Name: the barnacle balanus improvisus as a marine model - culturing and gene expression
Uploaded: 2018-08-08T14:00:00.000+00:00
Duration: 7 min 47 s
Description: Watch this Scientific Journal Video about The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression at JoVE.com

这项研究得到了瑞典研究理事会 (VR) 赠款2017-04559 的支持, 欧盟支持布隆博格。特别是, 多年来, 建立了培养设施, 得到以下供资机构的赠款支持: 琼森 (瑞典战略研究基金会), 通过海洋科学和技术方案和密斯特拉通过程序海洋漆。肯特 Berntsson 在建立 culturingfacility 的早期阶段发挥了作用。为建立培养设施提供的额外资金来自海洋进化生物学中心 (www.cemeb.science.gu.se), 由瑞典研究委员会 FORMAS 和 VR 资助。

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	<li>Darwin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A monograph of the sub-class Cirripedia, with figures of all species. The Balanidae (or sessile cirripedes); the Verrucidae, etc., etc., etc. , (1854).</li><li>Lind, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+aquaporins+from+the+euryhaline+barnacle+Balanus+improvisus+reveals+differential+expression+in+response+to+changes+in+salinity.">Analysis of aquaporins from the euryhaline barnacle Balanus improvisus reveals differential expression in response to changes in salinity.</a> PLoS ONE. 12 (7), 0181192 (2017).</li><li>Lind, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+the+alpha-subunit+of+Na+/K++ATPase+from+the+euryhaline+barnacle+Balanus+improvisus+reveals+multiple+genes+and+differential+expression+of+alternative+splice+variants.">Molecular characterization of the alpha-subunit of Na+/K+ ATPase from the euryhaline barnacle Balanus improvisus reveals multiple genes and differential expression of alternative splice variants.</a> PLoS ONE. 8, 77069 (2013).</li><li>Fyhn, H. 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作者没有什么要申报的。

