We thank James Maeda and Aaron Cooke for assistance with the filming and editing of this video. Funding was provided by the National Science Foundation (OCE-0550468, 1029515).

注：本协议的第3步涉及脊椎动物科目。该程序已被设计成动物获得了最人性化的治疗可能和已经批准的机构动物护理和使用委员会（IACUC）在北卡罗莱纳州威尔明顿的大学。 1）组织提取<ol><li>使用组织是在其天然水，而不是挤压，干涸的或过于潮湿的状态，因为这将改变次生代谢产物的体积浓度。切剁的组织，以片或片可插入到50ml离心管中。注意：新鲜组织可以在某些情况下使用，但它往往是更好地切剁冷冻组织，这是不受挤压时切。 </li><li>加组织片到30毫升1:1的二氯甲烷1:1混合物（DCM）和甲醇（MeOH）中一个刻度离心管中，直到40 ml的终体积达到。一定要进行涉及转移的所有步骤溶剂在通风橱中有足够的通风。 </li><li>帽筒并倒转几次，然后在4小时的提取周期反复搅动。注意：在此期间，水结合的MeOH和所得甲醇：水相从DCM相分离。组织被交替地暴露于DCM和MeOH：水作为乳液的试管进行搅拌。 </li><li>对DCM萃取转移到一个圆底烧瓶中，并使用低热量蒸发至干，在旋转蒸发器（&lt;40℃）。使用最少的溶剂，将干燥的提取物转移到20ml的闪烁瓶中。适合的小瓶用旋转蒸发器适配器和使用低热量再次蒸发至干，在旋转蒸发器（&lt;40℃）。 注：下一步需要使用自制的压缩器械可通过拧顺序以下项目上的螺纹杆的端部进行组装的：（1）螺母，（2）垫圈，和（3）橡子螺母。垫圈必须或者被穿孔或装，使得它小于50毫升的离心管的内直径。 </li><li>返回到包含组织和甲醇毕业的离心管：水提取物，通过压缩挤压中提取出来的组织。在甲醇转移：水提取到同一个圆底烧瓶中，并冷冻保存（&lt;10°C）。 </li><li>加入甲醇到该带刻度的离心管中，直至现在的脱水组织浸没为2至6小时的持续时间的第二提取，然后传输新的MeOH提取到含有甲醇的冷却圆底烧瓶：水提取物。如果有，该组织还没有得到充分提取的任何关注，重复2至6小时的MeOH萃取。 </li><li>干燥掉MeOH中在旋转蒸发器使用低热量（&lt;40℃）。从圆底烧瓶中含有干燥的非极性提取物中的闪烁小瓶转移剩余的含水提取物，使用MeOH的最小体积冲洗的圆底烧瓶中。 </li><li> Ëvaporate含水提取物用低热（&lt;40℃）在真空浓缩至干。闪烁小瓶现在包含10毫升组织的总干粗有机提取物。疏散用N 2气的小瓶中，以防止氧化的顶部空间，封紧，并储存冷冻（-20℃）。 </li></ol> 2）食品准备<ol><li>准备冷冻鱿鱼地幔粉末。 注意：鱿鱼地幔提供营养源即比得上其他底栖无脊椎动物，并且将被用作在2.2以下子步骤的成分。 <ol><li>在温暖的去离子（DI）水鱿鱼地幔解冻冷冻环，然后原浆他们在一个高速搅拌机。 </li><li>倾一薄层泥鱿鱼地幔到浅Cookie表并冷冻（-20℃），然后破冻鱿鱼原浆的片切成小片被冻干。 </li><li>冻干冷冻鱿鱼地幔原浆继FR的操作程序EEZE干燥器。 </li><li>粉碎所述冻干片鱿鱼地幔原浆在一个高速混合器以形成粉末。 </li><li>在通风橱中，倒入粉状鱿鱼地幔到旋转面粉筛子过筛并从细粉分离组织的大块。 </li><li>细粉状鱿鱼地幔转移到一个密封的容器。疏散用N 2气在容器顶部空间，以防止氧化和储存冷冻（-20℃）。 </li></ol></li><li>准备食物混合。 注意：当运行多个连续的试验中，它是实际制备约100毫升食物混合物的，但是本配方可以缩放到更小的体积，如果必要的。 <ol><li>结合3克藻酸和5g冻干鱿鱼地幔粉用100ml的去离子水的混合物在150毫升烧杯中。用microspatula几分钟剧烈搅拌，直至粉末完全水合和该混合物是均匀的。 注意：如果需要，食用色素可在此届添加EP：它是比较容易的染料添加到食品混合物，将产生两处理和对照混合物（掩蔽在提取处理的混合物中的提取物的天然色素），而不是试图通过将相匹配的提取物处理的混合物的颜色染到控制混合物。浅绿色或褐色食物颜色，通常希望以掩盖任何颜料的粗提取物。 </li><li>装载食品的混合物正是10毫升成毕业的注射器。小心避免在此过程中包含气泡。 </li><li>取出20mL闪烁小瓶从冷冻干燥的粗有机提取物。添加两滴MeOH中，然后搅拌该提取物与一microspatula均匀的混合物。 </li><li>弹出装载10毫升注射器食品基质的成20mL闪烁小瓶中，并用一个microspatula搅拌使其均匀化提取物的经处理的食品混合物。 注：它可以帮助排出以较小的增量（ 即注射器，弹出2毫升，均质，则R。EPEAT直到所有10毫升已经均匀）。 </li></ol></li><li>准备检测颗粒。 <ol><li>加载一个非常小的体积的提取物的混合物（约1毫升）中进注射器，并淹没注射器尖端中的0.25M的氯化钙溶液。喷射注射器的内容物，以形成一个长的，意大利面条样链。 </li><li>几分钟后，取出硬化链，把它切成长4毫米小球在玻璃菜板用刀片，然后用清水冲洗在海水中。 </li><li>重复步骤2.3.1和2.3.2不包括组织提取物，使控制颗粒。一定要善待控制颗粒与溶剂同等体积（参见步骤2.2.3除甲醇来处理混合物）来控制溶剂的加法。