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）为研究对象，投喂添加戊糖乳杆菌HC-2（Lactobacillus pentosus HC-2）和黄曲霉毒素B1(AFB1)的饲料，探讨在对虾养殖中，益生菌-戊糖乳杆菌HC-2对降低黄曲霉毒素损伤的保护作用，并从组织学、基因表达、酶活性以及肠道菌群变化方面初步研究戊糖乳杆菌HC-2对在AFB1刺激下的凡纳对虾肝胰腺和肠道的保护作用机制。

将健康的凡纳对虾（900尾）随机分成三组，分别投喂基础饲料、添加AFB1（500 μg/kg）饲料以及添加AFB1（500 μg/kg）+戊糖乳杆菌HC-2（5 × 10 ⁸ CFU/g）饲料6周。在试验结束后，统计分析对虾存活和生长状况，分别取三组对虾的肝胰腺和肠道进行显微观察、免疫相关基因表达的测定、肝胰腺酶活性的测定以及肠道菌群的分析。

在投喂AFB1后，AFB1+ HC-2组的存活率(AFB1组:37%，AFB1+ HC-2组:49%，对照组:61%)和增重百分比(AFB1组:98.28%，AFB1+ HC-2组:112.40%，对照组:111.27%)均高于AFB1组。三组对虾通过组织切片观察肝胰腺的组织变化，AFB1+HC-2组与AFB1组相比损伤程度较轻。同对照组相比，AFB1组和AFB1+HC-2组的肝胰腺免疫相关基因Rab、GST、mucin-like PM、Dorsal、Relish、Pro-PO的相对表达量均呈现显著下调（P<0.05），且与AFB1组相比，AFB1+HC-2组的免疫相关基因GST、Dorsal、Pro-Po的相对表达量下调幅度较小（P<0.05）。与对照组相比，AFB1组和AFB1+HC-2组的碱性磷酸酶活性（AKP）明显上升、且AFB1+HC-2组的AKP活性高于AFB1组。与对照组相比，AFB1组和AFB1+HC-2组中谷胱甘肽巯基转移酶（GST）活性明显升高，但两组间无显著性差异，超氧化物歧化酶（SOD）和过氧化氢酶（CAT）活性在AFB1组和AFB1+HC-2组间无显著性差异，且三组的总抗氧化能力（T-AOC）无显著性差异。结果表明，饲料中添加戊糖乳杆菌HC-2对AFB1刺激下的凡纳对虾肝胰腺组织结构有一定程度的保护作用，对部分免疫基因和碱性磷酸酶活性有显著影响，但对肝胰腺总抗氧化能力没有显著影响。

利用组织切片观察肠道组织的形态变化，AFB1+HC-2组肠道形态较AFB1组损伤程度轻。与对照组相比，AFB1组和AFB1+HC-2组的免疫相关基因Rab、GST、mucin-like PM、Dorsal、Pro-PO均呈现显著下调（P<0.05），并且AFB1+HC-2组的免疫相关基因Rab、GST、mucin-like PM、Dorsal、Pro-PO的表达水平均高于AFB1组（P<0.05），但Relish基因的表达在对照组和实验组间无显著变化。肠道菌群分析表明，对照组、AFB1组和AFB1+HC-2组的肠道菌群的群落结构存在明显差异。三组共同拥有的OTU（Operational Taxonomic Units ）有440个，其中在对照组中特有的OTU最少（522个OTU）。AFB1+HC-2组特有的OTU最多（862个OTU）。在三组中以变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)、拟杆菌门(Bacteroidetes)、放线菌门(Actinobacteria)为主，其中变形菌门丰度最高。与对照组相比，AFB1+HC-2组的变形菌门相对丰度显著增加，而厚壁菌门和拟杆菌门的相对丰度明显降低。AFB1组与对照组相比，变形菌门的相对丰度明显降低，而厚壁菌门和拟杆菌门的相对丰度明显增加。在属水平上，与对照组相比，实验组肠道菌群的相对丰度变化很大。AFB1+HC-2组的发光杆菌属(Photobacterium )和弧菌属(Vibrio)的相对丰度较对照组明显增加，而不动杆菌属(Acinetobacter)、脱硫球茎菌属(Desulfobulbus)、颤螺旋菌属(Oscillospira)的相对丰度降低。AFB1组颤螺旋菌属(Oscillospira)和瘤胃球菌属(Ruminococcus)的相对丰度较对照组明显增加，而不动杆菌属(Acinetobacter)和脱硫球茎菌属(Desulfobulbus)的相对丰度则明显降低。在AFB1组的肠道菌群中标志物种为脱硫弧菌目(Desulfovibrionales)差异最为显著，在AFB1+HC-2组中乳杆菌科(Lactobacillaceae)差异最为显著。结果表明，戊糖乳杆菌HC-2通过调节肠道菌群结构，对AFB1影响的凡纳对虾的生长性能和抗性有积极影响。

