Name: observation and quantification of mating behavior in the pinewood nematode, bursaphelenchus xylophilus
Uploaded: 2016-12-25T14:00:00.000+00:00
Duration: 9 min 55 s
Description: Watch this Scientific Journal Video about Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus at JoVE.com

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。虽然使用了不同的实验方法，本雌雄同体物种显示出相似性的吸引力松材线虫 ， 并观察它们之间的物种间的吸引力。这种雌雄同体的妹妹种可能被用来作为一种模式来进行对松材线虫的生物学进一步调查。 该协议提供了关于如何观察和调查交配期间松材线虫的行为特征的指导方针。我们的研究结果表明，使用处女成人线虫是用于增加成功观察交配过程（ 图1）的机会的关键。获取J4的幼虫，精确区分他们的性别是收购处女成年人两个重要的基本步骤。根据我们的实验条件下（ 即，在黑暗中25℃），52小时期间是适当的时候，以从J2幼虫得到J4。然而，在早期阶段J4区分性主要取决于个人的经验。为了确保准确性，对于不同的人要仔细检查性配置是必要的。 B.在交配过程中松材线虫的行为特征可以通过记录四的持续时间不同的定性描述和定量分析耳鼻喉科阶段，交配成功的第一次接触，交配的频率，以及不同组合的交配时间后的百分比。有趣的是，在第一次，我们由女性和男性和女性强帧内性竞争观察交配选择。未发现乱;然而，这可能是由于在我们的研究中所用的线虫的数量有限。这个协议不仅可以用于研究松材线虫的交配行为，但它也可以是有用的和有价值的作为其它线虫参考。

