Name: methods for the extraction of endosymbionts from the whitefly bemisia tabaci
Uploaded: 2017-06-19T12:30:00.000+00:00
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Description: Watch this Scientific Journal Video about Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci at JoVE.com

本研究的财政支持由国家重点研究发展计划（2016YFC1200601）和国家自然科学基金（31390421）提供。

白蛉饲养和隐蔽物种识别<ol><li>在27±1℃，70±10％湿度和14小时光:10小时黑暗条件的标准条件下，将棉花棉属 （Malvaceae）（马鞭草科）（cv。Zhe-Mian 1973）保存在笼中。 </li><li>收集单个成年粉虱，并在30μL裂解缓冲液（10mM Tris，pH 8.4,50mM KCl，0.45％[wt / vol] Tween-20,0.2％[wt / vol]明胶，0.45％[vol / vol] Nonidet P 40,60g / mL蛋白酶K）。 </li><li>将匀浆在65℃孵育1小时，然后100℃孵育10分钟。 注意:如有必要，可以增加65℃的孵育时间。应严格控制100℃下的孵育时间，以充分灭活蛋白酶K并避免DNA损伤。 </li><li> 基于线粒体细胞色素氧化酶I引物，使用粉虱DNA（在1.3节获得）进行聚合酶链反应（PCR）。 COI-F:5&#39;-TTGATTTTTTGGTCATCCAGAAGT-3&#39; COI-R:5&#39;-TAATATGGCAGATTAGTGCATTGGA-3&#39; <ol><li>进行PCR反应，最终体积为25μL，含有1U Taq DNA聚合酶，2.5μL10x Buffer，0.2 mM dNTP，0.2 mM各引物和2μL粉虱DNA。 </li><li>使用以下PCR程序条件:初始变性95℃3分钟，然后35个循环，95℃45秒（变性），50℃45秒（退火）和72℃1分钟（延伸）35个循环;在72℃另外10分钟进一步延伸。 </li></ol></li><li>使用推荐的方案使用DNA凝胶提取试剂盒清洗PCR扩增产物。 </li><li>按照制造商的说明，用DNA样品测序系统对纯化的DNA样品进行顺序。 </li><li>使用基本局部比对搜索工具（BLAST）分析序列，以识别粉虱的隐蔽物种，避免其他物种的污染ES。 </li></ol>内分泌鉴定和定位<ol><li>对粉虱DNA（步骤1.3获得）进行PCR，以扩增粉虱内每个内生子的特异性基因（PCR配方在1.4节中描述，PCR程序和引物列于表1 ）。 </li><li>根据先前发布的方案20 （1％琼脂糖凝胶，Tris-乙酸盐-EDTA作为运行缓冲液，电压为5V / cm）使扩增的样品进行琼脂糖凝胶电泳，以鉴定每种细菌的特异性基因。 </li><li>恢复和序列每个条带以确定粉虱内的细菌种类（见第1.5-1.7节）。 </li><li> 在粉虱上进行荧光原位杂交（FISH），以根据先前发布的方案19来识别内共生菌的位置。 注意:如有必要，增加荧光探针的浓度。 </li></oL&gt; 透射电子显微镜（TEM） <ol><li>将含有2.5％[vol / vol]戊二醛的磷酸盐缓冲液（0.1M，pH 7.0）中的粉虱浸泡并固定4小时。 </li><li>在磷酸盐缓冲液（0.1M，pH7.0）中洗涤样品三次，每次15分钟。 </li><li>在含有1％[wt / vol] OsO 4的磷酸盐缓冲液（0.1M，pH7.0）中沉淀并固定样品2小时。 </li><li>通过一系列乙醇（30％，50％，70％，80％，90％，95％和100％），每次15分钟使样品脱水。 </li><li>将样品转移并浸入100％丙酮中20分钟。 </li><li>将样品在100％丙酮和100％Spurr树脂（1:1）的混合物中放置1小时。 </li><li>将样品转移到100％丙酮和100％Spurr树脂（1:3）的混合物中3小时。 </li><li>将样品转移并浸入100％Spurr树脂（在70℃下孵育）超过9小时。 </li><li>通过使用微型部分粉虱样品对我来说。 </li><li>通过浸渍在绝对醋酸铀酰中5-10分钟，然后浸入50％碱性柠檬酸铅另外5-10分钟，将超薄膜（在3.9节中获得）染色。 注意:第3.1-3.10节中使用的试剂的体积取决于样品。确保样品浸入试剂中。 </li><li>观察透射电子显微镜（15,000X）下的样品，以确定进一步使用的细菌的定位，形状和大小（见第4.6节）。 </li></ol>白蛉细菌解剖和纯化<ol><li>在显微镜载玻片上加入100μL1x PBS溶液。从棉花叶中挑一些3或4龄的若虫，并将其浸入PBS溶液中。 </li><li> 在立体显微镜下使用精细的昆虫针，从粉虱身上拉出细菌。 <ol><li>在若虫的一边轻轻切开一个洞，轻轻按一下一边让细菌出来。 </li></ol></li><li>在0.5-10μL移液管上装入20μL微型加载器（前端切割以满足细菌大小）。将各种细菌从PBS溶液中提取到微型加载器中。 </li><li>洗涤细菌以通过将细菌吸移到PBS溶液中并提取细菌来消除其他粉虱组织的污染。重复三次。 </li><li>立即将洗涤的细菌移至含有60μL1x PBS溶液的离心管中。 </li><li>注射器通过5μm滤膜过滤组装的细菌（由粉虱中的细菌和细菌核的大小确定）。重复多次，彻底移动混合液通过过滤膜。 注意:为了获得更好的扩增和测序结果，组装超过100个细菌。首先通过使用1×PBS溶液来过滤膜以减少细菌的损失结合膜。 </li></ol>内分泌基因组的扩增<ol><li> 使用推荐的方案，使用DNA扩增试剂盒扩增滤液（主要含有粉虱的内共生体），并进行一些修改。 <ol><li>选择从血液或细胞中扩增基因组DNA的推荐方案，并制备缓冲液D2用于随后的实验。 </li><li>直接向离心管中加入1.5μL滤液（见第4.6节），然后加入1.5μLBuffer D2，充分混匀。 </li><li>在冰上孵育10分钟，并加入1.5μL的终止溶液。 </li><li>加入15μL反应缓冲液和1μLDNA聚合酶，轻轻混匀。 </li><li>将混合物在30℃下孵育16小时，然后在65℃下孵育3分钟。 </li></ol></li><li>在扩增的滤液上直接进行PCR（如果需要，稀释扩增的样品），使用专门为bac设计的特异性引物teria来确认细菌种类（见2.1节）。 </li><li>根据先前发表的方法21 ，使用粉虱基因β-肌动蛋白和EF1引物进行PCR以确认是否存在宿主基因组的污染（PCR配方在1.4节中描述）。 </li></ol> Endosymbiont Metagenome测序<ol><li>在测序前将扩增的滤液置于分光光度计和荧光计（根据制造商的说明书），以测试样品的质量以及样品是否符合测序标准。 </li><li>根据制造商的说明书将其扩增至基因组测序仪。 </li></ol>

