娜姐朱佰利群同等贡献这项工作。这项工作在财政的公益专项基金林业科学研究（201204501）和中国国家自然科学基金（31170604，31270688，31570638和）的支持。我们感谢Holighaus格里特博士和Danielle Hickford博士对英语写作的有益建议。

1.处女成人线虫收购<ol><li> 松材线虫分离和培养条件 注: 松材线虫隔离Nxy61最初是从浙江省在中国的宁波地区患病马尾松提取。 <ol><li>准备马铃薯葡萄糖琼脂（PDA，见材料表 ）种植灰霉病板。 </li><li>对于接种，冲了一块与从灰霉病垫子10毫米直径的真菌垫，并把它放在一个新的PDA板中间。文化在黑暗中灰霉病 5天在25℃。 </li><li>文化包含灰霉病在25°C黑暗准备的培养皿松木线虫。 </li></ol></li><li> 松材线虫鸡蛋收购 <ol><li>将一个漏斗下方的夹紧橡胶管按住准备漏斗在机架组装贝尔曼漏斗装置。放置滤纸代替两层纱布在漏斗口。 </li><li>转移真菌培养漏斗设置，直到真菌垫浸入加水。等待2小时，线虫会从真菌培养出来，穿过滤纸，并在贝尔曼漏斗的最低部分安顿下来。 </li><li>从管的底部成15毫升管收集第一对的水滴，并通过缓慢释放在管夹紧收集线虫。 </li><li>离心上述集合以3000 xg离心在RT 2分钟。 </li><li>用移液管除去水的上层，并用1毫升无菌双蒸（DD）的H 2 O重悬它</li><li>用移液管，在上述再悬浮转移至新的玻璃培养皿（直径6cm）。加入3毫升无菌DD 的 H 2 O至培养皿以确保线虫能够自由游泳。 </li><li>保持线虫在GLASš的Petri在黑暗盘10分钟在25℃。鸡蛋会被怀孕女性被解雇。因为它们的糖蛋白表面的，将卵粘在玻璃培养皿的底部。 </li><li>小心除去水和蠕虫，而不会干扰蛋。加入3毫升无菌DD 的 H 2 O至玻璃培养皿立即防止蛋干燥。 </li><li>重复步骤3倍1.2.8删除所有的幼虫和成虫，并获得纯净的鸡蛋。添加无菌DD 的 H 2的O的额外2毫升（5毫升计）的培养皿。 </li></ol></li><li> 第四阶段少年的采集（J4的幼虫） <ol><li>哈奇收集鸡蛋在黑暗中24小时在25℃，得到J2幼虫。 </li><li>用移液管转移J2幼虫到15毫升管中，离心以3000 xg离心2分钟，并除去水的上层。 </li><li>重悬用无菌DD H 2的O 200微升J2幼虫，然后将它们转移到含灰霉病的PDA板。 </li><li>文化在黑暗中J2幼虫52 h后25℃。在这个时间点，最J2幼虫会已发展成J4幼虫的早期阶段。 </li></ol></li><li> 处女的成年线虫的收购 <ol><li>准备通过在酒精灯加热后绘制的玻璃毛细管拿起线虫针。 注:此毛细管针的尖端通常需要为50微米的直径，大致成年线虫的主体的宽度，因此，它可以用作用于拾取单个线虫的工具。 </li><li>组装贝尔曼漏斗并提取从真菌培养物线虫2小时，如在步骤1.2.1描述 - 1.2.3。 </li><li>传输与水J4到一个干净的培养皿。 </li><li>观察在立体显微镜下的线虫和生殖器形态学男性和女性之间区分，如通过玛米亚和清原8（ 图1中描述</strong&gt;的）。拾取所需性别的线虫与制备毛细管针，并将其转移到合适的真菌培养板为男性或女性。 注:有女性多于男性，而女性在60百分比 - 70％。女性外阴是从男性毛刺明显不同。