Tjärnö海洋研究实验室 (瑞典) 的藤壶养殖已经运行了20年, 并被用于许多不同研究领域的研究。在过去的几年中, 已经出版了超过30份科学论文, 其中包括防污13、22、流体力学23、化学生态学24、气候变化16的研究., 进化生物学5和分子生物学2。
为了避免选择一些更适合实验室环境的个体 (可能不代表野生人口的个体), 建议每年从田间收集新的亲鱼。此外, 每年对文化进行振兴也是一种良好的做法, 因为在正常的一年里, 成年人的死亡率大约为 50-80%。然而, 如果目的是生产自交系或建立实验性进化研究, 只有实验室饲养的家庭才会被使用。
在 Tjärnö海洋研究实验室的面板上收集improvisus的好时间是在 June–August, 因为当时在海里有很好的 cyprid 幼虫供应。每周检查面板, 看看藤壶定居点何时开始, 并手动移除 improvisus (如贻贝、tunicates、苔藓虫和、hydroids、纽虫/tubeworms 和其他藤壶物种) 的其他已定居物种.面板 (例如, 用牙刷)。Tjärnö周围有三种浅水藤壶物种 (improvisus、 Semibalanus balanoides和Balanus crenatus)。然而, B. improvisus是 July–August 期间光滑坚硬表面的主要 fouler。balanoides在早春时有沉降期, 主要倾向于天然基质 (如石头)。crenatus在夏季可能会在面板上出现低数。
也有可能从培养的 cyprids 开始新的成年藤壶世代, 这将是必要的, 如果某些 linages 具有特定的特征已经建立, 或在研究的实验性进化。开始新一代成人最方便的方法是在实验室的热塑性塑料板上 cyprids。这些新定居的 cyprids 的面板也可以用于实验治疗或在野外暴露。在紧急情况下, 你也可以使用来自附近地点的巨石的成年人 (例如, Idefjorden 在 Tjärnö海洋研究实验室的情况下), 其中B. improvisus是常见的。这些已经建立成人的治疗方式与成人在面板上, 从而被放置在托盘和美联储通过流经系统。流动细胞也可以用来建立与藤壶25的面板。这些是流动的房间与浮游生物网在 cyprids 不安定的边, 以小组作为唯一的解决表面为幼虫。
有几个步骤, 对于建立一个长期运作的藤壶文化, 包括所有生命阶段至关重要。用于培养B. improvisus的方法在很大程度上也适用于其他具有自由游泳、planktotrophic 幼虫的海洋无脊椎动物。一些物种的培养程序已经被很好的描述 (例如, 对于蓝贻贝和不同种类的牡蛎)26, 而对于其他海洋无脊椎动物, 只有几个例子, 长期文化跨越他们的整个生命周期。第一次成功的文化藤壶 (B. 安菲特律特) 的尝试是由 Rittschof等人完成的。15. 在考虑设立藤壶养殖设施之前, 应建立长期财政和个人资源。这类藤壶文化的维护需要至少一个人工作一半时间。今后在生产线上一些步骤的自动化可能有一定的潜力, 主要是微藻的培养27。此外, 为了取得成功, 有必要获得大量高质量的海水。微藻、卤虫和藤壶的培养不涉及任何特定的安全程序。然而, 一些防污物质或有毒化学品的测试可能需要特别的预防措施。
这些小组每周检查几次以进行污染。养殖中使用的海水从 Tjärnö海洋研究实验室外的科斯特峡湾的40米深度抽水, 在进入实验室水系统之前经过两个滤砂过滤器。如果没有过滤的水已经做, 将有更多的污染, 在文化。必须定期清洁从碎屑和其他无脊椎动物 (例如stolon 建筑 hydroids 和捕食性纽虫) 进入该系统的文化中的面板, 从该领域供应海水。例如, 如果没有生产幼虫, 尽管这一文化已经得到了很好的喂养, 否则似乎状况良好, 问题可能是纽虫的存在, 似乎抑制交配。自然地, Tjärnö海洋研究实验室的文化中的许多污染生物是专门为瑞典西海岸, 和其他类型的污染生物将是普遍的和更大的挑战在其他地理区域。在瑞典的西海岸, 在面板上发现其他藤壶物种的污染是不寻常的。有时, balanoides的建立已经被发现, 但这是一个非常边缘的问题 (最多, 一个S balanoides污染物为 1万B. improvisus样品)。在夏季, 当 larvaefrom B. improvisus具有高度优势时, 缺乏污染物种很可能依赖于建立新文化的制度。此外, 还有一个明显的丰富的improvisus在面板上, 因为这个物种是选择性的表面光滑13。
清除死亡的成年藤壶是必不可少的。如果空壳留在面板上, 它们可以成为卤虫幼体以及各种污染物种的庇护所。此外, 人们还注意到, 死亡的个体会影响邻近个体的福祉, 可能是在分解过程中释放有毒化合物。成人死亡率的另一个后果是, 一些人将被单独和离任何其他成年人太远, 以允许交配 (即使藤壶有在动物世界中最长的阳具与它的大小)28。这些人将生存, 但不生产的幼虫。然而, 这些孤立的成年个体可以轻轻地移除不损害基板和被放置在水平接近他人, 以使交配。藤壶也可以配对通过放置面板与一个成年人在每个但足够接近, 以便交叉受精可能发生。这样, 基因线就可以产生14。
几乎每天都要生产高质量的饲料和喂养文化是至关重要的。即使几天没有食物, 也会减少幼虫的释放。以前的饮食成分测试表明, 硅藻对藤壶幼体的生长和存活是必不可少的。一些硅藻品种似乎足够作为饲料, 虽然小或单独的细胞 (直径小于10µm) 可能是必要的幼体的摄取。pseudonana 的marinoi、单纯形和T.均已证明是improvisus幼体的适宜饲料, 同时也易于培养。此外, 在呈指数增长的藻类中, 饲料质量一般较高。据报道, 硅藻对建立B. 安菲特律特15的生产文化是必不可少的。硅藻的重要性的理论是, 他们有一个独特的脂肪酸剖面, 特别丰富的高不饱和度20:5 脂肪酸29。结果表明, 某些脂肪酸对30牡蛎幼虫的成功发育具有重要意义。
多年来, 藤壶文化中没有发生过有害疾病的发病率。在许多商业无脊椎动物水产, 如牡蛎和贻贝, 疾病是相当普遍的, 可能是非常有害的。野生种群也报告了病毒的有害影响。在法国的当地牡蛎被葡萄牙牡蛎angulata在1925年替换了, 但这个种类由虹彩模识消灭了大约 197031。最近, 太平洋牡蛎在世界各地的文化中发生了大量的死亡事件, 这似乎与 ostreid 疱疹病毒 132有关。迄今为止, 还没有发表关于藤壶病原体、细菌或病毒的报告。然而, 在正在进行的improvisus基因组项目中, 发现了病毒序列 (Alm Rosenblad等, 未发表的数据), 但与疾病症状没有明显的联系。抗生素的混合物以前被应用到文化中, 以尽量减少细菌感染的风险;然而, 这一程序目前被遗弃, 到目前为止, 这并没有造成任何污染问题。
如果海水受热 (如上所述), 过热可能是养殖生产线上最严重的风险。当然, 尽管可以使用传感器和适当的警报系统 (例如, 向负责人员发送电子邮件或短信), 但防止过热是困难的。这种情况在过去造成了成年人在文化中的大量杀戮。当然, 这可能是毁灭性的, 破坏长期投资的时间和金钱。特别是, 如果建立了近亲繁殖的遗传线, 这将是灾难性的。为了保证这些线路的寿命, 并确保它们免受意外损失, 最好为藤壶开发一种冷冻保存方法。据报道, 来自太平洋牡蛎的幼虫可以冷冻下来, 并恢复部分成功33。Cryobanking 还是保护34种种类的遗传资源的宝贵工具。据报道, 即使是从B. 安菲特律特的幼体在冰冻35中存活下来, 也发现20% 的冰冻个体成功变质成 cyprids36。然而, 目前还没有采用冻结的方法来维持文化的长期可持续性, 但确实需要维护选定的路线;这将是坚定地建立improvisus成为强有力的海洋 modelsystem 的重要步骤。
在这里, 提出了一个协议, 以解剖不同的组织从成人的improvisus (即, cirri, 躯体, 和壁炉)。然而, 应该强调的是, 其他组织也可以提取。例如, Tetraclita的膜基种的外幔和内地幔之间的软组织被仔细隔离, 用于 rna 提取和 rna 序列分析的基因表达37。此处描述的概要优化提取协议提供了足够数量的高质量 RNA, 用于从极小的起始材料中进行排序。首先, 将单个幼虫直接收集到均匀化管中, 将从一管转移到另一管子的损失降到最低。此外, 在不同的试验方法中, 与超声波或杵均匀化相比, 与陶瓷珠的均匀性在 RNA 的产量和完整性方面是最有效的。在规划基因表达或基因组实验时, 你必须牢记藤壶中高遗传变异的挑战, 至少对improvisus。藤壶有一个遗传多样性的范围 3–5%, 甚至在编码区域 (Alm Rosenblad等, 未发布的数据)。当然, 这对 qPCR 分析的引物设计提出了具体要求, 其中应确定更多的保守区域, 并将其用作底漆的模板, 以获得一致的表达结果 betweenbatches。对目标基因的保守区域, 如水通道蛋白和 Na +/K + ATPases, 可以通过研究这些基因的序列变异性, 从含有数以百计的人的 cyprids 的种群中获得。对于基因组分析, DNA 将被取样。然而, 从B. improvisus获得高质量的 DNA 可能具有挑战性21。
最后, 建立的藤壶文化在不同的实验研究中被证明是有益的。特别是, 全年幼虫生产允许我们进行实验, 而不限于自然发生的产卵期 ( B. improvisus, 这是在夏季)。获得的幼虫可用于进行广泛的实验研究, 包括沉降分析, 行为分析, 对特定基因的表达研究, 以及全基因组的转录研究。