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Since the endosymbionts within whiteflies cannot be cultured in vitro, dissection and assembling bacteriocytes is an effective way to obtain enough genetic material of endosymbionts. Before dissection, the species of whitefly and endosymbionts involved should be explicitly confirmed. The whitefly B. tabaci is a species complex with more than 35 morphologically indistinguishable species and different cryptic species may contain different endosymbionts. Portiera is uniformly harbored as an obligate symbiont22, and secondary symbionts vary among species, even the individual whitefly17,21,23. Moreover, although many endosymbionts have been proven to be restrained within bacteriocytes, some may be distributed in the entire body of the whitefly24. Thus, analyzing the localization of different endosymbionts by FISH and TEM is necessary before bacteriome dissection. 
For bacteriome dissection and any subsequent operations, the utilized materials must be strictly sterile to eliminate possible contamination of other bacteria, particularly the PBS solution, the filtration equipment, and the centrifuge tubes. Special care should be taken when filtering the PBS solution containing the bacteriomes to avoid environmental contamination. It is best to use a relatively aseptic environment. In addition, whitefly nymphs can be stored in absolute ethanol at room temperature before dissection, which may be useful to send samples to other places. Nevertheless, dead or environmental stress treated insects should not be used because there would be declination of bacteria25,26. 
In this protocol, the 3rd or 4th instar nymphs were utilized to isolate bacteriome. Compared to other stages of the whitefly, there are several advantages of using the 3rd or 4th instar nymphs for dissection. Firstly, compared to the 1st and 2nd instar nymphs, whiteflies in this stage are obviously larger and not so fragile. Secondly, in the 3rd or 4th instar nymphs of whitefly, bacteriome can be easily distinguished from other tissues of the whitefly body with an obviously yellow color, and bacteriocytes always cluster firmly and can be conveniently separated from other tissues. For the bacteriome in adult whiteflies, once pulled out from the body, bacteriocytes would separate quite easily thus increasing the difficulty in assembly. Hence, the best choice for dissecting bacteriomes is the 3rd or 4th instar nymphs. Nevertheless, this protocol can be used with adult whiteflies for certain experiment needs, with more practice. 
Compared to a previously published method using a single bacteriocyte21, this protocol assembled more bacteriocytes used in the amplification, which contributed to the richness and diversity of bacteriocytes. Furthermore, we utilized filtration to separate symbiont cells from whitefly bacteriocytes, which is more convenient than another method using a micromanipulator and a microinjector. Nevertheless, there were some limitations of this protocol. Firstly, symbionts not confined to bacteriocytes cannot be separated by this method, since we only dissect the bacteriome of the whitefly. Secondly, this protocol cannot absolutely eliminate the contamination of the whitefly genome, since some fragments of whitefly tissues would pass through the filter membrane. Thirdly, clustered symbionts cannot be further separated into individual symbiont species. Hence, obtaining a single species of bacterium accurately and purely requires further study.
In summary, we reported a convenient method for the extraction and purification of endosymbionts from the whitefly B. tabaci that is suitable for subsequent sequencing and analysis. The protocol described here can be easily adapted for the isolation of endosymbionts from other insects for genome sequencing.