相比于成人，在早期J4的毛刺或外阴更小，但它们仍然可以很容易地看到和分辨。线虫总是在水中，这有时会增加在区分男性和女性的困难移动。 </li><li>培养J4的幼虫在暗处在25℃另外24小时。 注:所有线虫应发育为成虫，但因为它们是未交配，它们被认为是处女成年人。 </li><li>装配2贝尔曼漏斗和提取的真菌培养处女大人2小时，如步骤1.2.1描述 - 1.2.3，和处女的成年男性和女性转移到两个独立的，清洁的培养皿。 </li></o升&gt; </li></ol> 2.观察和交配行为的录像<ol><li>准备几个凹幻灯片，并添加无菌DD H 2的O 200μL到他们的每一个孔中。 </li><li>拿起准备好的处女的成年男性或女性，并用毛细管针，把只有一个线虫到每个凹幻灯片的水。 </li><li>保持井内的处女大人1小时，让他们适应新的水环境中。 </li><li>用吸管转移其中的一个从一个井到另一对处女的男性和女性。 </li><li>观察立体显微镜下的凹幻灯片1♀+1♂组合的交配行为。 </li><li>分析处女成年人的配合效率，计算出它们的交配成功率（R）和其与随机选择的成人对的比较。计算R相比研究重复的总数，其中观察到交配重复的百分比。 </li><li&gt;探讨交配选择和竞争性内，组和观察两种不同的组合处女线虫:1♀+3♂和3♀+1♂。 </li><li>为了收集数据进行定性和定量分析，电影利用视频显微镜的交配行为。通常情况下，整个交配过程，从时间，当线虫放置在凹幻灯片一时的好，当他们交配后搬走时，2小时内完成。因此，连续地记录了至少2小时作为一个复制的配合过程。完成至少30复制总数为每个组合。 注:添加更多无菌DD H 2 O（通常为50微升）每0.5小时至井的录制过程中，以弥补蒸发。 </li></ol> 3.数据收集<ol><li>仔细查看电脑屏幕上的所有视频和定性描述子的行为。 </li><li>通过测量次交配的时间Ë帧一帧的视频录像机功能。手动减去从结束点的开始时间。 </li><li>对于1♀+1♂组合，分析每一个视频录制和测量以下事件的持续时间: <ol><li>测量搜索的持续时间（ 即，从当阴和阳被引入凹形滑动到它们遇到的那一刻的阱和彼此接触的时间）。 </li><li>测量接触的持续时间（ 即 ，从第一接触的瞬间当阳被精确地定位在阴的外阴用于第一交配的时间）。 </li><li>测量交尾的持续时间（ 即 ，从当直到他射精后滑动从女性的身体向下阳被精确地定位在阴的外阴的时间）。注意:某些线虫可在2小时交配几次。在这种情况下，测量交配从第一交配期间向拉斯的端t的一，包括之间的时间。 </li><li>测量挥之不去的持续时间（ 即 ，交配后的期间，直到彼此雌雄游程，通过的距离超过所述线虫的身体长度被分离（通常1毫米））。 </li></ol></li><li>计数交配次数每交配的持续时间和所有交配的在2小时的平均持续时间为每个不同的组合（ 即，1♀+1♂，3♀+1♂和1♀+3♂）。 </li><li>计算第一接触（P），对于不同的组合作为第一接触，这是立即和没有任何中断进行后交配成功率的交配成功率。在接触阶段，几次接触可能实际交配后方可。 <ol><li>当两个个体之间的距离小于所述主体的长度以上，考虑一个接触到结束。对于一个复制，记录数p作为100％时，交配我小号马上第一次接触后，如果没有观察到交配或观察到的，没有休息，或0.00％，如果有交配但经过两个或两个以上的联系人。 注:有30个重复这里描述的每个组合。 </li></ol></li></ol>

