海洋生态与环境科学重点实验室知识库

刺参（Apostichopus japonicus）是我国重要的海洋经济物种，在我国北方海水养殖产业中刺参养殖产业的单一产值最大。刺参行为学研究可为刺参采捕设施的研发和增殖放流策略的制定提供参考，为刺参工厂化养殖、池塘养殖等增养殖模式创新提供参数支撑。行为内分泌学是行为学研究的一个重要分支，主要探究动物激素和行为之间的相互影响。本研究以褪黑激素和两种代表性神经肽为例，综合运用红外摄影技术、运动行为量化软件、代谢组学技术，系统探究了褪黑激素和两种神经肽对刺参运动行为的调控作用和行为变化的内在生理机制。此外，本研究还运用转录组学技术查明了刺参应激行为—吐肠行为的内在分子机制。

1. 褪黑激素对刺参运动行为的调控及内在机制研究

检测了褪黑激素在刺参体腔液中的含量，并采用体腔注射的方法研究了褪黑激素对刺参运动行为的影响。此外，使用超高效液相色谱和质谱联用技术（UPLC-Q-TOF-MS）检测了褪黑激素对刺参肌肉组织代谢活动的影响。研究结果表明褪黑激素在刺参体腔液中的浓度为135.0 ng/L左右，随着注射褪黑激素浓度的增加，注射后9小时内刺参运动的总距离和步数逐渐减少，且部分处理组差异显著，而平均和最大运动速度及步幅和步幅频率均有所下降，但无显著差异。因此褪黑激素对刺参具有镇静作用。在褪黑激素处理组刺参肌肉组织中检测到22种代谢物的浓度发生改变，其中5-羟色胺、9-顺式视黄酸、全反式视黄酸、黄素单核苷酸浓度明显下降。此外，处理组肌肉组织中游离脂肪酸（FFA）和腺苷5'-三磷酸（ATP）的浓度均降低。因此，褪黑激素抑制刺参运动行为的潜在生理机制包括运动调节剂—血清素浓度下降、降低脂肪酸氧化和氧化磷酸化过程。

2. Pedal神经多肽对刺参运动行为的调控及内在机制研究

采用体腔注射后记录刺参运动行为变化的方法研究Pedal神经多肽对刺参运动行为的影响，同时基于UPL-Q-TOF-MS代谢组学检测技术研究Pedal神经肽注射后刺参肌肉代谢物的变化情况。结果表明Pedal神经肽注射后刺参的步幅有所降低，表明该神经肽很可能参与调节刺参肌肉的收缩。此外，运动路程、步数的增加和运动速度的降低表明Pedal神经肽可增强刺参的运动耐力而降低其运动效率。肌肉代谢组学结果表明，磷脂酰乙醇胺（PE）和磷脂酰胆碱（PC）的下调、LysoPCs和LysoPEs的升高以及花生四烯酸（ARA）浓度的升高是Pedal神经肽对刺参运动行为产生这些影响的潜在生理机制。

3. SALMFamide神经多肽对刺参运动行为的调控及内在机制研究

体外合成刺参L型SALMFamide神经肽后，采用体腔注射的方法研究了SALMFamide神经肽对刺参运动行为的调控作用，并采用代谢组学技术检测了该神经肽注射后刺参肌肉生理的变化情况。实验结果表明SALMFamide神经肽使刺参运动步幅有一定提升，表明该神经肽可能参与刺参体壁肌肉松弛的调节。此外，运动路程、步数、时间和运动速度均增大表明SALMFamide神经肽不仅增强了刺参的运动耐力，而且提升了刺参的运动效率。代谢组学结果表明刺参肌肉泛酸含量的升高、磷脂酰乙醇胺（PE）和磷脂酰胆碱（PC）比例的变化、溶血磷脂酰乙醇胺（LysoPEs）和花生四烯酸（ARA）浓度的升高是SALMFamide神经肽调控刺参运动行为的潜在生理机制。