与节肢动物形成密切共生关系的细菌广泛存在于节肢动物中。在几乎每个发育阶段5中 ，内分泌已被证明影响宿主的方面，如营养代谢，繁殖，对环境胁迫2,3,4 等的反应。然而，支持协会的机制仍然很大程度上是未知的。研究细菌的潜在功能和作用时，基因组学是重中之重。一些基本信息， 即分类学状态，功能基因，代谢途径，分泌系统，可以从基因组序列推断出来，揭示了共生体在共生中的潜在作用。随着高通量测序的发展，大量的细菌基因组已被测序各种功能揭示6 。 内含子在半ter子中是至关重要的，如蚜虫7 ，臭虫8 ，木虱9 ，褐飞虱10和蝉11 。例如，作为专性共生体的蚜虫中的Buchnera已被证明参与必需氨基酸生物合成，以及来自蚜虫基因组12的基因。此外， Buchnera的转录调控也显示13 。在木麻黄中， 卡桑奈尔被测序，并且是发现的最小的细菌基因组14 。所有这些内含子的标志都是基于基因组序列推断的。由于这些内生子宫内膜不能在体外培养 ，因此已经采用了几种方法来分离足够的细菌uencing。在蚜虫中，通过离心和过滤提取内生子，并进行进一步的基因组和转录组学分析。在褐飞虱中，内共生连同整个昆虫基因组10进行测序。 烟粉虱是一种含有超过35种形态不可区分物种（隐蔽物种）的物种，其中两种入侵物种已经入侵到世界各地，对农业生产造成了巨大的危害15 。值得注意的是， 烟粉虱物种内的内生梭菌在害虫16的发育中已显示出重要性。到目前为止，在粉虱中已经鉴定出8个内含子，其中包括专性共生体， Candidatus Portiera aleyrodidarum和七个次生共生体Hamiltonella，立克次体 ， Arsenophonus ， Cardinium ，Wolbachia， Fritschea和Hemipteriphilus定义为17,18 。 与以前描述的半扁桃不同，粉虱烟粉虱是一只极小的昆虫，长度只有1毫米。大多数内生子体被限制在细菌19 （含有共生体的特殊细胞，其进一步在烟粉虱中形成细菌）。此外，这些内生子宫内膜不能在体外培养 。从烟粉虱获得内生菌的唯一方法是将细菌分解出来。然而，解剖有困难。首先，脆弱的细菌总是与粉虱的其他组织连接，这是很难分离的。其次，粉虱的微小尺寸限制了足够细菌的分离。第三，内生菌群聚集在细菌中，使获得单一种细菌非常复杂。

<table><tbody><tr><td>&lt;em&gt;Taq&lt;/em&gt; DNA polymerase</td><td>Takara</td><td>R001A</td><td>including rTaq, 10&amp;times;Buffer and dNTP</td></tr><tr><td>Gel DNA extraction kit</td><td>Qiagen</td><td>28704</td><td></td></tr><tr><td>DNA sample sequencing system</td><td>ABI</td><td>ABI-3730XL</td><td></td></tr><tr><td>Microtome</td><td>Leica</td><td>EM UC7</td><td></td></tr><tr><td>Transmission electron microscopy</td><td>Hitachi</td><td>H-7650 TEM</td><td></td></tr><tr><td>Stereo microscope</td><td>Zeiss</td><td>Stemi 2000-C</td><td></td></tr><tr><td>20 &amp;mu;L microloader</td><td>Eppendorf</td><td>F2771951</td><td></td></tr><tr><td>Filter holder</td><td>Millipore</td><td>SX0001300</td><td></td></tr><tr><td>Filter membrane filter</td><td>Millipore</td><td>SMWP001300</td><td>5.0 &amp;mu;m SMWP</td></tr><tr><td>REPLI-g UltraFast Mini Kit</td><td>Qiagen</td><td>150033</td><td>DNA amlification kit</td></tr><tr><td>NanoDrop</td><td>Thermo Scientific</td><td>NanoDrop 2000</td><td></td></tr><tr><td>Qubit Fluorometer</td><td>Thermo Fisher Scientific</td><td>Q33216</td><td></td></tr><tr><td>Genome Sequencer</td><td>Illumina</td><td>Hiseq 2000</td><td></td></tr></tbody></table>

