实验海洋生物学重点实验室知识库

白斑综合征病毒(white spot syndrome virus, WSSV)和副溶血弧菌(Vibrio parahaemolyticus, Vp)是引发对虾流行性疾病的两种主要病原微生物，系统开展对虾针对这两种病原的免疫响应特征研究是发展对虾病害防治技术的重要基础。本论文以凡纳滨对虾为研究对象，通过比较转录组分析其重要免疫组织包括血细胞(Hc)、淋巴器官(Oka)和肝胰腺(Hp)在WSSV和Vp免疫刺激早期的响应特征，以解析凡纳滨对虾不同免疫组织对不同病原早期响应的分子机制。研究结果不仅可丰富甲壳动物免疫学的基础理论，也可为对虾的健康养殖与病害防治提供新思路。具体进展如下：

（1）对虾三种重要免疫组织在Vp和WSSV免疫刺激早期的转录组分析：通过Illumina测序获得1,288,985,148条clean reads；通过de novo组装，得到59,583条Unigenes，其中有17,721 (29.74%)条有注释信息。GO富集分析发现，代谢过程(metabolic process)、细胞过程(cellular process)和单有机体过程(single-organism process)是富集基因最多的生物学过程；KEGG pathway分析发现大量与免疫相关的通路，包括内吞(Endocytosis)、溶酶体(Lysosome)、吞噬体(Phagosome)和过氧化物酶体(Peroxisome)等，表明了三个组织的生物学功能与免疫密切相关；通过WGCNA分析，将基因划分成17个模块，为后续关键基因的挖掘提供了重要参考。

（2）对虾血细胞对Vp和WSSV免疫刺激的早期响应特征分析：从Vp刺激前后的血细胞(VHc)转录组中获得2454个差异表达基因(differentially expressed genes, DEGs)，其中上调基因1935个，下调基因519个；从WSSV感染前后的血细胞(WHc)转录组中获得3444个DEGs，其中上调基因3240个，下调基因204个。GO富集分析显示，这两种病原刺激均影响了血细胞的RNA代谢(RNA metabolism)、细胞酰胺生物合成(amide biosynthetic process)及基因表达(gene expression)等生物学过程。KEGG pathway富集分析表明，真核生物核糖体的生物合成(Ribosome biogenesis in eukaryotes)和氨酰tRNA的生物合成(Aminoacyl-tRNA biosynthesis)通路在两种病原刺激时均显著富集，暗示血细胞的蛋白合成过程受到影响。对差异表达基因的进一步分析与聚类发现，Vp和WSSV刺激引起对虾血细胞的代谢、翻译、内吞、转运、免疫及神经内分泌等多种生理过程的改变。特别地，WSSV感染显著激活了对虾血细胞介导的神经内分泌免疫系统，引起18种神经肽前体基因表达水平的明显上调，并广泛改变该系统的相关过程，涉及神经肽前体加工过程、氨基酸神经递质代谢过程、生物胺合成途径及乙酰胆碱信号通路。与WSSV感染不同，Vp刺激对神经内分泌免疫系统的影响相对较小，仅有四种神经肽前体及其加工相关基因发生变化。这是第一个有关甲壳动物血细胞介导的神经内分泌免疫系统参与免疫刺激响应的报道，为对虾疾病的防治提供了新的思路。

（3）对虾淋巴器官对Vp和WSSV免疫刺激的早期响应特征分析：从Vp感染前后的淋巴器官(VOka)转录组中获得2127个DEGs，其中上调基因809个，下调基因1318个；从WSSV感染前后的淋巴器官(WOka)转录组中获得1569个DEGs，其中上调基因138个，下调基因1431个。GO富集分析显示，氨基聚糖代谢过程(aminoglycan metabolic process)在两种病原刺激时均显著富集，其中涉及到大量几丁质结合蛋白。KEGG pathway富集显示，溶酶体(Lysosome)通路仅在Vp刺激的转录组中显著富集，表明淋巴器官溶酶体在Vp刺激时入可能发挥重要的清除作用；而与碳水化合物代谢相关的三条通路，包括肌醇磷酸代谢(Inositol phosphate metabolism)、丙酸代谢(Propanoate metabolism)和糖酵解/糖异生(Glycolysis/Gluconeogenesis)只在WSSV刺激的转录组中显著富集，表明WSSV刺激明显影响了对虾淋巴器官的代谢过程。对差异表达基因的进一步分析与聚类发现，Vp和WSSV刺激均可影响淋巴器官的proPO激活系统、溶酶体、细胞外基质、细胞骨架蛋白、几丁质结合蛋白等。进一步，鉴定了两类特异性响应Vp刺激的关键免疫分子，包括组织蛋白酶和卵黄膜外层蛋白I，它们在受到Vp刺激时表达水平均明显上调，暗示了其在淋巴器官抗细菌免疫应答中的重要作用。

