Name: a fish-feeding laboratory bioassay to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms
Uploaded: 2015-01-11T14:00:00.000+00:00
Duration: 16 min 3 s
Description: 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

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。通才天敌是最好的选...

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><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 ...

a-fish-feeding-laboratory-bioassay-to-assess-antipredatory-activity

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Environment

9.4K 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

Laboratory Estimation of Net Trophic Transfer Efficiencies of PCB Congeners to Lake Trout (Salvelinus namaycush) from Its Prey

A technique for laboratory estimation of net trophic transfer efficiency (&#947;) of polychlorinated biphenyl (PCB) congeners to piscivorous fish from their prey is described herein. During a 135-day laboratory experiment, we fed bloater (Coregonus hoyi) that had been caught in Lake Michigan to lake trout (Salvelinus namaycush) kept in eight laboratory tanks. Bloater is a natural prey for lake trout. In four of the tanks, a relatively high flow rate was used to ensure relatively high activity by the lake trout, whereas a low flow rate was used in the other four tanks, allowing for low lake trout activity. On a tank-by-tank basis, the amount of food eaten by the lake trout on each day of the experiment was recorded. Each lake trout was weighed at the start and end of the experiment. Four to nine lake trout from each of the eight tanks were sacrificed at the start of the experiment, and all 10 lake trout remaining in each of the tanks were euthanized at the end of the experiment. We determined concentrations of 75 PCB congeners in the lake trout at the start of the experiment, in the lake trout at the end of the experiment, and in bloaters fed to the lake trout during the experiment. Based on these measurements, &#947; was calculated for each of 75 PCB congeners in each of the eight tanks. Mean &#947; was calculated for each of the 75 PCB congeners for both active and inactive lake trout. Because the experiment was replicated in eight tanks, the standard error about mean &#947; could be estimated. Results from this type of experiment are useful in risk assessment models to predict future risk to humans and wildlife eating contaminated fish under various scenarios of environmental contamination.

Laboratory Estimation of Net Trophic Transfer Efficiencies of PCB Congeners to Lake Trout Salvelinus namaycush from Its Prey

A technique for laboratory estimation of net trophic transfer efficiency of polychlorinated biphenyl (PCB) congeners to piscivorous fish from their prey is presented. To maximize applicability of the laboratory results to the field, the piscivorous fish should be fed prey fish that are typically eaten in the field.

多氯联苯同系物净营养转移效率湖鳟鱼实验室估计（<em&gt;红点鲑namaycush</em&gt;），从它的猎物

A technique for laboratory estimation of net trophic transfer efficiency (&#947;) of polychlorinated biphenyl (PCB) congeners to piscivorous fish from ...

科研

环境科学

Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes

This protocol provides multiple methods for the analysis and quantification of predatory feeding behaviors in nematodes. Many nematode species including Pristionchus pacificus display complex behaviors, the most striking of which is the predation of other nematode larvae. However, as these behaviors are absent in the model organism Caenorhabditis elegans, they have thus far only recently been described in detail along with the development of reliable behavioral assays 1. These predatory behaviors are dependent upon phenotypically plastic but fixed mouth morphs making the correct identification and categorization of these animals essential. In P. pacificus there are two mouth types, the stenostomatous and eurystomatous morphs 2, with only the wide mouthed eurystomatous containing an extra tooth and being capable of killing other nematode larvae. Through the isolation of an abundance of size matched prey larvae and subsequent exposure to predatory nematodes, assays including both "corpse assays" and "bite assays" on correctly identified mouth morph nematodes are possible. These assays provide a means to rapidly quantify predation success rates and provide a detailed behavioral analysis of individual nematodes engaged in predatory feeding activities. In addition, with the use of a high-speed camera, visualization of changes in pharyngeal activity including tooth and pumping dynamics are also possible.

Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes

Behavioral assays provide powerful tools for understanding neuronal function. Here we present several protocols for quantifying predatory feeding behavior found in the model nematode Pristionchus pacificus and its relatives. Additionally, we provide methods for analyzing predatory feeding adaptations including mouth structures and teeth.

测定掠夺取食行为<em&gt; Pristionchus</em&gt;及其他线虫

This protocol provides multiple methods for the analysis and quantification of predatory feeding behaviors in nematodes. Many nematode species including ...

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 ...