methods for the extraction of endosymbionts from the whitefly bemisia tabaci

细菌共生体与宿主形成亲密关系，在大多数情况下赋予宿主优势。基因组信息对于研究其宿主细菌共生体的功能和进化至关重要。由于大多数共生体不能在体外培养 ，所以分离足够量的细菌用于基因组测序的方法是非常重要的。在粉虱Bemisia tabaci中 ，已经确定了许多内生子，并且通过多种方法预测害虫的发育和繁殖是重要的。然而，支持协会的机制仍然很大程度上是未知的。障碍部分来自于粉虱中的内分泌物，主要是抑制细菌，难以与宿主细胞分离。在这里我们报告一个主要通过解剖的粉虱B.烟粉虱鉴定，提取和纯化内分泌物的分步骤方案开和过滤。通过该方法制备的内含子样品虽然仍然是不同内分泌物种的混合物，但适用于随后的基因组测序和分析烟粉虱内共生菌的可能作用。这种方法也可用于从其他昆虫中分离内分泌物。

以烟草烟草复合体的中东亚洲1号（MEAM1）为例进行说明。用于饲养粉虱的棉花和几个发育阶段的粉虱显示在图1中，包括棉花植物，成年粉虱和粉虱的第1， 第 2和第 4龄的若虫（3龄的若虫似乎与第4龄若虫相似） ）。显然，第4龄若虫比第1龄和第2龄的若虫大（ 图1 ）。 MEAM1中Portiera和Hamiltonella的 FISH分析如图2所示 。这两个内共生体被限制在粉虱的细菌细胞中，也观察到两种细菌的​​重叠。 图3显示了传输电路<html>白蛉的Portiera endosymbiont的tron显微镜图像，这表明Portiera可能会失去其细胞壁。 对于测序，构建了两个分别为200bp和2,000bp的插入片段的文库。测序后，两个文库分别生成1,626 Mb和1,302 Mb的原始数据。清理适配器和重复的污染数据后，获得了1,484 Mb和1,219 Mb的清洁数据。该组合成功地导致了专有共生体Portiera的完整基因组序列。此外，还获得了Hamiltonella的基因组草案。基于200bp和2,000bp文库的组合产生了138个重叠群。根据配对关系，将138个重叠群进一步组装成89个支架（ 表2 ）。 figimg"src ="/ files / ftp_upload / 55809 / 55809fig1.jpg"/&gt; 图1:本文使用的植物 - 昆虫系统概述。 （ A ）白花在棉花植物（ Gossypium hirsutum cv。Zhe -Mian 1793）上饲养。 （ B ）成年粉虱的看法。 （ C ）粉虱生命周期中若虫的若虫发育阶段。红箭指的是粉虱的蛋;黑色箭头指粉虱的第一龄若虫;白色箭头指粉虱的第二只若虫;蓝色箭头指粉虱的第 4龄若虫。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55809/55809fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/55809/55809fig2.jpg" /> 图2: Portiera ， Ham的 FISH分析il ella in in。。。。。。。。。 使用缀合有Cy3的Portiera特异性探针（红色），用Cy5标记的Hamiltonella特异性探针（绿色）。刻度棒=100μm。 （ A ）红色杂交信号代表黑暗场下的Portiera 。 （ B ）绿色杂交信号表示黑暗场下的Hamiltonella 。 （ C ） Portiera和Hamiltonella在暗场下合并信号。 （ D ） Portiera和Hamiltonella在明场合并信号。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55809/55809fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" class="xfigimg" src="/files/ftp_upload/55809/55809fig3.jpg" /> 图3:透射电子微风笛在白蛉的图像。箭头表示Portiera的位置。比例尺= 1μm。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55809/55809fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 目标共生体 </td><td> 靶基因 </td><td colspan="3"> 引物序列（5&#39;-3&#39;） </td><td> PCR程序 </td><td> 参考 </td></tr><tr><td> Portiera </td><td> 16S rRNA </td><td colspan="3"> Por-F:TGCAAGTCGAGCGGCATCAT </td><td> 95℃1分钟，60℃1分钟，72℃1分钟，5次循环; </td><td> 27 </td></tr><tr><td></td><td></td><td colspan="3"> Por-R:AAAGTTCCCGCCTTATGCGT </td><td colspan="2"> 9576; C 1分钟，58℃1分钟，72℃1分钟，30个循环; </td></tr><tr><td></td><td></td><td colspan="3"></td><td> 72℃20分钟</td><td></td></tr><tr><td> Hamiltonella </td><td> 16S rRNA </td><td colspan="3"> Ham-F:TGAGTAAAGTCTGGGAATCTGG </td><td> 95℃1分钟，60℃1分钟，72℃1分钟，5次循环; </td><td> 27 </td></tr><tr><td></td><td></td><td colspan="3"> Ham-R:CCCGGGAACGTATTCACCGTAG </td><td colspan="2"> 95℃1分钟，58℃1分钟，72℃1分钟，30个循环; </td></tr><tr><td></td><td></td><td colspan="3"></td><td> 72℃20分钟</td><td></td></tr><tr><td> 立克次体 </td><td> 16S rRNA </td><td colspan="3"> Ric-F:GCTCAGAACGAACGCTATC </td><td> 95℃2分钟; </td><td> 24 </td></tr><tr><td></td><td></td><td colspan="3"> Ric-R:GAAGGAAAGCATCTCTGC </td><td> 92°C 1分钟，58°C 1分钟，72℃90秒，30个循环; </td><td></td></tr><tr><td></td><td></td><td colspan="3"></td><td> 72℃5分钟</td><td></td></tr><tr><td> Arsenophonus </td><td> 23S rRNA </td><td colspan="3"> Ars-F:CGTTTGATGAATTCATAGTCAAA </td><td> 95℃5分钟; </td><td> 28 </td></tr><tr><td></td><td></td><td colspan="3"> Ars-R:GGTCCTCCAGTTAGTGTTACCCAAC </td><td> 95℃30秒，60.5℃30秒，72℃45秒，30个循环; </td><td></td></tr><tr><td></td><td></td><td colspan="3"></td><td> 72℃10分钟</td><td></td></tr><tr><td> Cardinium </td><td> 16S rRNA </td><td colspan="3"> Car-F:TACTGTAAGAATAAGCACCGGC </td><td> 95℃2分钟</td><td> 29 </td></tr><tr><td></td><td></td><td colspan="3"> Car-R:GTGGATCACTTAACGCTTTCG </td><td> 92℃1分钟，57℃1分钟，72℃90秒，30个循环; </td><td></td></tr><tr><td></td><td></td><td colspan="3"></td><td> 72℃5分钟</td><td></td></tr><tr><td> 沃尔巴克氏体 </td><td> 16S rRNA </td><td colspan="3"> Wol-F:TTGTAGCCTGCTATGGTATAACT </td><td> 94℃5分钟; </td><td> 30，31 </td></tr><tr><td></td><td></td><td colspan="3"> Wol-R:GAATAGGTATGATTTTCATGT </td><td colspan="2"> 94℃1分钟，55℃1分钟，72℃1分钟，35个循环</td></tr><tr><td> Fritschea </td><td> 23S rRNA </td><td colspan="3"> Fri-F:GATGCCTTGGCATTGATAGGCGATGAAGGA </td><td> 95℃5分钟; </td><td> 32 </td></tr><tr><td></td><td></td><td colspan="3"> Fri-R:TGGCTCATCATGCAAAAGGCA </td><td> 94℃1分钟，64℃1分钟，72℃90秒，35个循环; </td><td></td></tr><tr><td></td><td></td><td colspan="3"></td><td> 72℃5分钟</td><td></td></tr><tr><td> Hemipteriphilus </EM&gt; </td><td> groEL基因 </td><td colspan="3"> OR- groEL -F:CACCWAAAATTACTAAAGATGG </td><td> 94℃3分钟; </td><td> 18 </td></tr><tr><td></td><td></td><td colspan="3"> OR- groEL -R:TAGAARTCCATWCCKCCCATWC </td><td> 94℃30秒，52℃30秒，72℃2分钟，34个循环; </td><td></td></tr><tr><td></td><td></td><td colspan="3"></td><td> 72℃10分钟</td><td></td></tr></tbody></table> 表1:粉虱中内含子的PCR引物和程序。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td><td> Portiera </td><td> Hamiltonella </td></tr><tr><td> Contig号码 </td><td> 1 </td><td> 138 </td></tr><tr><td> 脚手架号a </str翁&gt; </td><td> 1 </td><td> 89 </td></tr><tr><td> 总长度，bp </td><td> 358232 </td><td> 1711449 </td></tr><tr><td> N50，bp </td><td> / </td><td> 118802 </td></tr><tr><td> N90，bp </td><td> / </td><td> 16753 </td></tr><tr><td> 最大长度，bp </td><td> / </td><td> 390511 </td></tr><tr><td> 最小长度，bp </td><td> / </td><td> 503 </td></tr><tr><td> GC含量，％ </td><td> 26.18 </td><td> 39.89 </td></tr><tr><td colspan="3">以下介绍的信息均基于脚手架。 </td></tr></tbody></table> 表2: Portiera 和 Hamiltonella 基因组的 一般特征 。

Watch this Scientific Journal Video about Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci at JoVE.com