如果阴性对照期望确认试验鱼可以从馈送却步，添加苯甲酸地那铵以2mg ml的浓度-1到生食混合物27。 </li></ol></li></ol> 3）适口性生物测定<ol><li>进行饲养试验与野生捕捞黄相双带锦鱼，Thalassoma bifasciatum，存放在三个实验室水族馆的透明双面车厢组。 </li><li>递送食物颗粒从海水的使用具有橡胶管的玻璃吸移管的烧杯中。注意：这可能需要几天时间来训练鱼领取食物以这种方式。甲条件刺激（在水族箱玻璃移液器例如几个抽头），该前面食物的递送可能是有帮助训练鱼期望在加入食品颗粒。 </li><li>进球颗粒。考虑接受粒料如果易于通过鱼消耗。考虑颗粒拒绝，如果至少三次尝试由一个或更多的鱼把它变成自己的口腔后没有吃过，或者沉淀走近后一个这样的尝试忽略。 </li><li>进球样品。注意：该测定过程被描绘为流程图在图1中的鱼的群组拒食。控制颗粒在协议中的任何一步都没有进一步考虑。有两个潜在的单个测定的运行的结果：样品被接受或拒绝。 <ol><li>以一个控制的丸粒，以确认该组的鱼是合作。提供了一个处理球。如果鱼接受治疗的小球，比分样品为接受。如果鱼拒绝治疗的颗粒，提供了后续的控制球，以确定是否鱼已经停止喂养。如果鱼接受后续的控制球，比分样品被否决。 </li></ol></li><li>复制。鱼的10对各提取物独立团重复检测程序。 </li></ol> 4）评估意义<ol><li>评估控制VS处理的颗粒与Fisher精确检验26改良版的消费差异的显着性。修改测试，使得边际总计控制和处理的粒料是固定，对待他们既作为随机样本。注：此为P = 0.057时，7丸吃;因此，任何提取物被认为是威慑，如果6个或更少的颗粒被吃掉，可口，如果7个或更多的颗粒被吃掉。 </li><li>比较提取物的群体之间的相对适口性，计算出颗粒每组内吃掉的平均数量。保持阈值在6粒料这样一组复制提取物被认为的威慑如果粒料的平均数目吃掉+标准误（SE）≤6。注意：在代表性的结果，组分配是物种，因此复制提取来自不同的个体和相对适口可以物种间比较。 </li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+the+skeleton+of+a+sponge+provide+a+defense+against+predatory+reef+fish.">Does the skeleton of a sponge provide a defense against predatory reef fish.</a> Oecologia. 107 (2), 225-231 (1996).</li><li>Chanas, B., Pawlik, J. R., Lindel, T., Fenical, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+defense+of+the+Caribbean+sponge+Agelas+clathrodes+(Schmidt).">Chemical defense of the Caribbean sponge Agelas clathrodes (Schmidt).</a> Journal of Experimental Marine Biology and Ecology. 208 (1-2), 185-196 (1997).</li><li>Wilson, D. M., Puyana, M., Fenical, W., Pawlik, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+defense+of+the+Caribbean+reef+sponge+Axinella+corrugata+against+predatory+fishes.">Chemical defense of the Caribbean reef sponge Axinella corrugata against predatory fishes.</a> Journal of Chemical Ecology. 25 (12), 2811-2823 (1999).</li><li>Chanas, B., Pawlik, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defenses+of+Caribbean+sponges+against+predatory+reef+fish+II.+Spicules,+tissue+toughness,+and+nutritional+quality.">Defenses of Caribbean sponges against predatory reef fish II. Spicules, tissue toughness, and nutritional quality.</a> Marine Ecology Progress Series. 127 (1), 195-211 (1995).</li><li>Albrizio, S., Ciminiello, P., Fattorusso, E., Magno, S., Pawlik, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphitoxin,+a+new+high+molecular+weight+antifeedant+pyridinium+salt+from+the+Caribbean+sponge+Amphimedon+compressa.">Amphitoxin, a new high molecular weight antifeedant pyridinium salt from the Caribbean sponge Amphimedon compressa.