We thank Dr. Daniel Bumbarger for the predation behavior picture. This work was funded by the Max-Planck Society.

1.口形式表型<ol><li> 嘴形鉴定琼脂糖片 注意:为了形象化线虫口摇身一变，用固定在琼脂糖垫温和的麻醉剂治疗的蠕虫和观察如下。 <ol><li>成长和维护线虫文化，如P. pacificus 6厘米标准线虫生长介质（NGM）板和大肠杆菌的细菌草坪饲料大肠杆菌 OP50 19。 </li><li>通过在一个15毫升管第一加法0.06克琼脂糖至3ml 的 H 2 O操作制作3个×2％琼脂糖溶液使琼脂糖垫。这可以在4°C被存储长达一年</li><li>混合并在微波炉彻底融化琼脂糖或选择使用加热块设置为&gt; 88°C。 </li><li>一旦完全熔化，加入10微升10％的叠氮化钠溶液的琼脂糖调匀。注意:干叠氮化钠是被动的和一切形式是有毒的。 </li><li>用1毫升微量，放置不小于300微升液体琼脂糖叠氮化物混合物一滴到一个标准的玻璃显微镜载片的中间。 </li><li>琼脂冷却之前，迅速地放置在滴的顶部的第二载玻片以弄平其在冷却时形成一个垫的琼脂糖。如需要重复尽可能多垫。 </li><li>只需通过滑动关闭它们彼此使用，果皮分开显微镜玻片之前。注意:如果琼脂糖焊盘提前太多制备它们可以是过于干燥，并可能损坏线虫。 </li><li>蠕虫转移到麻醉剂琼脂糖垫，放置的M9缓冲一滴（2 - 3微升）到垫上的中心位置。选择2 - 3年轻的P. pacificus大人到M9的在焊盘精心放置盖玻片前下降。该P. pacificus线虫会在琼脂糖固定化并准备进行可视化。 </li><li>传送含有麻醉蠕虫显微镜载玻片到适当的显微镜第二下63X光学诺马斯基观察。分类基于以下特征变形标识:一个附加的子腹侧齿，放大背侧齿和广口开口的存在标示着eurystomatous口变形动物的，而一个单一的背齿和窄口开口的存在指示stenostomatous动物（ 图1）。 注意:为了维持动物的健康，蠕虫应保持在不超过5分钟的琼脂垫。 </li><li>口变身经过鉴定，恢复的要求通过轻轻滑动它关闭琼脂糖垫取出盖玻片要么eurystomatous或stenostomatous线虫。仔细挑选从琼脂糖垫所选动物（ 大肠杆菌 OP50可以在拾取被用于帮助使它粘）到新鲜NGM板。允许从麻醉恢复正常之前，能动的行为已经恢复赖以动物准备作进一步化验掠夺。 </li></ol></li> &lt;李&gt; 快速口表型 注意:可替换地，具有更多的经验，口腔形式类型可以，而不需要通过以高倍率（150X）立体显微镜任何麻醉剂治疗测定。 <ol><li>标准NGM板的地方线虫具有E的细菌草坪大肠杆菌 OP50到显微镜的视野。 </li><li>检测口尺寸和宽度的差异。注:在此放大状结构无齿是可观测因此口变形识别是完全基于宽口窄与嘴巴。 </li></ol></li></ol> 2.咬分析注:咬测定允许一个详细的掠夺行为分析。 <ol><li>发展和维护标准NGM板（6厘米）线虫文化和大肠杆菌的细菌草坪饲料大肠杆菌 OP50 19。 </li><li>通过生长选定猎物线虫幼虫大量如C使测定平板线虫或人替换地适当的生态相关的猎物。注:成人C.线虫过大，是合适的猎物所以它使用幼虫阶段是重要的。 <ol><li> C.维护线虫或大肠杆菌的细菌草坪上标准NGM板和进给其他潜在的猎物大肠杆菌 OP50，直到人口新鲜饿死，造成丰盈的年轻L1幼虫。 注意:时间饥饿是依赖于许多环境和实验因素，包括用于启动培养线虫数， 大肠杆菌的量大肠杆菌 OP50，并将环境温度。 </li></ol></li><li>洗四个或更多的新鲜饥饿猎物板与M9和通过两个20微米的过滤器通过蜗杆溶液收集之前，除去所有大的动物和任何剩余的卵在一个15毫升管。只有小幼虫应保持在溶液中。 </li><li>为了形成一个幼虫粒料离心过滤猎物在377 xg离心1分钟。 </li><li&gt;移液器3纯虫丸微升到无E.将6cm NGM板大肠杆菌 OP50目前并等待至少30分钟的幼虫传播出去，足以产生一个检测板。 注:纯虫丸3微升的标准检测板含有&gt; 3000捕食幼虫。这是足以产生捕食者和猎物之间的频繁接触。 </li><li>屏幕掠夺线虫为所需嘴变形（方案1）。 </li><li>使用标准的蠕虫采摘技术和光立体显微镜19，到测定板转移正确分类捕食者。照顾以减少细菌污染转印天敌时尽可能少OP50细菌尽可能转移到测定板。等待15分钟，以允许蜗杆从被传送的应力恢复并检查野生型能动行为，以确保蠕虫没有被从转印损坏。 注:无需挨饿P. PACificus，因为它们是其他线虫幼虫高效大鳄在细菌甚至在吃得饱饱的。 </li><li>恢复后，使用光体视显微镜10分钟观察捕食者。有了这个设备，观察和描述不同的喂养活动，如咬 ，其特点是捕食者限制猎物的运动; 杀害 ，其中由猎物角质层被检测的开口;和馈送 ，由猎物内脏（ 图2A，B和电影1）的可观察到的消耗分类。 </li><li>通过筛选和观察至少10个人掠夺性线虫，以确保准确性重复检测。 </li></ol> 3.尸分析注:尸体化验便于掠夺行为更快速的定量分析。 <ol><li>发展和维护标准的NGM板线虫文化和在细菌L饲料E.芒的大肠杆菌 OP50 19。生成前面提到的测定板（协议2.1 - 2.5）的一式三份。 </li><li>为所需的口变形屏幕掠夺性线虫协议1所描述的使用标准蠕虫采摘技术和必要的口腔变形，以每块测定板轻体视显微镜转移5掠夺性线虫。与猎物2小时离开大鳄在一起。 </li><li> 2小时后屏幕的测定板为空尸体（ 图2B和C）的存在。由于缺乏运动与明显的形态缺陷，包括内脏泄漏或丢失的蠕虫病毒片段一起找出尸体。 </li></ol> 4.咽牙齿移动的分析<ol><li>发展和维护标准的NGM板线虫文化和大肠杆菌的细菌草坪饲料大肠杆菌 OP50 19。生成检测板如以前（协议2.1 - 2.5）提及。如果标准6厘米NGM板不物镜和显微镜载物台之间适合，可使用的含2ml NGM作为合适的替代小35毫米培养皿盖。 </li><li>为所需的口变形屏幕捕食性线虫如方案1中所述使用标准蜗杆采摘技术和光立体显微镜，对到测定板中传输单个正确分类捕食者。等待15分钟，使蠕虫，从被转移的压力中恢复。 </li><li>观察显微镜食肉动物在40 - 63X Normaski，用高速摄像机（ 电影2和3）。记录咽抽水，在15秒内牙移动，​​在至少20个动物50赫兹，以保证精确的定量。重播记录在所需速度的电影，以计算单个泵和牙齿的事件。 注意:泵送在语料库，位于咽的中间观察到的，而牙齿移动是在口开口可检测，仅作业从背齿服务。 </li></ol>