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

在这里，我们提出了通过解剖和过滤从粉虱烟粉虱中分离内分泌物的方案。扩增后，DNA样品适用于后续测序和研究内共生与粉虱之间的共生关系。

从白蛉提取内分泌物的方法<em&gt; Bemisia tabaci</em

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to ...

methods-for-extraction-endosymbionts-from-whitefly-bemisia

Nanyang Technological University

Research

JoVE Journal

Biology

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

9.8K 次观看.  Zhejiang University. 在这里，我们提出了通过解剖和过滤从粉虱烟粉虱中分离内分泌物的方案。扩增后，DNA样品适用于后续测序和研究内共生与粉虱之间的共生关系。 

视频: Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

Life tables provide a means of measuring the schedules of birth and death from populations over time. They also can be used to quantify the sources and rates of mortality in populations, which has a variety of applications in ecology, including agricultural ecosystems. Horizontal, or cohort-based, life tables provide for the most direct and accurate method of quantifying vital population rates because they follow a group of individuals in a population from birth to death. Here, protocols are presented for conducting and analyzing cohort-based life tables in the field that takes advantage of the sessile nature of the immature life stages of a global insect pest, Bemisia tabaci. Individual insects are located on the underside of cotton leaves and are marked by drawing a small circle around the insect with a non-toxic pen. This insect can then be observed repeatedly over time with the aid of hand lenses to measure development from one stage to the next and to identify stage-specific causes of death associated with natural and introduced mortality forces. Analyses explain how to correctly measure multiple mortality forces that act contemporaneously within each stage and how to use such data to provide meaningful population dynamic metrics. The method does not directly account for adult survival and reproduction, which limits inference to dynamics of immature stages. An example is presented that focused on measuring the impact of bottom-up (plant quality) and top-down (natural enemies) effects on the mortality dynamics of B. tabaci in the cotton system.

Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

Life tables allow quantification of the sources and rates of mortality in insect populations and contribute to understanding, predicting and manipulating population dynamics in agroecosystems. Methods for conducting and analyzing cohort-based life tables in the field for an insect with sessile immature life stages are presented.

用虱、烟粉虱在棉花中开发无梗昆虫生命表的方法作为模型系统

Life tables provide a means of measuring the schedules of birth and death from populations over time. They also can be used to quantify the sources and ...

科研

环境科学

Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions

Aphids are excellent experimental models for a variety of biological questions ranging from the evolution of symbioses and the development of polyphenisms to questions surrounding insect's interactions with their host plants. Genomic resources are available for several aphid species, and with advances in the next-generation sequencing, transcriptomic studies are being extended to non-model organisms that lack genomes. Furthermore, aphid cultures can be collected from the field and reared in the laboratory for the use in organismal and molecular experiments to bridge the gap between ecological and genetic studies. Last, many aphids can be maintained in the laboratory on their preferred host plants in perpetual, parthenogenic life cycles allowing for comparisons of asexually reproducing genotypes. Aphis nerii, the milkweed-oleander aphid, provides one such model to study insect interactions with toxic plants using both organismal and molecular experiments. Methods for the generation and maintenance of the plant and aphid cultures in the greenhouse and laboratory, DNA and RNA extractions, microsatellite analysis, de novo transcriptome assembly and annotation, transcriptome differential expression analysis, and qPCR verification of differentially expressed genes are outlined and discussed here.

Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions

The aphid Aphis nerii colonizes on highly-defended plants in the dogbane family (Apocyanaceae) and provides numerous opportunities to study plant-insect interactions. Here, we present a series of protocols for the maintenance of plant and aphid cultures, and the generation and analysis of molecular and -omic data for A. nerii.

nerii 蚜生物培养和基因表达的维持--植物-昆虫相互作用的非模型系统

Aphids are excellent experimental models for a variety of biological questions ranging from the evolution of symbioses and the development of polyphenisms ...

遗传学

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant viruses they vector. Despite numerous studies of the biology of these species in different environments, a key life history parameter, offspring sex ratios, has received little attention, yet is important for predicting population dynamics. The primary sex ratio (sex ratio at oviposition) of Bemisia tabaci has never been reported but can be found by determining the egg fertilization rate of this haplodiploid insect. The technique involves the dechorionation of eggs with bleach, a series of fixation steps, and the application of the general DNA fluorescent stain, DAPI (4&#8242;,6-diamidino-2-phenylindole, a DNA-binding fluorescent dye), to bind to female and male pronuclei. Here, we present the technique, and an example of its application, to test whether an endosymbiotic bacterium, Rickettsia sp. nr. bellii, influenced the primary sex ratio of B. tabaci. This method may assist in population studies of whiteflies, or in determining if sex allocation exists with certain environmental stimuli.

