海洋生态与环境科学重点实验室知识库

仿刺参（Apostichopus japonicus），又名刺参，属于棘皮动物门（Echinodermata），主要分布在中国北部沿海及北太平洋西部浅海地区，是我国重要的海水养殖物种之一，并且在世界贸易中占据越来越重要的地位。温度和溶解氧是刺参水产养殖中最重要的两个非生物限制因素。近年来，在中国沿海水域和池塘中的刺参由于极端环境大量死亡，造成极为惨重的经济损失和资源破坏，限制了刺参水产养殖业的可持续发展。且在全球气候变化及人类活动加剧的大背景下，水域高温低氧现象正呈现常态化趋势。因此，迫切需要了解海洋生物对环境变化的响应机制。为探明极端天气下水体高温低氧环境对机体生理行为及分子调控等各方面的影响，本研究将刺参作为一种典型的棘皮动物模型，研究其对逆境的响应机制。利用控温棒及水体溶解氧实时控制系统模拟水体高温和低氧环境，通过测序技术辅以实时荧光定量PCR（real-time quantitative polymerase chain reaction, real-time PCR或qPCR）验证，获得了刺参在应对高温低氧胁迫过程中mRNA、长非编码RNA（long non-coding RNA, lncRNA）、microRNA（miRNA）、蛋白质、代谢物及关键免疫因子的变动与调控特征，并通过测定关键酶活性以及行为观察等方法获得了刺参生理行为方面的响应特征。主要研究结果如下：

1. 生理行为响应特征

为探查高温低氧环境胁迫对刺参生理行为层面的响应特征，本项研究选取了与消化功能、免疫防御、氧化应激相关的16种酶进行活力测定。环境胁迫下刺参中α-淀粉酶（AMS）、胃蛋白酶（PEP）、胰蛋白酶（TRY）、脂肪酶（LPS）等消化相关酶活显著降低，消化功能可能受到潜在的负面影响。受到环境胁迫后刺参诱导了体内的免疫防御机制，酸性磷酸酶（ACP）和溶菌酶（LZM）活性显著增加，非特异性免疫增强。过氧化氢酶（CAT）、超氧化物歧化酶（SOD）、酚氧化酶（PPO）等氧化应激相关酶，在机体遭受环境胁迫时，活力发生改变，以清除O₂⁻、H₂O₂、OH⁻等物质，共同组成了刺参活性氧防御系统，减弱刺参氧化应激压力。此外，环境胁迫下刺参的分布状态、运动速度、排泄摄食等发生改变；低氧胁迫下刺参多集中于水表面分布，机体形态、体壁及内脏状况发生改变，包括吸水胀大、体表溃烂等。且较单因素胁迫相比，双因素协同作用下，刺参的耐受性急剧下降。实验结果表明刺参机体为适应极端环境综合调节消化、免疫、氧化应激等相关酶类，形成一系列适应性机制，同时改变行为以响应逆境。

2. mRNA（message RNA）和lncRNA（long non-coding RNA）响应特征

为探究lncRNA和mRNA的响应特征，本项研究获取了在高温胁迫（HT）、低氧胁迫（LO）、高温低氧胁迫（HL）以及正常对照处理条件（NC）下刺参呼吸树的lncRNA和mRNA表达谱。在高温、低氧和高温低氧胁迫下分别鉴定出159、355和495个显著上调的基因以及230、518和647个显著下调的基因，并基于此构建了差异lncRNA-mRNA共表达网络。其中，三种处理组中共识别出的关系对共有8对，并且根据连接程度，MSTRG.27265、MSTRG.19729和MSTRG.95524被证明是关键的lncRNA。基于测序数据和real-time PCR验证的显著变化的lncRNA中，有4个lncRNA（MSTRG.34610、MSTRG.10941、MSTRG.81281和MSTRG.93731）均与之前研究中鉴定出的刺参低氧响应的关键miRNA（Aja-miR-2013-3p）具有结合位点；低氧诱导因子（HIF-1α）也显示为Aja-miR-2013-3p的潜在靶向目标。双萤光素酶报告系统验证结果显示，“HIF-1α/Aja-miR-2013-3p/MSTRG.34610”网络和“HIF-1α/Aja-miR-2013-3p/MSTRG.10941”网络在刺参应对环境胁迫时可能发挥重要作用。环境胁迫通过改变lncRNA和mRNA的表达水平，进而影响刺参的免疫、能量代谢和细胞周期等各种生物学过程。与单因素胁迫相比，高温低氧协同胁迫能引起刺参分子水平上更加广泛的响应。