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><tbody><tr><td>&lt;em&gt;B. xylophilus&lt;/em&gt; isolate &lt;em&gt;Nxy61&lt;/em&gt;</td><td></td><td></td><td>Extracted from &lt;em&gt;Pinus massoniana&lt;/em&gt; in Ningbo area of China</td></tr><tr><td>Grey mold strain</td><td></td><td></td><td>Obtained from Forestry Academy of China</td></tr><tr><td>Baermann funnels</td><td>Sengong</td><td></td><td></td></tr><tr><td>falcon tubes</td><td>Sengong</td><td></td><td></td></tr><tr><td>centrifuge</td><td>Sengong</td><td></td><td></td></tr><tr><td>pipette</td><td>Sengong</td><td></td><td></td></tr><tr><td>sterile dd H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>Sterilized by high pressure of steam at 121 &amp;deg;C for 30 min.</td></tr><tr><td>Petri dishes</td><td>Sengong</td><td></td><td></td></tr><tr><td>PDA medium</td><td>Huankai Bio.</td><td>021050</td><td></td></tr><tr><td>incubator</td><td>Sengong</td><td></td><td></td></tr><tr><td>Laminar flow</td><td>Sengong</td><td></td><td></td></tr><tr><td>glass capillary</td><td>Sengong</td><td></td><td></td></tr><tr><td>Stereoscope</td><td>Leica</td><td>LED5000 RL</td><td></td></tr><tr><td>inverted stereo microscope</td><td>Zeiss</td><td>A1</td><td></td></tr><tr><td>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个不同的阶段来描述:寻找，联系，交配，而绵长。这四个阶段的持续时间分别为21.8±2.0分钟，28.0±1.9分钟，25.8±0.7分钟，和7.2±0.5分钟，分别为（ 图3）。在最近的一份出版物中，我们描述了11分行为作为巡航，接近，encounteriNG，感人，胡平，定位，安装，射精，分离，平静，和漫游9。 当一个男人同时满足两个或更多的女性，男性似乎与他们中的一个随机交配，没有任何选择。然而，观察到的女性松材线虫明显交配选择。当一个女性接触男性，女性往往她的头轻轻触摸男性的头，身，尾，和生殖器区域，似乎被她的评估潜在的合作伙伴。如果女性不接受男，女迅速离开了他，游到另一男性为接下来的评测。一项所述的男性然后通过阴交配选择。交配成功率的第一接触（P）之后的1♀+1♂，3♀+1♂和1♀+3♂组合分别为46.7±9.3％，80.0±7.4％，和23.4±7.4％，分别和WER它们之间的差异Ë显著（F = 8.21，DF = 2，P = 0.0124）（ 图4）。 在男性和女性中观察性内的竞争。当一个处女女与3处女男性混合，男性试图接近并联系女尽可能得到与她交配的机会。当外阴是由一名男性占据，其他的男性试图通过暴力体波动或拖动到它们分开来打扰交配男性。女性性内竞争被认为是或多或少类似男性性内的竞争。 交配成功主要取决于女性。当女性接受男性，男性移动更慢，卷曲，甚至形成一个圈，女性前移积极，使其更容易为男性找到她的外阴和避免其他男性的干扰。如果女性没有接受男性，女性试图甩开他，即使男性已经安装并通过她的阴户给自己定位。因为性内的竞争，所述1♀+1♂，3♀+1♂的交配的频率，和1♀+3♂组合为1.43±0.091，1.17±分别0.074和1.83±0.074，和它们之间的差异显著（F = 14.50，自由度= 2，P = 0.0001）（ 图5）。 在我们的调查中，交配持续时间差别很大，最小的20秒，最大值为42分钟。根据我们的数据，交配的持续时间可以明显分化成两组:短期交配，历时小于5分钟，和长期交配，历时长于10分钟。不同群体之间观察明显的差异短期交配（F = 3.81的平均期限，DF = 2，P </em&gt; = 0.0296），长期交配（F = 179.52，自由度= 2，P = 0.0001），以及所有交配（F = 2.93，DF = 2，P = 0.0367）（ 图6）。 <img alt="图1" src="/files/ftp_upload/54842/54842fig1.jpg" /> 图1:男，女松材线虫的生殖器。 （ 一 ）男（蓝色箭头）的毛刺。 （ 二 ）女性（红色箭头）的外阴。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54842/54842fig1large.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/54842/54842fig2.jpg" /> 图2:处女成人夫妇的交配成功率和松材线虫的随机选择成人夫妇。算出的平均值是从30个重复。竖线展S中的标准差。当杜克的试验比较了不同小写字母表示在P &lt;0.05显著差异。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54842/54842fig2large.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/54842/54842fig3.jpg" /> 图3: 松材线虫的交配过程的4个阶段的持续时间。算出的平均值是从30个重复。垂直条显示标准误差。当杜克的试验比较了不同小写字母表示在P &lt;0.05显著差异。在交配相的平均持续时间是25.80±0.71分钟，这是整个交配过程的31.17％。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54842/54842fig3large.jpg" target="_blank">请点击此处查看该图的放大版本</a>URE。 <img alt="图4" src="/files/ftp_upload/54842/54842fig4.jpg" /> 图4:交配成功对松材线虫的第一次接触。计算出的平均值是30±重复SE。当通过Tukey的诚实显着差异检验比较与每一列中的相同小写字母表示没有显著不同的P &lt;0.05。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54842/54842fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54842/54842fig5.jpg" /> 图5: 松材线虫不同群体交配的频率。计算出的平均值是30±重复SE。用相同的小写字母手段并不显著不同在P &lt;0.05时，补偿由杜克的诚实着性检验ARED。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54842/54842fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/54842/54842fig6.jpg" /> 图6: 松材线虫的交配时间。当杜克的诚实显着差异试验比较每一组列在同一个小写字母手段并不显著不同在P &lt;0.05。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54842/54842fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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. ...

observation-quantification-mating-behavior-pinewood-nematode

Universidad San Sebastian

Research

JoVE Journal

Biology

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

A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters (Homarus americanus)