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

We present a simple cytogenetic technique using 4&#8242;,6-diamidino-2-phenylindole (DAPI) to determine the fertilization rate and primary sex ratio of the haplodiploid invasive pest Bemisia tabaci.

用细胞遗传学技术测定芽孢杆菌的受精率

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant ...

A Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of Bemisia tabaci

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many countries around the world. Severe yield losses are caused by direct feeding, and even more importantly, also by the transmission of more than 100 harmful plant pathogenic viruses. As for other invasive pests, increased international trade facilitates the dispersal of B. tabaci to areas beyond its native range. Inspections of plant import products at points of entry such as seaports and airports are, therefore, seen as an important prevention measure. However, this last line of defense against pest invasions is only effective if rapid identification methods for suspicious insect specimens are readily available. Because the morphological differentiation between the regulated B. tabaci and close relatives without quarantine status is difficult for non-taxonomists, a rapid molecular identification assay based on the loop-mediated isothermal amplification (LAMP) technology has been developed. This publication reports the detailed protocol of the novel assay describing rapid DNA extraction, set-up of the LAMP reaction, as well as interpretation of its read-out, which allows identifying B. tabaci specimens within one hour. Compared to existing protocols for the detection of specific B. tabaci biotypes, the developed method targets the whole B. tabaci species complex in one assay. Moreover the assay is designed to be applied on-site by plant health inspectors with minimal laboratory training directly at points of entry. Thorough validation performed under laboratory and on-site conditions demonstrates that the reported LAMP assay is a rapid and reliable identification tool, improving the management of B. tabaci.

A Loop-mediated Isothermal Amplification LAMP Assay for Rapid Identification of Bemisia tabaci

This paper reports the protocol for a rapid identification assay for Bemisia tabaci based on loop-mediated isothermal amplification (LAMP) technology. The protocol requires minimal laboratory training and can, therefore, be implemented on-site at points of entry for plant imports such as seaports and airports.

循环介导等温扩增 (灯管) 法快速识别烟粉虱

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many ...

Fluorescence in situ Hybridizations (FISH) for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells and can be further modified to visualize other components in the cell such as infection with viruses and bacteria. Spatial localization and visualization of viruses and bacteria during the infection process is an essential step that complements expression profiling experiments such as microarrays and RNAseq in response to different stimuli. Understanding the spatiotemporal infections with these agents complements biological experiments aimed at understanding their interaction with cellular components. Several techniques for visualizing viruses and bacteria such as reporter gene systems or immunohistochemical methods are time-consuming, and some are limited to work with model organisms&#160;and involve complex methodologies. FISH that targets RNA or DNA species in the cell is a relatively easy and fast method for studying spatiotemporal localization of genes and for diagnostic purposes. This method can be robust and relatively easy to implement when the protocols employ short hybridizing, commercially-purchased probes, which are not expensive. This is particularly robust when sample preparation, fixation, hybridization, and microscopic visualization do not involve complex steps. Here we describe a protocol for localization of bacteria and viruses in insect and plant tissues. The method is based on simple preparation, fixation, and hybridization of insect whole mounts and dissected organs or hand-made plant sections, with 20 base pairs short DNA probes conjugated to fluorescent dyes on their 5&#39; or 3&#39; ends. This protocol has been successfully applied to a number of insect and plant tissues, and can be used to analyze expression of mRNAs or other RNA or DNA species in the cell.

Fluorescence in situ Hybridizations FISH for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

We describe here a simple fluorescence in situ hybridization (FISH) method for the localization of viruses and bacteria in insect and plant tissues. This protocol can be extended for the visualization of mRNA in whole mount and microscopic sections.

荧光<em&gt;原位</em&gt;杂交（FISH）技术对病毒和植物和昆虫组织内共生细菌的本土化

Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells ...

免疫与感染

Trichogramma dendrolimi 和毒液提取的人工宿主寄生

Extracting Venom from the Parasitoid Wasp Trichogramma dendrolimi Using an Artificial Host

Parasitoid wasps are a diverse group of hymenopteran insects that serve as invaluable resources for pest biocontrol. To ensure successful parasitism, parasitoid wasps inject venom into their hosts to suppress their hosts&#39; immunity, modulate hosts&#39; development, metabolism, and even behavior. With over 600,000 estimated species, the diversity of parasitoid wasps surpasses that of other venomous animals, such as snakes, cone snails, and spiders. Parasitoid wasp venom is an underexplored source of bioactive molecules with potential applications in pest control and medicine. However, collecting parasitoid venom is challenging due to the inability to use direct or electrical stimulation and the difficulty in dissection because of their small size. Trichogramma is a genus of tiny (~0.5 mm) egg parasitoid wasps that are widely used for the biological control of lepidopteran pests in both agriculture and forests. Here, we report a method for extracting venom from T. dendrolimi using artificial hosts. These artificial hosts are created with polyethylene film and amino acid solutions and then inoculated with Trichogramma wasps for parasitism. The venom was subsequently collected and concentrated. This method enables the extraction of large amounts of Trichogramma venom while avoiding contamination from other tissues caused by dissection, a common issue in venom reservoir dissection protocols. This innovative approach facilitates the study of Trichogramma venom, paving the way for new research and potential applications.