4. 刺参吐肠行为内在分子机制研究

为探究刺参应激行为—吐肠行为的分子机制，本研究采用Illumina（RNA-Seq）测序平台同时测试了刺参三种状态下的样品：正常（TCQ）、去除内脏时（TCZ）和去除内脏后3小时（TCH）。测试结果表明总共产生129,905个unigenes，N50长度为2651个碱基对，54787个unigenes可从7个功能数据库（KEGG，KOG，GO，NR，NT，Interpro和Swiss-Prot）得到注释。此外，在TCQ与TCZ、TCZ与TCH和TCQ与TCH的比较中，分别鉴定出190、191和320个发生差异表达基因（DEG）。这些DEG可映射到KEGG数据库中的157、113和190个信号传导途径。KEGG分析结果显示潜在的DEGs分别属于“环境信息处理”、“生物系统”、“新陈代谢”和“细胞过程”类别。这些DEGs与肌肉收缩、激素和神经递质分泌、神经和肌肉损伤、能量供应、细胞应激和细胞凋亡有关。这些相关的基因和信号通路有助于阐明刺参吐肠行为的内在分子机制。

Apostichopus japonicus is an important commercial marine species in China. The single production value of A. japonicus aquaculture industry is the largest one in all the marine aquaculture industries in northern China. The behavioral research of sea cucumber can provide reference for the development of sea cucumber catching facilities and sea cucumber enhancement and releasing strategies, and provide data support for the innovation of sea cucumber aquaculture pattern including industrial and pond aquaculture. Behavioral endocrinology is an important branch of behavioral research that mainly study the interaction between hormones and behavior in animals. In this study, melatonin and two representative neuropeptides were used to study their effects on locomotor behavior of A. japonicus by infrared camera and ethovision software. Meanwhile, metabolomics was used to systematically detect the changing of muscle physiology after melatonin and two neuropeptides administration. In part, we expected to clarify the intrinsic physiological mechanisms of the behavioral regulation of melatonin and two neuropeptides. In addition, transcriptomics techniques were used to identify the intrinsic molecular mechanisms of sea cucumber evisceration behavior.

1. The effect of melatonin on locomotor behavior and muscle physiology in A. japonicus

The goals of this part were to show the existence of melatonin in the sea cucumber A. japonicus and to evaluate its effect on locomotor activity. In addition, muscle tissues from control and melatonin-treated sea cucumbers were tested using ultra performance liquid chromatography and quadrupole time-offlight mass spectrometry (UPLC-Q-TOF-MS) to determine the changes of metabolic activity in muscle. Melatonin was present in the coelomic fluid of A. japonicus at a concentration of ∼135.0 ng/L. The total distance traveled and number steps taken over 9 h after melatonin administration decreased with increasing concentration of the melatonin dose. Mean and maximum velocity of movement and stride length and stride frequency also decreased, but their differences were not statistically significant. Overall, these results suggest that melatonin administration had a sedative effect on A. japonicus. The levels of 22 different metabolites were altered in the muscle tissues of melatonin-treated sea cucumbers. Serotonin, 9-cis retinoic acid, all-trans retinoic acid, flavin mononucleotide in muscles were downregulated after melatonin administration. Moreover, a high free fatty acid (FFA) concentration and a decrease in the adenosine 50-triphosphate (ATP) concentration in the muscle tissues of the melatonin-treated group were detected as well. These results suggest that the sedative effect of melatonin involves some other metabolic pathways, and the reduced locomotor modulator— serotonin, inhibited fatty acid oxidation and disturbed oxidative phosphorylation are potential physiological mechanisms that result in the inhibitory effect of melatonin on locomotion in sea cucumbers.

2. The effect of pedal neuropeptide on locomotor behavior and muscle physiology in A. japonicus

The changing of A. japonicus locomotor behavior was recorded after pedal neuropeptide administration in this part. In addition, UPL-Q-TOF-MS metabolomics was used to detect the changing of muscle metabolites after pedal neuropeptide injection. The results showed that the locomotor stride of A. japonicus after pedal neuropeptide injection was decreased, indicating that this neuropeptide was likely to participate in the regulation of muscle contraction in A. japonicus. Besides, the increase of moving distance and steps and the decrease of moving velocity indicated that pedal neuropeptide can enhance the moving tolerance and reduce the moving efficiency in sea cucumber. The data of muscle metabolomics suggested that down-regulation of phosphatidylethanolamine (PE) and phosphatidylcholine (PC), up-regulation of LysoPCs, LysoPEs and arachidonic acid (ARA) might be the underlying mechanisms that responsible for behavioral effects of pedal neuropeptide in A. japonicus.