Despite being one of the most productive fisheries in the Northwest Atlantic, much remains unknown about the natural reproductive dynamics of American lobsters. Recent work in exploited crustacean populations (crabs and lobsters) suggests that there are circumstances where mature females are unable to achieve their full reproductive potential due to sperm limitation. To examine this possibility in different regions of the American lobster fishery, a reliable and noninvasive method was developed for sampling large numbers of female lobsters at sea. This method involves inserting a blunt-tipped needle into the female&#39;s seminal receptacle to determine the presence or absence of a sperm plug and to withdraw a sample that can be examined for the presence of sperm. A series of control studies were conducted at the dock and in the laboratory to test the reliability of this technique. These efforts entailed sampling 294 female lobsters to confirm that the presence of a sperm plug was a reliable indicator of sperm within the receptacle and thus, mating. This paper details the methodology and the results obtained from a subset of the total females sampled. Of the 230 female lobsters sampled from George&#39;s Bank and Cape Ann, MA (size range = 71-145 mm in carapace length), 90.3% were positive for sperm. Potential explanations for the absence of sperm in some females include: immaturity (lack of physiological maturity), breakdown of the sperm plug after being used to fertilize a clutch of eggs, and lack of mating activity. The surveys indicate that this technique for examining the mating success of female lobsters is a reliable proxy that can be used in the field to document reproductive activity in natural populations.

A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters Homarus americanus

Fishery-induced changes to exploited crustacean fisheries, such as the American lobster fishery, could potentially influence their reproductive dynamics, leading to a reduction in mating success. This study&#39;s goal was to develop a noninvasive method for ascertaining the mating success of female lobsters that may be physiologically or functionally mature.

一种非侵入性的方法<em&gt;原位</em&gt;交配成功的女美国龙虾​​（测定<em&gt;美洲螯龙虾</em&gt;）

Despite being one of the most productive fisheries in the Northwest Atlantic, much remains unknown about the natural reproductive dynamics of American ...

科研

环境科学

Field Experiments of Pollination Ecology: The Case of Lycoris sanguinea var. sanguinea

Plant-pollinator interactions have been studied for approximately one hundred years. During that time, many field methods have been developed to clarify the pollination effectiveness of each pollinator for visited flowers. Pollinator observations have been one of the most common methods to identify pollinators, and bagging and cage experiments have been conducted to show the effectiveness of specific pollinators. In a previous study of Lycoris sanguinea var. sanguinea, its effective pollinators, the visitation frequencies of each floral visitor, and its reproductive strategies were not identified. This study reports the observation that small bees visited flowers that were partially opened (breaking buds). To the best of our knowledge, this phenomenon has not been reported previously. Further, this study investigates the hypothesis that small bees can pollinate at that flowering stage. This study demonstrates the basic methods of field experiments in pollination ecology using L. sanguinea var. sanguinea. Pollinator observations and digital video showed the visitation frequencies of each floral visitor. Bagging and cage experiments revealed that these flowers could be pollinated fully and that breaking-bud pollination could be important for the pollination of this plant species. The advantages and disadvantages of each method are discussed, and recent developments, including laboratory experiments, are described.

Field Experiments of Pollination Ecology: The Case of Lycoris sanguinea var. sanguinea

To reveal the pollinator effectiveness of a given plant species, multiple methods of field experiments have been developed. This study demonstrates the basic methods of field experiments for pollination ecology using the case study of Lycoris sanguinea var. sanguinea and the novel pollination mechanism, breaking-bud pollination.

传粉生态学的田间试验:为例<em&gt;血红石蒜</em&gt;变种。<em&gt;荪</em

Plant-pollinator interactions have been studied for approximately one hundred years. During that time, many field methods have been developed to clarify ...

