我们要感谢米歇尔. 月亮 (范德比尔特大学) 为摄影提供帮助。范德比尔特大学向 PA 和 SSLB 提供支持, DGE-1445197 支持。

1. 植物文化
<ol><li>从任何商业供应商购买种子或从该领域的成熟植物中收集。 注: 本协议适用于大多数商业上可用的马利筋物种 (如马利 incarnata、 syriaca、 curassavica、 Gomphocarpus physocarpus)。有些种子可能需要冷分层, 并检查种子供应商的指示。</li>
	<li>
植物种子在一个美好的发芽土壤 (60–70% 细泥煤青苔, 珍珠岩, 蛭石, 石灰石)。
	<ol>
<li>用发芽混合土填充标准苗盘;确保土壤到达水井的顶端。在每个井, 做一个压痕, 以创建一个洞在土壤约3厘米深。</li>
		<li>把一粒种子放在每个洞里, 水很好地与浇水的罐头, 这样土壤覆盖种子和饱和。</li>
		<li>在温室中种植种子 (见下面的条件, 1.5)。定期水, 每天到每一天;足够保持土壤湿度到中等水平。</li>
	</ol>
</li>
	<li>
当幼苗已经种植了他们的第一套完整的叶子, 重新盆栽幼苗的一般盆栽混合 (50–60% 泥炭苔藓, 树皮和石灰石) (图 1a)。
	<ol>
<li>使用4英寸圆盆, 适合与紧密封与杯笼。填充与一般灌封土壤约5厘米以下的边缘。</li>
		<li>在土壤深处创建一个洞, 足够深到罐子的底部。</li>
		<li>用手轻轻地从井里舀出成熟的幼苗, 把它深深地放在4寸锅里的洞里。用泥土覆盖幼苗。水很好。</li>
		<li>定期在温室和水中种植植物, 每日到每隔一天;足以保持适度的土壤水分。</li>
	</ol>
</li>
	<li>
温室条件。
	<ol>
<li>使用制造商的说明, 设置温室恒温器, 以保持18–28°c 和夜间温度之间的日间温度16–22°c。</li>
		<li>在冬季月份, 白天较短, 补充日光与 600 W 高压钠灯泡 (12 小时, 上午8点-下午8点)。</li>
	</ol>
</li>
	<li>
然而, 用叶面的有机肥皂溶液控制有害的害虫 (如蓟马、蚜虫), 请谨慎使用这些产品。
	<ol>
<li>根据制造商的建议制作 soap 解决方案, 并使用喷雾瓶进行应用。</li>
		<li>把肥皂放在植物上 4–24 h. 用清水轻轻冲洗植物, 除去皂 4–24 h 后应用, 并在实验室蚜虫培养前第二次用水冲洗。</li>
	</ol>
</li>
	<li>养殖平均蚜虫种群, 种植的植物至少有3–4组的全叶, 并且至少有10厘米高 (图 1B)。</li>
</ol>2. 蚜虫文化
<ol><li>
从现有实验室 isoclonal 种群开始实验室蚜虫种群, 或从田间采集的蚜虫开始, 按照下面的指示进行。
	<ol>
<li>当从现有实验室 isoclonal 人口中启动实验室人口时, 转移蚜虫, 如 2.3. 1–2.3 3。</li>
	</ol>
</li>
	<li>
当开始新的 isoclonal, 田间收集蚜虫种群, 并放置一个单一的, 繁殖, 成年蚜虫在适当的寄主植物, 如步骤1.6 所述。 注: 人群可从翼状 (迟到) 或无翅 (apterous) 成人开始。<ol>
<li>在使用实验室蚜虫之前, 从温室中手动检查植物的有害害虫。用不需要的蚜虫冷冻任何植物。如果需要, 使用乙醇真空瓶去除蓟马或其他害虫。 注: 请务必在使用步骤1.5.2 中的描述之前, 冲洗已用肥皂处理过的植物。</li>
		<li>使用用 3/16 "ID x 1/4" 外径塑料管、1000µL 吸管尖端和200µL 吸管尖 (图 2a) 创建的画笔或嘴吸管安全地转移单个成人蚜虫。</li>
		<li>安全地覆盖植物与蚜虫与一个杯子笼创建了一个塑料杯与顶部切断, 覆盖了良好的网格和固定胶带 (图 2B)。</li>
		<li>将蚜虫出没的植物放在托盘中, 并保持在受控环境室 (16 升: 8 d, 22 °c, 70% 湿度)。</li>
	</ol>
</li>
	<li>
为了维持种群数量, 将蚜虫转移到新鲜的新植物每周 (2.2. 1–2.2 3)。
	<ol>