</a> Journal of Natural Products. 58 (5), 647-652 (1995).</li><li>Assmann, M., Lichte, E., Pawlik, J. R., K&#246;ck, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chemical defenses of the Caribbean sponges Agelas wiedenmayeri and Agelas conifera. Marine Ecology Progress Series. 207, 255-262 (2000).</li><li>Kubanek, J., Fenical, W., Pawlik, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+antifeedant+triterpene+glycosides+from+the+Caribbean+sponge+Erylus+Formosus.">New antifeedant triterpene glycosides from the Caribbean sponge Erylus Formosus.</a> Natural Product Letters. 15 (4), 275-285 (2001).</li><li>Pawlik, J. R., McFall, G., Zea, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+the+odor+from+sponges+of+the+genus+Ircinia+protect+them+from+fish+predators.">Does the odor from sponges of the genus Ircinia protect them from fish predators.</a> Journal of Chemical Ecology. 28 (6), 1103-1115 (2002).</li><li>Waddell, B., Pawlik, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defenses+of+Caribbean+sponges+against+invertebrate+predators.+I.+Assays+with+hermit+crabs.">Defenses of Caribbean sponges against invertebrate predators. I. Assays with hermit crabs.</a> Marine Ecology Progress Series. 195, 125-132 (2000).</li><li>Waddell, B., Pawlik, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defense+of+Caribbean+sponges+against+invertebrate+predators.+II.+Assays+with+sea+stars.">Defense of Caribbean sponges against invertebrate predators. II. Assays with sea stars.</a> Marine Ecology Progress Series. 195, 133-144 (2000).</li><li>Burns, E., Ifrach, I., Carmeli, S., Pawlik, J. R., Ilan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+anti-predatory+defenses+of+Red+Sea+and+Caribbean+sponges.+I.+Chemical+defense.">Comparison of anti-predatory defenses of Red Sea and Caribbean sponges. I. Chemical defense.</a> Marine Ecology Progress Series. 252, 105-114 (2003).</li><li>Jones, A. C., Blum, J. E., Pawlik, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testing+for+defensive+synergy+in+Caribbean+sponges:+Bad+taste+or+glass+spicules.">Testing for defensive synergy in Caribbean sponges: Bad taste or glass spicules.</a> Journal of Experimental Marine Biology and Ecology. 322 (1), 67 (2005).</li></ol>

