实验海洋生物学重点实验室知识库

本研究以凡纳对虾（Penaeus vannamei）5日龄仔虾（PL5）为研究对象，分别通过密度、盐度、温度等三种环境训练方式进行30-40天的标粗生长实验，从补偿生长情况、体长均匀度表现、消化酶活性、渗透调节酶活性、抗氧化酶活性、热休克蛋白基因（Hsp）表达水平、mTOR信号通路、副溶血弧菌VPE1感染实验等多角度综合评估了凡纳对虾的补偿生长效应及生产应用可能。具体研究结果如下：

1、密度训练对凡纳对虾仔虾的补偿生长效应及潜在作用机制

根据工厂化养殖实践，在凡纳对虾标粗阶段，设计三种饲养密度进行了实验（D1（对照）= 10,000 PL/m³，D3 = 30,000 PL/m³，和 D5 = 50,000 PL/m³；17 days），随后将密度均调整至10,000 PL/m³，养殖13天。结果表明，D5组具有超补偿生长效应，而D3组则表现完全补偿生长效应。同时初步研究了对虾补偿生长效应发生的机制，研究结果显示，在动物补偿阶段，D5组的SGR（特殊生长率）和FCE（饲料转化效率）明显高于对照组（P<0.05）。T-SOD酶活力水平与对照组无显著的区别（P>0.05）。 CAT、GSH-PX、MDA等酶活性或含量均有补偿性变化。此外，测量了一些基因的相对mRNA表达。结果表明，生长相关基因（EGFR、MSTN）、蜕皮相关基因（MIH）、免疫相关基因（Toll）和消化相关基因（α-淀粉酶）的相对mRNA表达有补偿性变化。同时，对补偿阶段不同密度训练后的仔虾进行转录组分析。在D5和D3组处理的虾中分别鉴定出237和348 个差异表达基因 (DEGs)。在D5处理的虾中，DEGs被注释并分为与动物代谢反应相关的9个过程或途径。对于D3处理的虾，DEGs主要注释到与动物代谢反应相关的15个过程或途径。因此，上述结果为凡纳对虾仔虾标粗过程中补偿生长效应的理论和实践提供了依据。

2、盐度训练对凡纳对虾仔虾的补偿生长效应、生理变化和抗病力的影响

本研究评估了盐度训练（限制和补偿）是否可以促进仔虾的生长。在四个盐度处理组（SAL20（对照）= 20 ppt、SAL10 = 10 ppt、SAL30 = 30 ppt 和 SAL40 = 40 ppt）饲养20天（限制阶段），然后将它们均转移到盐度20的环境中饲养20天（补偿阶段）。SAL40组在体长表现出超补偿生长效应。在盐度限制阶段，SAL30和SAL40组虾的消化酶活性降低，渗透压调节酶活性升高；在补偿阶段，均出现了相反的变化趋势。这一结果表明，盐度限制可能增加了渗透调节所需的能量消耗，因此对虾通过增加食物利用率来补偿。从限制期到补偿期，tor、s6k、4e-bp基因表达水平先升高后降低。结果还表明，mTOR信号通路可能响应盐度限制阶段和补偿阶段的变化而调节生长。最后，使用不同的盐度训练组仔虾进行了副溶血性弧菌E1感染实验，发现SAL30组抗弧菌感染的能力较强。总之，上述数据表明，以盐度30培养仔虾20天后，然后将水体盐度降低到20 ppt不会影响存活率或最终体重，并且可能会提高仔虾的抗病能力和均匀度。

3、温度训练对凡纳对虾仔虾的补偿生长效应、生理变化和抗病力的影响

本研究在三个温度处理组（TEMP28（对照）= 28℃、TEMP25 = 25℃和TEMP31 = 31℃）饲养了20天（限制阶段），然后将它们转移到28℃的温度环境中饲养20天（补偿阶段）。研究结果发现TEMP25组处理不仅不影响凡纳对虾的最终体重和存活率，并且提高了它们的最终体重，即超补偿生长，并在一定程度上可能提高了其抗病能力。在VPE1攻毒实验中，TEMP31组对病原的抗感染能力较对照组弱，TEMP25组抗感染能力强于对照组。温度训练过程中仔虾的消化酶活性分析表明，α-淀粉酶活性存在显著的抑制-补偿变化，而脂肪酶并没有显著变化。温度训练可以激活mTOR信号通路，相关基因的表达上调。通过分析热休克蛋白基因的表达水平变化发现温度训练中胁迫水平可能会导致仔虾体长均一程度的变化。根据对实验组凡纳对虾仔虾的生长表现和抗病性的比较，发现以25℃培养仔虾20天后，温度逐渐提高到28℃后持续20天，可以获得更好的补偿生长潜力。。

