这项工作是由中国国家重点基础研究发展计划（编号2013CB127602）和中国国家自然科学基金（31321004号）的创新研究群体科学基金的支持。

1. HaDV2无棉铃虫殖民地的建设<ol><li>后棉铃虫幼虫（ 棉铃虫 ）上以受控的生长室或人工气候室中的人工饲料32在25±1℃，14小时光照/ 10小时黑暗和60％相对湿度。 </li><li>通过使用覆盖有棉纱布每对塑料笼子（10厘米的高度，直径5cm），保证良好的通风，并作为一个产卵基板帮助新eclosed蛾的单对配合。为了提高获得无病毒菌落的可能性，利用至少30对单对交配。用10％的糖和2％维生素溶液32供给成年蛾浸泡上棉球和每日补充。 </li><li>由于雌成虫产卵对棉花纱布约2天交配后，每天更换纱布。收集并维护在同一生长室中含有鸡蛋纱布（或人工气候湛BER）作为成蛾，直到卵孵化。 </li><li>立即新孵化的幼虫转移到新的24孔板中，与一个单独的每孔。 </li><li>第一产蛋日期之后的5天，收集家长飞蛾，并将它们存储在液氮中。 注:收集白蛾第一产蛋日期5天后确保父母在收集点活，因此避免了使用上的DNA提取精度死飞蛾的任何可能的影响。 </li><li>隔离使用根据制造商的说明书的DNA提取试剂盒在这些样品的基因组DNA。 <ol><li>扩增肌动蛋白基因的引物与肌动蛋白F /肌动蛋白R A 574-bp片段（肌动蛋白F:5- CATCTACGAGGGTTACGC-3和肌动蛋白R:5- CATCTGTTGGAAGGTGGA-3）。可视化的聚合酶链反应（PCR），使用根据标准方案琼脂糖凝胶电泳产物;肌动蛋白基因的成功扩增确保DNA的质量是好的。 </li><li>评估Ť他HaDV2的存在下，在使用PCR用特异性引物的父蛾:DVVPF（GGATTGGCCTGGGAAATGAC）和DVVPR（CGTTGTTTTTATATCCGAGG）31。 </li></ol></li><li>一旦一个干净的一对已被确定，从该单个HaDV2未感染的育种对至少五代后的后代，并保持此作为HaDV2 - 自由集落（NONINF应变）;所述HaDV2感染菌落被定义为INF-菌株。 </li><li>为了确认在NONINF应变的HaDV2感染状态（并排除已知细菌或主机开发病毒的效果），评估在八个随机选择的个人幼虫为HaDV2， 沃尔巴克氏体和棉铃虫核型（HaNPV）的感染状况的使用PCR的NONINF -应变（用特异性引物:DVVPF / DVVPR为HaDV2，NPVF / NPVR为HaNPV，和81F / 691R为沃尔巴克氏体 31，33）。 注:据了解，一些病毒可能迟到NT在被感染的宿主，使用基于PCR的技术，甚至测不出时的水平。 NGS技术可以用来确认没有病毒序列存在于基因组DNA样品英寸</li></ol> 2.含有HaDV2-液态流体的制备注:我们已经表明，HaDV2病毒颗粒的纯化试图阻止其感染性和活动31;因此，进行以下方案通过过滤HaDV2感染的个体，以实现感染HaDV2溶液。 <ol><li>单独收集成蛾，并立即将它们存储在液氮中。 </li><li>完全研磨使用杀菌研钵和杵在液氮中所收集的个人蛾。传递约10μg碎片到一个干净的1.5毫升管中。传输剩余的碎片到新的试管，并立即将其存储在-80℃。 </li><li>从碎片的10微克DNA提取，弗洛翼由制造商提供的方案。一旦蛾DNA已经被分离，检查HaDV2使用协议，如在步骤1.6中描述。 </li><li>根据在步骤2.3中获得的结果，分割剩余的碎片分成两组:HaDV2阳性和HaDV2阴性，这取决于它们的感染状态。加入1毫升的磷酸盐缓冲盐水（PBS; 137mM的氯化钠，2.7mM的氯化钾，8毫的Na 2 HPO 4，和1.5mM的KH 2 PO 4; pH7.5）中，每个单独的完全溶解剩余的幼虫碎片。 </li><li>离心匀浆物（来自步骤2.4）在6500×g的15分钟，4℃。过滤与0.22μm的膜过滤器的液体上清液。收集经过滤的液体（每管200μL），并立即将其存储在-80℃。 注:0.22μm的膜过滤器将过滤掉大部分不希望的颗粒，细菌和真菌的。第一预冷离心机至4℃。执行所有协议involvi纳克在冰上过滤后的液体的操作，以防止变成褐色和凝结匀浆。 </li></ol> 3. HaDV2感染棉铃虫殖民地的建设<ol><li> HaDV2水平传播能力<ol><li>生成HaDV2标准曲线。 <ol><li>扩增含有引物的片段（VPF:CTGGTGAGGCGATGGACATG; VPR:TGACAAGATCCAGCGTAGACATC）和探针（VP-探针:GCCACCACTAGAGGACCCACCAGATG），其用于HaDV2病毒的定量，与从头引物（PF:GGACAATGCTGGTGAGGC; PR:AATCCTCTTTGTCCGTTATCTATG）。使用下面的程序:30秒在94℃，在53℃30秒，和60秒72℃，40个循环PCR仪31上。添加包含HaDV2基因组的基因组DNA（步骤2.3）的1μL到作为模板DNA的25-μLPCR反应。根据制造商的方案克隆扩增片段到克隆载体。 </li><li>计算的拷贝数质粒用以下公式:拷贝/μL=（N A×C）/（N×M×10 9），其中N A是阿伏伽德罗数（6.02×10 23分子/摩尔）; C是质粒（毫微克/微升），其可以使用显微分光光度进行测定的质量浓度; n为含有PCR产物（5195 bp）的重组质粒的长度; M是平均碱基对的双链DNA（660克/摩尔）的分子量。 </li><li>制备6 10倍连续稀释在1.5毫升微量离心管中，用1.5×10 9个浓度，1.5×10 8 1.5×10 7，1.5×10 6，1.5×10 5，1.5×10 4拷贝数/ μL。 </li><li>执行使用特异性引物（VPF / VPR）和探针（VP-探针），以产生循环THRE实时定量聚合酶链式反应（qPCR的）在步骤3.1.1.3中描述的质粒的shold（CT）的值。执行三个独立的技术重复，取它们的平均值为进一步计算。 </li><li>使用上述的结果，产生标准曲线的log 2变换的HaDV2浓度现有平均CT值相关。 </li></ol></li><li>在过滤后的液体的流体量化HaDV2。 <ol><li>吸取含有HaDV2 DNA提取过滤的液体流体（步骤2.6）的100-μL测试样品。 </li><li>使用实时-qPCR的获得的CT值的液体流体和使用在步骤3.1.1标准曲线计算拷贝数。 </li></ol></li><li>确定与不同浓度的HaDV2感染过滤液体的口腔感染效率。 <ol><li>含有HaDV2液态流体稀释至下列浓度:10 8，10 7，10 6，10 5，10 4，和0拷贝数/μL。 </li><li>传播人工迪等均匀地凝固之前厘米直径9培养皿中。均匀地散布HaDV2含有过滤后的液体（200μL）用于固体人工饲料的表面上的各浓度。