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。

<ol>
	<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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Holeuryhalinity+and+its+mechanisms+in+a+cirriped+crustacean,+Balanus+improvisus.">Holeuryhalinity and its mechanisms in a cirriped crustacean, Balanus improvisus.</a> Comparative Biochemistry and Physiology - Part A: Comparative Physiology. 53 (1), 19-30 (1976).</li><li>Wrange, A. L., 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=The+story+of+a+hitchhiker:+population+genetic+patterns+in+the+invasive+barnacle+Balanus+(Amphibalanus)+improvisus+Darwin+1854.">The story of a hitchhiker: population genetic patterns in the invasive barnacle Balanus (Amphibalanus) improvisus Darwin 1854.</a> PLoS ONE. 11 (1), 0147082 (2016).</li><li>Berntsson, K. M., Jonsson, P. 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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。

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			<td height="21" style="height:21px;width:341px;">Plexiglas (poly-methyl methacrylate) panels</td>
			<td style="width:364px;">Plastic produkter, Bromma, Sweden</td>
			<td style="width:153px;"></td>
			<td style="width:385px;">transparent glas</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5-L PET bottle</td>
			<td style="width:364px;"></td>
			<td style="width:153px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Artemia</td>
			<td>INVE Aquaculture, Belgium</td>
			<td style="width:153px;"></td>
			<td>We have tested different companies; this is really the best one</td>
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		<tr height="42">
			<td height="42" style="height:42px;">Skeletonema marinoi (CCAP strain 1077/5)</td>
			<td style="width:364px;">CCAP (Culture Collection of Algae and Protozoa); Scotland&#160;</td>
			<td style="width:153px;">strain 1077/5</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Chaetoceros simplex var. gracilis (CCAP strain 1085/3)</td>
			<td style="width:364px;">CCAP (Culture Collection of Algae and Protozoa); Scotland&#160;</td>
			<td style="width:153px;">strain 1085/3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Millipore cartridge filter system</td>
			<td style="width:364px;">Millipore&#160;</td>
			<td style="width:153px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cartridge with a nominal pore size of 0.2 &#181;m</td>
			<td style="width:364px;">Millipore&#160;</td>
			<td style="width:153px;">cartridge CWSS01S03</td>
			<td>High capacity for large volumes</td>
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		<tr height="21">
			<td height="21" style="height:21px;">polycarbonate bottle</td>
			<td style="width:364px;">Nalgene</td>
			<td style="width:153px;"></td>
			<td>autoclavable</td>
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			<td height="21" style="height:21px;">RNA later</td>
			<td style="width:364px;">Qiagen</td>
			<td style="width:153px;">76106</td>
			<td>&#160;Fixation solution to preserve RNA</td>
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		<tr height="20">
			<td height="20" style="height:21px;">TURBO DNA-free Kit</td>
			<td style="width:364px;">Invitrogen/Thermofisher Scientific&#160;&#160;</td>
			<td>AM1907</td>
			<td>DNAse kit to remove DNA from prepared RNA</td>
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		<tr height="21">
			<td height="21" style="height:21px;">iScript cDNA Synthesis Kit</td>
			<td style="width:364px;">Biorad</td>
			<td>1708890</td>
			<td>cDNA synthesis kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SYBR Green supermix</td>
			<td style="width:364px;">Biorad</td>
			<td>1708880</td>
			<td>Dye for QPCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">RNeasy minikit</td>
			<td style="width:364px;">Qiagen</td>
			<td>74104</td>
			<td>RNA extraction of adults or many cyprids</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Soft tissue homogenising CK 14, 2 ml tubes</td>
			<td style="width:364px;">Precellys</td>
			<td style="width:153px;">KT03961-1-003.2</td>
			<td>Ceramic beads for homogenisation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">RNeasy micro kit</td>
			<td style="width:364px;">Qiagen</td>
			<td style="width:153px;">74004</td>
			<td>RNA extraction of few cyprids</td>
		</tr>
	</tbody>
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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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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 ...