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The authors declare no financial or competing interests.

线虫理解神经生物学和行为与C提供了一个功能强大的系统线虫迄今作为主要工具。然而，众多的线虫种类包括P. pacificus显示的行为，这是缺席或从模式生物C.复杂变化线虫 ，因此提高对这些行为的演变与调控引人入胜的问题。在许多其他线虫种类包括P.发现一个这样的附加 ​​行为pacificus是掠夺性喂养1，20从事以补充他们的饮食细菌的能力。因此，我们已开发出并描述了详细的协议线虫这些以前未经分析的掠夺行为，容易和快速鉴定。 首先，我们提供的方法中的线虫内口馈送装置用于筛选变体。正确口型的鉴定是必不可少的头sTEP成功捕食测定法，至少在该Pristionchus只属动物eurystomatous能够掠夺性喂养。最好是，以确定口腔中的协议1.2作为此方法中描述的"快速嘴表型"协议摇身一变是侵入性的要少得多，因此，它是不太可能掠夺行为可以被扰动。但是，建议首先成为通过识别与在琼脂焊盘麻醉动物（协议1.1）熟悉不同口结构。 以下所需口变形的鉴定，我们已经描述了两种测定用于定量掠夺馈送。这些是一种快速，高通量"尸测定"（协议3）和一个更费时，但更深入的通过"咬合测定"行为分析（方案2）。这两种协议是高度灵活的，允许若干修改，以优化取决于experimen测定法TAL要求。对于使用P.咬测定在C. pacificus大鳄线虫猎物，对于10分钟的时间窗掠夺行为相互作用的观测是足以与其他馈送事件沿量化叮咬的显著量。对于"尸实验"再利用P.在C. pacificus大鳄线虫猎物，5大鳄2小时制作容易量化和一致的尸体数允许快速的行为分析。但是，应该以不同的速度指出，掠夺性线虫的举动不同的物种，吃的速度不同，一般展示其他行为1大的多样性。此外，不同的猎物物种也可以在类似的原因不同速率吃掉。因此，建议以优化基于测试既作为天敌和猎物的线虫物种的测定法，也可用于在环境条件下的任何差异。在这两次"咬"和"军团E"试验是至关重要的捕食与被捕食者是健康的，因为压力或受伤的大鳄将不能有效地杀灭。此外，新鲜的检测板是较旧的板块可以成为干出该线虫导致错误的健康造成不利影响至关重要测定，它也希望这些掠夺性试验的未来迭代将能够采取技术最新进展的优势，以自动执行大部分的分析作为已经完成调查22 C.观察到的许多行为线虫 21，目前如出现敏感得多接触它们，使得在可能废除掠夺馈送的微流体腔室的隔离和固定化的问题有可能在线虫如铜绿pacificus出现。克服此可能被证明具有挑战性的，但会促进个体线虫被筛选为微妙掠夺的行为。 最后，我们还提供了FO方法ř检查通过使用高速摄像机（协议4）定量齿和咽泵送动力学促进捕食性和细菌馈送模式之间的比较的线虫馈送装置本身。在C.咽抽水率的量化线虫已用于监测摄食多年23，但是，C。线虫没有任何形式的口锯齿和也没啥掠夺性的行为。通过结合咽与牙齿活动的泵送的量化，具体到捕食牙齿的任何神经支配也可以观察到。由于观察牙齿移动的动物经常移出焦平面所需的倍率，因此，它通常只能观察为很短的时间窗的齿。此外，不同于C.线虫 ，P的咽pacificus不连续泵，而是在抽水和饲养的法术啮合。因此，对于准确咽PUM的而喂养平速率确定，它记录15秒的连续馈送的是重要的。 这里提出这些方法，因此在线虫系统调查掠夺行为提供第一框架。此外，它们也可以是适于用在调查线虫的生态系统，包括附加生态相关生物体上捕食包括微生物，真菌和螨虫的影响范围内的其他交互。因此，他们提供了剖析这些掠夺行为是如何调节的，他们怎么可能已经进化以及它们的生态意义的手段。

与他们的小而复杂的神经系统线虫已经证明了强大的工具，对于理解神经生物学的许多方面，包括行为。这类研究的大部分都集中在模式生物线虫在其中一个丰富 ​​不同的行为已经被成功地解剖和分析。这些措施包括mechanosensory 3，4趋化，thermotactic 5,6和7磁感影响交配8,9，学习10和取食行为11。然而，其他更远亲线虫物种显示哪些没有在rhabditid C.观察到的行为线虫或交替显示的复杂性，这引起了有关其发展和监管相关问题的其他级别。这方面的一个这样的实例可以在远亲diplogastrid线虫Pristionchus pacificus，其中显示更复杂的馈送是被观察haviors和节奏比C.观察线虫1。尽管这是两个物种同源共享咽神经元12。这些额外的供给行为，巧合的P. pacificus还显示扩展的饮食范围，因为他们是狂热的食肉动物，能够通过喂养也是在其他线虫幼虫补充他们的饮食细菌的。幸运的是，P。 pacificus已发展为进行比较和综合进化生物学模型，因此许多分子和遗传工具现已上市。这些措施包括一个完全测序和基因组注释13，分子和基因工具，包括转基因14和CRISPR / Cas9 15,16以及一个详细和系统发育注释17，拥有超过25密切相关的物种，包括其新发现的物种的姐妹。此外，众多的Pristionchus物种的生态，包括P. pacificus是我们现在有被描述了许多种定义LL与共享金龟子一个necromenic协会，他们经常与其他线虫种类18共享一台主机。P.因此pacificus提供与剖析小说行为的演变及其生态意义的优秀模型系统。 为了分析在线虫物种如铜绿掠夺取食行为pacificus我们开发，便于观察和掠夺行为的量化几种新的行为检测。为P. pacificus显示二形口结构，它强烈地影响掠夺行为，正确的形态类型的识别是必不可少的1,2。窄口stenostomatous变形包含一个钝齿背在任何掠夺性喂养不参与。可替代地，广口eurystomatous变形包括大得多的爪形背齿和一个额外的相对亚腹侧齿，它们一起工作以有效地打开他们的猎物的角质层。掠夺eurystomatous到非掠夺stenostomatous形式的比率Pristionchus物种的不同而不同，也内P. pacificus然而 ，eurystomatous口的百分比变形在P. pacificus野生型菌株（PS312）通常是70 - 90％2。此外，嘴形比可能出现波动取决于不同的环境因素（包括已知的，包括饥饿和一些小分子信号以及未知因素）掠夺eurystomatous口形式，从而正确识别和隔离是成功的掠夺性检测是必不可少的。 除了掠夺口形式的描述中，我们制定了"啃法"的直接观察和掠夺性的行为，包括咬，杀害和饲养活动的定量。这里猎物线虫是通过过滤器隔离ING新饿死文化并暴露在掠夺成人P. pacificus，这是在很短的时间跨度观察在一起。此外，我们还开发了高通量"尸实验"，通过掠夺性事件的间接观测便于掠夺行为快速筛选。这需要幼虫尸体的存在优势，筛选捕食的工具。这两个实验提供了方便和高度可重复的观察和线虫如铜绿测量掠夺行为的方法pacificus。