藤壶是海洋甲壳类与无梗的成人和自由游泳, 浮游幼虫。1200种藤壶中的大多数栖息在浅水中, 许多人经常接触到低盐度。一个物种, 海湾藤壶Balanus (Amphibalanus) 即兴创作(B. improvisus), 可以容忍几乎淡水和查尔斯达尔文描述这个物种从一条小河在1乌拉圭的里约热内卢河口。极端容忍 tolow 盐度使B. improvisus一个特别相关的模型为 osmoregulatory 机制的研究2,3。这种藤壶喜欢咸水条件, 但能够生活在矿化度的水域, 从大约1.6 个电源到高达 404。它是在淡咸的波罗的海中唯一发现的藤壶物种。improvisus被认为起源于美国大陆的东海岸, 但今天在全世界被发现由于运输5。它是一个主要的污垢有机体, 通常在岩石、码头和船体上发现, 因此, 了解生物污垢在海洋和微咸水中的构造机制6,7是普遍的兴趣。
与大多数其他藤壶相似, B. improvisus是两性与交叉受精;繁殖发生通过交配的相邻个体使用拉长的小弟弟和内部受精。生育期主要从9月。B. improvisus有七个中上层幼虫阶段 (六幼体其次是一个 cyprid 阶段8)。受精卵孵化成一个无节幼体幼虫, 这是自由游泳和饲料在水柱长达数周前, 蜕皮成一个非喂养的 cyprid 幼虫。cyprid 使用多个线索找到一个合适的地点定居, 然后经历蜕变成一个无梗的少年藤壶9。该物种可以在实验室中培养, 在海洋中具有多年的寿命 (在实验室培养中2–3年)。平均来说, B. improvisus长到10毫米直径 (最大约20毫米), 达到最大高度约6毫米 (虽然它可以在拥挤的条件下长得更高)。该物种可以由其平滑的钙质均匀壳 (白色或灰), 壳板的径向图案的钙质基, 和背部板的形状1,10。
藤壶B. improvisus有几个优势特征作为研究渗透调节的模型, 重点是分子和生理机制, 以及生态相互作用和进化后果。它也被广泛使用作为一个模型为解决生物的调查, 特别是关于防污研究和机制涉及7,11,12,13。然而, 自然季节性产卵产生不可预测的 cyprid 幼虫供研究。因此, 通过全年的整个生命周期培养这种藤壶的能力, 是实现各种分子和机械研究的主要资产。此外, 它在世界范围内的海洋/微咸水域中的存在, 使得野外和实验研究结合在一起。受控育种还可以为长期培养14的已知血统的家庭生产, 而在几个月的世代时间可能允许长期的实验性进化。还有一个基因组和几个 transcriptomes 可用, 这些资源被用于克隆几个基因 (例如, 渗透调节中的重要基因)2,3。
本议定书的目的是描述如何建立和保持一个文化的藤壶improvisus全年, 以执行基因表达研究的成年人或幼虫的这个有机体。Rittschof等。15简要描述了一种培养藤壶的方法, 从幼体的释放到 cyprids 的Balanus 安菲特律特的沉降。该议定书已适应了在 Tjärnö海洋研究实验室 (瑞典) 全年培养B. improvisus , 并详细描述了生产线的所有步骤, 包括藤壶的生产和饲养幼虫, 以及对成人和幼虫饲料的管理。有关完整过程的概述, 请参见图 1。使用该培养系统的例子是一些常见的实验设置, 并说明在功能基因组学研究的钠+/K+ atp 酶和水通道蛋白, 阐明其可能的功能在渗透调节2, 3。有时有必要检查特定组织中的基因表达, 并将覆盖藤壶解剖的一些基本知识。由于优质海水供应良好, 在世界各地的海洋实验室中, 藤壶B. improvisus和潜在的许多其他物种的培养应该是可能的。