在养殖实践中，戊糖乳杆菌HC-2可以用来减少黄曲霉毒素的危害。这可能与戊糖乳杆菌HC-2对维持肠粘膜结构完整性、促进免疫相关基因表达和调节肠道菌群组成的作用有关。

上述结果为应用戊糖乳杆菌HC-2降低对虾养殖中黄曲霉毒素损伤提供了数据支持，并从组织结构、基因表达、抗氧化系统以及肠道微生物等角度加深了对其保护作用机制的认识。相关数据为乳酸菌在水产养殖中的应用以及黄曲霉毒素毒性的降低提供理论依据和指导策略。

To investigate the positive effect of Lactobacillus pentosus HC-2 on protecting the Penaeus vannamei affected by AFB1 toxicity. Shrimp were respectively fed with formulated feed containing AFB1, AFB1 + L. pentosus HC-2, and a basal commercial diet. After feeding for 6 weeks, the survival and growth,the changes of histology morphology, gene expression and enzyme activity of the hepatopancreas and the changes of histology morphology, gene expression and intestinal flora of the intestine of P. vannamei were determined.

To explore the effects of L. pentosus HC-2 on the hepatopancreas morphology, immune-related genes and enzyme activity of P. vannamei affected by AFB1, shrimp were respectively fed with formulated feed containing AFB1 (500 μg/kg), AFB1 (500 μg/kg) + L. pentosus HC-2 (5 × 10⁸ CFU/g feed), and a basal commercial diet (Control group). After feeding for 6 weeks, the survival rate (AFB1 group: 37%, AFB1+ L. pentosus HC-2 group: 49%，Control group：61%) and the percent weight gain (AFB1 group: 98.28%, AFB1+ L. pentosus HC-2 group: 112.40%，Control group：111.27%) in the AFB1 + L. pentosus HC-2 group were higher than the AFB1 group.At the end of the experiment, the hepatopancreas morphology, the expression of immune-related genes, and the activity of hepatopancreas enzymes were detected. The hepatopancreas morphology of the AFB1 + L. pentosus HC-2 group suffered less damage than the AFB1 group. The relative expression of immune-related genes (Rab, GST, mucin-like PM, Dorsal, Relish, Pro–PO) in AFB1 group and AFB1 + HC-2 group was significantly lower than control group (P < 0.05), and the relative expression of immune-related genes (GST, Dorsal, Pro-PO) in AFB1 + HC-2 group was higher than the AFB1 group (P < 0.05). The activity of Alkaline Phosphatase (AKP) increased significantly in AFB1 group and AFB1+ HC-2 group compared with the control group, and the activity of AKP in AFB1+ HC-2 group was higher than the AFB1 group. The activity of Glutathione S- Transferase (GST) increased in AFB1 group and AFB1+ HC-2 group compared with the control group, and there have no significant difference in AFB1+ HC-2 group and AFB1group. The activity of Superoxide Dismutase (SOD) and Catalase (CAT) has no significant difference between the AFB1 group and AFB1+ HC-2 group, and the Total Antioxidant Capacity (T-AOC) has no significant difference between the control group and the experimental group. We concluded that L. pentosus HC-2 had a positive effect on the hepatopancreas structure of shrimp stimulated by AFB1, and induced a significant effect on some immune genes and AKP activity, but had no significant effect on the T-AOC of the hepatopancreas.