<table><tbody><tr><td>Nylon net filters (20 &amp;#181;m)</td><td>Merck Millipore Ltd</td><td>NY2004700</td><td>Used to filter worms just leaving larvae for use as prey.</td></tr><tr><td>PP Funnel for filter (54 mm)</td><td>Duran</td><td>292215003</td><td>Used to filter worms just leaving larvae for use as prey.</td></tr><tr><td>Small petri dish (35/10 mm)</td><td>Greiner Bio-One&amp;nbsp;</td><td>627102</td><td>For imaging on High speed camera</td></tr><tr><td>Zeiss SteREO Discovery V12</td><td></td><td></td><td>For mouth form identificaton</td></tr><tr><td>Axio-Imager A1</td><td></td><td></td><td>For mouth form identificaton</td></tr><tr><td>Glass Slides</td><td>Roth</td><td>H869</td><td></td></tr><tr><td>Cover Slips</td><td>Roth</td><td>657</td><td></td></tr><tr><td>Motion Scope M3 Highspeed camera</td><td>IDT</td><td></td><td>High speed camera</td></tr><tr><td>Video zoom 44 ENG 1/2&quot; 0.5X to 2.4X</td><td>Zeis</td><td>452984-0000-000</td><td>High speed camera zoom</td></tr><tr><td>Nematode Growth Medium (NGM) ingredients:</td><td></td><td></td><td></td></tr><tr><td>Agar</td><td>Roth</td><td>5210.2</td><td>CAS-Nr. 9002-18-0</td></tr><tr><td>Sodium chloride (NaCl)</td><td>Roth</td><td>3957</td><td>CAS-Nr. 7647-14-5</td></tr><tr><td>Bacto Tryptone</td><td>BD</td><td>211699</td><td>Lot 4316614</td></tr><tr><td>Calcium chloride dihydrate (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>C3306</td><td>CAS-Nr. 10035-04-8</td></tr><tr><td>Cholesterol from lanolin</td><td>Sigma-Aldrich</td><td>F 26732 00050</td><td>CAS-Nr. 57-88-5</td></tr><tr><td>Magnesium sulfate heptahydrate (MgSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Merck</td><td>1,058,861,000</td><td>CAS-Nr. 10034-99-8</td></tr><tr><td>Potassium dihydrogen phosphate (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>ACROS organics</td><td>271080025</td><td>CAS-Nr. 7778-77-0</td></tr><tr><td>6 cm petri dish</td><td>Greiner Bio-One</td><td>628102</td><td></td></tr><tr><td>3.5 cm petri dish</td><td>Greiner Bio-One</td><td>627102</td><td></td></tr><tr><td>M9 ingredients:</td><td></td><td></td><td></td></tr><tr><td>Potassium dihydrogen phosphate (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>ACROS organics</td><td>271080025</td><td>CAS-Nr. 7778-77-0</td></tr><tr><td>Sodium hydrogen phosphate heptahydrate (NaHPO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S9390-500G-D</td><td>CAS-Nr. 7782-85-6</td></tr><tr><td>Sodium chloride (NaCl)</td><td>Roth</td><td>3957</td><td>CAS-Nr. 7647-14-5</td></tr></tbody></table>

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.