<ol>
	<li>Tokushige, Y., Kiyohara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bursaphelenchus+sp.+in+the+wood+of+dead+pine+trees.">Bursaphelenchus sp. in the wood of dead pine trees.</a> J. Jap. For. Soc. 51, 193-195 (1969).</li><li>Sun, Y. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bursaphelenchus+xylophilus+was+discovered+in+Sun+Yet-sen's+mausoleum+in+Nanjing.">Bursaphelenchus xylophilus was discovered in Sun Yet-sen's mausoleum in Nanjing.</a> J. Jiangsu Fore. Sci. Tech. (in Chinese). 4, 47 (1982).</li><li>Mota, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+report+of+Bursaphelenchus+xylophilus+in+Portugal+and+in+Europe.">First report of Bursaphelenchus xylophilus in Portugal and in Europe.</a> Nematology. 1, 727-734 (1999).</li><li>Robertson, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+the+pine+wood+nematode+Bursaphelenchus+xylophlius+Steiner+&amp;+Buhrer,+1934+(Nickle,+1970)+in+Spain.">Incidence of the pine wood nematode Bursaphelenchus xylophlius Steiner &amp; Buhrer, 1934 (Nickle, 1970) in Spain.</a> Nematology. 13, 755-757 (2011).</li><li>Vicente, C., Espada, M., Vieira, P., Mota, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pine+wilt+disease:+a+threat+to+European+forestry.">Pine wilt disease: a threat to European forestry.</a> Euro. J. Plant Path. 133, 89-99 (2012).</li><li>Jones, J. T., Moens, M., Mota, M., Li, H., Kikuchi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bursaphelenchus+xylophilus:+opportunities+in+comparative+genomics+and+molecular+host-parasite+interactions.">Bursaphelenchus xylophilus: opportunities in comparative genomics and molecular host-parasite interactions.</a> Mol. Plant. Path. 9, 357-368 (2008).</li><li>Mamiya, Y., Furukawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fecundity+and+reproduction+of+Bursaphelenchus+lignicolus.">Fecundity and reproduction of Bursaphelenchus lignicolus.</a> Jap. J. Nemat. 7, 6-9 (1977).</li><li>Mamiya, Y., Kiyohara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Description+of+Bursaphelenchus+lignicolus.+n.+sp.+(Nematoda:+Aphelenchoididae)+from+pine+wood+and+histophathology+of+nematode-infested+trees.">Description of Bursaphelenchus lignicolus. n. sp. (Nematoda: Aphelenchoididae) from pine wood and histophathology of nematode-infested trees.</a> Nematologica. 18, 120-124 (1972).</li><li>Liu, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioural+features+of+Bursaphelenchus+xylophilus+in+the+mating+process.">Behavioural features of Bursaphelenchus xylophilus in the mating process.</a> Nematology. 16, 895-902 (2014).</li><li>Simon, J. M., Sternberg, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+a+mate+finding+cue+in+the+hermaphrodite+nematode+Caenorhabditis+elegans.">Evidence of a mate finding cue in the hermaphrodite nematode Caenorhabditis elegans.</a> Proc. Natl. Acad. Sci. USA. 99, 1598-1603 (2002).</li><li>Barr, M. M., Garcia, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Male+mating+behaviour.">Male mating behaviour.</a> The C. elegans Research Community Wormbook. , (2006).</li><li>Shinya, R., Chen, A., Sternberg, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+attraction+and+mating+in+Bursaphelenchus+okinawaensis+and+B.+xylophilus.">Sex attraction and mating in Bursaphelenchus okinawaensis and B. xylophilus.</a> J. Nematol. 47 (3), 176-183 (2015).</li></ol>