The authors declare that they have no competing financial interests.

这里所描述的程序提供了一个相对简单的，生态的相关实验室的协议，以评估海洋生物antipredatory化学防御。在这里，我们回顾了由这套方法满足重要的标准： （1）适当的捕食者。这种喂养法采用双带锦鱼，Thalassoma bifasciatum，珊瑚礁整个加勒比地区最丰富的鱼类之一。该bluehead是一个通才食肉动物称为采样底栖无脊椎动物19各式各样。通才天敌是最好的?...

通过化学家和生态学家的合作化学生态学的发展。虽然陆地化学生态学的分支学科已经有一段时间了，那海洋化学生态学的只有几十年的历史，但已经提供了重要的见解海洋生物1-8的进化生态学和群落结构。以水肺潜水和核磁共振光谱的新兴技术的优势，有机化学迅速产生，描述上世纪70年代和80年代，从9底栖海洋无脊椎动物和藻类代谢产物的小说出版了大量。假设次级代谢产物必须为某种目的，许多这些出版物没有经验证据冲高重要生态价值属性的新化合物。大约在同一时间，生态学家还采取水肺潜水的来临优势和描述的分布和底栖动物和植物的前身来回的丰度米相对无效的抽样方法，如疏浚。这些研究人员的假设是，任何无柄和软体必须捍卫化学，以避免掠食者10消费。在努力引进经验什么是关于物种丰度，否则描述工作中，一些生态学家开始从毒性试验11推断的化学防御。大多数毒性测定法涉及的整鱼或其他生物体的脊椎动物组织的粗有机萃取水性悬浮液的曝光，随后确定负责杀死半数试验生物提取物的干重的浓度。然而，毒性试验不效仿其潜在的掠食者自然条件下感知猎物的方式，和随后的研究发现毒性和12-13适口性之间没有任何关系。令人惊讶的是，在著名期刊出版物使用具有很少或没有。生态技术升相关14-15，今天，这些研究仍然被广泛引用。而更令人震惊的要注意的是基于毒性数据研究继续出版16-18。这里所描述的生物测定方法的开发，在20世纪80年代后期，为海洋化工生态学家的生态相​​关的方法来评估antipredatory化学防御。该方法需要一个模型捕食样品从目标生物体的粗有机提取物在营养食品可比矩阵天然浓度，从而提供适口的数据，比毒性数据更加生态有意义。 一般的方法来评估海洋生物的组织的antipredatory活动包括四个重要的标准：（1）一个适当的通才捕食必须馈送测定法中使用，（2）所有极性有机代谢物必须详尽地从所述的组织中提取靶生物，（3）的代谢物必须BE在相同体积浓度混入营养合适的实验食品如在先前从其中提取，并且（4）的实验设计和统计方法必须提供一个有意义的度量，以指示相对于令人厌恶的生物体中发现。 下面的程序是专为评估加勒比海海洋无脊椎动物antipredatory化学防御。我们采用了双带锦鱼，Thalassoma bifasciatum，作为一种模式掠食性鱼类，因为这是种常见的对加勒比海的珊瑚礁，被称为采样底栖无脊椎动物19各式各样。从目标生物体组织被首先提取出来，然后与一种食物混合物，最后提供给T的组bifasciatum，观察他们是否拒绝提取物治疗食品。使用这种方法测定的数据提供了重要的见解海洋生物12,20-21，L的化学防御IFE历史权衡22-24和群落生态学25-26。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Dichloromethane</td>
			<td>Fisher Scientific</td>
			<td>D37-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>A41220</td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrous Calcium Chloride</td>
			<td>Fisher Scientific</td>
			<td>C614-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryocool Heat Transfer Fluid</td>
			<td>Fisher Scientific</td>
			<td>20-548-146</td>
			<td>For vacuum concentrator</td>
		</tr>
		<tr>
			<td>Alginic Acid Sodium Salt High Viscosity</td>
			<td>MP Biomedicals</td>
			<td>154723</td>
			<td></td>
		</tr>
		<tr>
			<td>Squid mantle rings</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Can be purchased at grocery store</td>
		</tr>
		<tr>
			<td>Denatonium benzoate</td>
			<td>Aldrich</td>
			<td>D5765</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml graduated centrifuge tube</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>20 ml scintillation vial</td>
			<td>Fisher Scientific</td>
			<td>03-337-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Pasteur pipets</td>
			<td>Fisher Scientific</td>
			<td>13-678-20D</td>
			<td></td>
		</tr>
		<tr>
			<td>Rubber bulbs for Pasteur pipets</td>
			<td>Fisher Scientific</td>
			<td>03-448-24</td>
			<td></td>
		</tr>
		<tr>
			<td>Red bulbs for pellet delivery</td>
			<td>Fisher Scientific</td>
			<td>03-448-27</td>
			<td></td>
		</tr>
		<tr>
			<td>250 ml round-bottom flask</td>
			<td>Fisher Scientific</td>
			<td>10-067E</td>
			<td></td>
		</tr>
		<tr>
			<td>Scintillation vial adapter for rotavap</td>
			<td>Fisher Scientific</td>
			<td>K747130-1324</td>
			<td></td>
		</tr>
		<tr>
			<td>Weightboats</td>
			<td>Fisher Scientific</td>
			<td>02-202B</td>
			<td></td>
		</tr>
		<tr>
			<td>Microspatula</td>
			<td>Fisher Scientific</td>
			<td>21-401-10</td>
			<td></td>
		</tr>
		<tr>
			<td>5 ml graduated syringe</td>
			<td>Fisher Scientific</td>
			<td>14-817-53</td>
			<td></td>
		</tr>
		<tr>
			<td>10 ml graduated syringe</td>
			<td>Fisher Scientific</td>
			<td>14-817-54</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Fisher Scientific</td>
			<td>S17302</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