<html>继P.适当的口变形的识别成功pacificus，eurystomatous和stenostomatous动物之间明显的差异可以被检测（ 图3）仅与eurystomatous动物杀死行为接合。在stenostomatous动物这种行为似乎完全被抑制。此外，在eurystomatous动物对细菌和猎物齿活性和咽泵送差异（ 图4和影2和3）也很明显。而掠夺馈送，泵送速率降低低于在一比一的比例与咽泵送检测细菌馈送和牙齿移动过程中观察到。这是潜在的指示调节到不同的饮食行为反应的关键调节机制。 S / ftp_upload / 54404 / 54404fig1.jpg"/&gt; 图1. P. pacificus 有嘴异形从而影响摄食行为。 （一）eurystomatous口形态能捕食，并与大爪形背齿（假色红色）和（B）的大型对方钩形亚腹侧齿（假色蓝色）宽口的开口。 （三）stenostomatous口形式只能对细菌饲料，并与火石形背齿（假色红色）和（D）没有子腹齿（*）较窄张口。 Normaski图像63X和比例尺条为10微米。 <a href="https://www.jove.com/files/ftp_upload/54404/54404fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54404/54404fig2.jpg" /> <str翁&gt;图2. 捕食检测。（A）P. pacificus咬伤并杀死其他线虫，如C.幼虫线虫。（B）对于咬测定，可以使用光立体显微镜和成功的杀伤和喂食事件还记录观察bitesby食肉 ​​动物（*）的数量。尸体也清晰可见（圆圈）。（C）对于尸体实验，幼虫尸体（箭头）很容易识别相比，活幼虫。比例尺代表B中为1mm，150微米C. <a href="https://www.jove.com/files/ftp_upload/54404/54404fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54404/54404fig3.jpg" /> 图3. 咬尸体化验对C.结果线虫猎物。 （A）咬行为仅仅是显而易见的<html>与这种行为eurystomatous口形式stenostomatous动物不显示。错误条代表的10个重复的标准差。（B），没有咬行为从stenostomatous动物明显之际，尸体实验也表明只对eurystomatous动物实验板尸体。错误条代表5次重复的标准差。 <a href="https://www.jove.com/files/ftp_upload/54404/54404fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54404/54404fig4.jpg" /> 而eurystomatous动物从事掠夺性喂养图4 Eurystomatous掠夺饲养过程中抽气速率和牙齿移动，牙移动只观察到。这也与咽抽下降一致。错误条代表的10个重复的标准偏差。<html> HREF ="htt​​ps://www.jove.com/files/ftp_upload/54404/54404fig4large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="电影1" src="/files/ftp_upload/54404/54404movie1.jpg" /> <a href="https://www.jove.com/files/ftp_upload/54404/Movie_1_Killing_behaviour.mp4">电影1 杀害了使用轻体视显微镜测定撕咬行为的观察。（右键点击下载）。</a> <img alt="电影2" src="/files/ftp_upload/54404/54404movie2.jpg" /> <a href="https://www.jove.com/files/ftp_upload/54404/Movie_2_HS_Camera_Killing.mp4">P.的电影2.高速摄像机电影pacificus杀C.线虫幼虫。（右键点击下载）。</a> 广告/ 54404 / 54404movie3.jpg"/&gt; <a href="https://www.jove.com/files/ftp_upload/54404/Movie_3_HS_pumping_and_tooth.mp4">电影3.放大牙齿移动过程中捕食的高速摄像机。（右键点击下载）。</a>

Watch this Scientific Journal Video about Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes at JoVE.com

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 ...

assaying-predatory-feeding-behaviors-pristionchus-other

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Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes

9.7K 次观看.  Max-Planck Institute for Developmental Biology. 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. 

视频: Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes

Soil Sampling and Isolation of Entomopathogenic Nematodes (Steinernematidae, Heterorhabditidae)

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to two families: Steinernematidae and Heterorhabditidae. Until now, more than 70 species have been described in the Steinernematidae and there are about 20 species in the Heterorhabditidae. The nematodes have a mutualistic partnership with Enterobacteriaceae bacteria and together they act as a potent insecticidal complex that kills a wide range of insect species.
Herein, we focus on the most common techniques considered for collecting EPN from soil. The second part of this presentation focuses on the insect-baiting technique, a widely used approach for the isolation of EPN from soil samples, and the modified White trap technique which is used for the recovery of these nematodes from infected insects. These methods and techniques are key steps for the successful establishment of EPN cultures in the laboratory and also form the basis for other bioassays that consider these nematodes as model organisms for research in other biological disciplines. The techniques shown in this presentation correspond to those performed and/or designed by members of S. P. Stock laboratory as well as those described by various authors.

Soil Sampling and Isolation of Entomopathogenic Nematodes Steinernematidae, Heterorhabditidae

Entomopathogenic nematodes are soil-inhabiting roundworms that parasitize a wide range of insects. We demonstrate sampling methods used for the isolation of these nematodes from the soil using two techniques: the insect baiting and the modified White trap, for the recovery of nematodes from soil samples and infected insect cadavers, respectively.

土壤采样及昆虫病原线虫的分离（Steine​​rnematidae，异小）

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to ...