The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and identification of an active pheromone compound. This method has resulted in the successful characterization of the chemical signals that function as pheromones in a wide range of animal species. Sea lampreys rely on olfaction to detect pheromones that mediate behavioral or physiological responses. We use this knowledge of fish biology to posit functions of putative pheromones and to guide the isolation and identification of active pheromone components. Chromatography is used to extract, concentrate, and separate compounds from the conditioned water. Electro-olfactogram (EOG) recordings are conducted to determine which fractions elicit olfactory responses. Two-choice maze behavioral assays are then used to determine if any of the odorous fractions are also behaviorally active and induce a preference. Spectrometric and spectroscopic methods provide the molecular weight and structural information to assist with the structure elucidation. The bioactivity of the pure compounds is confirmed with EOG and behavioral assays. The behavioral responses observed in the maze should ultimately be validated in a field setting to confirm their function in a natural stream setting. These bioassays play a dual role to 1) guide the fractionation process and 2) confirm and further define the bioactivity of isolated components. Here, we report the representative results of a sea lamprey pheromone identification that exemplify the utility of the bioassay-guided fractionation approach. The identification of sea lamprey pheromones is particularly important because a modulation of its pheromone communication system is among the options considered to control the invasive sea lamprey in the Laurentian Great Lakes. This method can be readily adapted to characterize the chemical communication in a broad array of taxa and shed light on waterborne chemical ecology.

Here, we present a protocol to isolate and characterize the structure, olfactory potency, and behavioral response of putative pheromone compounds of sea lampreys.

利用生物测定引导分馏法鉴定海洋鳗信息素

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and ...

生物化学

Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and toxicological studies. Using automated digital tracking platforms, recent efforts in neuropharmacology are leveraging larval fish locomotor behaviors to identify potential therapeutic targets for novel small molecules. Similar to these efforts, research in the environmental sciences and comparative pharmacology and toxicology is examining various behaviors of fish models as diagnostic tools in tiered evaluation of contaminants and real-time monitoring of surface waters for contaminant threats. Whereas the zebrafish is a popular larval fish model in the biomedical sciences, the fathead minnow is a common larval fish model in ecotoxicology. Unfortunately, fathead minnow larvae have received considerably less attention in behavioral studies. Here, we develop and demonstrate a behavioral profile protocol using caffeine as a model neurostimulant. Though photomotor responses of fathead minnows were occasionally affected by caffeine, zebrafish&#160;were markedly more sensitive for photomotor and locomotor endpoints, which responded at environmentally relevant levels. Future studies are needed to understand comparative behavioral sensitivity differences among fish with age and time of day, and to determine whether similar behavioral effects would occur in nature and be indicative of adverse outcomes at the individual or population levels of biological organization.

Here, we present a protocol to examine larval zebrafish and fathead minnow locomotor activities and photomotor responses (PMR) using an automated tracking software. When incorporated in common toxicology bioassays, analyses of these behaviors provide a diagnostic tool to examine chemical bioactivity. This protocol is described using caffeine, a model neurostimulant.

检测幼虫行为反应剖面的实验协议: 在神经刺激咖啡因中的应用

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and ...

Identification of Hemolytic and Phospholipase Activity in Crude Extracts from Sea Anemones by Straightforward Bioassays

Sea anemone venom composition includes polypeptide and non-proteins molecules. Cytolytic components have a high biotechnological and biomedical potential for designing new molecular tools. Sea anemone venom locates in glandular cells from ectoderm and sub-cellular structures called nematocysts, both of which are distributed throughout the sea anemone body. This characteristic implies challenges because the cells and nematocyst must be lysed to release the venom components with other non-toxic molecules. Therefore, first, the venom is derived from a crude extract (mixture of different and diverse molecules and tissue debris). The next step is to detect polypeptides with specific bioactivities. Here, we describe an efficient strategy to obtain the sea anemone crude extract and bioassay to identify the presence of cytolysins. The first step involves inexpensive and straightforward techniques (stirred and freeze-thaw cycle) to release cytolysins. We obtained the highest cytolytic activity and protein (~500 mg of protein from 20 g of dry weight). Next, the polypeptide complexity of the extract was analyzed by SDS-PAGE gel detecting proteins with molecular weights between 10 kDa and 250 kDa. In the hemolytic assay, we used sheep red blood cells and determined HU50 (11.1 &plusmn; 0.3 &#181;g/mL). In contrast, the presence of phospholipases in the crude extract was determined using egg yolk as a substrate in a solid medium with agarose. Overall, this study uses an efficient and inexpensive protocol to prepare the crude extract and applies replicable bioassays to identify cytolysins, molecules with biotechnological and biomedical interests.

Here, we describe a protocol to obtain crude venom extract from sea anemone and detect its hemolytic and phospholipase activity.

通过直接生物测定法鉴定海葵粗提取物中的溶血和磷脂酶活性

Sea anemone venom composition includes polypeptide and non-proteins molecules. Cytolytic components have a high biotechnological and biomedical potential ...

Lethality Bioassay Using Artemia salina L.