<table><tbody><tr><td>Plexiglas (poly-methyl methacrylate) panels</td><td>Plastic produkter, Bromma, Sweden</td><td></td><td>transparent glas</td></tr><tr><td>1.5 L PET bottle</td><td></td><td></td><td></td></tr><tr><td>Artemia</td><td>INVE Aquaculture, Belgium</td><td></td><td>We have tested different companies; this is really the best one</td></tr><tr><td>&lt;em&gt;Skeletonema marinoi&lt;/em&gt; (CCAP strain 1077/5)</td><td>CCAP (Culture Collection of Algae and Protozoa); Scotland&amp;nbsp;</td><td>strain 1077/5</td><td></td></tr><tr><td>&lt;em&gt;Chaetoceros simplex&lt;/em&gt; var. &lt;em&gt;gracilis&lt;/em&gt; (CCAP strain 1085/3)</td><td>CCAP (Culture Collection of Algae and Protozoa); Scotland&amp;nbsp;</td><td>strain 1085/3</td><td></td></tr><tr><td>Millipore cartridge filter system</td><td>Millipore&amp;nbsp;</td><td></td><td></td></tr><tr><td>cartridge with a nominal pore size of 0.2 &amp;micro;m</td><td>Millipore&amp;nbsp;</td><td>cartridge CWSS01S03</td><td>High capacity for large volumes</td></tr><tr><td>polycarbonate bottle</td><td>Nalgene</td><td></td><td>autoclavable</td></tr><tr><td>RNA later</td><td>Qiagen</td><td>76106</td><td>&amp;nbsp;Fixation solution to preserve RNA</td></tr><tr><td>TURBO DNA-free Kit</td><td>Invitrogen/Thermofisher Scientific&amp;nbsp;&amp;nbsp;</td><td>AM1907</td><td>DNAse kit to remove DNA from prepared RNA</td></tr><tr><td>iScript cDNA Synthesis Kit</td><td>Biorad</td><td>1708890</td><td>cDNA synthesis kit</td></tr><tr><td>SYBR Green supermix</td><td>Biorad</td><td>1708880</td><td>Dye for QPCR</td></tr><tr><td>RNeasy minikit</td><td>Qiagen</td><td>74104</td><td>RNA extraction of adults or many cyprids</td></tr><tr><td>Soft tissue homogenising CK 14, 2 ml tubes</td><td>Precellys</td><td>KT03961-1-003.2</td><td>Ceramic beads for homogenisation</td></tr><tr><td>RNeasy micro kit</td><td>Qiagen</td><td>74004</td><td>RNA extraction of few cyprids</td></tr></tbody></table>

the barnacle balanus improvisus as a marine model - culturing and gene expression

藤壶是海洋甲壳类与无梗的成人和自由游泳, 浮游幼虫。藤壶Balanus (Amphibalanus) improvisus特别相关, 作为研究 osmoregulatory 机制的模型, 因为它对低盐度的极端耐受性。它也被广泛地用作沉降生物学的模型, 特别是在防污研究方面。然而, 自然季节性产卵产生不可预测的 cyprid 幼虫供研究。制定了一项全年培养improvisus的议定书, 概述了生产线上所有步骤的详细情况 (即, 在小组中建立成人文化, 收集和饲养藤壶幼虫, 并管理成人和幼虫饲料)。该描述还提供了关于故障排除和讨论关键参数的指导 (例如, 清除污染、生产高质量饲料、需要人力以及高质量海水的重要性)。每批从养殖系统最大产量约1.2万幼体, 可以提供四批在一周内, 所以多达5万幼虫每周可以生产。用于养殖B. improvisus的方法在很大程度上也适用于其他具有自由 swimminglarvae 的海洋无脊椎动物。本文介绍了各种组织的解剖方法, 并提出了高质量的 RNA 用于基因表达的研究。还描述了如何在广泛的实验设计中使用培养成人和饲养 cyprids, 以检查基因表达与外部因素的关系。利用培养的藤壶在基因表达中的应用研究了可能 osmoregulatory 作用的钠+/钾 atp 酶和水通道蛋白。