<li>使用口吸管安全转移 1–3 2 和 3路龄若虫和1个成年龄蚜虫 (图 2A, 3)。 注: 最好通过转移无翅个人来维持股票。</li>
		<li>安全地覆盖蚜虫出没的植物与一个杯子笼子创建了一个塑料杯与顶部切断, 覆盖了一个细网眼和固定胶带。</li>
		<li>将植物放在托盘中, 并将蚜虫保存在一个环境室内 (16 升: 8 d, 22 摄氏度, 70% 湿度)。</li>
		<li>或者, 如果需要, 如果寄主植物质量好, 使用乙醇真空瓶减少人口只留下一个繁殖的成人和二到三第二或第三龄的若虫。</li>
	</ol>
</li>
	<li>
为了创造同样年龄的人口用于实验, 将多达5的成年人 (最好是无翅) 从种群中安置到一个新的寄主植物上。
	<ol>
<li>24小时后移除成人。</li>
		<li>大约5–7天后, 一旦 F1的后代成熟到成年, 在一个新的寄主植物上放置多达5无翅 F1成人。24小时后移除成人。</li>
		<li>一旦 F2人口成熟到成年, 这个人口就可以在实验中使用了。 注意: 这一过程确保实验人群的年龄大致相同, 并且出生于年龄相仿的母亲。</li>
	</ol>
</li>
	<li>用微卫星基因分型 (如下所述, 3 & 4 节) 确认野外捕获 isoclonal 线间基因的差异。</li>
</ol>3. DNA 提取
<ol><li>
制备
	<ol>
<li>使用无菌技术制备 1 L 裂解缓冲液 (0.1 米氯化钠, 0.2 米蔗糖, 0.1 米三 (pH 9.1), 0.05 米 EDTA, 0.05% SDS)。</li>
		<li>将加热块或水浴预热至65摄氏度。</li>
	</ol>
</li>
	<li>
组织均匀化和裂解
	<ol>
<li>将蚜虫放在不育的1.5 毫升离心管的底部附近。</li>
		<li>将无菌杵与蚜虫放在管中, 将管子的底部浸入液氮中。 注: 当蚜虫位于杵和管侧之间时, 最佳组织崩解。</li>
		<li>用杵磨蚜虫, 最初溶解细胞。</li>
		<li>对于单一的成人蚜虫, 使用200µL (分裂成 2 x 100 µL 整除数) 的溶解缓冲。加入第一整除研磨和并用重悬粉碎蚜虫, 直到样品明显解体, 然后使用第二整除洗掉杵。</li>
		<li>在65摄氏度的水浴或热块中, 将被粉碎的蚜虫在溶解缓冲中孵化30分钟。</li>
	</ol>
</li>
	<li>
DNA 沉淀
	<ol>
<li>当管子是温暖的, 增加14µL 8 M KOAc。把管子颠倒来混合。将样品储存在冰上30分钟。 注意: 该协议可以在这里暂停, 样品可以存储在-20 °c 高达24小时。</li>
		<li>离心机在室温下为 1.3万 x克, 15 分钟。用吸管将上清液转到新的1.5 毫升管。小心不要去除任何颗粒碎片。</li>
		<li>为了改善 DNA 颗粒的可视化, 添加2µL 糖原 (20 µg/毫升) 到上清。如果样本大小足够大, 请省略此步骤。</li>
		<li>将200µL 的冷100% 分子级乙醇添加到上清。倒置管在室温下混合和孵育至少15分钟。 注意: 该协议可以在这里暂停, 样品可以存储在-20 °c 高达24小时。</li>
		<li>离心机在室温下为 1.3万 x克, 15 分钟。用吹打去除乙醇。</li>
	</ol>
</li>
	<li>
DNA 洗涤和洗脱
	<ol>
<li>加入200µL 冷70% 分子级乙醇。然后轻轻地把管子并用重悬, 然后把球团洗干净。</li>
		<li>离心机在 1.3万 x g 5 分钟。在可视化颗粒的同时, 小心地去除乙醇, 吹打200µL 的冷100% 分子级乙醇。</li>
		<li>离心机在 1.3万 x g 5 分钟。在可视化颗粒时, 用吹打小心地去除乙醇。 注: 如有必要, 重复100-70-100 乙醇清洗。</li>
		<li>空气干燥的颗粒为5–10分钟, 管铺设水平打开在纸巾上。</li>
		<li>并用重悬80µL 的 DNA 颗粒 (10 毫米三盐酸, 0.1 毫米 EDTA)。</li>
		<li>使用分光光度计量化悬浮 DNA。</li>
		<li>存储在4摄氏度。</li>
	</ol>
</li>
</ol>4. 微卫星 PCR 与蚜虫基因分型排序