在这里，我们报告该生物测定6种常见加勒比海绵（ 图2）的结果。这些数据最初发表于1995年由Pawlik 等 12并证明了这种方法的力量调查共发生类群之间的化学防御策略的差异。结果被报告为标准误差（SE），针对每个物种食用+食物颗粒的平均数量。几乎没有颗粒被吃掉的测定与象耳海绵，Amphimedon牛鞭 ，并Aplysina cauliformis粗制有机提取物。与此相反，从Ca...

Watch this Scientific Journal Video about A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms at JoVE.com

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

这种生物测定采用模型肉食性鱼类，以评估喂食威慑代谢产物从海洋生物的天然浓度的组织有机提取物用营养可比食品基质的存在。

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 ...

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9.3K 次观看.  University of North Carolina Wilmington. 这种生物测定采用模型肉食性鱼类，以评估喂食威慑代谢产物从海洋生物的天然浓度的组织有机提取物用营养可比食品基质的存在。 

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

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 ...

科研

环境科学

A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated (WATER-PAM) Fluorometry

Conventional methods for experimental manipulation of microalgae have employed large volumes of culture (20 ml to 5 L), so that the culture can be subsampled throughout the experiment1&#8211;7. Subsampling of large volumes can be problematic for several reasons: 1) it causes variation in the total volume and the surface area:volume ratio of the culture during the experiment; 2) pseudo-replication (i.e., replicate samples from the same treatment flask8) is often employed rather than true replicates (i.e., sampling from replicate treatments); 3) the duration of the experiment is limited by the total volume; and 4) axenic cultures or the usual bacterial microbiota are difficult to maintain during long-term experiments as contamination commonly occurs during subsampling.
The use of microtiter plates enables 1 ml culture volumes to be used for each replicate, with up to 48 separate treatments within a 12.65 x 8.5 x 2.2 cm plate, thereby decreasing the experimental volume and allowing for extensive replication without subsampling any treatment. Additionally, this technique can be modified to fit a variety of experimental formats including: bacterial-algal co-cultures, algal physiology tests, and toxin screening9&#8211;11. Individual wells with an alga, bacterium and/or co-cultures can be sampled for numerous laboratory procedures including, but not limited to: WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry, microscopy, bacterial colony forming unit (cfu) counts and flow cytometry. The combination of the microtiter plate format and WATER-PAM fluorometry allows for multiple rapid measurements of photochemical yield and other photochemical parameters with low variability between samples, high reproducibility and avoids the many pitfalls of subsampling a carboy or conical flask over the course of an experiment.

A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated WATER-PAM Fluorometry

The goal of this procedure is to demonstrate the reproducibility and adaptability of using a microtiter plate format for microalgal screening. This rapid screen combines WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry to measure photosynthetic yield as an indicator of Photosystem II (PSII) health with small volume bacterial-algal co-cultures.

小体积的生物测定评估细菌/浮游植物共培养使用水脉冲幅度调制（WATER-PAM）荧光

Conventional methods for experimental manipulation of microalgae have employed large volumes of culture (20 ml to 5 L), so that the culture can be ...

Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA libraries are needed for optimal results from next-generation sequencing. Environmental samples such as water may have low quality and quantities of DNA as well as contaminants that co-precipitate with DNA. The mechanical and enzymatic processes involved in extraction and library preparation may further damage the DNA. Gel size selection enables purification and recovery of DNA fragments of a defined size for sequencing applications. Nevertheless, this task is one of the most time-consuming steps in the DNA library preparation workflow. The protocol described here enables complete automation of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments. 
In this study, we describe a high-throughput approach to prepare high quality DNA libraries from freshwater samples that can be applied also to other environmental samples. We used an indirect approach to concentrate bacterial cells from environmental freshwater samples; DNA was extracted using a commercially available DNA extraction kit, and DNA libraries were prepared using a commercial transposon-based protocol. DNA fragments of 500 to 800 bp were gel size selected using Ranger Technology, an automated electrophoresis workstation. Sequencing of the size-selected DNA libraries demonstrated significant improvements to read length and quality of the sequencing reads.

This manuscript describes an automated gel size selection approach for purifying DNA fragments for next-generation sequencing. The Ranger Technology provides complete automation of the entire process of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments allowing for high-throughput and high quality next-generation sequencing libraries.

自动凝胶尺寸选择，以提高下一代测序文库的质量，从环境水样准备

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA ...

Laboratory Simulation of an Iron(II)-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria

A conventional concept for the deposition of some Precambrian Banded Iron Formations (BIF) proceeds on the assumption that ferrous iron [Fe(II)] upwelling from hydrothermal sources in the Precambrian ocean was oxidized by molecular oxygen [O2] produced by cyanobacteria. The oldest BIFs, deposited prior to the Great Oxidation Event (GOE) at about 2.4 billion years (Gy) ago, could have formed by direct oxidation of Fe(II) by anoxygenic photoferrotrophs under anoxic conditions. As a method for testing the geochemical and mineralogical patterns that develop under different biological scenarios, we designed a 40 cm long vertical flow-through column to simulate an anoxic Fe(II)-rich marine upwelling system representative of an ancient ocean on a lab scale. The cylinder was packed with a porous glass bead matrix to stabilize the geochemical gradients, and liquid samples for iron quantification could be taken throughout the water column. Dissolved oxygen was detected non-invasively via optodes from the outside. Results from biotic experiments that involved upwelling fluxes of Fe(II) from the bottom, a distinct light gradient from top, and cyanobacteria present in the water column, show clear evidence for the formation of Fe(III) mineral precipitates and development of a chemocline between Fe(II) and O2. This column allows us to test hypotheses for the formation of the BIFs by culturing cyanobacteria (and in the future photoferrotrophs) under simulated marine Precambrian conditions. Furthermore we hypothesize that our column concept allows for the simulation of various chemical and physical environments &#8212; including shallow marine or lacustrine sediments.

Laboratory Simulation of an IronII-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria

We simulated a Precambrian ferruginous marine upwelling system in a lab-scale vertical flow-through column. The goal was to understand how geochemical profiles of O2 and Fe(II) evolve as cyanobacteria produce O2. The results show the establishment of a chemocline due to Fe(II) oxidation by photosynthetically produced O2.

一个铁（II）的实验室模拟的前寒武纪富海洋上升流系统探索光合细菌生长

A conventional concept for the deposition of some Precambrian Banded Iron Formations (BIF) proceeds on the assumption that ferrous iron [Fe(II)] upwelling ...

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food webs. Toxicity testing based on microorganisms are widely used because they are relatively quick, reproducible, cheap, and are not associated with ethical issues. Here, we describe an ecotoxicological method to evaluate the biological response of the marine bacterium Vibrio anguillarum. This method assesses the acute toxicity of chemical compounds, including new contaminants such as nanoparticles, as well as environmental samples. The endpoint is the reduction of bacterial culturability (i.e., the capability to replicate and form colonies) due to exposure to a toxicant. This reduction can be generally referred to as mortality. The test allows for the determination of the LC50, the concentration that causes a 50% decrease of bacteria actively replicating and forming colonies, after a 6 h exposure. The culturable bacteria are counted in terms of colony forming units (CFU), and the &#34;mortality&#34; is evaluated and compared to the control. In this work, the toxicity of copper sulphate (CuSO4) was evaluated. A clear dose-response relationship was observed, with a mean LC50 of 1.13 mg/L, after three independent tests. This protocol, compared to existing methods with microorganisms, is applicable in a wider range of salinity and has no limitations for colored/turbid samples. It uses saline solution as the exposure medium, avoiding any possible interferences of growth medium with the investigated contaminants. The LC50 calculation facilitates comparisons with other bioassays commonly applied to ecotoxicological assessments of the marine environment.