3. The effect of L-SALMFamide neuropeptide on locomotor behavior and muscle physiology in A. japonicus

After synthesizing L-type SALMFamide neuropeptide in vitro, the regulation of SALMFamide neuropeptide on locomotor behavior of A. japonicus was studied by neuropeptide coelom injection. Besides, the changes of muscle physiology were detected by metabolomics. Our results showed that SALMFamide neuropeptide can increase the moving stride of sea cucumber, which indicates that the neuropeptide may take part in the muscle relaxation of A. japonicus. In addition, the increase of moving distance, number of steps, cumulative duration of moving and moving velocity indicated that SALMFamide neuropeptide enhanced not only the moving endurance, but also the moving efficiency of A. japonicus. Metabolomics results suggested that the increase of pantothenic acid, LysoPEs and arachidonic acid (ARA) concentration, as well as the changing of PC : PE ratio, were the potential physiological mechanisms underlying the regulation of this neuropeptides on locomotor behavior in A. japonicus.

4. Molecular mechanisms responsible for evisceration behavior in A. japonicus

In this part, Illumina sequencing (RNA-Seq) was performed on A. japonicus specimens in three states: normal (TCQ), eviscerating (TCZ), and 3 h after evisceration (TCH). In total, 129,905 unigenes were generated with an N50 length of 2651 base pairs, and 54,787 unigenes were annotated from seven functional databases (KEGG, KOG, GO, NR, NT, Interpro, and Swiss-Prot). Additionally, 190, 191, and 320 genes were identifed as differentially expressed genes (DEGs) in the comparisons of TCQ vs. TCZ, TCZ vs. TCH, and TCQ vs. TCH, respectively. These DEGs mapped to 157, 113, and 190 signaling pathways in the KEGG database, respectively. KEGG analyses also revealed that potential DEGs enriched in the categories of “environmental information processing,” “organismal system,” “metabolism,” and “cellular processes,” and they were involved in evisceration behavior in A. japonicus. These DEGs are related to muscle contraction, hormone and neurotransmitter secretion, nerve and muscle damage, energy support, cellular stress, and apoptosis. In conclusion, through our comparative analysis of A. japonicus in different stages, we identifed many candidate evisceration-related genes and signaling pathways that likely are involved in evisceration behavior. These results should help further elucidate the mechanisms underlying evisceration behavior in sea cucumbers.

第1章  文献综述... 1

1.1  刺参及其养殖产业介绍... 1

1.1.1  刺参分类地位、分布及生物学特征... 1

1.1.2  我国刺参养殖产业发展现状... 3

1.1.3  我国刺参产业发展存在问题... 4

1.1.4  我国刺参产业发展对策及方向... 5

1.2  刺参行为学研究进展... 6

1.2.1  刺参运动行为研究进展... 6

1.2.2  刺参摄食行为研究进展... 6

1.2.3  刺参繁殖行为研究进展... 8

1.2.4  刺参吐肠行为研究进展... 8

1.2.5  刺参夏眠行为研究进展... 9

1.3  刺参神经内分泌学研究进展... 10

1.3.1  刺参体腔液激素对环境的应答... 10

1.3.2  刺参神经多肽与多肽激素的鉴别分析... 10

1.3.3  刺参神经多肽功能研究现状... 11

1.4  转录和代谢组学技术在刺参研究中的应用... 12

1.4.1  转录组学技术在刺参研究中的应用... 12

1.4.2  代谢组学技术在刺参研究中的应用... 13

1.5  本研究的目的、意义以及思路... 14

1.5.1  研究目的及意义... 14

1.5.2  科学问题... 15

1.5.3  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  褪黑激素对刺参运动行为的调控作用研究... 18