通过对improvisus成人藤壶培养过程的描述, 每周可生产多达四批无节幼体幼虫。几乎每晚都可以收集无节幼体幼虫, 但这需要更多的人和基础设施 (在亲鱼中有许多藤壶, 一种文化会不断地释放幼虫)。幼虫生产的另外一个限制因素似乎是优质饲料的供应, 特别是关于硅藻中肋骨条。最大地, 每批从养殖系统包括大约1.2万幼体, 因此5万幼体每周可以养殖。然而, 在几个星期内, 可能会有更多的幼虫产生。一个成年人每天可以生产多达7000只幼虫14, 这意味着, 在每个批次中, 大人们都会释放幼虫。在一周内, 约70–90% 的收集幼体将发展成 cyprids (每星期产生大约 3万 cyprids, 最大), 可用于解决的化验和分子研究。
应该强调的是, 批次之间的 cyprid 特性有变化, 一般来说, 批次之间的差异比批处理中的大 。例如, 在沉降检测中沉降的成功率在30和70% 之间因不同批次而异。最有可能的是, 这是由特定对的成年人在不同的取样期内释放幼虫的个体遗传变异引起的。当然, 建议重复的实验 (生物复制) 应该包括一些批次的 cyprids, 如果要对结果作更多的一般性陈述的话。批量变异对实验设计提出了要求, 应在基因表达研究中应用适当的控制和标准化。然而, 即使实施了若干统计规范化程序, 大大减少了批处理之间的差异, 批处理的某些影响通常仍然很明显 (未发布的数据)。
根据提供的协议, 平均来说, 可以获得500的高质量 RNA 从多达 20 cyprids, 无论藤壶解决阶段 (表 1)。RNA 的质量通常被测量作为18S 和28S 峰顶的比率 (二个峰值的期望的位置在图 4被表明)。然而, 在藤壶和许多其他节肢动物的情况下, 28S rRNA 在加热时分解 (作为分析方法的一部分), 并与18S 峰值20一起迁移。这就是为什么在这种类型的藤壶分析中, 原则上有一个单一的 rRNA 峰。从这个测试 (图 4) 可以清楚地看出, 陶瓷珠子的均匀化为 RNA 提供了最高的完整性, 因此是选择的方法。RNA 在数量和质量上是足够的, 可以生成高质量的顺序库来进行排序, 从而平均每个样本读取7000万个 (当然, 读取的次数取决于排序过程中的多路复用程度)。RNA 的数量也足以用于基因的 cDNA 合成和 qPCR 表达分析。
图 5显示了水通道蛋白的 qPCR 分析和 Na+/K+ atp 酶 (NAK1) 拼接变体的结果, 其中的表达变化是针对环境线索2,3的变化进行调查的。对 NAK1 长、短拼接变形的相对表达式的比较表明, 低盐度的长 NAK1 mRNA 与短 NAK 的关系有双重增加 (图 5a)。因此, 数据表明, 在低盐度条件下, 交替拼接可使长形占优势。在水通道蛋白的情况下, 明显的两个输水 paralogs AQP1 和 AQP2 显示差分表达式 (图 5B)。特别是, 在地幔组织中, 明显的是, AQP1 在较低的矿化度, 这是没有看到 AQP2。相反, AQP2 显示在较低的矿化度, 但在躯体中的表达略有增加。这些发现为研究不同improvisus离子转运体和水通道蛋白在藤壶渗透调节中的功能作用提供了基础。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57825/57825fig1.jpg"> 图 1: 对成人基因表达研究的全培养过程和 RNA 提取进行综述.为了启动一种新的文化, 面板连接到一个框架, 并部署在海 1-3 m 深度。几个星期后, 与成人/青少年的面板被垂直放置在托盘在实验室的架子上。每个托盘有大约40个面板与成人。每个面板大约有100个成年人, 总≈4000成人个体被培养每托盘。成年藤壶用卤虫喂养, 可以常年保存。无节幼体幼虫每周收集几次从托盘通过过滤通过筛子。收集的幼体被转移到桶保持在26°c 在水浴和哺养与微藻。幼体被饲养, 直到他们蜕皮成非喂养 cyprids, 这是收集的过滤。新的面板可以建立在实验室中, 解决 cyprids 在面板上, 要么提供新的面板, 为全年文化或用于特定的实验设置与改变的外部条件。然后在实验结束或特定的时间点从青少年/成人中提取 RNA。<a href="https://www.jove.com/files/ftp_upload/57825/57825fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57825/57825fig2.jpg"> 图 2: 培养过程中一些重要步骤的图像.(A) 该图像显示了从该领域收集新人口的面板框架。(b) 这张图显示了improvisus的成人藤壶, 放在托盘中的架子上。面板被放置在大约 2 cm 分开。(C) 该图像显示了托盘与藤壶面板和喂养池的左侧。从每个托盘, 有一个出口, 其中的筛子放置在收集无节幼体幼虫。(D) 该图像显示了为成人藤壶生产卤虫饲料。(E) 这张图显示了幼体在水浴中放置的桶中的饲养, 设置为26摄氏度。<a href="https://www.jove.com/files/ftp_upload/57825/57825fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57825/57825fig3.jpg"> 图 3: 描述成人藤壶的初步解剖步骤: 从壳体中取出身体.(A) 通过光圈轻轻插入镊子, 抓住其中一个鳃盖板。轻轻拉取出盘子, 露出动物。(B) 抓住 cirri 下方的躯体部分, 拔出动物。(C) 这个小组显示橡子藤壶的整体解剖。当身体被拉出时, 地幔和可能受精的卵 (在卵巢中) 停留在壳腔内。关于藤壶解剖学的说明 (对于一个更深入的帐户, 见安德森)9: 藤壶的墙板向内倾斜, 并在一起, 形成一个火山状锥。开口, 光圈, 由两个鳃盖板覆盖, 形成一个门, 或笼盖, 以关闭光圈。橡子藤壶一般有一个石灰基板, 牢固地粘附在底层;然而, 有些藤壶缺乏这种钙质板块 (例如, balanoides)。藤壶从深色的地幔 (甲壳) 中分泌外骨骼。双层地幔的外表面钙化成刚性, 而地幔的内表面不钙化, 因此具有弹性。在光圈内, cirri 存在于缩回位置。这些是胸附件, 藤壶用于悬浮喂养。卵巢靠近藤壶的底部, 而睾丸位于躯体中。这一数字已被帕诺娃等所采用。21 , 并已发表的许可, 从 Springer。<a href="https://www.jove.com/files/ftp_upload/57825/57825fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57825/57825fig4.jpg"> 图 4: 毛细管凝胶电泳法测定 rRNA 的完整性.RNA 是由两种不同的均匀化方法制备的: (A) 超声波和 (B) 陶瓷珠。y 轴上的单位, 傅, 代表荧光单位。<a href="https://www.jove.com/files/ftp_upload/57825/57825fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57825/57825fig5.jpg"> 图 5: 基因表达的结果来自两个不同的基因研究对渗透调节在 improvisus 的重要. (A) 本小组显示的差异表达式, 由 qPCR 的剪接变种的钠+/钾+ atp 酶NAK1 , 以回应各种矿化度和2级3。在低盐度处理中, 长异构体 (NAK1-L) 的表达相对于短 (方差分析, P < 0.001) 增加。(b) 本小组显示在improvisus的成人中, 水通道蛋白在不同矿化度2期间的表现。qPCR 被用来确定水通道蛋白的表达水平相对于肌动。成年个体在3个不同的矿化度 (3, 20 和 33) 孵化了14天。为 RNA 准备, 成人的躯体、cirri 和地幔被分离了。在这两个数字中, 误差线表示标准偏差。** 和 ** 分别表明了意义的程度 (方差分析), 0.01 和0.001。这些数字已经从林德等。2,3. 这两个数字都是在公共图书馆的许可下公布的。<a href="https://www.jove.com/files/ftp_upload/57825/57825fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>结算阶段</td>
			<td>总 RNA 量 (ng)</td>
		</tr>
<tr>
<td>免费游泳</td>
			<td>512</td>
		</tr>
<tr>
<td>探索: 近距离搜索</td>
			<td>518</td>
		</tr>
<tr>
<td>附加 cyprid</td>
			<td>550</td>
		</tr>
<tr>
<td>新变质的少年</td>
			<td>832</td>
		</tr>
</tbody></table>表 1: RNA 的产量.本表显示了在沉降过程中不同阶段收集的 20 cyprid 个体的 rna 制备试剂盒提取的 rna 数量。

