culture environment. The results provide new insights into the individual growth differences of A. japonicus, and provide a theoretical basis for improving the efficiency of A. japonicus culture, which is conducive to the sustainable and healthy development of A. japonicus culture industry.

第1章  绪论... 1

1.1  刺参介绍及个体生长差异研究进展... 1

1.1.1  刺参介绍... 1

1.1.2  刺参个体生长差异研究进展... 1

1.2  刺参行为学研究进展... 3

1.2.1  刺参标记技术研究进展... 3

1.2.2  刺参运动行为研究进展... 3

1.2.3  刺参摄食行为研究进展... 4

1.3  影响动物个体生长差异的因素... 5

1.3.1  个体属性... 5

1.3.2  种内竞争... 7

1.4  动物个性研究进展... 9

1.4.1  动物个性研究的起源及定义... 9

1.4.2  动物个性的生理基础... 10

1.4.3  动物个性的适应性解释模型... 13

1.5  研究目的、意义及思路... 15

1.5.1  目的及意义... 15

1.5.2  科学问题... 15

1.5.3  研究内容与技术路线... 15

1.5.4  预期成果... 16

1.6  本章小结... 16

第2章  不同生长速率刺参的行为及生理比较... 17

2.1  研究背景... 17

2.2  材料与方法... 18

2.2.1  刺参来源及养殖环境... 18

2.2.2  实验设计及样品采集... 18

2.2.3  静息代谢率测定... 18

2.2.4  行为视频采集及处理... 19

2.2.5  消化酶测定... 19

2.2.6  DNA提取及16SrDNA测序... 20

2.2.7  数据处理及统计分析... 20

2.3  实验结果... 21

2.3.1  刺参的生长差异... 21

2.3.2  运动行为和摄食行为差异... 23

2.3.3  消化酶活性和静息代谢率差异... 23

2.3.4  肠道微生物差异... 25

2.4  讨论... 30

2.4.1  不同生长速率刺参的生理结构差异... 30

2.4.2  不同生长速率刺参的行为差异... 30

2.4.3  不同生长速率刺参的消化能力和能量代谢差异... 31

2.4.4  不同生长速率刺参的肠道微生物差异... 31

2.5  小结... 33

第3章  不同生长速率刺参的分子调控特征... 35

3.1  研究背景... 35

3.2  材料与方法... 36

3.2.1  刺参来源及暂养... 36

3.2.2  刺参养殖及样品采集... 36

3.2.3  代谢物提取、检测及鉴定... 36

3.2.4  代谢组学数据分析... 37

3.2.5  RNA测序及数据分析... 37

3.2.6  代谢组学和转绿组学关联分析... 38

3.2.7  数据处理与分析... 38

3.3  实验结果... 38

3.3.1  刺参生长表现... 38

3.3.2  不同生长速率刺参的代谢组学分析... 39

3.3.3  不同生长速率刺参的转录组学分析... 42

3.3.4  转录组学和代谢组学关联分析... 46

3.4  讨论... 49

3.4.1  胶原蛋白编码基因在缓慢生长个体中显著下调... 49

3.4.2  缓慢生长个体生长受到抑制... 49

3.4.3  缓慢生长个体中耗能增加... 50

3.4.4  缓慢生长个体中氧化应激加剧... 50

3.5  小结... 51

第4章  个性因素对刺参个体生长差异的影响... 53

4.1  研究背景... 53

4.2  材料与方法... 54

4.2.1  刺参来源及养殖条件... 54

4.2.2  实验设计... 54

4.2.3  个性行为特征评价指标... 55

4.2.4  行为视频采集及处理... 55

4.2.5  皮质醇测定... 55

4.2.6  数据处理与分析... 55

4.3  结果... 56

4.3.1  刺参生长情况... 56

4.3.2  刺参个性行为特征和能量代谢的可重复性... 57

4.3.3  刺参生长与个性行为特征和生理的关系... 58

4.3.4  刺参个性行为特征与生理的关系... 59

4.4  讨论... 60

4.4.1  刺参生长差异与摄食和能量转化的差异有关... 60

4.4.2  刺参个性行为特征和静息代谢率的可重复性... 61

4.4.3  刺参生长与个性行为特征和生理的关系... 62

4.4.4  刺参个性行为特征与生理的关系... 63

4.5  小结... 63

第5章  社会互动因素对刺参个体生长差异的影响... 65

5.1  研究背景... 65

5.2  材料与方法... 66

5.2.1  刺参来源及养殖环境... 66

5.2.2  实验设计及样品采集... 66

5.2.3  行为视频采集及处理... 66

5.2.4  DNA提取及16SrDNA测序... 67

5.2.5  数据处理与分析... 67

5.3  结果... 68

5.3.1  隔离养殖和群体养殖刺参的生长情况... 68

5.3.2  隔离养殖和群体养殖刺参的行为差异... 71

5.3.3  隔离养殖和群体养殖刺参的肠道微生物... 72

5.4  讨论... 75

5.4.1  隔离养殖和群体养殖刺参的生长比较... 75

5.4.2  隔离养殖和群体养殖刺参的行为和能量代谢... 76

5.4.3  隔离养殖和群体养殖刺参的肠道微生物比较... 76

5.5  小结... 78

第6章  研究总结与展望... 79

6.1  研究总结... 79

6.2  主要创新点... 80

6.3  存在问题... 80

6.4  研究展望... 81

参考文献... 83

致  谢... 111

作者简历及攻读学位期间发表的学术论文与其他相关学术成果  113