2.1  前言... 18

2.2  材料与方法... 20

2.2.1  刺参采集、暂养及褪黑激素检测... 20

2.2.2  褪黑激素处理及刺参运动行为视频采集与分析... 20

2.2.3  肌肉样品采集及UPLC-Q-TOF-MS检测... 21

2.2.4  肌肉ATP和FFA检测... 22

2.2.5  数据分析... 23

2.3  实验结果... 23

2.3.1  刺参体腔液褪黑激素的含量... 23

2.3.2  褪黑激素对刺参运动行为的影响... 24

2.3.3  褪黑激素对刺参肌肉生理的影响... 26

2.3.4  褪黑激素处理前后刺参肌肉FFA和ATP含量... 29

2.4  讨论... 30

2.4.1  刺参体内的褪黑激素... 30

2.4.2  褪黑激素对刺参运动行为的影响... 30

2.4.3  褪黑激素抑制刺参运动行为的潜在机制... 31

2.5  本章小结... 34

第3章  Pedal神经肽对刺参运动行为的调控作用研究.... 36

3.1  前言... 36

3.2  材料与方法... 38

3.2.1  Pedal神经肽合成... 38

3.2.2  刺参采集、暂养... 39

3.2.3  Pedal神经肽处理及刺参运动行为视频采集与分析... 39

3.2.4  肌肉样品采集、UPLC-Q-TOF-MS检测及数据分析... 40

3.3  实验结果... 40

3.3.1  刺参Pedal神经肽合成结果... 40

3.3.2  Pedal神经肽对刺参运动行为的影响... 41

3.3.3  Pedal神经肽对刺参肌肉生理的影响... 43

3.4  讨论... 48

3.4.1  Pedal神经肽对刺参运动行为的影响... 48

3.4.2  Pedal神经肽促进刺参运动行为的潜在机制... 49

3.5  本章小结... 51

第4章  SALMFamide神经肽对刺参运动行为的调控作用研究.... 52

4.1  前言... 52

4.2  材料与方法... 53

4.2.1  SALMFamide神经肽合成... 53

4.2.2  实验刺参采集、暂养... 54

4.2.3  SALMFamide神经肽处理及运动行为视频采集与分析... 54

4.2.4  肌肉样品采集、代谢组学检测及数据分析... 55

4.3  实验结果... 55

4.3.1  刺参SALMFamide神经肽合成结果... 55

4.3.2  SALMFamide神经肽对刺参运动行为的影响... 56

4.3.3  SALMFamide神经肽对刺参肌肉生理的影响... 57

4.4  讨论... 62

4.4.1  SALMFamide神经肽对刺参运动行为的影响... 63

4.4.2  SALMFamide神经肽促进刺参运动行为的潜在生理机制... 64

4.5  本章小结... 66

第5章  刺参吐肠行为分子机制研究.... 67

5.1  前言... 67

5.2  材料与方法... 68

5.2.1  刺参采集及暂养... 68

5.2.2  吐肠行为诱导及取样... 68

5.2.3  RNA提取及Illumina测序... 69

5.2.4  转录组数据组装及基因功能注释... 69

5.2.5  差异表达基因筛选及富集分析... 70

5.2.6  RT-qPCR验证... 70

5.2.7  数据分析... 71

5.3  实验结果... 71

5.3.1  样品测序结果... 71

5.3.2  转录组序列组装、注释及分类结果... 71

5.3.3  刺参吐肠行为相关差异表达基因... 77

5.3.4  DEGs功能富集分析... 79

5.3.5  RT-qPCR验证结果... 84

5.4  讨论... 85

5.4.1  刺参吐肠行为与信号传导和刺激反应相关基因... 85

5.4.2  刺参吐肠行为与动物有机系统相关基因... 86

5.4.3  刺参吐肠行为与代谢相关基因... 88

5.4.4  刺参吐肠行为与其他功能相关基因... 88

5.5  本章小结... 89

第6章  总结与展望.... 92

6.1  总结... 92

6.2  创新性... 92

6.3  存在问题... 92

6.4  研究展望... 93

参考文献... 95

致谢... 117

作者简历及攻读学位期间发表的学术论文与研究成果    119