<ol><li>为微卫星测序提供适当的 F 和 R 引物 (表 120)。 注: 反向底漆序列应修改与 5 '-6-或 5 '-5 十六进制荧光标签, 以允许多路采样的微卫星测序。</li>
	<li>
用单蚜虫 DNA 样品 (3 节中描述) 和荧光标记微卫星引物进行 PCR。
	<ol>
<li>根据制造商的协议 (0.2 µM, 2.5 毫米氯化镁2, 50–200 ng DNA 模板) 混合 PCR 反应。</li>
		<li>使用以下 thermocycler 设置: 初始变性在94°c 为4分钟, 35 周期94°c 为三十年代, 58 °c 三十五年代, 72 °c 为四十五年代, 并且最后伸长步在72°c 为10分钟。</li>
	</ol>
</li>
	<li>将 PCR 样品与不同的荧光标记结合在一起, 减少样品的数量, 并在基因分型装置上序列微卫星样品。</li>
	<li>使用微卫星分析软件分析. fsa 原始示例文件。</li>
</ol>5. RNA 提取
<ol><li>收集1.5 毫升 RNase/无 DNase 管中 RNA 提取的蚜虫样品, 并立即在液氮中冷冻。 注: 如果不立即执行以下步骤, 则可以将样品存储在摄氏-80 摄氏度。</li>
	<li>
组织均匀化
	<ol>
<li>用无菌杵在管中用蚜虫, 冻在液氮中10–15秒, 直至试样停止咝作响。在步骤3.2 中描述的杵粉碎蚜虫。 注: 当蚜虫位于杵和管侧之间时, 最佳组织崩解。</li>
		<li>在油烟机中, 添加800µL 胍硫氰酸盐-苯酚-氯仿萃取试剂到样品 (1–5成虫蚜虫)。用杵融汇样品, 并处理杵。</li>
	</ol>
</li>
	<li>
相分离 注意: 所有步骤都应在通风罩中进行。<ol>
<li>在室温下孵育匀质样品5分钟。添加160µL 的氯仿样品。在十五年代用手大力摇动。</li>
		<li>室温下孵育2-3 分钟。离心机为15分钟在 1.2万 x g在4°c。 注: 离心后, 混合物分为3层: 一个较低的, 红色的苯酚-氯仿相, 一个界面和一个无色的上水相。RNA 在水相中完全保持。水的体积将是480µL。</li>
	</ol>
</li>
	<li>
RNA 降雨雪
	<ol>
<li>在通风罩中, 将水相转移到一个新鲜的无 RNase 管。请勿扰乱中间阶段。</li>
		<li>添加400µL 异丙醇, 并在-20 摄氏度孵化样品10分钟, 使 RNA 沉淀。 注意: 该协议可以在这里暂停, 样品可以存储在-20 °c 高达24小时。</li>
		<li>离心样品为10分钟在 1.2万 x g在4°c。</li>
	</ol>
</li>
	<li>
RNA 洗涤和洗脱
	<ol>
<li>移除上清;注意 RNA 颗粒。</li>
		<li>用1毫升75% 乙醇在 DEPC 处理的水中清洗 RNA 颗粒。用慢涡流混合。离心机为5分钟在 7500 x g在4°c。</li>
		<li>重复步骤 5.5. 1–5.5 2, 以帮助去除苯酚污染物。</li>
		<li>去除上清液和空气干燥颗粒5-10 分钟, 管铺设水平打开在无菌长凳上。不要让 RNA 颗粒完全干燥。</li>
		<li>在30µL 的 RNase 或 DEPC 处理的水中溶解 RNA 颗粒。轻轻地把吸管向上和向下混合。在55–60°c 孵化10–15分钟。</li>
	</ol>
</li>
</ol>6. RNAseq 转录汇编、注释和差异表达式分析
<ol><li>
用芯片为基础的毛细管电泳系统分析 RNA 样品的浓度和质量。 注: 基于芯片的毛细管电泳系统比分光光度计分析更可取, 因为它为 RNA 浓度和质量提供了更准确、更灵敏的测量方法。<ol>
<li>如果样品是适当的质量 (≥ 250 ng 总, 玲 (RNA 完整性号) ≥ 5), 执行 RNA 排序。 注意: 重要的是, 由于此排序数据将用于表达式分析和转录程序集, 因此更高的读取深度将导致更高质量的转录。对于使用 Illumina 测序技术的合理综合装配, 100–200百万 100 bp, 配对端读将是一个推荐的出发点。总 mRNA 库的准备和 RNA 测序是由测序设施完成的。</li>