（4）对虾肝胰腺对Vp和WSSV免疫刺激的早期响应特征分析：从Vp感染前后的肝胰腺(VHp)转录组中获得373个DEGs，而从WSSV感染前后的肝胰腺(WHp)转录组中仅获得86个DEGs。GO富集分析发现，丙酮酸代谢过程(pyruvate metabolic process)在Vp和WSSV两种免疫刺激下均显著富集。KEGG pathway分析显示，糖酵解/糖异生(Glycolysis/Gluconeogenesis)通路在两个转录组中均显著富集，进一步证实了上述结果。另外，谷胱甘肽代谢(Glutathione metabolism)、药物代谢-细胞色素P450 (Drug metabolism-cytochrome P450)和外源物质代谢-细胞色素P450 (Metabolism of xenobiotics by cytochrome P450)也被显著富集，表明了肝胰腺的关键解毒功能。对差异表达基因的进一步分析与聚类发现，Vp刺激引起一些免疫相关基因发生明显变化，包括模式识别受体、丝氨酸蛋白酶及丝氨酸蛋白酶抑制剂等。另外，鉴定了一些转运及解毒相关的分子，如ABC转运体和细胞色素P450，它们可能在肝胰腺的免疫过程中扮演重要角色。

（5）对虾重组激活基因和免疫致敏相关分子的鉴定：首次在对虾中鉴定了脊椎动物抗体基因重排过程中起关键作用的重组激活基因RAGs的同源基因，即LvRAG1L和LvRAG2L，它们在蛋白序列、表达模式及可变剪接等方面与几种已报道的高等无脊椎动物和头索动物RAGs具有明显的相似性，为RAGs的起源与进化提供新的分子证据。另外，鉴定了对虾中免疫致敏相关分子DSCAMs和FREPs，分析了它们的基因结构及对病原感染的响应特征，结果显示：对虾DSCAMs具有多个旁系同源基因，参与对免疫刺激的响应；对虾FREPs可能通过多个基因座、可变剪接来增加其多样性，且广泛参与对Vp和WSSV免疫刺激的响应。上述结果可为对虾免疫致敏的分子机制研究提供重要参考。

White spot syndrome virus (WSSV) and Vibrio parahaemolyticus (Vp) are two common pathogenic microorganisms causing shrimp epidemic diseases. Therefore, the systematic study on the immune response characteristics of shrimp against these two pathogens could pave the way to develop technologies for prevention and control of shrimp disease. In this dissertation, comparative transcriptome analysis was performed to investigate the features of the immune responses of shrimp primary immune tissues including hemocytes (Hc), lymphoid organ (Oka) and hepatopancreas (Hp), at the early stage of WSSV and Vp immunostimulations, with the aim to understand the mechanism of different immune tissues of Litopenaeus vannamei in response to different pathogens. The findings could enrich the fundamental theory of crustacean immunology and provide a new perspective for the healthy breeding and disease control of shrimp. The main results of this dissertation are as follows:

(1) Transcriptome analysis of three primary immune tissues at the early stage of Vp and WSSV immunostimulations. A total of 1,288,985,148 clean reads were obtained by Illumina sequencing and 59,583 Unigenes were produced by de novo assembly with 17,721 annotated Unigenes (29.74%). “Metabolic process”, “cellular process” and “single-organism process” are three most enriched biological processes by GO enrichment analysis. KEGG pathway analysis found a large number of immune-related pathways, including “Endocytosis”, “Lysosome”, “Phagosome” and “Peroxisome”, suggesting the biological functions of these tissues are closely related to immunity. All Unigenes in the transcriptome were divided into 17 modules through WGCNA analysis, which provided an important reference for the subsequent mining of hub genes.