Watch this Scientific Journal Video about The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression at JoVE.com

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression

藤壶Balanus (Amphibalanus) improvisus是研究渗透调节和防污的典范。然而, 自然季节性产卵产生不可预知的 cyprid 幼虫供应。本文介绍了improvisus的全年培养方案, 包括幼虫的生产。阐述了培养的藤壶在基因表达研究中的应用。

藤壶Balanus improvisus作为海洋模型的培养和基因表达

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly ...

the-barnacle-balanus-improvisus-as-marine-model-culturing-gene

Research

JoVE Journal

Environment

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression

14.6K 次观看.  University of Gothenburg. 藤壶Balanus (Amphibalanus) improvisus是研究渗透调节和防污的典范。然而, 自然季节性产卵产生不可预知的 cyprid 幼虫供应。本文介绍了improvisus的全年培养方案, 包括幼虫的生产。阐述了培养的藤壶在基因表达研究中的应用。

视频: The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations.

荧光蛋白的表达<em&gt;披文昌鱼</em&gt;通过基因注射到未受精的卵母细胞

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

科研

发育生物学

Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

Cnidarians, and specifically Hydra, were the first animals shown to regenerate damaged or severed structures, and indeed such studies arguably launched modern biological inquiry through the work of Trembley more than 250 years ago. Presently the study of regeneration has seen a resurgence using both &#34;classic&#34; regenerative organisms, such as the Hydra, planaria and Urodeles, as well as a widening spectrum of species spanning the range of metazoa, from sponges through mammals. Besides its intrinsic interest as a biological phenomenon, understanding how regeneration works in a variety of species will inform us about whether regenerative processes share common features and/or species or context-specific cellular and molecular mechanisms. The starlet sea anemone, Nematostella vectensis, is an emerging model organism for regeneration. Like Hydra, Nematostella is a member of the ancient phylum, cnidaria, but within the class anthozoa, a sister clade to the hydrozoa that is evolutionarily more basal. Thus aspects of regeneration in Nematostella will be interesting to compare and contrast with those of Hydra and other cnidarians. In this article, we present a method to bisect, observe and classify regeneration of the aboral end of the Nematostella adult, which is called the physa. The physa naturally undergoes fission as a means of asexual reproduction, and either natural fission or manual amputation of the physa triggers re-growth and reformation of complex morphologies. Here we have codified these simple morphological changes in a Nematostella Regeneration Staging System (the NRSS). We use the NRSS to test the effects of chloroquine, an inhibitor of lysosomal function that blocks autophagy. The results show that the regeneration of polyp structures, particularly the mesenteries, is abnormal when autophagy is inhibited.

Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

Here we demonstrate how to induce and monitor regeneration in the Starlet Sea Anemone Nematostella vectensis, a model cnidarian anthozoan. We demonstrate how to amputate and categorize regeneration using a morphological staging system, and we use this system to reveal a requirement for autophagy in regenerating polyp structures.

从斯塔利特海葵的反口Physa完全诱导再生息肉<em&gt; Nematostella vectensis</em

Cnidarians, and specifically Hydra, were the first animals shown to regenerate damaged or severed structures, and indeed such studies arguably launched ...

Cultivation of the Marine Pelagic Tunicate Dolioletta gegenbauri (Uljanin 1884) for Experimental Studies

Gelatinous zooplanktons play a crucial role in ocean ecosystems. However, it is generally difficult to investigate their physiology, growth, fecundity, and trophic interactions primarily due to methodological challenges, including the ability to culture them. This is particularly true for the doliolid, Dolioletta gegenbauri. D. gegenbauri commonly occurs in productive subtropical continental shelf systems worldwide, often at bloom concentrations capable of consuming a large fraction of daily primary production. In this study, we describe cultivation approaches for collecting, rearing, and maintaining D. gegenbauri for the purpose of conducting laboratory-based studies. D. gegenbauri and other doliolid species can be captured live using obliquely towed conical 202 &micro;m mesh plankton nets from a drifting ship. Cultures are most reliably established when water temperatures are below 21 &deg;C and are started from immature gonozooids, maturing phorozooids, and large nurses. Cultures can be maintained in rounded culture vessels on a slowly rotating plankton wheel and sustained on a diet of cultured algae in natural seawater for many generations. In addition to the ability to establish laboratory cultures of D. gegenbauri, we demonstrate that the collection condition, algae concentration, temperature, and exposure to naturally conditioned seawater are all critical to the culture establishment, growth, survival, and reproduction of D. gegenbauri.