The authors have nothing to disclose.

行为测试是化学生态学，神经生物学，分子生物学和遗传学的一个基本方面。线虫，特别是线虫 ，被广泛地用于这些领域的研究。 线虫的交配行为之前已10-11调查。但是， 松材线虫是雌雄异体，并从雌雄同体线虫不同，它的交配过程有不同的行为特征9。近日， 松材线虫okinawaensis， 松材线虫的雌雄同体的妹妹种的交配行为报12。...

The pinewood nematode, Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle, is one of the most destructive invasive species, causing the wilting and eventual death of pine trees. This pathogenic nematode is believed to be native to the USA, but it was transported to several countries, including China, Japan, South Korea, and Portugal. Recently, it has also been reported in Spain, killing millions of pine trees at huge economic losses and threatening forest production and ecological stability1-5.
Once a host pine tree is infected by pinewood nematodes, thousands of millions of offspring are propagated rapidly within the trunk. This causes xylem dysfunction, resulting in wilting and eventually in the death of the host tree6. However, there is currently no efficient way to control this disease. Mating behavior might play an important role in the high fecundity of this nematode7. We therefore investigated the mating behavior of B. xylophilus in the laboratory, trying to find an effective way of disturbing its mating and decreasing its fecundity. 
This protocol is intended to introduce the detailed methods of how to obtain the virgin adults of B. xylophilus and how to observe and analyze the mating behavior using a video recorder and a stereomicroscope. This protocol can also be used as a reference for behavioral studies of other nematodes.

<table border="0" cellpadding="0" cellspacing="0" width="944">
	<tbody>
		<tr height="18">
			<td height="18" style="height:18px;width:233px;">B. xylophilus isolate Nxy61</td>
			<td style="width:123px;"></td>
			<td style="width:125px;"></td>
			<td style="width:463px;">Extracted from Pinus massoniana in Ningbo area of China</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Grey mold strain</td>
			<td></td>
			<td></td>
			<td>Obtained from Forestry Academy of China</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Baermann funnels</td>
			<td>Sengong</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">falcon tubes</td>
			<td>Sengong</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">centrifuge</td>
			<td>Sengong</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pipette</td>
			<td>Sengong</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">sterile dd H2O</td>
			<td></td>
			<td></td>
			<td>Sterilized by high pressure of steam at 121&#176;C for 30 min.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri dishes</td>
			<td>Sengong</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PDA medium</td>
			<td>Huankai Bio.</td>
			<td>021050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">incubator</td>
			<td>Sengong</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Laminar flow</td>
			<td>Sengong</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">glass capillary</td>
			<td>Sengong</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stereoscope</td>
			<td>Leica</td>
			<td>LED5000 RL</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">inverted stereo microscope</td>
			<td>Zeiss</td>
			<td>A1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">concave slides</td>
			<td>Sengong</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

observation and quantification of mating behavior in the pinewood nematode, bursaphelenchus xylophilus

用于观察和量化的松材线虫， 松材线虫的交配行为的方法，是立体显微镜下成立的。以提高松材线虫的配合效率和增加的交配观测的几率，处女成年人中培养并用于调查。卵通过保持线虫在水中，并允许雌性产卵10分钟获得的。第二级幼体（J2）通过在黑暗中在25℃温育卵24小时同步，和J4早期通过培养用灰霉病， 灰霉病在J2，另一个52 h条件。在这个时间点，最J4线虫可以作为是男性或女性使用其生殖器形态明确区分。男性和女性的J4收集并在两个不同的培养皿中24小时分别培养以获得处女成人线虫。一个处女男和处女女在笏一滴配对器中的凹滑动的孔中。交配行为与立体显微镜下录像机拍摄。配合过程的整个时间为82.8±3.91分钟（平均值±SE）和可分为4个不同的阶段:搜索，接触，交尾，和挥之不去。持续时间的平均分者21.8±2.0，28.0±1.9，25.8±0.7和7.2±0.5，分别为。 11个子行为进行了描述:巡航，接近，遭遇，触摸，胡平，定位，安装，射精，分离，平静，和漫游。有趣的是，当一个女性与男性3个或一个男性，女性3分组观察到明显的内部性的竞争。这个协议是有用的，有价值的，不仅在调查松材线虫的交配行为，而且在充当其他线虫行为学研究的参考。

处女成人的交配成功率（R）为86.67％，平均，比该随机选择的成人显著更高:13.33±4.65％（F = 1301.71，DF = 1，P = 0.0001）（ 图2）。这一发现表明，它更容易观察松材线虫与处女比成年人随机选取成年人交配行为。 当一名男性与一名女性配对，整个交配过程持续了平均82.8分钟。整个过程可以通过四...

Watch this Scientific Journal Video about Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus at JoVE.com

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. ...

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

9.3K 次观看.  Zhejiang Agricultural & Forestry University. 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.