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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

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5&#8217; mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work11, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

使用吉布森大会和基因枪烟草中瞬时基因表达

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

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Unraveling the Unseen Players in the Ocean - A Field Guide to Water Chemistry and Marine Microbiology

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling protocols include the assessment of resources available to the microbial community (dissolved organic carbon, particulate organic matter, inorganic nutrients), and a comprehensive description of the viral and bacterial communities (via direct viral and microbial counts, enumeration of autofluorescent microbes, and construction of viral and microbial metagenomes). We use a combination of methods, which represent a dispersed field of scientific disciplines comprising already established protocols and some of the most recent techniques developed. Especially metagenomic sequencing techniques used for viral and bacterial community characterization, have been established only in recent years, and are thus still subjected to constant improvement. This has led to a variety of sampling and sample processing procedures currently in use. The set of methods presented here provides an up to date approach to collect and process environmental samples. Parameters addressed with these protocols yield the minimum on information essential to characterize and understand the underlying mechanisms of viral and microbial community dynamics. It gives easy to follow guidelines to conduct comprehensive surveys and discusses critical steps and potential caveats pertinent to each technique.

Here, we present a comprehensive protocol to assess the organic and inorganic nutrient availability and the abundance and structure of microbial and viral communities in remote marine environments.

揭开看不见的玩家在海洋 - 一个领域指南水化学和海洋微生物

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling ...

A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with the goal of examining the ecological functions of secondary metabolites from the tissues of marine organisms. The result has been a progression of protocols that have increasingly refined the ecological relevance of the experimental approach. Here we present the most up-to-date version of a fish-feeding laboratory bioassay that enables investigators to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms. Organic metabolites of all polarities are exhaustively extracted from the tissue of the target organism and reconstituted at natural concentrations in a nutritionally appropriate food matrix. Experimental food pellets are presented to a generalist predator in laboratory feeding assays to assess the antipredatory activity of the extract. The procedure described herein uses the bluehead, Thalassoma bifasciatum, to test the palatability of Caribbean marine invertebrates; however, the design may be readily adapted to other systems. Results obtained using this laboratory assay are an important prelude to field experiments that rely on the feeding responses of a full complement of potential predators. Additionally, this bioassay can be used to direct the isolation of feeding-deterrent metabolites through bioassay-guided fractionation. This feeding bioassay has advanced our understanding of the factors that control the distribution and abundance of marine invertebrates on Caribbean coral reefs and may inform investigations in diverse fields of inquiry, including pharmacology, biotechnology, and evolutionary ecology.

This bioassay employs a model predatory fish to assess the presence of feeding-deterrent metabolites from organic extracts of the tissues of marine organisms at natural concentrations using a nutritionally comparable food matrix.

了鱼喂食实验室生物测定，从海洋生物的组织评估次生代谢产物的Antipredatory活动

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with ...

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and detritus, and excreting particulate and dissolved compounds, they serve as important agents for benthic-pelagic coupling. Accurately measuring the compounds removed and excreted by suspension feeders (such as sponges, ascidians, polychaetes, bivalves) is crucial for the study of their physiology, metabolism, and feeding ecology, and is fundamental to determine the ecological relevance of the nutrient fluxes mediated by these organisms. However, the assessment of the rate by which suspension feeders process particulate and dissolved compounds in nature is restricted by the limitations of the currently available methodologies. Our goal was to develop a simple, reliable, and non-intrusive method that would allow clean and controlled water sampling from a specific point, such as the excurrent aperture of benthic suspension feeders, in situ. Our method allows simultaneous sampling of inhaled and exhaled water of the studied organism by using minute tubes installed on a custom-built manipulator device and carefully positioned inside the exhalant orifice of the sampled organism. Piercing a septum on the collecting vessel with a syringe needle attached to the distal end of each tube allows the external pressure to slowly force the sampled water into the vessel through the sampling tube. The slow and controlled sampling rate allows integrating the inherent patchiness in the water while ensuring contamination free sampling. We provide recommendations for the most suitable filtering devices, collection vessel, and storing procedures for the analyses of different particulate and dissolved compounds. The VacuSIP system offers a reliable method for the quantification of undisturbed suspension feeder metabolism in natural conditions that is cheap and easy to learn and apply to assess the physiology and functional role of filter feeders in different ecosystems.

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

We introduce the VacuSIP, a simple, non-intrusive, and reliable method for clean and accurate point sampling of water. The system was developed and evaluated for the simultaneous collection of the water inhaled and exhaled by benthic suspension feeders in situ, to cleanly measure removal and excretion of particulate and dissolved compounds.