在通风橱中做到这一点。 </li><li>在培养皿中转移100新鲜孵化的幼虫与含HaDV2人工食物。轻轻敲击棉纱布（包括新鲜孵化的幼虫），以幼虫从纱布转移到白纸片材。轻轻敲打纸，使幼虫吐丝和"气球"断纸。使用消毒镊子触摸丝绸和移动幼虫培养皿中。 </li><li> 48小时后，转移的人工饲料至24孔板（每孔一个个体）的幼虫。 </li><li>在后方上述浓度到成年阶段与HaDV2接种幼虫。 </li><li>收集成年蛾，提取DNA，并用PCR检测HaDV2，如在步骤1.5中描述。 注:在这里，得到助的100％HaDV2感染率使用10 8拷贝数/μL，从而建立HaDV2感染细胞集落（INF-株）tton棉铃虫。 </li></ol></li></ol></li><li> HaDV2的垂直传播能力<ol><li>从NONINF和INF-菌株选择成年蛾，以产生单对♀-/♂-，♀+ /♂-，♀-/♂+和♀+ /♂+（+:阳性HaDV2感染; - :阴性HaDV2感染）。 </li><li>传送从上述对向无病毒的24孔板的后代;后幼虫到第三龄。 </li><li>提取从所收集的第三龄幼虫的DNA作为模板使用，以确定HaDV2的存在，如步骤1.5中所述。 </li></ol></li></ol> 4. HaDV2的组织分布<ol><li>为了防止病毒和细菌污染，消毒镊子和剪刀在75％的乙醇三次，然后在醇灯的火焰加热它们。 注:镊子sterilization是在必要时污染发生。 </li><li>用分析天平，称量空管将被用于存储组织。 </li><li>从放置在冰上的INF-应变幼虫和成虫蛾约5分钟。 </li><li>解剖在幼虫，成年女性，和成年男性在含有PBS缓冲液中的干净的培养皿在立体显微镜下以下的组织。 <ol><li>对于幼虫，吸管血淋巴和解剖前肠，肠，肠，脂肪体，和马氏管（ 图1A）。立即在液氮中的每个组织转移到管中。 <ol><li>固定使用两个昆虫针（直径为200μm，20mm长），插入到所述头部附近的节间膜和最后腹部段（ 图1A）上的泡沫板冷冻幼虫。 </li><li>使得幼虫血淋巴流出切幼虫的后腹部前脚。移液器使用microp 10秒内幼虫血淋巴ipette防止血淋巴的褐变和凝固。 注:苯基硫脲（50微克/毫升）可以1:20体积/体积比混合与血淋巴，以防止黑化。 </li><li>用一把剪刀，做一个切口在角质层从最后间膜头部。解剖使用两个镊子在立体显微镜下所有剩余的组织;对于每个组织图像示于图1A中 。 </li><li>从三米个人混合组织样本一起作为一个重复。 </li><li>为了防止剥离过程中与血淋巴剩余组织的污染，洗净在PBS中解剖组织缓冲液三次。然后，测试用于洗涤的PBS的HaDV2感染状态。如果最终的洗涤缓冲液是HaDV2 - 自由，组织应该是从与血淋巴污染干净。 </li></ol></li><li>对于成年女性，剖析头部（大脑），肌肉，脂肪体，肠，马氏管和卵巢（图1B）在PBS缓冲液中在立体显微镜下。对于成年男性，在PBS缓冲液在立体显微镜下解剖头部（大脑），肌肉，脂肪体，肠，马氏管，和睾丸（ 图1C）。立即在液氮中的每个组织转移到管中。 <ol><li>使用镊子和剪刀，取出成蛾的翅膀和洗剩余身体三次以清除鳞屑。 </li><li>用剪刀分离的头部，胸部和腹部。 </li><li>取出胸部的内骨骼和收集的肌肉。 </li><li>使用镊子，除去腹部的角质层和解剖图1B和1C之后的其他组织。 </li><li>从三个人的混合组织样本一起作为一个重复。洗在PBS中解剖组织缓冲液三次。 </li></ol></li></ol></li><li>称量管和上述组织时，他们已被冻结，并确保米不同组织的驴几乎相等。 质谱（组织）=质量（管/组织样品） - 质谱（仅管） </li><li>提取从这些组织的DNA并进行定量PCR，如在步骤3.1.2说明。 </li><li>计算每使用在步骤3.1.1中产生的标准曲线的每个mg组织的拷贝数。使用病毒滴度每单位质量，以校正由在组织的用于DNA提取量的任何差异的样品偏差。 </li><li>计算在特定组织HaDV2的百分比如下:HaDV2的百分比对于每个组织= HaDV2复制每测试组织/（的拷贝数占所有组织毫克总和）的毫克数。 </li></ol> 5.生物测定测试HaDV2对主机发展的影响<ol><li>地方至少100蛹或从NONINF应变在棉花纱布覆盖，5-L，金属丝网笼衍生新出现的成年蛾。 2天后，交配成年女性会开始对棉花纱布产卵。收集和维护含有蛋的棉纱布;鸡蛋将在三天孵化。 </li><li>与HaDV2新生儿幼虫接种。 <ol><li>转移NONINF应变的新生幼虫，以含有HaDV2（10 8拷贝数/μL）以建立INF-应变的人工饲料。 </li><li>类似地，新生幼虫转移到覆盖有HaDV2无过滤的液体（步骤2.4），以建立对照组（NONINF株）的人工饲料。包括在各处理（ 图2A）为至少240的幼虫。 注:幼虫被用于构造NONINF-和INF-菌株从在同一时间相同的父和队列舱口衍生，确保相同的遗传背景。确保NONINF-和INF-菌株都保存在相同的气候室（25±1℃，14小时光照/ 10小时黑暗和60％相对湿度）发展的同步速度;速率可以取决于温度和湿度而变化。处理德利奇轻轻地吃幼虫。 </li></ol></li><li> 48小时后，单独传送幼虫24孔板（每孔一个个体）（ 图2B）。 <ol><li>顺序号在每孔各幼虫，并记录每天的龄期和生存状态。 </li><li>约5天后，当人工饮食恶化，同时这些个体转移到含有新鲜人工饲料（ 图2C）一个新的24孔板中。 </li><li>使用分析天平以0.0001克精密称取从第7天幼虫11孵化后。称重后，幼虫单独返回到24孔板。 </li></ol></li><li>一旦第四蜕皮后蜕皮至第五龄（孵化后十天左右），转移幼虫玻璃管（10厘米高，2cm直径）（ 图2D）;第五龄幼虫也可通过头胶囊（约2.6mm）的宽度确定的。 注意事项:传送个人至在同一时间的每一天的玻璃管中。 <ol><li>码每个幼虫与上使用涂料或标记玻璃管其原始参考号。每日记录幼虫状态;在玻璃试管，之后五天左右-蛹阶段持续大约11天幼虫化蛹会。 </li><li>记录用于在玻璃管每个单独的蛹羽化和日期。记录3日龄蛹的质量。 </li></ol></li><li>羽化后，把一只雌一张雄与10％蔗糖和2％的维生素溶液（ 图2E）的单一笼。每天补充蔗糖溶液。记录死亡的每个蛾的日期。 </li><li>当产卵，每天更换纱布。立即计算蛋的数量。算上刚孵出的后代，而他们孵化。 </li><li>在生物测定，随机选择八个人每个处理确认HaDV2的感染状况。提取使用市售胰蛋白酶试剂这些样品的总RNA按照制造商的协议。合成第一链cDNA按照制造商的协议，并以此为模板，以测试是否病毒成功转录。 </li></ol>