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

This work describes a new protocol to assess the ecotoxicity of pollutants, including emerging contaminants such as nanomaterials, using the marine bacterium Vibrio anguillarum. This method allows for the determination of the LC50, or mortality, the concentration that causes a 50% decrease in bacteria culturability, after a 6 h exposure.

生物毒理学方法与海洋细菌<em&gt;鳗弧菌</em&gt;评估环境污染物的急性毒性

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food ...

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 ...

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

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly relevant as a model for the studies of osmoregulatory mechanisms because of its extreme tolerance to low salinity. It is also widely used as a model of settling biology, in particular in relation to antifouling research. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae for studies. A protocol for the all-year-round culturing of B. improvisus has been developed and a detailed description of all steps in the production line is outlined (i.e., the establishment of adult cultures on panels, the collection and rearing of barnacle larvae, and the administration of feed for adults and larvae). The description also provides guidance on troubleshooting and discusses critical parameters (e.g., the removal of contamination, the production of high-quality feed, the manpower needed, and the importance of high-quality seawater). Each batch from the culturing system maximally yields roughly 12,000 nauplii and can deliver four batches in a week, so up to almost 50,000 larvae per week can be produced. The method used to culture B. improvisus is, probably, to a large extent also applicable to other marine invertebrates with free-swimminglarvae. Protocols are presented for the dissection of various tissues from adults as well as the production of high-quality RNA for studies on gene expression. It is also described how cultured adults and reared cyprids can be utilized in a wide array of experimental designs for examining gene expression in relation to external factors. The use of cultured barnacles in gene expression is illustrated with studies of possible osmoregulatory roles of Na+/K+ ATPase and aquaporins.

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

The barnacle Balanus (Amphibalanus) improvisus is a model for studying osmoregulation and antifouling. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae. Here, a protocol for the all-year-round culturing of B. improvisus is described, including the production of larvae. The use of cultured barnacles in gene expression studies is illustrated.

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 ...

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.

生态动物的长期视频跟踪: 挪威龙虾 (诺菲氏肾病) 日生物活动的案例研究

We present a protocol related to a video-tracking technique based on the background subtraction and image thresholding that makes it possible to ...

Microfocus X-ray CT (microCT) Imaging of Actinia equina (Cnidaria), Harmothoe sp. (Annelida), and Xenoturbella japonica (Xenacoelomorpha)

Traditionally, biologists have had to rely on destructive methods such as sectioning in order to investigate the internal structures of opaque organisms. Non-destructive microfocus X-ray computed tomography (microCT) imaging has become a powerful and emerging protocol in biology, due to technological advancements in sample staining methods and innovations in microCT hardware, processing computers, and data analysis software. However, this protocol is not commonly used, as it is in the medical and industrial fields. One of the reasons for this limited use is the lack of a simple and comprehensible manual that covers all of the necessary steps: sample collection, fixation, staining, mounting, scanning, and data analyses. Another reason is the vast diversity of metazoans, particularly marine invertebrates. Because of marine invertebrates&#8217; diverse sizes, morphologies, and physiologies, it is crucial to adjust experimental conditions and hardware configurations at each step, depending on the sample. Here, microCT imaging methods are explained in detail using three phylogenetically diverse marine invertebrates: Actinia equina (Anthozoa, Cnidaria), Harmothoe sp. (Polychaeta, Annelida), and Xenoturbella japonica (Xenoturbellida, Xenacoelomorpha). Suggestions on performing microCT imaging on various animals are also provided.

Microfocus X-ray CT microCT Imaging of Actinia equina Cnidaria, Harmothoe sp. Annelida, and Xenoturbella japonica Xenacoelomorpha

Here, protocols for performing microfocus X-ray computed tomography (microCT) imaging of three marine invertebrate animals are explained in detail. This study describes steps such as sample fixation, staining, mounting, scanning, image reconstruction, and data analyses. Suggestions on how the protocol can be adjusted for different samples are also provided.

微聚焦 X 射线 CT (微CT) 阿克蒂尼亚 equina (Cnidaria),哈莫特霍sp. (安妮利达) 和Xenoturbella japonica (西纳科洛莫卡) 成像

Traditionally, biologists have had to rely on destructive methods such as sectioning in order to investigate the internal structures of opaque organisms. ...