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

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas) has become a popular organism for research in diverse areas of cell biology and genetics because of its simple life cycle, ease of growth and manipulation for genetic analysis, genomic resources, and transformability of the nucleus and both organelles. Mating strains is a common practice when genetic approaches are used in Chlamydomonass, to create vegetative diploids for analysis of dominance, or following tetrad dissection to ascertain nuclear vs. organellar inheritance, to test allelism, to analyze epistasy, or to generate populations for the purpose of map-based cloning. Additionally, genetic crosses are routinely used to combine organellar genotypes with particular nuclear genotypes. Here we demonstrate standard methods for gametogenesis, mating, zygote germination and tetrad separation. This protocol consists of an easy-to-follow series of steps that will make genetic approaches amenable to scientists who are less familiar with Chlamydomonas. Key parameters and trouble spots are explained. Finally, resources for further information and alternative methods are provided.

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

Mating and tetrad separation are required for genetic analysis in Chlamydomonas reinhardtii. Here we demonstrate standard methods for gametogenesis, mating, zygote germination and tetrad dissection. This protocol consists of an easy-to-follow series of steps that will make genetic approaches amenable to scientists who are less familiar with Chlamydomonas.

交配和四分体分离衣藻遗传分析

The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas) has become a popular organism for research in diverse areas of cell biology and ...

科研

生物学

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

电离辐射的反应定量的γH2AX灶

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression analyses by RNA interference and stable transgenesis remain limited in many species due to the particular anatomy and molecular cellular biology of the organism. For example, outside of the crown group Caenorhabditis that includes Caenorhabditis elegans3, stably transmitted transgenic lines in non-Caenorhabditis species have not been reported in this specious phylum (Nematoda), with the exception of Strongyloides stercoralis4 and Pristionchus pacificus5. To facilitate the expanding role of P. pacificus in the study of development, evolution, and behavior6-7, we describe here the current methods to use microinjection for making transgenic animals and gene knock down by RNAi. Like the gonads of C. elegans and most other nematodes, the gonads of P. pacificus is syncitial and capable of incorporating DNA and RNA into the oocytes when delivered by direct microinjection. Unlike C. elegans however, stable transgene inheritance and somatic expression in P. pacificus requires the addition of self genomic DNA digested with endonucleases complementary to the ends of target transgenes and coinjection markers5. The addition of carrier genomic DNA is similar to the requirement for transgene expression in Strongyloides stercoralis4 and in the germ cells of C. elegans. However, it is not clear if the specific requirement for the animals' own genomic DNA is because P. pacificus soma is very efficient at silencing non-complex multi-copy genes or that extrachromosomal arrays in P. pacificus require genomic sequences for proper kinetochore assembly during mitosis. The ventral migration of the two-armed (didelphic) gonads in hermaphrodites further complicates the ability to inject both gonads in individual worms8. We also demonstrate the use of microinjection to knockdown a dominant mutant (roller,tu92) by injecting double-stranded RNA (dsRNA) into the gonads to obtain non-rolling F1 progeny. Unlike C. elegans, but like most other nematodes, P. pacificus PS312 is not receptive to systemic RNAi via feeding and soaking and therefore dsRNA must be administered by microinjection into the syncitial gonads. In this current study, we hope to describe the microinjection process needed to transform a Ppa-egl-4 promoter::GFP fusion reporter and knockdown a dominant roller prl-1 (tu92) mutant in a visually informative protocol.

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

In model organisms, transgenesis can manipulate gene functions while RNAi can knockdown specific mRNA transcripts 1-2. This protocol aims to illustrate the techniques needed to introduce stably transmitted DNA and transient double stranded RNA into the necromenic nematode Pristionchus pacificus for studies in evolutionary, developmental, and behavioral biology.

通过显微注射介导的RNAi Necromenic线虫基因敲除和转基因 Pristionchus pacificus</em

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

使用屏幕在车身尺寸规例中的线虫基因介导的RNA干扰取食策略<em&gt; C.线虫</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here&#160;we describe the design and analysis of TPR in Drosophila.