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作者宣称，他们没有竞争的经济利益。

在过去的几十年中，以昆虫病毒相互作用的大多数研究都集中在蜜蜂健康34，35，36，人类疾病37的载体，植物病毒38，和一些昆虫致病病毒有很大的潜力作为生物控制剂39。很少有人注意支付给隐蔽病毒的昆虫，特别是鳞翅目害虫。在这里，我们提出了一个协议的隐蔽病毒与...

在过去的几十年中，测序技术的发展，如下一代测序（NGS）提供了便利的许多新的DNA和RNA病毒，特别是非致病性病毒，而且先前已知的病毒1，2，3新颖菌株的发现， 4，5，6，7，8，9，10，11，12。在模式生物果蝇 ，已使用的宏基因组技术13检测超过20个新的部分病毒基因组。许多病毒序列，包括新颖的病毒，也已在其它昆虫，如蜜蜂，米识别osquitoes，亚洲柑橘木虱，蜻蜓，和多个鳞翅目种类14，15，16，17，18，19，20，21。 在未来，可以预计将有更多的新病毒会使用这些先进技术的昆虫被发现;因此，我们对病毒与宿主相互作用的理解可能会相应地改变6,9。例如，病毒与宿主相互作用被认为是比以前认为的更加复杂，因为许多新的病毒被定义为互惠的合作伙伴，而不是严格的病原体22。例如，在Dysaphis plantaginea的共生浓核病毒DplDNV诱导翅变形和<html>增加流动性，有利于主机的扩散以及病毒23。此外，互惠病毒已与关于哺乳动物健康，干旱和植物的耐寒性，和细菌感染24的碰撞描述。塞内卡山谷病毒-001被证明介导选择性细胞毒性对肿瘤细胞具有神经内分泌癌特征25。 A型肝炎病毒感染抑制丙型肝炎病毒的复制，并可能导致丙型肝炎26恢复。疱疹病毒潜伏期赋予免受细菌感染27共生保护。人内源性逆转录病毒HERV-W的包膜糖蛋白诱导对脾坏死病毒28的细胞抗性。从内生真菌弯孢热耐受性病毒（CThTV）参与这种真菌和热带恐慌草之间的互惠互动REF"&gt; 29。因此，新发现的病毒和其宿主之间的相互作用的知识应该产生自己的生物学和管理的新观点。然而，新的病毒，尤其是隐蔽病毒，显示典型的急性感染无明显体征，有很少被查处，我们需要一个管道和协议来调查他们的主人的新发现的病毒的影响。 此前，我们已经报道的棉铃虫， 棉铃虫的流行新monosense浓核病毒棉铃虫浓核病毒（HaDV2），并提出了HaDV2和棉铃虫30,31之间的互惠关系的证据。在本文中，我们将介绍实验室实验方案进行详细研究HaDV2和棉铃虫宿主之间的相互作用。这里介绍的协议也可能是研究人员对第r高度相关OLE的其他口服传播的病毒，特别是在鳞翅目害虫。

<table><tbody><tr><td>24-well plate</td><td>Corning</td><td>07-200-740</td><td>Multiple suppliers available.</td></tr><tr><td>DNA extraction kit</td><td>TIANGEN</td><td>DP304-03</td><td>Multiple suppliers available.</td></tr><tr><td>thermal cycler</td><td>Veriti; Applied Biosystems</td><td>4375786</td><td></td></tr><tr><td>PBS</td><td>Corning</td><td>21-040-CV</td><td></td></tr><tr><td>0.22 &amp;micro;m membrane filter</td><td>Millipore</td><td>SLGS025NB</td><td></td></tr><tr><td>pEASY-T Cloning Vector</td><td>TransGen, Beijing, China</td><td>CT301-02</td><td></td></tr><tr><td>Tweezers</td><td>IDEAL-TEK</td><td>2.SA</td><td></td></tr><tr><td>Premix Ex Taq (Probe qPCR)</td><td>Takara</td><td>RR390A</td><td></td></tr><tr><td>Probes</td><td>Invitrogen</td><td>Custom order</td><td></td></tr><tr><td>Primers</td><td>Invitrogen</td><td>Custom order</td><td></td></tr><tr><td>microspectrophotometry NanoDrop 2000c&amp;nbsp;</td><td>Thermo scientific&amp;nbsp;</td><td>not available</td><td></td></tr><tr><td>7500 Real-Time PCR system</td><td>Applied Biosystems</td><td>not available</td><td></td></tr><tr><td>stereomicroscope SZX-16</td><td>Olympus</td><td>not available</td><td></td></tr><tr><td>sucrose</td><td></td><td></td><td>Multiple suppliers available.</td></tr><tr><td>vitamin complex</td><td></td><td></td><td>Multiple suppliers available.</td></tr></tbody></table>

protocols for investigating the host-tissue distribution, transmission-mode, and effect on the host fitness of a densovirus in the cotton bollworm