Induction and Evaluation of Inbreeding Crosses Using the Ant, Vollenhovia Emeryi

The genetic and molecular components of the sex-determination cascade have been extensively studied in the honeybee, Apis mellifera, a hymenopteran model organism. However, little is known about the sex-determination mechanisms found in other non-model hymenopteran taxa, such as ants. Because of the complex nature of the life cycles that have evolved in hymenopteran species, it is difficult to maintain and conduct experimental crosses between these organisms in the laboratory. Here, we describe the methods for conducting inbreeding crosses and for evaluating the success of those crosses in ant Vollenhovia emeryi. Inducing inbreeding in the laboratory using V. emeryi, is relatively simple because of the unique biology of the species. Specifically, this species produces androgenetic males, and female reproductives exhibit wing polymorphism, which simplifies identification of the phenotypes in genetic crosses. In addition, evaluating the success of inbreeding is straightforward as males can be produced continuously by inbreeding crosses, while normal males only appear during a well-defined reproductive season in the field. Our protocol allow for using V. emeryi as a model to investigate the genetic and molecular basis of the sex determination system in ant species.

Induction and Evaluation of Inbreeding Crosses Using the Ant, Vollenhovia Emeryi

In this protocol, methods for conducting inbreeding crosses, and for evaluating the success of those crosses, are described for the ant Vollenhovia emeryi. These protocols are important for experiments aimed at understanding the genetic basis of sex determination systems in Hymenoptera.

利用蚂蚁Vollenhovia Emeryi对近亲繁殖杂交的诱导和评价

The genetic and molecular components of the sex-determination cascade have been extensively studied in the honeybee, Apis mellifera, a hymenopteran model ...

遗传学

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors remains poorly understood. In Drosophila melanogaster, environmental cues, such as food availability, affect mating activity offering a tractable system to investigate the mechanisms modulating sexual behavior. In D. melanogaster, environmental cues are often sensed via the chemosensory gustatory and olfactory systems. Here, we present a method to test the effect of environmental chemical cues on mating behavior. The assay consists of a small mating arena containing food medium and a mating couple. The mating frequency for each couple is continuously monitored for 24 h. Here we present the applicability of this assay to test environmental compounds from an external source through a pressurized air system as well as manipulation of the environmental components directly in the mating arena. The use of a pressurized air system is especially useful to test the effect of very volatile compounds, while manipulating components directly in the mating arena can be of value to ascertain a compound's presence. This assay can be adapted to answer questions about the influence of genetic and environmental cues on mating behavior and fecundity as well as other male and female reproductive behaviors.

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

We demonstrate an assay to analyze the environmental and genetic cues that influence mating behavior in the fruit fly Drosophila melanogaster.

测试环境线索对交配行为的影响的方法<em&gt;黑腹果蝇</em

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors ...

行为学

Individual Culturing of Tigriopus Copepods and Quantitative Analysis of Their Mate-guarding Behavior

Copepods of the genus Tigriopus, which are common zooplankton in rocky tide pools, show precopulatory mate-guarding behavior where a male clasps a potential mate to form a pair. While this phenomenon has attracted interest of researchers, methods for its analysis have not been well described. Here we describe procedures for: 1) individual culturing and staging of Tigriopus juveniles and adults, and 2) video-based analysis of their mate-guarding behavior. The culturing method enables experimental control of paring experience of animals as well as the ability to track their development before behavioral tests. The analysis method allows quantitative evaluation of several aspects of the mate-guarding behavior, including capturing attempts by males and swimming trajectory of mate-guarding pairs. Although these methods were originally established for ethological studies on Tigriopus, with proper modifications they can also be applied to studies of other zooplankton in different research fields, such as physiology, toxicology, and ecological genetics.

Individual Culturing of Tigriopus Copepods and Quantitative Analysis of Their Mate-guarding Behavior

Mate-guarding behavior plays an important role in reproduction of intertidal copepods of the genus Tigriopus. However, methods for studying this behavior have not been well described. Here we describe methods for: 1) individual culture of virgin Tigriopus animals, and 2) quantitative analysis of their mate-guarding behavior.