3. microRNA响应特征

为探究三种类型的环境胁迫对刺参中miRNA表达的影响，本项研究鉴定并表征了差异表达的miRNA，并查明了其主要参与的生物学过程。与对照组相比，高温、低氧和高温低氧胁迫处理组分别鉴定出21、26和22个差异表达的miRNA（DE-miRNA），在三种处理组中共有54种非重复性差异表达miRNA。利用比较组学分析及real-time PCR，鉴定并验证代表性响应miRNA。表征了在刺参应对高温应激（热应激）中发挥潜在关键作用的五个miRNA包括Aja-miR-novel-299、Aja-let-7b-3p、Aja-miR-71b-5p、Aja-miR-novel-13218和Aja-miR-2004；以及在刺参应对低氧应激中可能发挥关键作用的Aja-miR-92b-3p、Aja-miR-210-5p和Aja-miR-novel-26331。鉴定出的差异表达miRNA参与了与生物合成、代谢、免疫、细胞凋亡和信号转导有关的多个关键生物学过程，从而影响了刺参的机体状态。

4. 蛋白水平响应特征

为阐明环境胁迫下刺参中蛋白质水平的响应，本项研究基于比较蛋白质组学分析，利用同位素标记相对和绝对定量技术（isobaric tags for relative and absolute quantification, iTRAQ），获取了高温和低氧单个及协同胁迫下的刺参呼吸树蛋白质表达谱，共鉴定出8437种蛋白质。其中高温、低氧及高温低氧胁迫下分别有262、155和433个差异响应蛋白。在高温低氧双因子共胁迫下，在刺参中鉴定出的差异调节蛋白多余单个胁迫，表明在蛋白质组水平上双因素协同胁迫具有累加效应。基于差异蛋白的基因本体论（Gene ontology, GO）分析表明，环境胁迫下,免疫响应相关蛋白上调，蛋白合成能力受到抑制；能量类物质如氨基酸、碳水化合物、脂类代谢过程发生改变；低氧胁迫诱导刺参中参与铁稳态的蛋白上调，糖原合成相关蛋白下调；高温胁迫及高温低氧胁迫下刺参中糖异生过程上调。刺参采取不同的策略应对不同的环境胁迫，主要包括“物质生物合成、运输和代谢”、“信号转导”、“蛋白质合成”、“免疫和防御反应”以及“能量产生和转化”等方面。 

5. 代谢物响应特征

为探究逆境下刺参中代谢物变动特征，本项研究利用超高效液相色谱-质谱（Ultra-Performance Liquid Chromatography-Mass Spectrometry, UPLC-MS）获得刺参响应于不同形式的环境胁迫的代谢组学变化。在高温、低氧和高温低氧双胁迫下的刺参中分别观察到84、68和417种代谢物浓度发生显著改变，说明双环境因子协同胁迫对刺参呼吸树代谢产物的影响比单一胁迫更为广泛。研究确定了刺参中潜在的十种高温胁迫生物标志物和十种低氧胁迫生物标志物，包括德尔塔林、镰刀菌素C、环吡阿尼酸、顶羽菊内酯、蛇孢菌素A、软海绵素B、苦艾素、紫金牛醌和杀菌素等。结果表明，环境胁迫下刺参中氨基酸、碳水化合物、脂质、辅酶因子和维生素以及核苷酸有关的代谢过程产生了响应，使刺参得以维持基本的生存。具体而言，高温胁迫导致涉及氨基酸、脂类、辅酶和维生素代谢的代谢产物发生变化、碳水化合物代谢减少；当暴露于低氧环境时，刺参中涉及氨基酸、碳水化合物代谢和脂肪酸代谢的产物水平显著增加。此外，三羧酸循环（tricarboxylic acid cycle, TCA cycle）中的关键代谢物及参与能量代谢调节的代谢物水平发生了显著变化。推定热应激是干扰刺参中谷氨酸-谷氨酰胺代谢过程的主要因素。这些结果将有助于了解水生动物对不利环境的响应机制。