许多新的病毒已经在用新一代测序技术的动物宿主被发现。此前，我们报道了互惠病毒， 棉铃虫浓核病毒（HaDV2），在鳞翅目的物种，棉铃虫， 棉铃虫 （棉铃虫）。在这里，我们描述了目前用于研究HaDV2其主机上的效果的协议。首先，我们从单一的养殖对建立自由HaDV2，棉铃虫落。然后，我们口服接种用含有HaDV2-过滤的液体以产生两个菌落用该遗传背景一些新生儿幼虫后代:一个HaDV2感染，其他未感染的。一个协议到HaDV2感染和-uninfected个体还提出之间比较生命表参数（ 例如，幼虫，蛹和成虫期和繁殖力），作为用于确定HaDV2的宿主组织分布和传输效率的协议。这些协议をULD也适用于研究在其昆虫宿主，特别是鳞翅目的主机其他口头传播病毒的影响。

从分别HaDV2 -自由（ 图3A）的父母后代饲养作为NONINF应变。我们使用相同的DNA模板，这表明DNA模板质量良好（ 图3B）成功地扩增肌动蛋白基因。此外，随机选择的8个后代也是免费HaDV2（ 图3C），HaNPV（ 图3D），并沃尔巴克氏体 （ 图3E）的。再一次，我们的结论是，后代的DNA样品的质量好，因为?...

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棉铃虫浓核病毒宿主组织分布、传播模式及其对宿主适应性影响的研究方案生物学 |视频

Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm

在这里，我们提出了一个协议，调查宿主组织分布，传输模式和效果上的浓核病毒的宿主健身鳞翅目物种内，棉铃虫。该协议也可以用于研究其他经口传播的病毒和它们的昆虫宿主之间的相互作用。

协议侦办主机组织分布，传播模式和效果上浓核病毒的主机健身的棉铃虫

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, ...

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8.4K 次观看.  Chinese Academy of Agricultural Sciences. 在这里，我们提出了一个协议，调查宿主组织分布，传输模式和效果上的浓核病毒的宿主健身鳞翅目物种内，棉铃虫。该协议也可以用于研究其他经口传播的病毒和它们的昆虫宿主之间的相互作用。 

视频: Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm

Remote Sensing Evaluation of Two-spotted Spider Mite Damage on Greenhouse Cotton

The objective of this study was to evaluate a ground-based multispectral optical sensor as a remote sensing tool to assess foliar damage caused by the two-spotted spider mite (TSSM), Tetranychus urticae Koch, on greenhouse grown cotton. TSSM is a polyphagous pest which occurs on a variety of field and horticultural crops. It often becomes an early season pest of cotton in damaging proportions as opposed to being a late season innocuous pest in the mid-southern United States. Evaluation of acaricides is important for maintaining the efficacy of and preventing resistance to the currently available arsenal of chemicals and newly developed control agents. Enumeration of spider mites for efficacy evaluations is laborious and time consuming. Therefore, subjective visual damage rating is commonly used to assess density of spider mites. The NDVI (Normalized Difference Vegetation Index) is the most widely used statistic to describe the spectral reflectance characteristics of vegetation canopy to assess plant stress and health consequent to spider mite infestations. Results demonstrated that a multispectral optical sensor is an effective tool in distinguishing varying levels of infestation caused by T. urticae on early season cotton. This remote sensing technique may be used in lieu of a visual rating to evaluate insecticide treatments.

This manuscript describes a multispectral optical sensor that effectively detected damage to early season cotton artificially infested with varying densities of the two-spotted spider mite populations.

温室棉二斑叶螨危害的遥感评价

The objective of this study was to evaluate a ground-based multispectral optical sensor as a remote sensing tool to assess foliar damage caused by the ...

科研

环境科学

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 ...