Cultivation of the Marine Pelagic Tunicate Dolioletta gegenbauri Uljanin 1884 for Experimental Studies

Doliolids, including the species Dolioletta gegenbauri, are small gelatinous marine zooplankton of ecological significance found on productive subcontinental shelf systems worldwide. The difficulty of culturing these delicate organisms limits their investigation. In this study, we describe cultivation approaches for collecting, rearing, and maintaining the doliolid Dolioletta gegenbauri.

用于实验研究的海洋可点多利奥莱塔·格根巴里(Uljanin 1884)的培养

Gelatinous zooplanktons play a crucial role in ocean ecosystems. However, it is generally difficult to investigate their physiology, growth, fecundity, ...

环境科学

Streamlined Sampling and Cultivation of the Pelagic Cosmopolitan Larvacean, Oikopleura dioica

Oikopleura dioica is a planktonic chordate with exceptional filter-feeding ability, rapid generation time, conserved early development, and a compact genome. For these reasons, it is considered a useful model organism for marine ecological studies, evolutionary developmental biology, and genomics. As research often requires a steady supply of animal resources, it is useful to establish a reliable, low-maintenance culture system. Here we describe a step-by-step method for establishing an O. dioica culture. We describe how to select potential sampling sites, collection methods, target animal identification, and the set-up of the culturing system. We provide troubleshooting advice based on our own experiences. We also highlight critical factors that help sustain a robust culture system. Although the culture protocol provided here is optimized for O. dioica, we hope our sampling technique and culture setup will inspire new ideas for maintaining other fragile pelagic invertebrates.

Streamlined Sampling and Cultivation of the Pelagic Cosmopolitan Larvacean, Oikopleura dioica

Oikopleura dioica is a tunicate model organism in various fields of biology. We describe sampling methods, species identification, culturing setup, and culturing protocols for the animals and algal feed. We highlight key factors that helped strengthen the culture system and discuss the possible problems and resolutions.

优化取样和种植的佩拉吉大都会拉瓦切安，奥科普拉迪奥卡

Oikopleura dioica is a planktonic chordate with exceptional filter-feeding ability, rapid generation time, conserved early development, and a compact ...

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their intercommunication, are paramount for the protection against infections. The interactions of epithelial and innate immune cells with a pathogen are best investigated in vivo, where complex behavior unfolds over time and space. However, existing models do not allow for easy spatio-temporal imaging of the battle with pathogens at the mucosal level.
The model developed here creates a mucosal infection by direct injection of the fungal pathogen, Candida albicans, into the swimbladder of juvenile zebrafish. The resulting infection enables high-resolution imaging of epithelial and innate immune cell behavior throughout the development of mucosal disease. The versatility of this method allows for interrogation of the host to probe the detailed sequence of immune events leading to phagocyte recruitment and to examine the roles of particular cell types and molecular pathways in protection. In addition, the behavior of the pathogen as a function of immune attack can be imaged simultaneously by using fluorescent protein-expressing C. albicans. Increased spatial resolution of the host-pathogen interaction is also possible using the described rapid swimbladder dissection technique.
The mucosal infection model described here is straightforward and highly reproducible, making it a valuable tool for the study of mucosal candidiasis. This system may also be broadly translatable to other mucosal pathogens such as mycobacterial, bacterial or viral microbes that normally infect through epithelial surfaces.

In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

造型粘膜念珠菌在斑马鱼幼虫通过鳔注射

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their ...

免疫与感染

Ablation of a Single Cell From Eight-cell Embryos of the Amphipod Crustacean Parhyale hawaiensis

The amphipod Parhyale hawaiensis is a small crustacean found in intertidal marine habitats worldwide. Over the past decade, Parhyale has emerged as a promising model organism for laboratory studies of development, providing a useful outgroup comparison to the well studied arthropod model organism Drosophila melanogaster. In contrast to the syncytial cleavages of Drosophila, the early cleavages of Parhyale are holoblastic. Fate mapping using tracer dyes injected into early blastomeres have shown that all three germ layers and the germ line are established by the eight-cell stage. At this stage, three blastomeres are fated to give rise to the ectoderm, three are fated to give rise to the mesoderm, and the remaining two blastomeres are the precursors of the endoderm and germ line respectively. However, blastomere ablation experiments have shown that Parhyale embryos also possess significant regulatory capabilities, such that the fates of blastomeres ablated at the eight-cell stage can be taken over by the descendants of some of the remaining blastomeres. Blastomere ablation has previously been described by one of two methods: injection and subsequent activation of phototoxic dyes or manual ablation. However, photoablation kills blastomeres but does not remove the dead cell body from the embryo. Complete physical removal of specific blastomeres may therefore be a preferred method of ablation for some applications. Here we present a protocol for manual removal of single blastomeres from the eight-cell stage of Parhyale embryos, illustrating the instruments and manual procedures necessary for complete removal of the cell body while keeping the remaining blastomeres alive and intact. This protocol can be applied to any Parhyale cell at the eight-cell stage, or to blastomeres of other early cleavage stages. In addition, in principle this protocol could be applicable to early cleavage stage embryos of other holoblastically cleaving marine invertebrates.

Ablation of a Single Cell From Eight-cell Embryos of the Amphipod Crustacean Parhyale hawaiensis

The amphipod Parhyale hawaiensis is a promising model organism for studies of crustacean embryology and comparative arthropod development and evolution. This protocol describes a method for manual removal of single blastomeres from early cleavage stage embryos of Parhyale.