VacuSIP，对改进的方法INEX<em&gt;原位</em&gt;颗粒物与测量溶解的化合物加工后主动悬挂给料机

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment during manufacturing and transport. Because of their environmental persistence, they are widely distributed in the oceans and on beaches all over the world. They can act as a vector of potentially toxic organic compounds (e.g., polychlorinated biphenyls) and might consequently negatively affect marine organisms. Their possible impacts along the food chain are not yet well understood. In order to assess the hazards associated with the occurrence of plastic pellets in the marine environment, it is necessary to develop methodologies that allow for rapid determination of associated organic contaminant levels. The present protocol describes the different steps required for sampling resin pellets, analyzing adsorbed organochlorine pesticides (OCPs) and identifying the plastic type. The focus is on the extraction of OCPs from plastic pellets by means of a pressurized fluid extractor (PFE) and on the polymer chemical analysis applying Fourier Transform-InfraRed (FT-IR) spectroscopy. The developed methodology focuses on 11 OCPs and related compounds, including dichlorodiphenyltrichloroethane (DDT) and its two main metabolites, lindane and two production isomers, as well as the two biologically active isomers of technical endosulfan. This protocol constitutes a simple and rapid alternative to existing methodology for evaluating the concentration of organic contaminants adsorbed on plastic pieces.

Microplastics act as vector of potentially toxic organic contaminants with unpredictable effects. This protocol describes an alternative methodology for assessing the levels of organochlorine pesticides adsorbed on plastic pellets and identifying the polymer chemical structure. The focus is on pressurized fluid extraction and attenuated total reflectance Fourier transform infrared spectroscopy.

从塑料颗粒中提取有机氯农药和塑料类型分析

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...

Development of New Methods for Quantifying Fish Density Using Underwater Stereo-video Tools

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and estimating fish abundance. We developed and implemented a rotating stereo-video camera tool that covers a full 360 degrees of sampling, which maximizes sampling effort compared to stationary camera tools. A variety of studies have detailed the ability of static, stereo-camera systems to obtain highly accurate and precise measurements of fish; the focus here was on the development of methodological approaches to quantify fish density using rotating camera systems. The first approach was to develop a modification of the metric MaxN, which typically is a conservative count of the minimum number of fish observed on a given camera survey. We redefine MaxN to be the maximum number of fish observed in any given rotation of the camera system. When precautions are taken to avoid double counting, this method for MaxN may more accurately reflect true abundance than that obtained from a fixed camera. Secondly, because stereo-video allows fish to be mapped in three-dimensional space, precise estimates of the distance-from-camera can be obtained for each fish. By using the 95% percentile of the observed distance from camera to establish species-specific areas surveyed, we account for differences in detectability among species while avoiding diluting density estimates by using the maximum distance a species was observed. Accounting for this range of detectability is critical to accurately estimate fish abundances. This methodology will facilitate the integration of rotating stereo-video tools in both applied science and management contexts.

We describe a new method for counting fishes, and estimating relative abundance (MaxN) and fish density using rotating stereo-video camera systems. We also demonstrate how to use distance from camera (Z distance) to estimate species-specific detectability.

利用水下立体视频工具量化鱼类密度的新方法研究进展

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and ...

Methods for Image-based Surveys of Benthic Macroinvertebrates and Their Habitat Exemplified by the Drop Camera Survey for the Atlantic Sea Scallop

Underwater imaging has long been used in the field of marine ecology but decreasing costs of high-resolution cameras and data storage have made the approach more practical than in the past. Image-based surveys allow for initial samples to be revisited and are non-invasive compared to traditional survey methods that typically involve nets or dredges. Protocols for image-based surveys can vary greatly but should be driven by target species behavior and survey objectives. To demonstrate this, we describe our most recent methods for an Atlantic sea scallop (Placopecten magellanicus) drop camera survey to provide a procedural example and representative results. The procedure is divided into three critical steps that include survey design, data collection, and data products. The influence of scallop behavior and the survey goal of providing an independent assessment of the U.S. sea scallop resource on the survey procedure are then discussed in the context of generalizing the method. Overall, the broad applicability and flexibility of the University of Massachusetts Dartmouth School for Marine Science and Technology (SMAST) drop camera survey demonstrates the method could be generalized and applied to a variety of sessile invertebrates or habitat focused research.

Image based surveying is an increasingly practical, non-invasive method to sample the marine environment. We present the protocol of a drop camera survey that estimates the abundance and distribution of the Atlantic sea scallop (Placopecten magellanicus). We discuss how this protocol can be generalized for application to other benthic macroinvertebrates.

基于图像的海底无脊椎动物及其栖息地调查方法大西洋扇贝落相机调查

Underwater imaging has long been used in the field of marine ecology but decreasing costs of high-resolution cameras and data storage have made the ...