Tigriopus桡足的个体培养及其交配行为的定量分析

Copepods of the genus Tigriopus, which are common zooplankton in rocky tide pools, show precopulatory mate-guarding behavior where a male clasps a ...

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

对检测方法果蝇行为

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

神经科学

通过卵巢生殖分析鉴定迁徙昆虫的解剖

Dissection and Grading of Ovarian Development in Wild-Type Female Insects

Migratory insect pests pose serious challenges to food production and security all over the world. The migratory pests can be monitored and captured using searchlight traps. One of the most important techniques for migratory pest forecasting is to identify the migratory species. However, in most cases, it is difficult to get the information just by appearance. Therefore, using knowledge acquired by systematic analysis of the female reproductive system can help to understand the combined anatomic morphology of the ovarian mating sac and ovary developmental grading of wild-type migratory insects captured with searchlight traps. To demonstrate the applicability of this method, ovarian development status and egg grain development stages were directly assessed in Helicoverpa armigera, Mythimna separata, Spodoptera litura, and Spodoptera exigua for the ovarian anatomy, and the ovarian mating sacs were studied in Agrotis ipsilon, Spaelotis valida, Helicoverpa armigera, Athetis lepigone, Mythimna separata, Spodoptera litura, Mamestra brassicae, and Spodoptera exigua, to explore their relationships. This work shows the specific dissection method to predict wild-type migratory insects, comparing the unique reproductive system of different migratory insects. Then, both tissues, namely, the ovary and mating sacs, were further investigated. This method helps to predict the dynamics and the structural development of reproductive systems in wild-type female migratory insects.

作者聚焦:分析迁徙昆虫的卵巢解剖结构以克服实验挑战 

The protocol demonstrates a simple and easy dissection method, suitable for wild-type migratory female insects captured with searchlight traps. This technique can significantly clarify the same species by comparing both reproductive tissues, namely the mating sac and ovarian development of wild-type female insects.

野生型雌性昆虫卵巢发育的解剖和分级

Migratory insect pests pose serious challenges to food production and security all over the world. The migratory pests can be monitored and captured using ...

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy and cheap methods to distinguish between individuals in a trial. A standard technique for this is CO2 anaesthesia and then labelling or wing clipping each fly. However, this is invasive and has been shown to affect behavior. Other techniques have used coloration to identify flies. This article presents a simple and non-invasive method for labelling Drosophila that allows them to be individually identified within experiments, using food coloring. This method is used in trials where two males compete to mate with a female. Dyeing allowed quick and easy identification. There was, however, some difference in the strength of the coloration across the three species tested. Data is presented showing the dye has a lower impact on mating behavior than CO2 in Drosophila melanogaster. The impact of CO2 anaesthesia is shown to depend on the species of Drosophila, with D. pseudoobscura and D. subobscura showing no impact, whereas D. melanogaster males had reduced mating success. The dye method presented is applicable to a wide range of experimental designs.

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

This protocol describes a simple, cost effective way to individually identify Drosophila or other insects. Demonstration data investigating mating success across three species of Drosophila show that this method is comparable or better than the use of CO2 anaesthesia.

染色昆虫行为检测:麻醉的交配行为<em&gt;果蝇</em

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy ...

观察和交配行为的量化在我国松材线虫，

摘要

探索更多视频

此视频中的章节

一种非侵入性的方法

传粉生态学的田间试验:为例

利用蚂蚁Vollenhovia Emeryi对近亲繁殖杂交的诱导和评价

测试环境线索对交配行为的影响的方法

Tigriopus桡足的个体培养及其交配行为的定量分析

对检测方法果蝇行为

野生型雌性昆虫卵巢发育的解剖和分级

野生型雌性昆虫卵巢发育的解剖和分级

野生型雌性昆虫卵巢发育的解剖和分级

染色昆虫行为检测:麻醉的交配行为