海洋生态与环境科学重点实验室知识库

脉红螺（Rapana venosa），自然分布于我国的渤海、黄海和东海以及日本海等海域，是我国重要的经济贝类，但在欧洲黑海、爱琴海、美国切萨皮克湾、阿根廷拉普拉塔河等海域为生物入侵种，对当地的双壳贝类资源造成破坏。变态过程是贝类生活史中重要的发育阶段，变态的成功与否直接关系到贝类种群资源变动。因此，研究脉红螺幼虫变态机理，对于促进其苗种繁育、资源恢复、生物入侵防控等工作的开展具有重要的现实和理论意义。

本研究利用RNA-seq、iTRAQ、GC-MS、Real time PCR等技术对脉红螺幼虫变态过程分子机理展开研究，从转录组水平、蛋白质组水平和代谢组水平揭示了幼虫变态过程调控特征，筛选了脉红螺变态过程中的差异表达的关键转录本/蛋白组/代谢物，并对它们在变态中发挥的潜在生物学功能进行了探讨；开展了脉红螺幼虫变态过程microRNA的响应特征研究，筛选了变态中的差异表达的microRNA并对它们潜在调控的靶基因进行预测，揭示其在变态过程中所发挥的功能；筛选了在脉红螺变态发育过程中和在不同组织中稳定表达的内参基因，为将来进一步研究关键基因在变态过程中的表达水平提供基础；获得关键基因5-HT receptor和NOS的cDNA序列，探讨了其在脉红螺变态过程中表达特点及调控机理。主要研究结果如下：

1、早期发育阶段的转录组建库

利用第二代高通量测序技术对其转录组进行测序，建立了脉红螺转录组数据库，为后继的发育基因表达谱研究和分子调控机理研究提供了必要的条件。该转录组数据库包涵212049条Unigene，平均大小为619bp。其中有70877条Unigene至少在一个数据库内成功注释，占所有序列的33.42%。对所有Unigene按功能进行了分类，讨论了表达量最高的前20条基因潜在的生物学功能，并挑选了6条与神经内分泌相关的基因研究其在变态发育过程中的表达情况。

2、变态过程的基因表达谱特征

利用数字基因表达谱分析技术研究脉红螺5个不同阶段的基因表达的时空动态变化趋势，并筛选发育阶段特异性表达基因，构建发育调控相关的重要功能基因网络，系统解析了调控脉红螺变态过程的相关分子机理。通过对差异基因的聚类热图分析和GO富集分析发现，在变态过程中发生显著改变的基因大部分与其生长、凋亡、神经内分泌系统、消化系统和免疫系统密切相关。对20条在变态前后显著改变的差异表达基因进行qPCR实验，发现数字基因表达谱的数据与qPCR的分析结果具有很好的一致性，说明RNA-seq的数据较为真实可靠。

3、变态过程中蛋白组响应特征

利用iTRAQ技术，开展脉红螺变态前后差异蛋白组学研究，共鉴定到5312个蛋白。有1138个蛋白在幼虫变态过程发生差异表达，其中470个蛋白在变态后发生了显著上调，668个蛋白在变态后发生了显著下调。变态前后的差异蛋白富集在77个生物功能、27细胞组分和63分子功能相关的GO条目下；有7条KEGG通路发生显著富集。这些差异蛋白主要影响细胞骨架和细胞粘附、摄食和消化、应激反应和免疫、以及特异的组织发育等生理活动

4、变态过程中代谢组响应特征

利用GC-MS技术，开展脉红螺变态前后差异代谢组学研究，共鉴定到263个代谢物。有53种代谢产物在幼虫变态过程发生差异表达，其中29种代谢产物在变态后发生了显著上调，24种代谢产物在变态后发生了显著下调。在29种变态后上调的代谢物中，喹啉-4-羧酸指示了变态后免疫系统的进一步成熟，阿那啶可能参与幼虫变态过程中NO和多巴胺的调控。在24种变态后下调的代谢产物中，麦芽三糖、葡萄糖-6-磷酸、纤维二糖和麦芽糖等储能物质发生了显著下调，反应了变态前后能量策略的不同。