6. 免疫基因时序响应特征

前几部分的实验结果显示，环境胁迫下刺参的免疫发生了响应。为探究刺参免疫基因对环境胁迫的时序响应特征，本项研究分别分析了暴露于高温和低氧两种环境下8个时间点的刺参中与核因子κB（NF-κB）通路、蛋白酶家族、补体系统、热休克蛋白（HSPs）和转铁蛋白家族有关的17个基因的表达水平。这些基因具有相互关联的作用，共同构成环境胁迫下刺参的免疫防御网络。实验结果表明刺参中与NF-κB通路和热休克蛋白家族相关的基因表达水平受到高温胁迫的强烈影响；而黑素转铁蛋白（Mtf）、铁蛋白（Ft）和甘露聚糖结合C型凝集素（MBCL）的表达水平则受到低氧的显著影响。相比之下，在环境胁迫初期，补体因子B（Bf）、肌球蛋白V（Mys）和丝氨酸蛋白酶抑制剂（SPI）基因在早期对胁迫并不敏感。在刺参受到高温和低氧胁迫时，一些基因具有相似的表达模式，包括Hsp90和溶菌酶（LZM）的表达增加以及主要蛋黄蛋白（MYP）和组织蛋白酶C（CTLC）的表达减少；与之相反，NF-κB和Hsp70在两种胁迫处理下的表达趋势不同。另外LZM诱导的免疫防御在刺参应对胁迫时可较为灵活地发挥作用。实验结果表明，刺参对不同类型的胁迫表现出复杂且多样的免疫反应。这些结果为研究环境胁迫下海洋生物响应提供免疫指标参考，有助于了解刺参在全球气候变化下的适应策略。

Sea cucumber Apostichopus japonicus belongs to Echinodermata, was mainly distributed in the coastal areas of northern China and the shallow seas of the western North Pacific. It is one of the important economic marine aquaculture species in China, and it is an increasingly important part of world trade. Temperature and dissolved oxygen are two of the most important abiotic limiting factors in aquaculture of sea cucumber. In recent years, many sea cucumbers have died due to extreme environments in coastal areas and ponds in China, causing severe losses of resource and economy. That limited the sustainable development of aquaculture of sea cucumbers. Moreover, in the context of global climate change and intensified human activities, the phenomenon of high temperature and low oxygen in oceans is showing a normalization trend. Therefore, it is urgent to understand the response mechanisms of marine organisms to environmental changes. In order to ascertain the effects of high temperature and low dissolved oxygen on the body's physiological behavior and molecular regulation in extreme environment, the present study used sea cucumber as a typical echinoderm model to study its response mechanism to adverse environment. The temperature controller and self-designed real-time control system of dissolved oxygen simulated the high-temperature and low-oxygen environment in the experiments, respectively. The sequencing technique and real-time quantitative polymerase chain reaction (real-time PCR or qPCR) were used to verify and validate the changes and effects of mRNAs, long non-coding RNAs (lncRNAs), microRNAs (miRNAs), proteins, metabolites and key immune factors in sea cucumbers in response to thermal and hypoxic stress, as well as the physiological and behavioral response by measuring key enzyme activities and behavior observation. The main findings are as follows:

1. Physiological and behavioral responses of sea cucumbers under thermal and hypoxic stress

Here, the physiological and behavioral responses of A. japonicus to high temperature and oxygen deficiency was illustrated. Sixteen enzymes related with digestion, immune defense and oxidative response were measured. AMS, PEP, TRY and LPS were significantly depressed in sea cucumber under environmental stresses, thus leading to a negative impact on digestive function. Moreover, the immune defense mechanism was induced based on the increased ACP and LZM. Furthermore, the activities of antioxidant enzymes (CAT, SOD and PPO) were changed to scavenge harmful substance like O₂⁻, H₂O₂ and OH⁻. The antioxidant system was formed to reduce the oxidative stress. Moreover, the distribution, move speed, excretion and feeding of sea cucumbers were changed under environmental stress. Sea cucumbers were mainly distributed near water surface, and the body became edematous and the body wall became ulcerated and dissolved. In addition, compared with single stress factor, the tolerance of A. japonicus decreased sharply under the combined stress of heat and hypoxia. A. japonicus regulated digestion, immunity, oxidative stress and other related enzymes, forming a series of adaptive mechanisms and changed physical behavior to survive and adapt to the extreme environment.

2. Changed mRNAs (message RNAs) and lncRNAs (long non-coding RNAs) in sea cucumbers in response to thermal and hypoxic stress

Here, differentially expressed (DE) lncRNAs and mRNAs were identified as responses to environmental stresses in the economically important sea cucumber, Apostichopus japonicus. Based on a total of 159, 355 and 495 significantly upregulated genes and 230, 518 and 647 significantly downregulated genes identified in the thermal (HT), hypoxic (LO) and combination thermal + hypoxic (HL) stress treatments, respectively, we constructed DE-lncRNA-mRNA coexpression networks. Among the networks, eight shared pairs were identified from the three treatments, and based on the connectivity degree, MSTRG.27265, MSTRG.19729 and MSTRG.95524 were shown to be crucial lncRNAs. Among all the significantly changed lncRNAs identified by real-time PCR and sequencing data, binding sites were found in four other lncRNAs (MSTRG.34610, MSTRG.10941, MSTRG.81281 and MSTRG.93731) with Aja-miR-2013-3p, a key miRNA that responds to hypoxia in sea cucumbers. The hypoxia-inducible factor (HIF-1α) was also shown as the possible targeted mRNA of Aja-miR-2013-3p. As indicated by a dual-luciferase reporter assay system, “HIF-1α gene/Aja-miR-2013-3p/MSTRG.34610” network and the “HIF-1α gene/Aja-miR-2013-3p/MSTRG.10941” network may play important roles in sea cucumbers under environmental stresses. Moreover, environmental stress altered the expression of multiple lncRNAs and mRNAs, thus affecting various biological processes in A. japonicus, including immunity, energy metabolism and the cell cycle. At the molecular level, more comprehensive responses were elicited by the combined thermal/hypoxic stress treatment than by individual stresses alone in sea cucumbers.

3. Changed microRNAs in sea cucumbers in response to thermal and hypoxic stress

Here, based on the construction of miRNA profiles in sea cucumber under varied environmental stress, the DE-miRNA, as well as the biological processes they involved in, were identified and characterized. Compared with normal condition, a total of 21, 26 and 22 differentially expressed (DE) miRNAs were clarified in sea cucumber A. japonicus under thermal, hypoxic and the combined stresses. In all, 54 non-repetitive miRNAs were identified to be differentially expressed in the pairwise comparison among three treatments with significant levels. The comparative miRNA-seq and real-time PCR were used to identify and validate the representative miRNAs, including Aja-miR-novel-299, Aja-let-7b-3p, Aja-miR-71b-5p, Aja-miR-novel-13218, Aja-miR-2004 in response to high temperature and Aja-miR-92b-3p, Aja-miR-210-5p, Aja-miR-novel-26331 in response to oxygen limitation. The potential targeted genes of DE-miRNAs mainly involved in biosynthesis, metabolism, immunity, apoptosis and signaling transduction, suggesting that these processes are potentially impact the status of A. japonicus.