	</ol>
</li>
	<li>使用快速 QC22检查读数的质量。</li>
	<li>结合所有的样本读取和组装转录使用三位一体23,24 (Trimmomatic 质量过滤启用)。</li>
	<li>
细化程序集
	<ol>
<li>使用 Transdecoder25识别开放阅读框架 (码), 其长度至少为100个氨基酸。</li>
		<li>分别使用 BLASTP28和 HMMER29, 执行对 Pfam26和 UniProt27数据库的翻译码的同源搜索。</li>
		<li>删除细菌记录 (任何被翻译的序列, 其最佳的爆炸命中是一个细菌基因的分数超过300和最低氨基酸序列标识为 50%)。</li>
		<li>使用 CD 命中30折叠所有在氨基酸水平上至少99% 相同的完整的、翻译的码。</li>
		<li>使用 CD 命中30折叠在核苷酸水平上至少95% 相同的剩余的不完整码。</li>
		<li>分配剩余的核苷酸序列与独特的, 物种特定的标识符 (例如, APHNE 0001)。</li>
	</ol>
</li>
	<li>使用 BUSCO (http://busco.ezlab.org/) 和节肢动物基因数据集31来评估精制组装的完整性。</li>
	<li>
转录注释
	<ol>
<li>首先, 用 HMMER 对 Pfam 数据库26,29注释精致的转录。</li>
		<li>第二, 使用 BLASTP 对 UniProt 数据库27、28注释转录。</li>
		<li>第三, 用 BLASTP 对所选昆虫的编码序列, 用已发布的、带注释的基因组注释转录。</li>
		<li>最后, 对转录使用 BLASTP 对豌豆蚜蛋白数据库进行注释。</li>
		<li>使用 Trinotate 从 UniProt 加入中生成 "转到" 注释。</li>
		<li>使用 Trinotate 将所有注释结果组织到 SQLite 数据库中, 并生成注释报告。</li>
	</ol>
</li>
	<li>
差分表达式分析 注意: 使用精致的转录作为参考, 分别对齐和量化每个库。<ol>
<li>使用 Trimmomatic 来质量过滤和修剪原始读取文件32。 注意: 如果在三位一体程序集之后执行此步骤, 则可以改用该步骤中的 Trimmomatic 输出。</li>
		<li>使用 Bowtie233对每个示例执行局部对齐。</li>
		<li>使用 SAMtools34分别从每个示例中提取读取计数。</li>
		<li>使用 DESeq2 与缺省参数和参数拟合35计算感兴趣的样本之间的微分表达式。</li>
	</ol>
</li>
</ol>7. 差异表达基因的 qPCR 验证
注: 如果用户对差异表达的基因感兴趣, 从他们的 RNAseq 实验, 下面的协议可以用来验证差异表达模式。
<ol><li>如上文所述生成 RNA 样本 (步骤 5)。</li>
	<li>定量 RNA 提取用分光光度计检查质量和获得浓度。</li>
	<li>根据制造商的建议, 使用商业上可用的试剂盒合成 cDNA 样本。</li>
	<li>
确定有兴趣的基因的底漆效率, 以确保准确的双倍 PCR 放大。
	<ol>
<li>根据原始 RNA 浓度, 执行序列稀释 (101) 获得 3 cDNA 浓度。</li>
		<li>使用定量 PCR 大师组合, 根据制造商的协议混合三 qPCR 反应使用三底漆浓度 (例如100 毫微米, 200 毫微米, 300 毫微米) 与三连续稀释的 cDNA 浓度 (例如, 0.1 ng/µL, 10ng/µL, 100 吴/µL)。</li>
		<li>对于每个目标基因, 计算使用平均 Ct值为每个样本所创建的直线的斜率 (m) 作为依赖变量和日志 (cDNA 浓度) 作为独立变量 (三点总计)。</li>
		<li>使用以下等式计算底漆效率 (E), 其中 m 是7.4.3 计算的斜率: E = 10 ^ (-1/m) 注: 90-110% 之间的底漆效率适用于分析。这一过程确保了计算中包括的所有基因的同等放大。</li>
	</ol>
</li>
	<li>使用ΔΔCt方法与管家基因量化的差异表达基因的兴趣36。</li>
</ol>