科研

环境科学

Automated Analysis of a Nematode Population-based Chemosensory Preference Assay

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection of the neural circuits underlying these behaviors, the development of robust and reproducible behavioral assays is necessary. Previous C. elegans behavioral studies have used variations of a "drop test", a "chemotaxis assay", and a "retention assay" to investigate the response of C. elegans to soluble compounds. The method described in this article seeks to combine the complementary strengths of the three aforementioned assays. Briefly, a small circle in the middle of each assay plate is divided into four quadrants with the control and experimental solutions alternately placed. After the addition of the worms, the assay plates are loaded into a behavior chamber where microscope cameras record the worms' encounters with the treated regions. Automated video analysis is then performed and a preference index (PI) value for each video is generated. The video acquisition and automated analysis features of this method minimizes the experimenter's involvement and any associated errors. Furthermore, minute amounts of the experimental compound are used per assay and the behavior chamber's multi-camera setup increases experimental throughput. This method is particularly useful for conducting behavioral screens of genetic mutants and novel chemical compounds. However, this method is not appropriate for studying stimulus gradient navigation due to the close proximity of the control and experimental solution regions. It should also not be used when only a small population of worms is available. While suitable for assaying responses only to soluble compounds in its current form, this method can be easily modified to accommodate multimodal sensory interaction and optogenetic studies. This method can also be adapted to assay the chemosensory responses of other nematode species.

We present a behavior recording setup and protocol that enables automated analysis of the nematode, Caenorhabditis elegans' preference for soluble compounds in a population-based assay. This article describes the construction of a behavior chamber, the behavioral assay protocol, and video analysis software usage.

基于线虫人群的化学感应优选测定的自动分析

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection ...

行为学

Rapid Isolation of Wild Nematodes by Baermann Funnel

Beyond being robust experimental model organisms, Caenorhabditis elegans and its relatives are also real animals that live in nature. Studies of wild nematodes in their natural environments are valuable for understanding many aspects of biology, including the selective regimes in which distinctive genomic and phenotypic characters evolve, the genetic basis for complex trait variation, and the natural genetic diversity fundamental to all animal populations. This manuscript describes a simple and efficient method for extracting nematodes from their natural substrates, including rotting fruits, flowers, fungi, leaf litter, and soil. The Baermann funnel method, a classical nematology technique, selectively isolates active nematodes from their substrates. Because it recovers nearly all active worms from the sample, the Baermann funnel technique allows for the recovery of rare and slow-growing genotypes that co-occur with abundant and fast-growing genotypes, which might be missed in extraction methods that involve multiple generations of reproduction. The technique is also well suited to addressing metagenetic, population-genetic, and ecological questions. It captures the entire population in a sample simultaneously, allowing an unbiased view of the natural distribution of ages, sexes, and genotypes. The protocol allows for deployment at scale in the field, rapidly converting substrates into worm plates, and the authors have validated it through fieldwork on multiple continents.

This protocol outlines a method for efficiently extracting live nematodes from natural substrates in the field.

由Baermann Funnel快速分离野生线虫

Beyond being robust experimental model organisms, Caenorhabditis elegans and its relatives are also real animals that live in nature. Studies of wild ...

遗传学

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in cellular and mammalian models. Here, we describe detailed methods to perform stable isotopic profiling of intermediary metabolism and metabolic flux in the nematode, Caenorhabditis elegans. Methods are described for profiling whole worm free amino acids, labeled carbon dioxide, labeled organic acids, and labeled amino acids in animals exposed to stable isotopes either from early development on nematode growth media agar plates or beginning as young adults while exposed to various pharmacologic treatments in liquid culture. Free amino acids are quantified by high performance liquid chromatography (HPLC) in whole worm aliquots extracted in 4% perchloric acid. Universally labeled 13C-glucose or 1,6-13C2-glucose is utilized as the stable isotopic precursor whose labeled carbon is traced by mass spectrometry in carbon dioxide (both atmospheric and dissolved) as well as in metabolites indicative of flux through glycolysis, pyruvate metabolism, and the tricarboxylic acid cycle. Representative results are included to demonstrate effects of isotope exposure time, various bacterial clearing protocols, and alternative worm disruption methods in wild-type nematodes, as well as the relative extent of isotopic incorporation in mitochondrial complex III mutant worms (isp-1(qm150)) relative to wild-type worms. Application of stable isotopic profiling in living nematodes provides a novel capacity to investigate at the whole animal level real-time metabolic alterations that are caused by individual genetic disorders and/or pharmacologic therapies.

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Stable isotopic profiling by gas chromatography mass spectrometric analysis of intermediary metabolic flux is described in the nematode, Caenorhabditis elegans. Methods are detailed for assessing isotopic enrichment in carbon dioxide, organic acids, and amino acids following isotope exposure either during development on agar plates or during adulthood in liquid culture.

在开发和成年阶段的中介代谢通量的稳定同位素分析线虫</em

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in ...

