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 border="0" cellpadding="0" cellspacing="0" width="573">
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		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Nylon net filters (20 um)</td>
			<td style="width:71px;">Merck Millipore Ltd</td>
			<td style="width:119px;">NY2004700</td>
			<td style="width:167px;">Used to filter worms just leaving larvae for use as prey.</td>
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			<td height="21" style="height:21px;width:216px;">PP Funnel for filter (54mm)</td>
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			<td>Used to filter worms just leaving larvae for use as prey.</td>
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			<td height="42" style="height:42px;">Small petri dish (35/10 mm)</td>
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			<td>For imaging on High speed camera</td>
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			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>For mouth form identificaton</td>
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			<td height="42" style="height:42px;width:216px;">Nematode Growth Medium (NGM) ingredients:</td>
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			<td>CAS-Nr. 9002-18-0</td>
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			<td height="21" style="height:21px;width:216px;">Sodium chloride (NaCl)</td>
			<td style="width:71px;">Roth</td>
			<td align="right" style="width:119px;">3957</td>
			<td>CAS-Nr. 7647-14-5</td>
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			<td style="width:71px;">Sigma-Aldrich</td>
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			<td>CAS-Nr. 10035-04-8</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cholesterol from lanolin</td>
			<td style="width:71px;">Sigma-Aldrich</td>
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			<td>CAS-Nr. 10034-99-8</td>
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			<td>CAS-Nr. 7778-77-0</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">6 cm petri dish</td>
			<td style="width:71px;">Greiner Bio-One</td>
			<td align="right" style="width:119px;">628102</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">3.5 cm petri dish</td>
			<td style="width:71px;">Greiner Bio-One</td>
			<td align="right" style="width:119px;">627102</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">M9 ingredients:</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Potassium dihydrogen phosphate (KH2PO4)</td>
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			<td align="right" style="width:119px;">271080025</td>
			<td>CAS-Nr. 7778-77-0</td>
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			<td height="42" style="height:42px;width:216px;">Sodium hydrogen phosphate heptahydrate (NaHPO4)</td>
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			<td>CAS-Nr. 7782-85-6</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium chloride (NaCl)</td>
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			<td align="right" style="width:119px;">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 ...

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JoVE Journal

Environment

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

Optimize Flue Gas Settings to Promote Microalgae Growth in Photobioreactors via Computer Simulations

Flue gas from power plants can promote algal cultivation and reduce greenhouse gas emissions1. Microalgae not only capture solar energy more efficiently than plants3, but also synthesize advanced biofuels2-4. Generally, atmospheric CO2 is not a sufficient source for supporting maximal algal growth5. On the other hand, the high concentrations of CO2 in industrial exhaust gases have adverse effects on algal physiology. Consequently, both cultivation conditions (such as nutrients and light) and the control of the flue gas flow into the photo-bioreactors are important to develop an efficient &ldquo;flue gas to algae&rdquo; system. Researchers have proposed different photobioreactor configurations4,6 and cultivation strategies7,8 with flue gas. Here, we present a protocol that demonstrates how to use models to predict the microalgal growth in response to flue gas settings. We perform both experimental illustration and model simulations to determine the favorable conditions for algal growth with flue gas. We develop a Monod-based model coupled with mass transfer and light intensity equations to simulate the microalgal growth in a homogenous photo-bioreactor. The model simulation compares algal growth and flue gas consumptions under different flue-gas settings. The model illustrates: 1) how algal growth is influenced by different volumetric mass transfer coefficients of CO2; 2) how we can find optimal CO2 concentration for algal growth via the dynamic optimization approach (DOA); 3) how we can design a rectangular on-off flue gas pulse to promote algal biomass growth and to reduce the usage of flue gas. On the experimental side, we present a protocol for growing Chlorella under the flue gas (generated by natural gas combustion). The experimental results qualitatively validate the model predictions that the high frequency flue gas pulses can significantly improve algal cultivation.

Flue gas from power plants is a cheap CO2 source for algal growth. We have built prototype &#34;flue gas to algal cultivation&#34; systems and described how to scale up the algal cultivation process. We have demonstrated the use of a mass-transfer bio-reaction model to simulate and to design the optimal operation of flue gas for the growth of Chlorella sp. in algal photo-bioreactors.

优化烟气设置通过电脑模拟来促进微藻生长的光生物反应器中

Flue gas from power plants can promote algal cultivation and reduce greenhouse gas emissions1. Microalgae not only capture solar energy more efficiently ...

科研

环境科学

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 ...