<ol>
	<li>Brisson, J. A., Stern, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pea+aphid,+Acyrthosiphon+pisum.:+an+emerging+genomic+model+system+for+ecological,+developmental+and+evolutionary+studies.">The pea aphid, Acyrthosiphon pisum.: an emerging genomic model system for ecological, developmental and evolutionary studies.</a> BioEssays. 28, 747-755 (2006).</li><li>Dixon, A. F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Aphid Ecology: An Optimization Approach. , (1985).</li><li>Consortium, T. I. A. 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作者没有什么可透露的。

长期以来, 人们已经认识到, aposematic A. nerii可以提供对抵抗植物防御的模式和机制的洞察力, 特别是化学封存18,37。一些基因组资源最近出现在. nerii10, 为生态和功能基因组研究提供了新的机会, 利用nerii作为模型。我们概述了蚜虫和植物培养的基本协议, 以及分子/基因组技术, 假设未来这一物种的工作可能涉及利用基...

蚜虫是小型的, hemimetabolous 昆虫, 在世界各地的不同的植物家庭殖民。它们是独特的几个特点, 最明显的是他们复杂的生命周期涉及周期性的孤雌和离散 polyphenisms, 和他们的义务营养共生与细菌或酵母 endosymbionts 提供营养缺乏从他们的饮食植物汁液1。虽然大多数蚜虫都是寄主植物专家, 但一些通才物种是重要的农作物害虫, 直接或通过其载体2的病原体和病毒对农作物造成相当大的经济损失。2010年的第一个蚜虫基因组的出版, 豌豆蚜虫Acyrthosiphon 豌豆3, 标志着蚜虫生物学研究的一个重要里程碑, 因为它提供了基因组资源, 以解决有关昆虫的问题对草食生活方式的适应, 包括那些可能导致更好的控制策略4。自那时以来, 额外的基因组资源已经积累了为大豆蚜虫蚜线虫5的注明基因组, 和公开提供的全基因组资源为另三-蚜虫种类 (桃cerasi (黑樱桃蚜虫),桃蚜(桃马铃薯蚜虫),蚜配弟(鸟樱桃燕麦蚜虫)6。有价值的 transcriptomic 资源, 以及其他一些蚜虫种 (例如,棉蚜 (棉花蚜虫)7,麦管蚜管蚜(谷物蚜虫)8, Cinarapinitabulaeformis (松蚜)9,蚜虫 nerii (马利筋-夹竹桃蚜虫)10)。
蚜虫也为我们对植物-昆虫相互作用和植物生命生态学的理解提供了持久的贡献11。蚜虫作出特别重要贡献的一个领域是我们对寄主植物相互作用的化学生态学的理解。草食昆虫为克服植物防御能力提供了多样的适应, 一些甚至为自己的利益选择了植物防御12,13,14。例如, 马利筋-夹竹桃蚜虫, nerii, 是一个明亮的黄色, 侵入性蚜虫发现在全球温带和热带地区, 殖民植物在马利筋家庭 (夹竹桃科)。夹竹桃科家族中的植物进化出多样的化学防御, 包括乳白色乳胶和心苷 cardenolides, 它结合了阳离子载体 Na, K atp 酶, 是对通才草食动物15的有效威慑,16. 马利筋专家表达了各种抵抗 cardenolides 的方式, 有些人有选择地或被动地积累或修改其组织中的 cardenolides, 以此来阻止捕食或其他好处17。nerii被认为以这种方式封存 cardenolides, 虽然机制和功能效益仍然不清楚10,18。
鉴于目前的基因组资源, nerii提供了一个优秀的实验模型, 用于研究有毒寄主植物与其专门草食动物之间的化学-生态相互作用的分子和遗传机制。值得注意的是, 虽然一些早期的研究nerii集中于 cardenolides19的封存, 但从那时起, 对一. nerii的研究提供了对一系列进化和生态学问题的洞察力,包括入侵昆虫的遗传结构20以及自底向上和自上而下调节对草食动物密度的相互作用21。因此, nerii是一个很好的候选者, 作为实验模型, 对昆虫-植物相互作用进行了特别广泛的研究。对任何与nerii的研究的成功至关重要的是蚜虫群体的细致文化, 其中包括蚜虫赖以生存的植物的文化, 以及高效生成高质量涨价可能数据。我们的目标是通过两者来引导读者。下面概述了在温室和实验室, DNA 和 RNA 提取, 微卫星分析, 转录组装和注释的产生和维持植物和蚜虫文化的方法转录差异表达分析和差异表达基因的 qPCR 验证。虽然这些方法是为. nerii编写的, 但一般的培养、提取和分析方法可以扩展到各种蚜虫种类。