Screening Cotton Genotypes for Reniform Nematode Resistance

A rapid non-destructive reniform nematode (Rotylenchulus reniformis) screening protocol is needed for the development of resistant cotton (Gossypium hirsutum) varieties to improve nematode management. Most protocols involve extracting vermiform nematodes or eggs from the cotton root system or potting soil to determine population density or reproduction rate. These approaches are generally time-consuming with a small number of genotypes evaluated. An alternative approach is described here in which the root system is visually examined for nematode infection. The protocol involves inoculating cotton seedling 7 days after planting with vermiform nematodes and determining the number of females attached to the root system 28 days after inoculation. Data are expressed as the number of females per gram of fresh root weight to adjust for variation in root growth. The protocol provides an excellent method for evaluating host-plant resistance associated with the ability of the nematode to establish an infection site; however, resistance that hinders nematode reproduction is not assessed. As with other screening protocols, variation is commonly observed in nematode infection among individual genotypes within and between experiments. Data are presented to illustrate the range of variation observed using the protocol. To adjust for this variation, control genotypes are included in experiments. Nonetheless, the protocol provides a simple and rapid method to evaluate host-plant resistance. The protocol has been successfully used to identify resistant accessions from the G. arboreum germplasm collection and evaluate segregating populations of more than 300 individuals to determine the genetics of resistance. A vegetative propagation method for recovering plants for resistance breeding was also developed. After removal of the root system for nematode evaluation, the vegetative shoot is replanted to allow the development of a new root system. More than 95% of the shoots typically develop a new root system with plants reaching maturity.

Here, a protocol is presented for the rapid non-destructive screening of cotton genotypes for reniform nematode resistance. The protocol involves visually examining the roots of nematode-infected cotton seedlings to determine infection response. The vegetative shoot from each plant is then propagated to recover plants for seed production.

筛选抗内虫抗性棉基因型

A rapid non-destructive reniform nematode (Rotylenchulus reniformis) screening protocol is needed for the development of resistant cotton (Gossypium ...

遗传学

Protocols for Oral Infection of Lepidopteran Larvae with Baculovirus

Baculoviruses are widely used both as protein expression vectors and as insect pest control agents. This video shows how lepidopteran larvae can be infected with polyhedra by droplet feeding and diet plug-based bioassays. This accompanying Springer Protocols section provides an overview of the baculovirus lifecycle and use of baculoviruses as insecticidal agents, including discussion of the pros and cons for use of baculoviruses as insecticides, and progress made in genetic enhancement of baculoviruses for improved insecticidal efficacy.

In this video, we demonstrate oral infection techniques of lepidopteran larvae with baculovirus in order to determine insecticidal efficiency.

口腔感染杆状病毒鳞翅目幼虫的协议

Baculoviruses are widely used both as protein expression vectors and as insect pest control agents. This video shows how lepidopteran larvae can be ...

生物学

Protocols for Microapplicator-assisted Infection of Lepidopteran Larvae with Baculovirus

Baculoviruses are widely used both as protein expression vectors and as insect pest control agents. . This video shows how lepidopteran larvae can be infected with microapplicator techniques in the gut with baculovirus polyhedra and in the hemolymph with budded virus. This accompanying Springer Protocols section provides an overview of the baculovirus lifecycle and use of baculoviruses as insecticidal agents.  Formulation and application of baculoviruses for pest control purposes are described elsewhere.

In this video, we demonstrate two microapplicator techniques used to infect of lepidopteran larvae with baculovirus in order to determine insecticidal efficiency.

Microapplicator辅助鳞翅目幼虫与杆状病毒感染的协议

Baculoviruses are widely used both as protein expression vectors and as insect pest control agents. . This video shows how lepidopteran larvae can be ...