4. Changed proteins in sea cucumbers in response to thermal and hypoxic stress

Here, based on isobaric tags for relative and absolute quantification (iTRAQ), comparative proteomic analysis was used to illustrated proteins responded to varied environmental stress in sea cucumber. A total of 8437 proteins were identified, 262, 155 and 433 proteins were differentially regulated in response to heat, hypoxia, and heat plus hypoxia, respectively. More differentially regulated proteins were identified under two environmental stress factors than single factor, indicating that the interaction between these two stressors has an additive effect at the proteome level. Based on the Gene Ontology (GO) analysis, proteins related with immune and defense response were largely induced and protein synthesis ability was depressed. Proteins involved in energy metabolism were significantly changed, including amino acid, carbohydrate and lipid metabolic processes. Additionally, hypoxic stress induced proteins involved in iron homeostasis. Gluconeogenesis was induced under heat and the combined stress. Alterations of defense strategies in sea cucumber are revealed to be normally dependent on the type of stressor. These proteome-wide alterations, including “substance biosynthesis, transport and metabolism”, “signal transduction”, “protein synthesis”, “immune and defense response” and “energy production and conversion” reflect an altered strategy that may enable A. japonicus to survive under global climate change in the future.

5. Changed metabolites in sea cucumbers in response to thermal and hypoxic stress

Here, ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) was utilized to obtain metabolic profiles of A. japonicus under environmental stresses. Changes in the concentrations of 84, 68, and 417 metabolites related to the responses of sea cucumbers to heat, hypoxia and the combined stress, respectively, were observed and analyzed. Synergistic effect of environmental stressors has an additive impact on the primary metabolome in sea cucumbers. Ten representative potential biomarkers of heat and hypoxia in sea cucumber are illustrated, including deltaline, fusarin C, cyclopiazonic acid, acroptilin, ophiobolin A, halichondrin B, absinthin, rapanone, volicitin. Results showed that metabolic processes related to amino acids, carbohydrates, lipids, cofactors and vitamins, and nucleotides responded to the environmental stresses, allowing the sea cucumber to maintain basic subsistence. As the results showed that, heat stress leads to a decreased carbohydrate and metabolism and changes in metabolites involved in amino acid, lipid, cofactors and vitamins metabolism. Moreover, when exposed to hypoxia, metabolites involved in amino acid, carbohydrate metabolism and the levels of fatty acids were significantly increased in sea cucumbers. The concentration of metabolites involved in the regulation of energy metabolism and the tricarboxylic acid (TCA)-cycle was significantly altered. Based on the decreased accumulation of glutamine, we hypothesized that heat stress is the main factor that interferes with the process of glutamic acid-glutamine metabolism in sea cucumbers. These results would facilitate further research on the adaptation of marine organisms to adverse environment.

6. Time course analysis of immunity-related gene expression in the sea cucumbers in response to thermal and hypoxic stress

The results of the previous parts showed that the immunity of A. japonicus responded to the environmental stresses. Here, time course analysis of immunity-related gene expression in the sea cucumber A. japonicus during exposure to thermal stress and hypoxic stress were carried out. The expression trends of 17 genes related to the nuclear factor κB (NF-κB) pathway, the protease family, the complement system, heat shock proteins (HSPs) and the transferrin family during exposure to two stresses (high temperature and low dissolved oxygen) at eight time points were concluded. These genes have interconnected roles in immunity of sea cucumber defensing against environmental stresses. The expression levels of genes relating to the NF-κB pathways and HSPs were strongly affected in the sea cucumber thermal stress response, while melanotransferrin (Mtf), ferritin (Ft) and mannan-binding C-type lectin (MBCL) were affected by hypoxia. In contrast, complement factor B (Bf), myosin V (Mys) and serine protease inhibitor (SPI) were not that sensitive during the initial period of environmental stress. Similar expression patterns under both thermal and hypoxic stress for certain genes, including an increase in Hsp90 and decreases in lysozyme (LZM), major yolk protein (MYP) and cathepsin C (CTLC) were observed in sea cucumbers. Conversely, NF-κB and Hsp70 were differentially affected by the two stress treatments. Lysozyme-induced immune defense was inconstant in sea cucumbers coping with stress. Our analysis suggests that sea cucumbers demonstrate complex and varied immune responses to different types of stresses. These results provide information about the innate immunity of A. japonicus and represent a key immune index of a marine organism under environmental stress. The findings in this study facilitate an understanding of the adaptive strategies of holothurians in coping with global climate changes.