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

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

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

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

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

Quantification of Heavy Metals and Other Inorganic Contaminants on the Productivity of Microalgae

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages include high potential yield, use of non-arable land and integration with waste streams. The nutrient requirements of a large-scale microalgae production system will require the coupling of cultivation systems with industrial waste resources, such as carbon dioxide from flue gas and nutrients from wastewater. Inorganic contaminants present in these wastes can potentially lead to bioaccumulation in microalgal biomass negatively impact productivity and limiting end use. This study focuses on the experimental evaluation of the impact and the fate of 14 inorganic contaminants (As, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Sb, Se, Sn, V and Zn) on Nannochloropsis salina growth. Microalgae were cultivated in photobioreactors illuminated at 984 &#181;mol m-2 sec-1 and maintained at pH 7 in a growth media polluted with inorganic contaminants at levels expected based on the composition found in commercial coal flue gas systems. Contaminants present in the biomass and the medium at the end of a 7 day growth period were analytically quantified through cold vapor atomic absorption spectrometry for Hg and through inductively coupled plasma mass spectrometry for As, Cd, Co, Cr, Cu, Mn, Ni, Pb, Sb, Se, Sn, V and Zn. Results show N. salina is a sensitive strain to the multi-metal environment with a statistical decrease in biomass yieldwith the introduction of these contaminants. The techniques presented here are adequate for quantifying algal growth and determining the fate of inorganic contaminants.

Integration of microalgal cultivation with industrial flue gas will ultimately introduce heavy metals and other inorganic compounds into the growth media. This study presents a procedure used to determine the end fate and impact of heavy metals and inorganic contaminants on the growth of Nannochloropsis salina grown in photobioreactors.

重金属和微藻的产其他无机污染物的量化

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages ...

Double-stranded RNA Oral Delivery Methods to Induce RNA Interference in Phloem and Plant-sap-feeding Hemipteran Insects

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran insects account for a number of economically substantial pests of plants that cause damage to crops by feeding on phloem sap. The brown marmorated stink bug (BMSB), Halyomorpha halys (Heteroptera: Pentatomidae) and the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae) are hemipteran insect pests introduced in North America, where they are an invasive agricultural pest of high-value specialty, row, and staple crops and citrus fruits, as well as a nuisance pest when they aggregate indoors. Insecticide resistance in many species has led to the development of alternate methods of pest management strategies. Double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) is a gene silencing mechanism for functional genomic studies that has potential applications as a tool for the management of insect pests. Exogenously synthesized dsRNA or small interfering RNA (siRNA) can trigger highly efficient gene silencing through the degradation of endogenous RNA, which is homologous to that presented. Effective and environmental use of RNAi as molecular biopesticides for biocontrol of hemipteran insects requires the in vivo delivery of dsRNAs through feeding. Here we demonstrate methods for delivery of dsRNA to insects: loading of dsRNA into green beans by immersion, and absorbing of gene-specific dsRNA with oral delivery through ingestion. We have also outlined non-transgenic plant delivery approaches using foliar sprays, root drench, trunk injections as well as clay granules, all of which may be essential for sustained release of dsRNA. Efficient delivery by orally ingested dsRNA was confirmed as an effective dosage to induce a significant decrease in expression of targeted genes, such as juvenile hormone acid O-methyltransferase (JHAMT) and vitellogenin (Vg). These innovative methods represent strategies for delivery of dsRNA to use in crop protection and overcome environmental challenges for pest management.

This article demonstrates novel techniques developed for oral delivery of double-stranded RNA (dsRNA) through the vascular tissues of plants for RNA interference (RNAi) in phloem sap feeding insects.

双链 rna 口服法诱导韧皮部和植物-sap 喂养 Hemipteran 昆虫 rna 干扰

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran ...

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current measures to combat such pathogens have proved too conservative and, thus, not sufficiently effective, and they can potentially pose environmental risks. Therefore, it is extremely necessary to identify host-resistance factors to assist in controlling plant diseases naturally through the identification of resistant germplasm, the isolation and characterization of resistance genes, and the molecular breeding of resistant cultivars. In this regard, there is need to establish an accurate, rapid, and large-scale inoculation method to breed and develop plant resistance genes. The rice blast fungal pathogen Magnaporthe grisea causes severe disease symptoms and yield losses. Recently, M. grisea has emerged as a model organism for studying the mechanisms of plant-fungal pathogen interactions. Hence, we report the development of a plant virulence test method that is specific for M. grisea. This method provides for both spray inoculation with a conidial suspension and wounding inoculation with mycelium cubes or droplets of conidial suspension. The key step of the wounding inoculation method for detached rice leaves is to make wounds on plant leaves, which avoids any interference caused by host penetration resistance. This spray/wounding protocol contributes to the rapid, accurate, and large-scale screening of the pathotypes of M. grisea isolates. This integrated and systematic plant infection method will serve as an excellent starting point for gaining a broad perspective of issues in plant pathology.