4、比较转录组分析温度和盐度训练对凡纳对虾产生积极效果的原因

通过对产生超补偿生长效应的两个处理组，即SAL40（对照组为SAL10组）和TEMP25组（对照组为TEMP28组）的对虾进行了转录组测序和生物信息学分析，初步分析了在温度和盐度训练后，凡纳对虾补偿生长过程中的肝胰腺组织的差异表达基因。SAL40和TEMP25组相对于各自对照组的差异表达，分别对应于236和327个DEGs，并对DEGs进行两组联合分析，筛选了15个DEGs，显著富集的通路有8条，希望可以借此进一步挖掘和探究凡纳对虾补偿生长过程中产生积极生理后果的分子机制。

In this study, the PL5 shrimp Penaeus vannamei (post-larvae, 5-day-old) were used in this study, and a 30-40-day nursery rearing growth experiment was carried out through three environmental training methods, such as stocking density, salinity, and temperature. Some factors were examined, such as the growth performance, digestive enzyme activity, osmotic regulation, antioxidant enzyme activity, heat shock protein gene (Hsp) expression level, mTOR signaling pathway, Vibrio parahaemolyticus VPE1 infection experiment, and compensatory growth effects and application have been evaluated . The specific research results are as follows:

1. Compensatory growth and potential mechanism of stocking density training on post-larvae Penaeus vannamei

Based on industry standards for indoor shrimp farming, three densities for Penaeus vannamei nursery rearing were examined (D1 (control) = 10,000 PL/m³, D3 = 30,000 PL/m³, and D5 = 50,000 PL/m³)). The results showed that the D5 group had an over-compensation growth effect, whereas the D3 group showed a full-compensation growth effect. A preliminary investigation of the mechanism of compensatory growth was explored. The results showed that during the compensation stage, the SGR (Special Growth Rate) and FCE (Feed Conversion Efficiency) of the D5 group were significantly higher (P < 0.05) than the control. T-SOD enzyme activity was not significantly different (P > 0.05) from the control. Antioxidants such as CAT, GSH-PX, and MDA all had compensatory changes. In addition, the relative mRNA expression of several genes was measured. The results showed that growth-related genes (EGFR, MSTN), molting-related genes (MIH), immune-related genes (Toll), and digestion-related genes (α-amylase) had a relative compensation of mRNA expression. Meanwhile, transcriptome analysis of shrimps after different density restrictions in the compensation stage was performed. Totally 237 and 348 differentially expression genes (DEGs) were identified in the D5- and D3-treated shrimps, respectively. In D5-treated shrimps, DEGs were annotated and grouped into 9 processes or pathways related to animal metabolic response. With respect to D3-treated shrimps, DEGs were mainly annotated to 15 processes or pathways related to animal metabolic response. Therefore, this study provides a basis for the theory and practice of compensatory growth during the nursery rearing of commercial shrimp.

2. Effects of salinity training on the compensatory growth effect, physiological changes and disease resistance of post-larvae Penaeus vannamei

In this study, whether salinity training (restriction and compensation) could promote growth of P. vannamei were assessed during shrimp nursery farming. Post-larval shrimp were cultured in four salinity treatment groups (SAL20 (control) = 20 ppt (parts per thousand), SAL10 = 10 ppt, SAL30 = 30 ppt, and SAL40 = 40 ppt) for 20 days (restriction stage) and then transferred them to the control salinity environment (20 ppt) for 20 days (compensation stage). The SAL40 group exhibited an over-compensation growth effect in body length. During the salinity restriction stage, shrimp in the SAL30 and SAL40 groups showed decreased activities of digestive enzymes and increased activities of osmoregulation enzymes; the opposite trends occurred in these groups during the compensation phase. This result suggests that salinity limitation increased energy consumption required for osmoregulation and thus the shrimp compensated by increasing food utilization. From the restriction stage to the compensation stage, the gene expression levels of tor, s6k, and 4e-bp first increased and then decreased. Results also suggested that the mechanistic target of rapamycin signaling pathway may regulate growth in response to changes in the salinity restriction stage and compensation stage. Finally, Vibrio parahaemolyticus E1 challenge experiments were conducted using the different salinity training groups and found that the SAL30 group may have the greatest ability to fight infection. In conclusion, the data suggest that increasing the culture salinity from 20 to 30 ppt for a 20 day restriction stage and then decreasing it to 20 ppt for a 20 day compensation stage would not affect survival rate or final weight and may improve disease resistance and uniformity.