The intestinal morphology of AFB1 + HC-2 group was less damaged than that of AFB1 group. The relative expression of immune-related genes including Rab, GST, mucin-like-PM, Dorsal, Relish and Pro-PO was detected. Expression of most immune-related genes in the experimental group significantly decreased compared with the control group, while the expression levels of immune genes (Rab, GST, mucin-like-PM, Dorsal and Pro-PO) in AFB1+ L. pentosus HC-2 group was higher than the AFB1 group, but there was no significant change in Relish gene expression between the control group and the experimental group. Intestinal microbiota analysis demonstrated that the community structure of intestinal microbiota was obviously different between the control group and experimental group. There were 440 OTUs in the three groups, among which 522 were the least in the control group, AFB1 + HC-2 group had the most unique OTUs (862 OTUs). Among the three groups, Proteobacteria, Firmicutes, Bacteroidetes and actinobacteria were the most abundant. At the phylum level, the relative abundance of Proteobacteria in AFB1 + HC-2 group increased significantly compared with the control group, while the relative abundance of Firmicutes and Bacteroides decreased significantly. At the genus level, The abundance of the intestinal microbiota in the experimental group changed greatly at the genus level compared with the Control group. The relative abundance of Photobacterium and Vibrio in the AFB1 + HC-2 group increased compared with the Control group, while the relative of Acinetobacter, Desulfobulbus, and Oscillospira decreased. The relative abundance of Oscillospira and Ruminococcus in the AFB1 group increased compared with the Control group, while the relative abundance of Acinetobacter and Desulfobulbus decreased. Desulfovibrionales was the most significant difference in the intestinal flora of group AFB1, and lactobacillus was the most significant difference in AFB1+ HC-2 group. These results indicated that L. pentosus HC-2 had a positive effect on the growth performance and resistance of P. vannamei affected by AFB1 according to regulate the structure of intestinal flora. In breeding practice, probiotics can be used to reduce the harm of aflatoxin. This may be related to the probiotic’s contribution to maintaining the structural integrity of the intestinal mucosa, promoting the expression of immune related genes and regulating the composition of intestinal microflora. It was concluded that the effect of L. pentosus HC-2 on regulating the imbalance of intestinal flora of P. vannamei affected by AFB1 was not obvious, but it reduced the content of some bacteria representing an unhealthy state. This change may be related to the reduction of shrimp mortality when challenged by AFB1.

The results provide data support for further study on the mechanism of L. pentosus HC-2 reducing AFB1 toxicity, and provide scientific theoretical basis for probiotics in the healthy culture of P. vannamei and other aquatic organisms. On the other hand, the mechanism of probiotics weakening the toxicity of aflatoxin remains to be further verified. For example, whether probiotics degrade aflatoxin directly or by adjusting the intestinal microflora, or whether they neutralize the toxicity of aflatoxin or directly activate the immune activity of the host, remains to be elucidated.

目录

第一章 绪论... 1

1.1 凡纳对虾的养殖现状... 1

1.2 黄曲霉毒素研究现状... 1

1.2.1黄曲霉毒素的理化性质... 2

1.2.2黄曲霉毒素的毒性作用机制... 2

1.2.3黄曲霉毒素对养殖生物的影响... 5

1.2.4黄曲霉毒素的防控措施... 5

1.3 乳酸菌的研究现状... 7

1.3.1乳酸菌的益生机理... 7

1.3.2乳酸菌在水产养殖中的应用... 9

1.3.3戊糖乳杆菌HC-2的研究现状及展望... 10

1.4 本研究的目的与意义... 10

第二章 戊糖乳杆菌HC-2对 AFB1刺激凡纳对虾生长及肝胰腺影响的研究... 11

2.1 引言... 11

2.2 材料方法... 11

2.2.1 实验动物及培养条件... 12

2.2.2 菌株及培养条件... 12

2.2.2 实验饲料的制备及饲养管理... 12

2.2.3 样品采集及生长指标的计算... 12

2.2.4 组织切片的制备... 13

2.2.5 免疫基因表达的测定... 13

2.2.6 抗氧化酶活的测定... 14

2.2.7 统计分析... 15

2.3 实验结果... 15

2.3.1 生长和存活指标分析... 15

2.3.2 肝胰腺组织形态学分析... 15

2.3.3 肝胰腺免疫相关基因表达... 16

2.3.4 肝胰腺相关酶活性变化... 17

2.4 讨论... 17

2.5 小结... 21

第三章 戊糖乳杆菌HC-2对AFB1刺激的凡纳对虾肠道影响的研究... 23

3.1 引言... 23

3.2 材料方法... 23

3.2.1实验动物及培养条件... 24

3.2.2实验饲料的制备及饲养管理... 24

3.2.3样品采集... 24

3.2.4组织切片的制备... 24

3.2.5免疫基因表达的测定... 24

3.2.6微生物总DNA提取及高通量测序分析... 24

3.2.7统计分析... 26

3.3 实验结果... 26

3.3.1肠道组织形态学分析... 26

3.3.2肠道免疫相关基因表达... 27

3.3.3肠道菌群分析... 28

3.4 讨论... 35

3.5 小结... 40

第四章 结论、创新点与展望... 41

4.1 主要结论... 41

4.2 创新点... 41

4.3 展望... 42

参考文献... 43

致 谢... 53

作者简历及攻读学位期间发表的学术论文与研究成果... 54