<table><tbody><tr><td>Sun Gro Fafard Germination Mix</td><td>Hummert International</td><td>10-0952-2</td><td></td></tr><tr><td>Sun Gro Fafard 3B/ Metro Mix</td><td>Hummert International</td><td>10-0951-2</td><td></td></tr><tr><td>2&quot; x 4&quot; Round Standard Pot</td><td>Anderson Pots</td><td>1503</td><td></td></tr><tr><td>DreamTaq DNA Polymerase</td><td>ThermoFisher Scientific</td><td>EP0701</td><td></td></tr><tr><td>Trizol</td><td>ThermoFisher Scientific</td><td>15596026</td><td></td></tr><tr><td>SuperScript&amp;reg; III First-Strand Synthesis kit</td><td>ThermoFisher Scientific</td><td>18080051</td><td></td></tr><tr><td>Power SYBR Green PCR Master Mix</td><td>ThermoFisher Scientific</td><td>4367659</td><td></td></tr></tbody></table>

maintaining biological cultures and measuring gene expression in aphis nerii: a non-model system for plant-insect interactions

蚜虫是一个优秀的实验模型的各种生物学问题, 从进化的共生和发展 polyphenisms 的问题周围昆虫与他们的寄主植物的相互作用。基因组资源可用于几种蚜虫物种, 随着下一代测序的进展, transcriptomic 研究正在扩展到缺乏基因组的非模型生物。此外, 蚜虫的培养可以从田间收集, 并在实验室饲养, 用于生物体和分子实验, 以弥合生态和遗传学研究之间的差距。最后, 许多蚜虫可以保持在实验室对他们的首选寄主植物在永久, 孤雌生殖生命周期允许比较无性繁殖基因型。蚜虫nerii, 马利筋-夹竹桃蚜, 提供一个这样的模型, 研究昆虫与有毒植物的相互作用, 利用生物体和分子实验。温室植物和蚜虫培养方法及实验室、DNA 和 RNA 提取、微卫星分析、转录组装和注释、转录差异表达本文对差异表达基因的分析和 qPCR 验证进行了概述和讨论。

植物文化:种子将需要大约四周, 取决于季节, 增长足够大, 以重新盆栽 (图 1)。再盆栽苗将需要另外四周的时间才能长成蚜虫培养的最佳大小 (图 1B)。
蚜虫文化:成人A. nerii的区别是一些黑暗的马尾, 可能是无翅 (apterous,<strong cla...