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ocean temperatures and ocean acidification, corals are increasingly under threat. To tackle these challenges, advances in cell and molecular biology have proven to be crucial for diagnosing the health of corals. Modifying some of the techniques commonly used in human medicine could greatly improve researchers&#39; ability to treat and save corals. To address this, a protocol for in situ hybridization used primarily in human medicine and evolutionary developmental biology has been adapted for use in adult corals under stress.
The purpose of this method is to visualize the spatial expression of an RNA probe in adult coral tissue that has been embedded in paraffin and sectioned onto glass slides. This method focuses on removal of the paraffin and rehydration of the sample, pretreatment of the sample to ensure permeability of the sample, pre-hybridization incubation, hybridization of the RNA probe, and visualization of the RNA probe. This is a powerful method when using non-model organisms to discover where specific genes are expressed, and the protocol can be easily adapted for other non-model organisms. However, the method is limited in that it is primarily qualitative, because expression intensity can vary depending on the amount of time used during the visualization step and the concentration of the probe. Furthermore, patience is required, as this protocol can take up to 5 days (and in many cases, longer) depending on the probe being used. Finally, non-specific background staining is common, but this limitation can be overcome.

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

The goal of this protocol is to perform in situ hybridization on adult coral samples that have been embedded in paraffin and sectioned onto glass slides. This is a qualitative method used to visualize the spatial expression of an RNA anti-sense probe in paraffin-embedded tissues.

就地石蜡镶嵌成人珊瑚样品的杂交技术

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ...

Individual Culturing of Tigriopus Copepods and Quantitative Analysis of Their Mate-guarding Behavior

Copepods of the genus Tigriopus, which are common zooplankton in rocky tide pools, show precopulatory mate-guarding behavior where a male clasps a potential mate to form a pair. While this phenomenon has attracted interest of researchers, methods for its analysis have not been well described. Here we describe procedures for: 1) individual culturing and staging of Tigriopus juveniles and adults, and 2) video-based analysis of their mate-guarding behavior. The culturing method enables experimental control of paring experience of animals as well as the ability to track their development before behavioral tests. The analysis method allows quantitative evaluation of several aspects of the mate-guarding behavior, including capturing attempts by males and swimming trajectory of mate-guarding pairs. Although these methods were originally established for ethological studies on Tigriopus, with proper modifications they can also be applied to studies of other zooplankton in different research fields, such as physiology, toxicology, and ecological genetics.

Individual Culturing of Tigriopus Copepods and Quantitative Analysis of Their Mate-guarding Behavior

Mate-guarding behavior plays an important role in reproduction of intertidal copepods of the genus Tigriopus. However, methods for studying this behavior have not been well described. Here we describe methods for: 1) individual culture of virgin Tigriopus animals, and 2) quantitative analysis of their mate-guarding behavior.

Tigriopus桡足的个体培养及其交配行为的定量分析

Copepods of the genus Tigriopus, which are common zooplankton in rocky tide pools, show precopulatory mate-guarding behavior where a male clasps a ...

Long-term Video Tracking of Cohoused Aquatic Animals: A Case Study of the Daily Locomotor Activity of the Norway Lobster (Nephrops norvegicus)

We present a protocol related to a video-tracking technique based on the background subtraction and image thresholding that makes it possible to individually track cohoused animals. We tested the tracking routine with four cohoused Norway lobsters (Nephrops norvegicus) under light-darkness conditions for 5 days. The lobsters had been individually tagged. The experimental setup and the tracking techniques used are entirely based on the open source software. The comparison of the tracking output with a manual detection indicates that the lobsters were correctly detected 69% of the times. Among the correctly detected lobsters, their individual tags were correctly identified 89.5% of the times. Considering the frame rate used in the protocol and the movement rate of lobsters, the performance of the video tracking has a good quality, and the representative results support the validity of the protocol in producing valuable data for research needs (individual space occupancy or locomotor activity patterns). The protocol presented here can be easily customized and is, hence, transferable to other species where the individual tracking of specimens in a group can be valuable for answering research questions.

Long-term Video Tracking of Cohoused Aquatic Animals: A Case Study of the Daily Locomotor Activity of the Norway Lobster Nephrops norvegicus

Here we present a protocol to individually track animals over a long period of time. It uses computer vision methods to identify a set of manually constructed tags by using a group of lobsters as case study, simultaneously providing information on how to house, manipulate, and mark the lobsters.