温度偏好行为及其在德罗索菲拉的昼夜节律的设计与分析

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization (FISH) with PNA Probes in Caenorhabditis elegans

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase or an alternative pathway, known as alternative lengthening of telomeres (ALT)1. Recently, C. elegans has emerged as a multicellular model organism for the study of telomere and ALT2. Visualization of repetitive sequences in the genome is critical in understanding the biology of telomeres. While telomere length can be measured by telomere restriction fragment assay or quantitative PCR, these methods only provide the averaged telomere length. On the contrary, fluorescence in situ hybridization (FISH) can provide the information of the individual telomeres in cells. Here, we provide protocols and representative results of the method to determine telomere length of C. elegans by fluorescent in situ hybridization. This method provides a simple, but powerful, in situ procedure that does not cause noticeable damage to morphology. By using fluorescently labeled peptide nucleic acid (PNA) and digoxigenin-dUTP-labeled probe, we were able to visualize two different repetitive sequences: telomere repeats and template of ALT (TALT) in C. elegans embryos and gonads.

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans

We report a concise procedure of fluorescence in situ hybridization (FISH) in the gonad and embryos of Caenorhabditis elegans for observing and quantifying repetitive sequences. We successfully observed and quantified two different repetitive sequences, telomere repeats and template of alternative lengthening of telomeres (TALT).

观察与端粒的定量和重复序列用荧光<em&gt;原位</em&gt;杂交（FISH）与PNA探针<em&gt;秀丽隐杆线虫</em

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase ...

Quantification of Proliferative and Dead Cells in Enteroids

The intestinal epithelium acts as a barrier that prevents luminal contents, such as pathogenic microbiota and toxins, from entering the rest of the body. Epithelial barrier function requires the integrity of intestinal epithelial cells. While epithelial cell proliferation maintains a continuous layer of cells that forms a barrier, epithelial damage leads to barrier dysfunction. As a result, luminal contents can across the intestinal barrier via an unrestricted pathway. Dysfunction of intestinal barrier has been associated with many intestinal diseases, such as inflammatory bowel disease. Isolated mouse intestinal crypts can be cultured and maintained as crypt-villus-like structures, which are termed intestinal organoids or &ldquo;enteroids&rdquo;. Enteroids are ideal to study the proliferation and cell death of intestinal epithelial cells in vitro. In this protocol, we describe a simple method to quantify the number of proliferative and dead cells in cultured enteroids. 5-ethynyl-2&rsquo;-deoxyuridine (EdU) and propidium iodide are used to label proliferating and dead cells in enteroids, and the proportion of proliferating and dead cells are then analyzed by flow cytometry. This is a useful tool to test the effects of drug treatment on intestinal epithelial cell proliferation and cell survival.

The presented protocol uses flow cytometry to quantify the number of proliferating and dead cells in cultured mouse enteroids. This method is helpful to evaluate the effects of drug treatment on organoid proliferation and survival.

肠内增殖细胞和死细胞的定量

The intestinal epithelium acts as a barrier that prevents luminal contents, such as pathogenic microbiota and toxins, from entering the rest of the body. ...

Culturing and Screening the Plant Parasitic Nematode Ditylenchus dipsaci

Plant-parasitic nematodes (PPNs) destroy over 12% of global food crops every year, which equates to roughly 157 billion dollars (USD) lost annually. With a growing global population and limited arable land, controlling PPN infestation is critical for food production. Compounding the challenge of maximizing crop yields are the mounting restrictions on effective pesticides because of a lack of nematode selectivity. Hence, developing new and safe chemical nematicides is vital to food security. In this protocol, the culture and collection of the PPN species Ditylenchus dipsaci are demonstrated. D. dipsaci is both economically damaging and relatively resistant to most modern nematicides. The current work also explains how to use these nematodes in screens for novel small molecule nematicides and reports on data collection and analysis methodologies. The demonstrated pipeline affords a throughput of thousands of compounds per week and can be easily adapted for use with other PPN species such as Pratylenchus penetrans. The techniques described herein can be used to discover new nematicides, which may, in turn, be further developed into highly selective commercial products that safely combat PPNs to help feed an increasingly hungry world.