Use of a Recombinant Mosquito Densovirus As a Gene Delivery Vector for the Functional Analysis of Genes in Mosquito Larvae

In vivo microinjection is the most commonly used gene transfer technique for analyzing the gene functions in individual mosquitoes. However, this method requires a more technically demanding operation and involves complicated procedures, especially when used in larvae due to their small size, relatively thin and fragile cuticle, and high mortality, which limit its application. In contrast, viral vectors for gene delivery have been developed to surmount extracellular and intracellular barriers. These systems have the advantages of easy manipulation, high gene transduction efficiency, long-term maintenance of gene expression, and the ability to produce persistent effects in vivo. Mosquito densoviruses (MDVs) are mosquito-specific, small single-stranded DNA viruses that can effectively deliver foreign nucleic acids into mosquito cells; however, the replacement or insertion of foreign genes to create recombinant viruses typically causes a loss of packaging and/or replication abilities, which is a barrier to the development of these viruses as delivery vectors.
Herein, we report using an artificial intronic small-RNA expression strategy to develop a non-defective recombinant Aedes aegypti densovirus (AaeDV) in vivo delivery system. Detailed procedures for the construction, packaging and quantitative analysis of the rAaeDV vectors, and for larval infection are described.
This study demonstrates, for the first time, the feasibility of developing a non-defective recombinant MDV micro RNA (miRNA) expression system, and thus providing a powerful tool for the functional analysis of genes in mosquito and establishing a basis for the application of viral paratransgenesis for controlling mosquito-borne diseases. We demonstrated that Aedes albopictus 1st instar larvae could be easily and effectively infected by introducing the virus into the water body of the larvae breeding site and that the developed rAaeDVs could be used to overexpress or knock down the expression of a specific target gene in larvae, providing a tool for the functional analysis of mosquito genes.

We report using an artificial intronic small RNA expression strategy to develop a non-defective recombinant Aedes aegypti densovirus (AaeDV) in vivo delivery system. A detailed procedure for the construction, packaging, and quantitative analysis of the rAaeDV vectors as well as for larval infection is described.

应用重组蚊子核作为基因载体, 用于蚊幼虫基因功能分析

In vivo microinjection is the most commonly used gene transfer technique for analyzing the gene functions in individual mosquitoes. However, this method ...

发育生物学

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

农杆菌介导的病毒诱导的基因沉默含量在棉花

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

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 ...

免疫与感染

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 ...

Localization and Quantification of Begomoviruses in Whitefly Tissues by Immunofluorescence and Quantitative PCR

Begomoviruses (genus Begomovirus, family Geminiviridae) are transmitted by whiteflies of the Bemisia tabaci complex in a persistent, circulative manner. Considering the extensive damage caused by begomoviruses to crop production worldwide, it is imperative to understand the interaction between begomoviruses and their whitefly vector. To do so, localization and quantification of the virus in the vector tissues is crucial. Here, using tomato yellow leaf curl virus (TYLCV) as an example, we describe a detailed protocol to localize begomoviruses in whitefly midguts, primary salivary glands, and ovaries by immunofluorescence. The method is based on the use of specific antibodies against a virus coat protein, dye-labeled secondary antibodies, and a confocal microscope. The protocol can also be used to colocalize begomoviral and whitefly proteins. We further describe a protocol for the quantification of TYLCV in whitefly midguts, primary salivary glands, hemolymph, and ovaries by quantitative PCR (qPCR). Using primers specifically designed for TYLCV, the protocols for quantification allow the comparison of the amount of TYLCV in different tissues of the whitefly. The described protocol is potentially useful for the quantification of begomoviruses in the body of a whitefly and a virus-infected plant. These protocols can be used to analyze the circulation pathway of begomoviruses in the whitefly or as a complement to other methods to study whitefly-begomovirus interactions.

We describe an immunofluorescence and quantitative PCR method for the localization and quantification of begomoviruses in insect tissues. The immunofluorescence protocol can be used to colocalize viral and vector proteins. The quantitative PCR protocol can be extended to quantify viruses in whole whitefly bodies and virus-infected plants.

免疫荧光和定量PCR对白蝇组织中白蝇病毒的定位和定量

Begomoviruses (genus Begomovirus, family Geminiviridae) are transmitted by whiteflies of the Bemisia tabaci complex in a persistent, circulative manner. ...