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Here, we present a protocol to test plant virulence with the plant pathogen Magnaporthe grisea. This report will contribute to the large-scale screening of the pathotypes of fungi isolates and serve as an excellent starting point for understanding the resistant mechanisms of plants during molecular breeding.

植物感染试验: 以植物病原菌为载体的喷雾和伤口介导接种

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current ...

Harvesting Venom Toxins from Assassin Bugs and Other Heteropteran Insects

Heteropteran insects such as assassin bugs (Reduviidae) and giant water bugs (Belostomatidae) descended from a common predaceous and venomous ancestor, and the majority of extant heteropterans retain this trophic strategy. Some heteropterans have transitioned to feeding on vertebrate blood (such as the kissing bugs, Triatominae; and bed bugs, Cimicidae) while others have reverted to feeding on plants (most Pentatomomorpha). However, with the exception of saliva used by kissing bugs to facilitate blood-feeding, little is known about heteropteran venoms compared to the venoms of spiders, scorpions and snakes.
One obstacle to the characterization of heteropteran venom toxins is the structure and function of the venom/labial glands, which are both morphologically complex and perform multiple biological roles (defense, prey capture, and extra-oral digestion). In this article, we describe three methods we have successfully used to collect heteropteran venoms. First, we present electrostimulation as a convenient way to collect venom that is often lethal when injected into prey animals, and which obviates contamination by glandular tissue. Second, we show that gentle harassment of animals is sufficient to produce venom extrusion from the proboscis and/or venom spitting in some groups of heteropterans. Third, we describe methods to harvest venom toxins by dissection of anaesthetized animals to obtain the venom glands. This method is complementary to other methods, as it may allow harvesting of toxins from taxa in which electrostimulation and harassment are ineffective. These protocols will enable researchers to harvest toxins from heteropteran insects for structure-function characterization and possible applications in medicine and agriculture.

Although many insects in the suborder Heteroptera (Insecta: Hemiptera) are venomous, their venom composition and the functions of their venom toxins are mostly unknown. This protocol describes methods to harvest heteropteran venoms for further characterization, using electrostimulation, harassment, and gland dissection.

从刺客臭虫和其他 Heteropteran 昆虫身上采集毒液毒素

Heteropteran insects such as assassin bugs (Reduviidae) and giant water bugs (Belostomatidae) descended from a common predaceous and venomous ancestor, ...

Design and Use of an Apparatus for Quantifying Bivalve Suspension Feeding at Sea

As shellfish aquaculture moves from coastal embayments and estuaries to offshore locations, the need to quantify ecosystem interactions of farmed bivalves (i.e., mussels, oysters, and clams) presents new challenges. Quantitative data on the feeding behavior of suspension-feeding mollusks is necessary to determine important ecosystem interactions of offshore shellfish farms, including their carrying capacity, the competition with the zooplankton community, the availability of trophic resources at different depths, and the deposition to the benthos. The biodeposition method is used to quantify feeding variables in suspension-feeding bivalves in a natural setting and represents a more realistic proxy than laboratory experiments. This method, however, relies upon a stable platform to satisfy the requirements that water flow rates supplied to the shellfish remain constant and the bivalves are undisturbed. A flow-through device and process for using the biodeposition method to quantify the feeding of bivalve mollusks were modified from a land-based format for shipboard use by building a two-dimensional gimbal table around the device. Planimeter data reveal a minimal pitch and yaw of the chambers containing the test shellfish despite boat motion, the flow rates within the chambers remain constant, and operators are able to collect the biodeposits (feces and pseudofeces) with sufficient consistency to obtain accurate measurements of the bivalve clearance, filtration, selection, ingestion, rejection, and absorption at offshore shellfish aquaculture sites.

A flow-through device for using the biodeposition method to quantify filtration and feeding behavior of bivalve mollusks was modified for shipboard use. A two-dimensional gimbal table built around the device isolates the apparatus from boat motion, thereby allowing the accurate quantification of bivalve filtration variables at offshore shellfish aquaculture sites.

海上双壳贝类悬浮饲料定量装置的设计与应用

As shellfish aquaculture moves from coastal embayments and estuaries to offshore locations, the need to quantify ecosystem interactions of farmed bivalves ...