生物学

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their locomotory patterns upon detection of diverse environmental stimuli and can modulate their suite of behavioral responses in response to starvation conditions. Nematodes typically exhibit a decreased aversive response when removed from a food source for over 30 min. Observation of behavioral changes in response to a changing nutritional status can provide insight into the mechanisms that regulate the transition from a well-fed to starved state.
We have developed an assay that measures a nematode&#39;s ability to cross an aversive barrier (i.e. copper) then reach a food source over a prolonged period of time. This protocol builds upon previous work by integrating multiple variables in a manner that allows for continued data collection as the organisms shift towards an increasingly starved condition. Moreover, this assay permits an increased sample size so that larger populations of nematodes can be simultaneously evaluated.
Organisms defective for the ability to detect or respond to copper immediately cross the chemical barrier, while wild type nematodes are initially repelled. As wild type worms are increasingly starved, they begin to cross the barrier and reach the food source. We designed this assay to evaluate a mutant that is incapable of responding to diverse environmental cues, including food sensation or detection of aversive chemicals. When evaluated via this protocol, the defective organisms immediately crossed the barrier, but were also incapable of detecting a food source. Hence, these mutants repeatedly cross the chemical barrier despite temporarily reaching a food source. This assay can straightforwardly test populations of worms to evaluate potential pathway defects related to aversion and starvation.

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

Here, we present a Caenorhabditis elegans-specific assay designed to evaluate changes in copper aversion behavior and the ability to locate a common food source, as the organism progresses from a well-fed to starved nutritional state.

一个<em秀丽隐杆线虫</em基于营养状态的铜反转测定

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their ...

神经科学

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. To improve the mating efficiency of B. Xylophilus and to increase the chances of mating observation, virgin adults were cultured and used for the investigation. Eggs were obtained by keeping the nematodes in water and allowing the females to lay eggs for 10 min. The second-stage juveniles (J2) were synchronized by incubating the eggs for 24 h at 25 &#176;C in the dark, and the early J4 were obtained by culturing the J2 with grey mold, Botrytis cinerea, for another 52 h. At this time point, most J4 nematodes could be clearly distinguished as being male or female using their genital morphology. The male and female J4 were collected and cultured separately in two different Petri dishes for 24 h to get virgin adult nematodes. A virgin male and a virgin female were paired in a drop of water in the well of a concave slide. The mating behavior was filmed with a video recorder under a stereomicroscope. The whole period of the mating process was 82.8 &#177;3.91 min (mean &#177;SE) and could be divided into 4 different phases: searching, contacting, copulating, and lingering. The mean minutes of duration were 21.8 &#177; 2.0, 28.0 &#177; 1.9, 25.8 &#177; 0.7 and 7.2 &#177; 0.5, respectively. Eleven sub-behaviors were described: cruising, approaching, encountering, touching, hooping, locating, attaching, ejaculating, separating, quiescence, and roaming. Interestingly, obvious intra-sexual competition was observed when one female was grouped with 3 males or one male with 3 females. This protocol is useful and valuable, not only in investigating the mating behavior of B. xylophilus, but also in acting as a reference for ethological studies of other nematodes.

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

A protocol for investigating the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, is presented. Behavioral features of B. xylophilus are described in the mating process.

观察和交配行为的量化在我国松材线虫，<em&gt;松材线虫</em

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. ...

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

用基团特异 rDNA 引物对水生无脊椎动物进行遗传肠道含量分析的实验室规程

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...

A Highly Scalable Approach to Perform Ecological Surveys of Selfing Caenorhabditis Nematodes

Caenorhabditis elegans is one of the major model organisms in biology, but only recently have researchers focused on its natural ecology. The relative sparsity of information about C. elegans in its natural context comes from the challenges involved in the identification of the small nematode in nature. Despite these challenges, an increasing focus on the ecology of C. elegans has caused a wealth of new information regarding its life outside of the laboratory. The intensified search for C. elegans in nature has contributed to the discovery of many new Caenorhabditis species and revealed that congeneric nematodes frequently cohabitate in the wild, where they feed on microbial blooms associated with rotting plant material. The identification of new species has also revealed that the androdioecious mating system of males and self-fertilizing hermaphrodites has evolved three times independently within Caenorhabditis. The other two selfing species, C. briggsae and C. tropicalis, share the experimental advantages of C. elegans and have enabled comparative studies into the mechanistic basis of important traits, including self-fertilization. Despite these advances, much remains to be learned about the ecology and natural diversity of these important species. For example, we still lack functional information for many of their genes, which might only be attained through an understanding of their natural ecology. To facilitate ecological research of selfing Caenorhabditis nematodes, we developed a highly scalable method to collect nematodes from the wild. Our method makes use of mobile data collection platforms, cloud-based databases, and the R software environment to enhance researchers&#39; ability to collect nematodes from the wild, record associated ecological data, and identify wild nematodes using molecular barcodes.

A Highly Scalable Approach to Perform Ecological Surveys of Selfing Caenorhabditis Nematodes

This protocol can be used to perform large-scale ecological surveys of selfing Caenorhabditis nematodes. The primary advantage of this method is the efficient organization and analysis of ecological and molecular data associated with the nematodes collected from nature.