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

蚜虫蚜虫nerii殖民在罗布麻家族 (Apocyanaceae) 高度防卫的植物上, 为研究植物-昆虫相互作用提供了许多机会。在这里, 我们提出了一系列维护植物和蚜虫文化的协议, 以及对nerii的分子和涨价可能数据的生成和分析。

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 ...

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Genetics

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

7.5K 次观看.  Vanderbilt University. 蚜虫蚜虫nerii殖民在罗布麻家族 (Apocyanaceae) 高度防卫的植物上, 为研究植物-昆虫相互作用提供了许多机会。在这里, 我们提出了一系列维护植物和蚜虫文化的协议, 以及对nerii的分子和涨价可能数据的生成和分析。

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

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. This is due to its reproductive behavior: females damage the external surface of fruits and vegetables when they lay eggs and the hatched larvae feed on their pulp. Wild C. capitata populations are traditionally controlled through insecticide spraying and/or eco-friendly approaches, the most successful being the Sterile Insect Technique (SIT). The SIT relies on mass-rearing, radiation-based sterilization and field release of males that retain their capacity to mate but are not able to generate fertile progeny. The advent and the subsequent rapid development of biotechnological tools, together with the availability of the medfly genome sequence, has greatly boosted our understanding of the biology of this species. This favored the proliferation of new strategies for genome manipulation, which can be applied to population control.
In this context, embryo microinjection plays a dual role in expanding the toolbox for medfly control. The ability to interfere with the function of genes that regulate key biological processes, indeed, expands our understanding of the molecular machinery underlying medfly invasiveness. Furthermore, the ability to achieve germ-line transformation facilitates the production of multiple transgenic strains that can be tested for future field applications in novel SIT settings. Indeed, genetic manipulation can be used to confer desirable traits that can, for example, be used to monitor sterile male performance in the field, or that can result in early life-stage lethality. Here we describe a method to microinject nucleic acids into medfly embryos to achieve these two main goals.

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Diptera: Tephritidae) is a worldwide pest of agriculture. A deeper understanding of its biology is key to control medfly populations and thus reduce economic impact. Embryo microinjection is a fundamental tool allowing both germ-line transformation and reverse genetics studies in this species.

核酸的传递通过显微注射胚胎在世界范围内的农业害虫，<em&gt;地中海实蝇</em

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. ...

科研

遗传学

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

饲养并在隐藏甲壳虫双链RNA介导的基因敲除，<em&gt;白腹皮蠹</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

Identification of Critical Conditions for Immunostaining in the Pea Aphid Embryos: Increasing Tissue Permeability and Decreasing Background Staining

The pea aphid Acyrthosiphon pisum, with a sequenced genome and abundant phenotypic plasticity, has become an emerging model for genomic and developmental studies. Like other aphids, A. pisum propagate rapidly via parthenogenetic viviparous reproduction, where the embryos develop within egg chambers in an assembly-line fashion in the ovariole. Previously we have established a robust platform of whole-mount in situ hybridization allowing detection of mRNA expression in the aphid embryos. For analyzing the expression of protein, though, established protocols for immunostaining the ovarioles of asexual viviparous aphids did not produce satisfactory results. Here we report conditions optimized for increasing tissue permeability and decreasing background staining, both of which were problems when applying established approaches. Optimizations include: (1) incubation of proteinase K (1 &#181;g/ml, 10 min), which was found essential for antibody penetration in mid- and late-stage aphid embryos; (2) replacement of normal goat serum/bovine serum albumin with a blocking reagent supplied by a Digoxigenin (DIG)-based buffer set and (3) application of methanol rather hydrogen peroxide (H2O2) for bleaching endogenous peroxidase; which significantly reduced the background staining in the aphid tissues. These critical conditions optimized for immunostaining will allow effective detection of gene products in the embryos of A. pisum and other aphids.

A protocol of whole-mount immunostaining on aphid embryos is presented, in which critical conditions for decreasing background staining and increasing tissue permeability are addressed. These conditions are specifically developed for effective detection of protein expression in the embryonic tissues of aphids.