(2) Analysis of the early response characteristics of shrimp hemocytes to Vp and WSSV immunostimulations. 2454 differentially expressed genes (DEGs) were obtained from the transcriptome of hemocytes before and after Vp stimulation (named VHc), including 1935 differentially up-regulated genes (DUGs) and 519 differentially down-regulated genes (DDGs). Accordingly, 3444 DEGs were acquired from the transcriptome of hemocytes before and after WSSV stimulation (named WHc), including 3240 DUGs and 204 DDGs. GO enrichment analysis revealed that both pathogenic stimuli changed several biological processes of hemocytes, such as “RNA metabolism”, “amide biosynthetic process” and “gene expression”. KEGG pathway analysis demonstrated that “Ribosome biogenesis in eukaryotes” and “Aminoacyl-tRNA biosynthesis” were significantly enriched in both pathogenic stimuli, indicating a change in the protein synthesis process of hemocytes. Further analysis and clustering of DEGs showed that Vp and WSSV stimulations caused changes in various physiological processes of hemocytes, including metabolism, translation, endocytosis, transport, immunity and neuroendocrine. In particular, WSSV infection significantly activated the hemocytes-mediated neuroendocrine-immune (NEI) system of shrimp. On the one hand, 18 genes encoded putative neuropeptide precursors were significantly induced by WSSV infection. On the other hand, WSSV infection affected several typical processes in the NEI system, in which comprised proteolytic processing of prohormones, amino acid neurotransmitter pathways, biogenic amine biosynthesis and acetylcholine signaling pathway. Unlike WSSV, Vp stimulation had little extensive effect on the NEI system that only four neuropeptide precursors and their processing enzymes were changed. It is the first report in crustaceans on the involvement of the hemocytes-mediated NEI system in the immune response, providing a new idea for the prevention of shrimp diseases.

(3) Analysis of the early response characteristics of shrimp lymphoid organ to Vp and WSSV immunostimulations. 2127 DEGs were obtained from the transcriptome of lymphoid organ before and after Vp stimulation (named VOka), including 809 DUGs and 1318 DDGs. Correspondingly, 1569 DEGs were acquired from the transcriptome of lymphoid organ before and after WSSV stimulation (named WOka), including 138 DUGs and 1431 DDGs. GO enrichment analysis showed that “aminoglycan metabolic process” was significantly enriched in both pathogenic stimuli and there were a large number of chitin-binding proteins (CBPs) found in the process. KEGG pathway analysis revealed that “Lysosome” were significantly enriched only in the VOka group, indicating that lysosomes played an important role in the clearance of Vp by shrimp lymphoid organ. And three pathways related to carbohydrate metabolism including “Inositol phosphate metabolism”, “Propanoate metabolism”, “Glycolysis/Gluconeogenesis” were significantly enriched only in the WOka group, suggesting that WSSV infection altered significantly the metabolic process of shrimp lymphoid organ. Further analysis and clustering of DEGs showed that immunostimulations apparently affected prophenoloxidase (proPO) activation system, lysosomes, extracellular matrix, cytoskeletal proteins and chitin-binding proteins in lymphoid organ. In addition, two key immune molecules that specifically responded to Vp stimulation, including cathepsin and VMO-I (vitelline membrane outer layer protein I), were identified, and their expression levels were clearly up-regulated upon Vp stimulation, suggesting their important roles in anti-bacterial immune response in lymphoid organ.

(4) Analysis of the early response characteristics of shrimp hepatopancreas to Vp and WSSV immunostimulations. 373 DEGs were obtained from the transcriptome of hepatopancreas before and after Vp stimulation (named VHp) whereas only 86 DEGs were acquired from the transcriptome of hepatopancreas before and after WSSV stimulation (named WHp). GO enrichment analysis found that the “pyruvate metabolic process” was significantly enriched under both treatments. KEGG pathway analysis showed that “Glycolysis/Gluconeogenesis” was significantly enriched both in VHp and WHp, further confirming the above results. Moreover, “Glutathione metabolism” “Drug metabolism-cytochrome P450” and “Metabolism of xenobiotics by cytochrome P450” were also significantly enriched, indicating the critical detoxification of hepatopancreas. Further analysis and clustering of DEGs revealed that some immune-related genes, including pattern recognition receptors, serine proteases and serine protease inhibitors, were changed significantly caused by Vp stimulation. Besides, several transport and detoxification-related molecules, such as ABC transporters and cytochrome P450, were identified, indicating that they might play an important role in the immune system of shrimp hepatopancreas.

(5) Identification of recombination activating genes (RAGs) and the molecules related to immune priming in shrimp. The homologous genes of RAGs involved in the gene rearrangement in vertebrates, were firstly identified from shrimp, namely LvRAG1L and LvRAG2L, which showed apparent similarity with higher invertebrates and cephalochordata RAGs in terms of protein sequence, expression pattern and alternative splicing, providing new molecular evidence for the origin and evolution of RAGs. Furthermore, the molecules related to immune priming like DSCAMs and FREPs in shrimp were identified, and their gene structure and function in response to pathogen infection were analyzed. The results showed that shrimp DSCAMs had multiple paralogs and FREPs might increase their diversities through multiple loci and alternative splicing. They were involved in the immune responses of Vp and WSSV immunostimulations. Our results would offer an important reference for the study of immune priming mechanism in shrimp.