Leaf Area Index Estimation Using Three Distinct Methods in Pure Deciduous Stands

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for describing the vegetation structure in the fields of ecology, forestry, and agriculture. Therefore, procedures of three commercially used methods (litter traps, needle technique, and a plant canopy analyzer) for performing LAI estimation were presented step-by-step. Specific methodological approaches were compared, and their current advantages, controversies, challenges, and future perspectives were discussed in this protocol. Litter traps are usually deemed as the reference level. Both the needle technique and the plant canopy analyzer (e.g., LAI-2000) frequently underestimate LAI values in comparison with the reference. The needle technique is easy to use in deciduous stands where the litter completely decomposes each year (e.g., oak and beech stands). However, calibration based on litter traps or direct destructive methods is necessary. The plant canopy analyzer is a commonly used device for performing LAI estimation in ecology, forestry, and agriculture, but is subject to potential error due to foliage clumping and the contribution of woody elements in the field of view (FOV) of the sensor. Eliminating these potential error sources was discussed. The plant canopy analyzer is a very suitable device for performing LAI estimations at the high spatial level, observing a seasonal LAI dynamic, and for long-term monitoring of LAI.

An accurate estimation of leaf area index (LAI) is crucial for many models of material and energy fluxes within plant ecosystems and between an ecosystem and the atmospheric boundary layer. Therefore, three methods (litter traps, needle technique, and PCA) for taking precise LAI measurements were in the presented protocol.

在纯落叶支架中使用三种截然不同的方法估算叶面积指数

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for ...

A Small-Scale Setup for Algal Toxicity Testing of Nanomaterials and Other Difficult Substances

Ecotoxicity data is a requirement for pre- and post-market registration of chemicals by European and international regulations (e.g., REACH). The algal toxicity test is frequently used in regulatory risk assessment of chemicals. In order to achieve high reliability and reproducibility the development of standardized guidelines is vital. For algal toxicity testing, the guidelines require stable and uniform conditions of parameters such as pH, temperature, carbon dioxide levels and light intensity. Nanomaterials and other so-called difficult substances can interfere with light causing a large variation in results obtained hampering their regulatory acceptance. To address these challenges, we have developed LEVITATT (LED Vertical Illumination Table for Algal Toxicity Tests). The setup utilizes LED illumination from below allowing for a homogenous light distribution and temperature control while also minimizing intra-sample shading. The setup optimizes the sample volume for biomass quantification and does at the same time ensure a sufficient influx of CO2 to support exponential growth of the algae. Additionally, the material of the test containers can be tailored to minimize adsorption and volatilization. When testing colored substances or particle suspensions, the use of LED lights also allows for increasing the light intensity without additional heat generation. The compact design and minimal equipment requirements increase the possibilities for implementation of the LEVITATT in a wide range of laboratories. While compliant with standardized ISO and OECD guidelines for algal toxicity testing, LEVITATT also showed a lower inter-sample variability for two reference substances (3,5-Dicholorophenol and K2Cr2O7) and three nanomaterials (ZnO, CeO2, and BaSO4) compared to Erlenmeyer flasks and microtiter plates.

We demonstrate algal toxicity testing for difficult substances (e.g., colored substances or nanomaterials) using a setup illuminated vertically with an LED.

纳米材料和其他难物质藻类毒性试验的小型装置

Ecotoxicity data is a requirement for pre- and post-market registration of chemicals by European and international regulations (e.g., REACH). The algal ...

测定掠夺取食行为

摘要

探索更多视频

此视频中的章节

优化烟气设置通过电脑模拟来促进微藻生长的光生物反应器中

土壤采样及昆虫病原线虫的分离（Steinernematidae，异小）

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

重金属和微藻的产其他无机污染物的量化

双链 rna 口服法诱导韧皮部和植物-sap 喂养 Hemipteran 昆虫 rna 干扰

植物感染试验: 以植物病原菌为载体的喷雾和伤口介导接种

从刺客臭虫和其他 Heteropteran 昆虫身上采集毒液毒素

海上双壳贝类悬浮饲料定量装置的设计与应用

在纯落叶支架中使用三种截然不同的方法估算叶面积指数

纳米材料和其他难物质藻类毒性试验的小型装置

测定掠夺取食行为

摘要

探索更多视频

此视频中的章节

优化烟气设置通过电脑模拟来促进微藻生长的光生物反应器中

土壤采样及昆虫病原线虫的分离（Steine​​rnematidae，异小）

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

重金属和微藻的产其他无机污染物的量化

双链 rna 口服法诱导韧皮部和植物-sap 喂养 Hemipteran 昆虫 rna 干扰

植物感染试验: 以植物病原菌为载体的喷雾和伤口介导接种

从刺客臭虫和其他 Heteropteran 昆虫身上采集毒液毒素

海上双壳贝类悬浮饲料定量装置的设计与应用

在纯落叶支架中使用三种截然不同的方法估算叶面积指数

纳米材料和其他难物质藻类毒性试验的小型装置

土壤采样及昆虫病原线虫的分离（Steinernematidae，异小）