Natural products have been used since ancient times to produce medicines. Nowadays, there are plenty of chemotherapeutic drugs obtained from natural sources and used against a plethora of diseases. Unfortunately, most of these compounds often display systemic toxicity and adverse effects. In order to better evaluate the tolerability of selected potentially bioactive samples, brine shrimp (Artemia salina) is generally used as a model in lethality studies. The A. salina test is based on the ability of the studied bioactive compounds to kill the microcrustaceans in their larval stage (nauplii). This method represents a convenient starting point for cytotoxicity studies, as well as for the general toxicity screening of synthetic, semisynthetic, and natural products. It can be considered a simple, quick, and low-cost assay, compared to many other assays (in vitro cells or yeast strains, zebrafish, rodents) generally suitable for the aforementioned purposes; moreover, it can be easily performed even without any specific training. Overall, A. salina assay represents a useful tool for the preliminary toxicity evaluation of selected compounds and the bio-guided fractionation of natural product extracts.

Lethality Bioassay Using Artemia salina L.

This work aims to evaluate and review the Artemia salina lethality bioassay procedure, also identified as brine shrimp lethality assay. This simple and cheap method gives information about the general toxicity (considered as a preliminary toxicity evaluation) of samples, namely, natural products.

使用 卤虫 盐L的致死性生物测定。

Natural products have been used since ancient times to produce medicines. Nowadays, there are plenty of chemotherapeutic drugs obtained from natural ...

化学

行为的：评估成年斑马鱼神经毒性作用的测试电池

Neurotoxicity Assessment in Adult Danio rerio using a Battery of Behavioral Tests in a Single Tank

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. However, in the last 50 years, the effects of chemicals on the behavior of model species have been actively investigated. Progressively, behavioral endpoints were incorporated into neurotoxicological screening protocols, and these functional outcomes are now routinely used to identify and determine the potential neurotoxicity of chemicals. Behavioral assays in adult zebrafish provide a standardized and reliable means to study a wide range of behaviors, including anxiety, social interaction, learning, memory, and addiction. Behavioral assays in adult zebrafish typically involve placing the fish in an experimental arena and recording and analyzing their behavior using video tracking software. Fish can be exposed to various stimuli, and their behavior can be quantified using a variety of metrics. The novel tank test is one of the most accepted and widely used tests to study anxiety-like behavior in fish. The shoaling and social preference tests are useful in studying the social behavior of zebrafish. This assay is particularly interesting since the behavior of the entire shoal is studied. These assays have proven to be highly reproducible and sensitive to pharmacological and genetic manipulations, making them valuable tools for studying the neural circuits and molecular mechanisms underlying behavior. Additionally, these assays can be used in drug screening to identify compounds that may be potential modulators of behavior.
We will show in this work how to apply behavioral tools in fish neurotoxicology, analyzing the effect of methamphetamine, a recreational drug, and glyphosate, an environmental pollutant. The results demonstrate the significant contribution of behavioral assays in adult zebrafish to the understanding of the neurotoxicological effects of environmental pollutants and drugs, in addition to providing insights into the molecular mechanisms that may alter neuronal function.

作者聚焦：成年斑马鱼环境和药物诱导的行为变化评估 

Here, we present a comprehensive behavioral test battery, including the novel tank, Shoaling, and social preference tests, to effectively determine the potential neurotoxic effects of chemicals (e.g., methamphetamine and glyphosate) on adult zebrafish using a single tank. This method is relevant to neurotoxicity and environmental research.

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

摘要

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多氯联苯同系物净营养转移效率湖鳟鱼实验室估计（

测定掠夺取食行为

生物毒理学方法与海洋细菌

利用生物测定引导分馏法鉴定海洋鳗信息素

检测幼虫行为反应剖面的实验协议: 在神经刺激咖啡因中的应用

通过直接生物测定法鉴定海葵粗提取物中的溶血和磷脂酶活性

使用卤虫盐L的致死性生物测定。

在单个水箱中使用一系列行为测试对成年 Danio rerio 进行神经毒性评估

在单个水箱中使用一系列行为测试对成年 Danio rerio 进行神经毒性评估

在单个水箱中使用一系列行为测试对成年 Danio rerio 进行神经毒性评估

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

摘要

探索更多视频

此视频中的章节

多氯联苯同系物净营养转移效率湖鳟鱼实验室估计（

测定掠夺取食行为

生物毒理学方法与海洋细菌

利用生物测定引导分馏法鉴定海洋鳗信息素

检测幼虫行为反应剖面的实验协议: 在神经刺激咖啡因中的应用

通过直接生物测定法鉴定海葵粗提取物中的溶血和磷脂酶活性

使用 卤虫 盐L的致死性生物测定。

在单个水箱中使用一系列行为测试对成年 Danio rerio 进行神经毒性评估

在单个水箱中使用一系列行为测试对成年 Danio rerio 进行神经毒性评估

在单个水箱中使用一系列行为测试对成年 Danio rerio 进行神经毒性评估

使用卤虫盐L的致死性生物测定。