5、变态过程中microRNA响应特征

利用Hi-seq技术，开展了脉红螺变态过程中的差异microRNA组学研究，共鉴定到195条差异表达显著的miRNA。对所有差异表达的miRNA进行了靶基因预测，并根据靶基因的注释结果进行了GO和KEGG pathway富集分析，从而研究差异miRNA的潜在生物学作用。鉴定了变态过程中，调控“消化和吸收”、“细胞骨架和细胞粘附”和“细胞凋亡”等过程的miRNA，并进行了qPCR验证。

6、荧光实时定量PCR内参基因的筛选

在脉红螺发育基因表达谱中筛选了13个内参候选基因（EF-1α、ACT、COX1、NDUFA7、RLRL28、GAPDH、TUBB、RS25、RS8、UBE2和HH3）作为qPCR内参候选基因。利用BestKeeper、NormFinder和GeNorm等内参筛选程序，评估了其稳定性。在脉红螺组织特异性分析中，EF-1α是最稳定的内参基因，而RL5和RL28可以作为第二备选内参基因。在脉红螺幼虫发育特异性分析中，RL28是最佳的内参基因候选，COX1和RL5可以作为第二备选的内参基因。

7、变态过程中关键基因的克隆和表达

首次克隆并获得了脉红螺变态信号转导路径中的关键基因（5-HT receptor和NOS）的cDNA 全长，并进行了物种间序列比对和系统进化树的构建。通过qPCR结果发现，5-HT receptor和NOS的表达量均在变态后发生了显著下调。通过免疫组化染色揭示了5-HT receptor在脉红螺幼虫体内的早期发生规律，发现面盘器官上有以5-HT 为信号传导的三条主神经纤维和纤毛基部感受器所连接的复杂神经网络结构，提示其可能与变态密切相关。

The veined rapa whelk (Rapana venosa) is an economically important sea snail in China, and since 1992, there has been interest in its commercial aquaculture. However, sea-ranching efforts have been hampered by difficulties cultivating larvae during the settlement and metamorphosis stages. In countries that do not consume R. venosa, such as the United States, Argentina, and France, this predatory species has become an invasive pest due to unintended worldwide transport and severely disrupts the survival of native bivalves. Because R. venosa population dynamics and spatial expansion are dominated by recruitment and survival rate during metamorphosis, which is a vital process in the species’ biphasic life cycle, understanding the mechanisms behind this process is necessary for both successful aquaculture and invasion control. Moreover, the metamorphosis of R. venosa is unusual compared with other lifelong phytophagous gastropods for exhibiting considerable developmental specificity; the planktonic, pelagic larvae go from filter-feeding on microalgae to carnivorous juveniles that prey on bivalves. This transition occurs rapidly, despite fundamental changes in morphology including velum degeneration and reabsorption, foot reorientation and elongation, as well as secondary-shell growth. Thus, clarifying R. venosa metamorphosis is also of theoretical interest to gastropod researchers.

In our study, we used RNA-seq, iTRAQ sequencing, GC-MS, Real time PCR et al. to study the molecular mechanism of R. venosa metamorphosis from transcriptomic, proteomic, metabonomic insight, the key transcripts/proteins/metabolite during this pelagic to benthic transition were identified and their potential functions were analyzed by bioinformatics methods. We also investigated the microRNA during metamorphosis and the taget gene of differential expressed microRNAs were predicted to reveal their potential function in this process. Besides, the stable reference genes for qPCR were selected to provide a solid base for studying metamorphic key genes. Furthermore, we cloned the cDNAs of key genes (5-HT receptor and NOS) and analyzed their expression during this metamorphic development and we used immunohistochemical method to locate the 5-HT receptor in larvae. Main results are as follows:

1. De novo transcriptome sequencing and analysis of Rapana venosa from six different developmental stages

De novo sequencing was performed to obtain a comprehensive transcriptome profile during early development. A Hi-seq 2500 sequencing run produced 148,737,902 raw reads that were assembled into 212,049 unigenes (average length of 619 nucleotides, of which 70,877 could be annotated). The unigenes were assigned to biological processes and functions after annotation in Gene Ontology, eukaryotic Ortholog Groups and Kyoto Encyclopedia of Genes and Genomes. We also identified 93,196 simple sequence repeats among the unigenes. Six unique sequences associated with neuroendocrine function were analyzed by quantitative real-time PCR. Our data represent the first comprehensive transcriptomic resource for R. venosa. Functional annotation of the unigenes involved in various biological processes could stimulate research on the mechanisms of early development in this species.