3. Effects of temperature training on the compensatory growth effect, physiological changes and disease resistance of post-larvae Penaeus vannamei

In this study, post-larval shrimp were cultured in three temperature treatment groups (TEMP28 (control) = 28°C, TEMP25 = 25°C, and TEMP31 = 31°C) for 20 days (restriction phase) and to the control salinity environment (28℃) for 20 days (compensation stage). In this study, we found that the TEMP25 treatment not only did not affect the final body weight and survival rate of P. vannamei, but also increased their final body weight, that is, TEMP25 exhibited an over-compensation growth effect in weight, and improved disease resistance to a certain extent. In the VPE1 challenge experiment, the anti-infection ability of the TEMP31 group was weaker than that of the control group, and the anti-infection ability of the TEMP25 group was stronger than that of the control group. Analysis of digestive enzyme activities of post-larvae during temperature training showed that there was a significant compensatory change in α-amylase activity, but no significant change in lipase activity. Temperature training can activate the mTOR signaling pathway, and the expression of related genes is up-regulated. By analyzing the changes in the expression levels of heat shock protein genes, it was found that the stress level during temperature training may lead to changes in the uniformity of body length of post-larval. According to the comparison of the growth performance and disease resistance of post-larval in the experimental group, the data suggest that after culturing shrimp at 25℃ for 20 days, the temperature should be gradually increased to 28℃ for 20 days may get better comensatory growth potential.

4. Comparative transcriptome analysis of the positive effect of temperature and salinity training on post-larval Penaeus vannamei

In this study, it was conducted that transcriptome sequencing and bioinformatics analysis of SAL40 and TEMP25 groups, which produced over-compensatory growth effects. The study preliminarily analyzed the differentially expressed genes (DEGs) of SAL40 and TEMP25 groups in hepatopancreas after temperature and salinity training, corresponding to 236 and 327 DEGs, respectively. The combined analysis of DEGs between the two groups showed that 15 DEGs were chose, and 8 pathways were significantly enriched. This can help to further explore the molecular mechanisms behind the positive physiological consequences during the compensatory growth of Penaeus vannamei.

目  录

第1章  引言

1.1  凡纳对虾养殖现状

1.2  补偿生长的研究进展

1.3  免疫训练

1.4  凡纳对虾标粗期进行环境训练的可行性

1.5  本文主要研究内容

1.6  本研究的目的与意义

第2章  密度训练对凡纳对虾补偿生长及潜在作用机制的研究

2.1  引言

2.2  材料和方法

2.2.1  实验设计及养殖管理

2.2.2  仔虾生长表现

2.2.3  样品采集

2.2.4  酶活性测定

2.2.5  RNA提取和基因表达测定

2.2.6  肝胰腺转录组测序分析

2.2.7  统计分析

2.3  实验结果

2.3.1  密度训练对仔虾补偿生长的影响

2.3.1  仔虾在不同实验阶段的生长表现

2.3.2  抗氧化酶和消化酶活性

2.3.2  仔虾生长、蜕皮、免疫和消化相关基因的表达量分析

2.3.3  仔虾肝胰腺转录组分析

2.4  讨论

2.5  小结

第3章  盐度训练对凡纳对虾补偿生长、生理变化和抗病力的影响

3.1  引言

3.2  材料和方法

3.2.1  实验设计及养殖管理

3.2.2  仔虾生长表现

3.2.3  样品收集

3.2.4  酶活性测定

3.2.5  RNA提取和基因表达测定

3.2.6  组织切片镜检

3.2.7  数据分析

3.3  实验结果

3.3.1  40天的标粗生长实验

3.3.2  72小时VPE1攻毒实验

3.4  讨论

3.5  小结

第4章  温度训练对凡纳对虾补偿生长、生理变化和抗病力的影响

4.1  引言

4.2  材料和方法

4.2.1  实验设计及养殖管理

4.2.2  仔虾生长表现

4.2.3  样品收集

4.2.4  酶活性测定

4.2.5  RNA提取和基因表达测定

4.2.6  组织切片镜检

4.2.7  数据分析

4.3  实验结果

4.3.1  40天的标粗生长实验

4.3.2  72小时VPE1攻毒实验

4.4  讨论

4.5  小结

第5章  比较转录组分析温度和盐度训练对凡纳对虾产生积极效果的原因

5.1  引言

5.2  材料与方法

5.2.1  总RNA提取和cDNA文库构建

5.2.2  Illumina测序、基因组装和注释

5.2.3  生物信息学比较分析

5.2.4  统计分析

5.3  结果

5.3.1  仔虾的肝胰腺差异表达基因 (DEGs) 的鉴定

5.3.2  GO功能富集分析和KEGG pathway分析

5.3.3  联合分析共有的潜在作用通路

5.4  讨论

5.5  小结

第6章  主要结论与创新点

6.1  主要结论

6.2  创新点

6.3  展望

参考文献

致  谢

作者简历及攻读学位期间发表的学术论文与研究成果