一个单细胞消融从甲壳动物端足八细胞胚胎<em&gt; Parhyale hawaiensis</em

The amphipod Parhyale hawaiensis is a small crustacean found in intertidal marine habitats worldwide. Over the past decade, Parhyale has emerged as a ...

生物学

Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e.&#160;teleost fish) and full regeneration following appendage loss (i.e.&#160;urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4.

In vitro culture systems have proven indispensible to our understanding of vertebrate myogenesis. However, much remains to be learned about nonmammalian skeletal muscle development and growth, particularly in basal taxa. An efficient and robust protocol for isolating the adult stem cells of this tissue, the myogenic precursor cells (MPCs), and maintaining their self-renewal, proliferation, and differentiation in a primary culture setting allows for the identification of conserved and divergent regulatory mechanisms throughout the vertebrate lineages.

原发性生肌前体细胞/成肌细胞从基底哺乳动物支系文化准备

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem ...

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a &#39;whole mount&#39; tissue preparation, or developmental stage (such as an embryo or larva) of interest. WMISH is extremely powerful because it can significantly contribute to the functional characterization of complex metazoan genomes, a challenge that is becoming more of a bottleneck with the deluge of next generation sequence data. Despite the conceptual simplicity of the technique much time is often needed to optimize the various parameters inherent to WMISH experiments for novel model systems; subtle differences in the cellular and biochemical properties between tissue types and developmental stages mean that a single WMISH method may not be appropriate for all situations. We have developed a set of WMISH methods for the re-emerging gastropod model Lymnaea stagnalis that generate consistent and clear WMISH signals for a range of genes, and across all developmental stages. These methods include the assignment of larvae of unknown chronological age to an ontogenetic window, the efficient removal of embryos and larvae from their egg capsules, the application of an appropriate Proteinase-K treatment for each ontogenetic window, and hybridization, post-hybridization and immunodetection steps. These methods provide a foundation from which the resulting signal for a given RNA transcript can be further refined with probe specific adjustments (primarily probe concentration and hybridization temperature).

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

The goal of this protocol is to provide users with a set of methods for the high-throughput decapsulation of Lymnaea stagnalis embryos and larvae in preparation for whole mount in situ hybridization, and for subsequent pre- and post-hybridization treatments.

整个山<em&gt;原位</em&gt;对腹足软体动物杂交方法<em&gt;静水椎实螺</em

Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a ...

Apoptosis Induction and Detection in a Primary Culture of Sea Cucumber Intestinal Cells

Primary cultured cells are used in a variety of scientific disciplines as exceptionally important tools for the functional evaluation of biological substances or characterization of specific biological activities. However, due to the lack of universally applicable cell culture media and protocols, well described cell culture methods for marine organisms are still limited. Meanwhile, the commonly occurring microbial contamination and polytropic properties of marine invertebrate cells further impede the establishment of an effective cell culture strategy for marine invertebrates. Here, we describe an easy-to-handle method for culturing intestinal cells from sea cucumber Apostichopus japonicus; additionally, we provide an example of in vitro apoptosis induction and detection in primary cultured intestinal cells. Moreover, this experiment provides details about the appropriate culture medium and cell collection method. The described protocol is compatible with a variety of widely available tissue samples from marine organisms including Echinodermata, Mollusca, and Crustacea, and it can provide sufficient cells for multiple in vitro experimental applications. This technique would enable researchers to efficiently manipulate primary cell cultures from marine invertebrates and to facilitate the functional evaluation of targeted biological materials on cells.

This protocol provides an easy-to-handle method to culture the intestinal cells from sea cucumber Apostichopus japonicus and is compatible with a variety of widely available tissue samples from marine organisms including Echinodermata, Mollusca, and Crustacea.

海参肠细胞原培养的凋亡诱导与检测

Primary cultured cells are used in a variety of scientific disciplines as exceptionally important tools for the functional evaluation of biological ...

Photogrammetric Three-dimensional Modeling and Printing of Cetacean Skeleton using an Omura's Whale Stranded in Hong Kong Waters as an Example

The preparation of cetacean, in particular baleen whale, skeletons presents a great challenge due to their high lipid content and uncommon size. Documentation of the skeletal morphology is important to produce accurate and reliable models for both research and educational purposes. In this paper, we used a 10.8-meter long Omura&#8217;s whale stranded in Hong Kong waters in 2014 as an example for the illustration. This rare and enormous specimen was defleshed, macerated, and sun-dried to yield the skeleton for research and public display. Morphology of each bone was then documented by photogrammetry. The complex contour of the skeleton made automated photoshoot inadequate and 3 manual methods were used on bones of different sizes and shapes. The captured photos were processed to generate three-dimensional (3D) models of 166 individual bones. The skeleton was printed half-size with polylactic acid for display purposes, which was easier to maintain&#160;than the actual cetacean bones with high residual fat content. The printed bones reflected most anatomical features of the specimen, including the bowing out rostral region and the caudal condylar facet that articulated with Ce1, yet the foramina on the parieto-squamosal suture, which are diagnostic character of Balaenoptera omurai, and an indented groove on the frontal bone at the posterior end of the lateral edge were not clearly presented. Extra photoshoots or 3D surface scanning should be performed on areas with meticulous details to improve precision of the models. The electronic files of the 3D skeleton were published online to reach a global audience and facilitate scientific collaboration among researchers worldwide.

The bone morphology of a semi-degreased baleen whale was documented by photogrammetry with a DSLR camera to generate three-dimensional (3D) models by computer, which were 3D printed as half-sized replicas of the original for display and educational purposes.

藤壶Balanus improvisus作为海洋模型的培养和基因表达

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