2. Transcriptomic analysis of differentially expressed genes during larval metamorphic development by digital gene expression profiling

Fifteen digital gene expression (DGE) libraries from five developmental stages of R. venosa were constructed and sequenced on the IIIumina Hi-Seq 2500 platform. Bioinformatics analysis identified numerous differentially and specifically expressed genes which revealed some genes associated with growth, nervous system, digestive system, immune system and apoptosis participate in important developmental processes. The functional analysis of differentially expressed genes was further implemented by gene ontology and Kyoto encyclopedia of genes and genomes enrichment. DGE profiling provided a general picture of the transcriptomic activities during the early development of R. venosa, which may provide interesting hints for further study.

3. Comprehensive and quantitative proteomic analysis of metamorphosis-related proteins

We analyzed the proteomes of competent R. venosa larvae and post-larvae, resulting in the identification of 5312 proteins, including 470 that were downregulated and 668 that were upregulated after metamorphosis. The differentially expressed proteins reflected multiple processes involved in metamorphosis, including cytoskeleton and cell adhesion, ingestion and digestion, stress response and immunity, as well as specific tissue development.

4. Metabolomic analysis of larval metamorphosis from pelagic to benthic

The present study examined the metabolic profiles of competent larval and post-larval stages of R. venosa using GC−MS. A total of 263 metabolites was detected, 53 of which had different concentrations in larvae and post-larvae: 29 that were apparently higher following metamorphosis and 24 that were lower. Among the metabolites whose concentrations were higher in post-larvae, quinoline-4-carboxylic acid, the dipeptide cysteinylglycine, and anandamide were the most abundant. The metabolites present in higher concentrations in the competent larvae were a suite of oligosaccharides (maltotriose, glucose-6-phosphate, cellobiose, and maltose), L-homoserine, adrenosterone, and sarcosine. Although the roles of these and other metabolites in whelk development are not yet completely known, they provide some clues to the changes in energy metabolism and cell signaling that take place during metamorphosis.

5. Understanding microRNA regulation involved in the metamorphosis

A total of 195 differentially expressed miRNAs was obtained. Sixty-five of these were expressed during the transition from pre-competent- to competent larvae. Thirty-three of these were upregulated and the others were downregulated. Another 123 miRNAs were expressed during the transition from competent- to post-larvae. Ninety-six of these were upregulated and the remaining 27 were downregulated. The expression of miR-276-y, miR-100-x, miR-183-x, and miR-263-x showed an over 100-fold change during development, while the miR-242-x and novel-m0052-3p expression levels changed over 3000-fold. Putative target gene co-expression, gene ontology, and pathway analyses suggest that these miRNAs play important roles in cell proliferation, migration, apoptosis, metabolic regulation, and energy absorption. Twenty miRNAs and their target genes involved in ingestion, digestion, cytoskeleton, cell adhesion, and apoptosis were identified. Nine of them were analysed with real-time PCR, which showed an inverse correlation between the miRNA and their relative expression levels.

6. Selection of housekeeping genes as internal controls for quantitative RT-PCR analysis

We selected 13 candidate genes for suitability as internal controls and measured their expression levels in eight different tissues and twelve larvae developmental stages by qRT-PCR. As a result of further analysis of the expression stability by GeNorm and RefFinder algorithms, we found that EF-1α was the most stable internal control gene in almost all adult tissue samples investigated with RL5 and RL28 as secondary choices. From a developmental perspective, we found that RL28 was the most stable gene in all developmental stages measured, and COX1 and RL5 were appropriate secondary choices.

7. Molecular cloning of key genes cDNAs and their expression analysis during metamorphosis

We obtained full-length cDNAs of 5-HT receptor and NOS in R. venosa for the first time. Analysis by real-time PCR showed the expression level of 5-HT receptor and NOS has a ~2-fold decrease after metamorphosis. The development of serotonin-containing cells in the mollusk R. venosa was also examined using immunohistochemical and histofluorescent methods. The first immunoreactive signal of 5-HT receptor firstly appeared in trochophora stage at the base of the velum and the future eyes position. The 5-HT receptor immunoreactive signals began to appear in the surface of the plate at the bottom of the foot primordial. In competent larvae, the complex nerve network is clearly visible on velum, with three main nerve fibers in the middle of each velum petal and receptors in cilia base. We speculate the 5-HT receptor participate the metamorphic signal transduction.