Culturing and Screening the Plant Parasitic Nematode Ditylenchus dipsaci

The present protocol describes a reliable and straightforward method for culturing, collecting, and screening Ditylenchus dipsaci.

植物寄生线虫的培养和筛选

Plant-parasitic nematodes (PPNs) destroy over 12% of global food crops every year, which equates to roughly 157 billion dollars (USD) lost annually. With ...

Application of RNA Interference in the Pinewood Nematode, Bursaphelenchus xylophilus

The pinewood nematode, Bursaphelenchus xylophilus, is one of the most destructive invasive species worldwide, causing the wilting and eventual death of pine trees. Despite the recognition of their economic and environmental significance, it has thus far been impossible to study the detailed gene functions of plant-parasitic nematodes (PPNs) using conventional forward genetics and transgenic methods. However, as a reverse genetics technology, RNA interference (RNAi) facilitates the study of the functional genes of nematodes, including B. xylophilus. 
This paper outlines a new protocol for RNAi of the ppm-1 gene in B. xylophilus, which has been reported to play crucial roles in the development and reproduction of other pathogenic nematodes. For RNAi, the T7 promoter was linked to the 5&#8242;-terminal of the target fragment by polymerase chain reaction (PCR), and double-stranded RNA (dsRNA) was synthesized by in vitro transcription. Subsequently, dsRNA delivery was accomplished by soaking the nematodes in a dsRNA solution mixed with synthetic neurostimulants. Synchronized juveniles of B. xylophilus (approximately 20,000 individuals) were washed and soaked in dsRNA (0.8 &#181;g/mL) in the soaking buffer for 24 h in the dark at 25 &#176;C.
The same quantity of nematodes was placed in a soaking buffer without dsRNA as a control. Meanwhile, another identical quantity of nematodes was placed in a soaking buffer with green fluorescent protein (gfp) gene dsRNA as a control. After soaking, the expression level of the target transcripts was determined using real-time quantitative PCR. The effects of RNAi were then confirmed using microscopic observation of the phenotypes and a comparison of the body size of the adults among the groups. The current protocol can help advance research to better understand the functions of the genes of B. xylophilus and other parasitic nematodes toward developing control strategies through genetic engineering.

Application of RNA Interference in the Pinewood Nematode, Bursaphelenchus xylophilus

Here, we introduce a detailed soaking method of RNA interference in Bursaphelenchus xylophilus to facilitate the study of gene functions.

RNA干扰在松林线虫、木犀牛线虫中的应用

The pinewood nematode, Bursaphelenchus xylophilus, is one of the most destructive invasive species worldwide, causing the wilting and eventual death of ...

Observation of Photobehavior in Chlamydomonas reinhardtii

For the survival of the motile phototrophic microorganisms, being under proper light conditions is crucial. Consequently, they show photo-induced behaviors (or photobehavior) and alter their direction of movement in response to light. Typical photobehaviors include photoshock (or photophobic) response and phototaxis. Photoshock is a response to a sudden change in light intensity (e.g., flash illumination), wherein organisms transiently stop moving or move backward. During phototaxis, organisms move toward the light source or in the opposite direction (called positive or negative phototaxis, respectively). The unicellular green alga Chlamydomonas reinhardtii is an excellent organism to study photobehavior because it rapidly changes its swimming pattern by modulating the beating of cilia (a.k.a., flagella) after photoreception. Here, various simple methods are shown to observe photobehaviors in C. reinhardtii. Research on C. reinhardtii&#39;s photobehaviors has led to the discovery of common regulatory mechanisms between eukaryotic cilia and channelrhodopsins, which may contribute to a better understanding of ciliopathies and the development of new optogenetics methods.

Observation of Photobehavior in Chlamydomonas reinhardtii

Most swimming photoautotrophic organisms show photo-induced behavioral changes (photobehavior). The present protocol observes the said photobehavior in the model organism Chlamydomonas reinhardtii.