作者聚焦：Trichogramma 寄生黄蜂的高效毒液提取方法 

Here, we present a protocol for extracting venom from Trichogramma dendrolimi using an artificial host created with polyethylene film and amino acid solution.

使用人工宿主从寄生蜂 Trichogramma dendrolimi 中提取毒液

Parasitoid wasps are a diverse group of hymenopteran insects that serve as invaluable resources for pest biocontrol. To ensure successful parasitism, ...

生物化学

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

果蝇病毒感染的建立及宿主病毒相互作用分析

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

Transmitting Plant Viruses Using Whiteflies

Whiteflies, Hemiptera: Aleyrodidae, Bemisia tabaci, a complex of morphologically indistinquishable species5, are vectors of many plant viruses. Several genera of these whitefly-transmitted plant viruses (Begomovirus, Carlavirus, Crinivirus, Ipomovirus, Torradovirus) include several hundred species of emerging and economically significant pathogens of important food and fiber crops (reviewed by9,10,16). These viruses do not replicate in their vector but nevertheless are moved readily from plant to plant by the adult whitefly by various means (reviewed by2,6,7,9,10,11,17). For most of these viruses whitefly feeding is required for acquisition and inoculation, while for others only probing is required. Many of these viruses are unable or cannot be easily transmitted by other means. Therefore maintenance of virus cultures, biological and molecular characterization (identification of host range and symptoms)3,13, ecology2,12, require that the viruses be transmitted to experimental hosts using the whitefly vector. In addition the development of new approaches to management, such as evaluation of new chemicals14 or compounds15, new cultural approaches1,4,19, or the selection and development of resistant cultivars7,8,18, requires the use of whiteflies for virus transmission. The use of whitefly transmission of plant viruses for the selection and development of resistant cultivars in breeding programs is particularly challenging7. Effective selection and screening for resistance employs large numbers of plants and there is a need for 100% of the plants to be inoculated in order to find the few genotypes which possess resistance genes. These studies use very large numbers of viruliferous whiteflies, often several times per year.
Whitefly maintenance described here can generate hundreds or thousands of adult whiteflies on plants each week, year round, without the contamination of other plant viruses. Plants free of both whiteflies and virus must be produced to introduce into the whitefly colony each week. Whitefly cultures must be kept free of whitefly pathogens, parasites, and parasitoids that can reduce whitefly populations and/or reduce the transmission efficiency of the virus. Colonies produced in the manner described can be quickly scaled to increase or decrease population numbers as needed, and can be adjusted to accommodate the feeding preferences of the whitefly based on the plant host of the virus.
There are two basic types of whitefly colonies that can be maintained: a nonviruliferous and a viruliferous whitefly colony. The nonviruliferous colony is composed of whiteflies reared on virus-free plants and allows the weekly availability of whiteflies which can be used to transmit viruses from different cultures. The viruliferous whitefly colony, composed of whiteflies reared on virus-infected plants, allows weekly availability of whiteflies which have acquired the virus thus omitting one step in the virus transmission process.

Improving our understanding of and our ability to manage many of the insect-transmitted plant viruses requires the use of the vector. Insect transmission of plant viruses is a tritrophic interaction, and as such requires the manipulation of insects, virus, and plant. Vectors must be reared in large numbers and manipulated in such a way as to insure high rates of transmission to test plants. The basics of rearing and manipulating the whitefly, Bemisa tabaci (Hemiptera: Aleyrodidae), a vector of plant viruses belonging to several emerging plant virus genera, representing a large number of economically significant viruses is presented.

发射使用粉虱的植物病毒

Whiteflies, Hemiptera: Aleyrodidae, Bemisia tabaci, a complex of morphologically indistinquishable species5, are vectors of many plant viruses. Several ...

从白蛉提取内分泌物的方法

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用虱、烟粉虱在棉花中开发无梗昆虫生命表的方法作为模型系统

nerii 蚜生物培养和基因表达的维持--植物-昆虫相互作用的非模型系统

用细胞遗传学技术测定芽孢杆菌的受精率

循环介导等温扩增 (灯管) 法快速识别烟粉虱

荧光

使用人工宿主从寄生蜂 Trichogramma dendrolimi 中提取毒液

使用人工宿主从寄生蜂 Trichogramma dendrolimi 中提取毒液

使用人工宿主从寄生蜂 Trichogramma dendrolimi 中提取毒液

果蝇病毒感染的建立及宿主病毒相互作用分析

发射使用粉虱的植物病毒