一种高度可扩展的方法，用于对自发 性Caenorhabditis 线虫进行生态调查

Caenorhabditis elegans is one of the major model organisms in biology, but only recently have researchers focused on its natural ecology. The relative ...

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant matter, especially rotten fruits like apples or on compost heaps. It is also associated with certain invertebrate hosts such as slugs and woodlice. These habitats are rich in microbes, which serve as food for C. elegans and which can also persistently colonize the nematode gut. To date, the exact diversity and consistency of the native C. elegans microbiota across habitats and geographic locations is not fully understood. Here, we describe a suitable approach for isolating C. elegans from nature and characterizing the microbiota of worms. Nematodes can be easily isolated from compost material, rotting apples, slugs, or attracted by placing apples on compost heaps. The prime time for finding C. elegans in the Northern Hemisphere is from September until November. Worms can be washed out of collected substrate material by immersing the substrate in buffer solution, followed by the collection of nematodes and their transfer onto nematode growth medium or PCR buffer for subsequent analysis. We further illustrate how the samples can be used to isolate and purify the worm-associated microorganisms and to process worms for 16S ribosomal RNA analysis of microbiota community composition. Overall, the described methods may stimulate new research on the characterization of the C. elegans microbiota across habitats and geographic locations, thereby helping to obtain a comprehensive understanding of the diversity and stability of the nematode's microbiota as a basis for future functional research.

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

Caenorhabditis elegans is one of the main model species in biology, yet almost all research is performed in the absence of its naturally associated microbes. The methods described here will help to improve our understanding of the diversity of associated microbes as a basis for future functional C. elegans research.

模型线虫秀丽隐杆线虫天然微生物群的分离和表征

The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant ...

使用狗粮琼脂培养基研究线虫的集体行为

The Large-Scale Cultivation of Nematodes to Study Their Collective Behaviors

Animals exhibit dynamic collective behaviors, as observed in flocks of birds, schools of fish, and crowds of humans. The collective behaviors of animals have been investigated in the fields of both biology and physics. In the laboratory, researchers have used various model animals such as the fruit fly and zebrafish for approximately a century, but it has remained a major challenge to study large-scale complex collective behavior orchestrated by these genetically tractable model animals. This paper presents a protocol to create an experimental system of collective behaviors in Caenorhabditis elegans. The propagated worms climb on the lid of the Petri plate and show collective swarming behavior. The system also controls worm interactions and behaviors by changing the humidity and light stimulation. This system allows us to examine the mechanisms underlying collective behaviors by changing environmental conditions and examining the effects of individual-level locomotion on collective behaviors using mutants. Thus, the system is useful for future research in both physics and biology.

作者聚焦：线虫 Caenorhabditis elegans 的集体行为分析

Here, a system is reported for studying the collective behaviors of nematodes by culturing them in bulk using dog food agar medium. This system allows researchers to propagate large numbers of dauer worms and can be applied to Caenorhabditis elegans and other related species.

大规模培养线虫以研究其集体行为

Animals exhibit dynamic collective behaviors, as observed in flocks of birds, schools of fish, and crowds of humans. The collective behaviors of animals ...

测定掠夺取食行为

摘要

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此视频中的章节

土壤采样及昆虫病原线虫的分离（Steinernematidae，异小）

基于线虫人群的化学感应优选测定的自动分析

由Baermann Funnel快速分离野生线虫

在开发和成年阶段的中介代谢通量的稳定同位素分析线虫

一个

观察和交配行为的量化在我国松材线虫，

用基团特异 rDNA 引物对水生无脊椎动物进行遗传肠道含量分析的实验室规程

一种高度可扩展的方法，用于对自发性Caenorhabditis 线虫进行生态调查

模型线虫秀丽隐杆线虫天然微生物群的分离和表征

大规模培养线虫以研究其集体行为

测定掠夺取食行为

摘要

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此视频中的章节

土壤采样及昆虫病原线虫的分离（Steine​​rnematidae，异小）

基于线虫人群的化学感应优选测定的自动分析

由Baermann Funnel快速分离野生线虫

在开发和成年阶段的中介代谢通量的稳定同位素分析线虫

一个

观察和交配行为的量化在我国松材线虫，

用基团特异 rDNA 引物对水生无脊椎动物进行遗传肠道含量分析的实验室规程

一种高度可扩展的方法，用于对自发 性Caenorhabditis 线虫进行生态调查

模型线虫秀丽隐杆线虫天然微生物群的分离和表征

大规模培养线虫以研究其集体行为

土壤采样及昆虫病原线虫的分离（Steinernematidae，异小）

一种高度可扩展的方法，用于对自发性Caenorhabditis 线虫进行生态调查