| 袁海晴,金荣胜,王宝燃,陈代强,张双勇,常志强.肠道灌注微囊藻毒素对凡纳对虾肝胰腺转录组的影响.渔业科学进展,2025,46(3):170-182 |
| 肠道灌注微囊藻毒素对凡纳对虾肝胰腺转录组的影响 |
| Investigating the effect of microcystins on the transcriptomics of hepatopancreas in Penaeus vannamei using reverse-gavage |
| 投稿时间：2024-02-24 修订日期：2024-04-07 |
| DOI：10.19663/j.issn2095-9869.20240224001 |
| 中文关键词: 微囊藻毒素-LR 凡纳对虾 肠道灌注 肝胰腺 转录组 |
| 英文关键词: Microcystins-LR Penaeus vannamei Reverse-gavage Hepatopancreas Transcriptome |
| 基金项目:宁波市“科创甬江2035”专项项目“海水池塘健康养殖新模式关键技术体系构建”、国家虾蟹产业技术体系(CARS-48)和中国水产科学研究院院级基本科研业务费(2023TD50)共同资助 |
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| 中文摘要: |
| 为探究微囊藻毒素亮氨酸–精氨酸异构体(microcystins-LR, MC-LR)处理下凡纳对虾(Penaeus vannamei)的基因表达特征与调控机制，本研究对凡纳对虾进行了MC-LR染毒处理。传统的急性攻毒实验多采用注射法，但此方法存在给对虾造成机体创伤的弊端，本研究首次采用肠道灌注的染毒方式，完成感染并规避了创伤导致的影响。同时，采用高通量测序技术对肝胰腺组织进行转录组测序与组装、功能注释与分类、差异基因筛选与分析，并采用实时荧光定量PCR (real-time quantitative PCR，RT-qPCR)技术对转录组测序结果进行验证。结果显示，MC-LR诱导后的肝胰腺中显著变化基因数为1 994个。MC-LR胁迫后，凡纳对虾肝胰腺差异表达基因代谢通路主要富集到核糖体生物合成、脂质和动脉粥样硬化、内质网中的蛋白质加工等通路中。差异基因统计显示，多个锌指蛋白基因显著下调，推测锌指蛋白参与了凡纳对虾MC-LR的应答反应。本研究结果为揭示凡纳对虾对MC-LR侵染的响应和分子调控机制提供了数据支撑，同时也为对虾染毒实验方法提供了参考。 |
| 英文摘要: |
| Whiteleg shrimp (Penaeus vannamei) significantly contributes to the Chinese aquatic product market. However, continuous intensification has led to eutrophication in water bodies, resulting in frequent cyanobacterial blooms. These blooms release microcystins, which pollute water and cause substantial death rates among cultured organisms, posing a serious threat to agriculture and public health. Microcystin-LR (MC-LR) is the most prevalent and highly toxic variant. MC-LR is toxic to aquatic organisms and impacts terrestrial organisms and human health in several ways. These significant effects have attracted widespread attention in the academic community. MC-LR is hepatotropic and accumulates in the hepatopancreas after entering the body. Numerous reports have described the toxicological effects of MC-LR from various perspectives; however, its potential biological processes remain highly complex and may involve multiple signaling pathways in P. vannamei.
Most experiments investigating the acute exposure of shrimp to compounds use blood sinus injection or intramuscular injection. However, these methods can potentially cause tissue damage and change immune indicators. Reverse-gavage reduces tissue damage and directly affects the hepatopancreas. Furthermore, reverse-gavage can also simulate reactions to toxic substance under natural conditions. First, the MC-LR solution was mixed with red edible dye, which was then slowly dripped into the anal cavity of the experimental group using an automatic pipette. The inoculation solution entered the midgut from the hindgut and ceased when the red color reached the hepatopancreas. After 24 h, the cephalothoraxes was dissected to obtain the hepatopancreas tissue. Subsequently, transcriptome sequencing technology was used to identify the differentially expressed genes related signaling pathways and metabolic pathways in the hepatopancreas of P. vannamei under MC-LR treatment. The results of the transcriptome sequencing were validated using quantitative real-time PCR.
MC-LR induced significant differential expression of 1 994 genes compared to the control group. Of these, 1 164 were up-regulated and 830 were down-regulated. The differentially expressed genes were categorized based on biological processes, cellular components, and molecular functions using the Gene Ontology (GO) database, resulting in 33 functional entries. Kyoto Encyclopedia of Genes and Genomes (KEGG) database analysis revealed that the transcriptome of P. vannamei had 240 differentially expressed genes annotated across six categories: metabolism, genetic information processing, environmental information processing, cellular processes, biological systems, and human disease. The main metabolic pathways included carbohydrate metabolism, lipid metabolism, protein translation, signal transduction, cell growth and apoptosis, transport and catabolism, immune system, and endocrine system. GO functional enrichment analysis revealed that the functions of significantly differentially expressed genes were primarily enriched in catalytic activity, heterocyclic compound binding, carbohydrate derivatives, small molecule binding, protein folding, and RNA metabolism. Among the top 20 enriched KEGG pathways, the ribosome biosynthesis pathway was significantly enriched. Additionally, pathways related to lipids, atherosclerosis, endoplasmic reticulum protein processing, and purine metabolism were also enriched. The number of differentially expressed genes was more than those of the untreated control. These pathways may be involved in metabolism, environmental information processing, and cellular processes of P. vannamei under microcystin stress. Consequently, the top ten genes with the most significant differential expression were further screened. Notably, zinc finger protein 761-like expression was down-regulated. Differential gene expression statistical analysis indicated that several genes belonging to the zinc finger protein family were significantly down-regulated. This suggests their potential involvement in the microcystin response of P. vannamei.
In conclusion, this study provides a methodological reference for shrimp exposure experiments and the molecular regulation mechanism of P. vannamei in response to microcystins. However, the reverse-gavage method requires improvement, and a methodological comparison of different approaches is also necessary to understand the advantages and disadvantages of reverse-gavage methods. Simultaneously, further verification of the differentially expressed genes is required to determine their close relationship with the response of P. vannamei to microcystin stress. These findings will enable in-depth exploration and improvement of the toxicological mechanism in P. vannamei. |
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