临界条件在免疫染色豌豆蚜虫胚胎鉴定:增加组织通透性和降低背景染色

The pea aphid Acyrthosiphon pisum, with a sequenced genome and abundant phenotypic plasticity, has become an emerging model for genomic and developmental ...

发育生物学

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and tomato. The susceptibility of M. sexta larvae to a variety of entomopathogenic bacterial species1-5, as well as the wealth of information available regarding the insect's immune system6-8, and the pending genome sequence9 make it a good model organism for use in studying host-microbe interactions during pathogenesis. In addition, M. sexta larvae are relatively large and easy to manipulate and maintain in the laboratory relative to other susceptible insect species. Their large size also facilitates efficient tissue/hemolymph extraction for analysis of the host response to infection.
The method presented here describes the direct injection of bacteria into the hemocoel (blood cavity) of M. sexta larvae. This approach can be used to analyze and compare the virulence characteristics of various bacterial species, strains, or mutants by simply monitoring the time to insect death after injection. This method was developed to study the pathogenicity of Xenorhabdus and Photorhabdus species, which typically associate with nematode vectors as a means to gain entry into the insect. Entomopathogenic nematodes typically infect larvae via natural digestive or respiratory openings, and release their symbiotic bacterial contents into the insect hemolymph (blood) shortly thereafter10. The injection method described here bypasses the need for a nematode vector, thus uncoupling the effects of bacteria and nematode on the insect. This method allows for accurate enumeration of infectious material (cells or protein) within the inoculum, which is not possible using other existing methods for analyzing entomopathogenesis, including nicking11 and oral toxicity assays12. Also, oral toxicity assays address the virulence of secreted toxins introduced into the digestive system of larvae, whereas the direct injection method addresses the virulence of whole-cell inocula.
The utility of the direct injection method as described here is to analyze bacterial pathogenesis by monitoring insect mortality. However, this method can easily be expanded for use in studying the effects of infection on the M. sexta immune system. The insect responds to infection via both humoral and cellular responses. The humoral response includes recognition of bacterial-associated patterns and subsequent production of various antimicrobial peptides7; the expression of genes encoding these peptides can be monitored subsequent to direct infection via RNA extraction and quantitative PCR13. The cellular response to infection involves nodulation, encapsulation, and phagocytosis of infectious agents by hemocytes6. To analyze these responses, injected insects can be dissected and visualized by microscopy13, 14.

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

The method described here utilizes direct injection of entomopathogenic bacteria into the hemocoel of Manduca sexta insect larvae. M. sexta is a commercially available and well-studied insect. Thus, this method represents a simple approach to analyzing host-bacterial interactions from the perspective of one or both partners.

饲养和注射<em&gt;烟草天蛾</em&gt;幼虫评估细菌的毒力

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and ...

免疫与感染

Mass-Rearing and Molecular Studies in Tortricidae Pest Insects

Tortricidae (Lepidoptera), commonly known as tortrix or leafroller moths, comprises many agricultural and forestry pests, which cause serious agricultural losses. To understand the biology of such pest moths, fundamental techniques have been in high demand. Here, methods for mass-rearing, observations, and molecular studies are developed using two tea tortrix, Homona magnanima and Adoxophyes honmai (Lepidoptera: Tortricidae). Insects were mass-reared with sliced artificial diet and maintained by inbreeding for over 100 generations by considering their biological characteristics. Insects have various sex dimorphisms; hence it is difficult to distinguish the sex during the developing stages, which have prevented subsequent assays. The present work highlighted that the sex of tortricids larvae could be determined by observing testes or lactic-acetic orcein staining to visualize the female-specific W chromosome. Moreover, using the sex determination methods, the present study enabled nucleic acid extractions from sex determined embryos and application toward high throughput sequencing. These tips are applicable for other pest insects and will facilitate further morphological and genetic studies.

The present protocol describes the rearing method of tortricid pest insects in the laboratories. The procedures to distinguish insects' sex and extract nucleic acids for high throughput sequencing are established using two tortricid pests.

龟科害虫的大规模饲养与分子研究

Tortricidae (Lepidoptera), commonly known as tortrix or leafroller moths, comprises many agricultural and forestry pests, which cause serious agricultural ...

环境科学

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)

This video demonstrates novel techniques of RNA interference (RNAi) which downregulate two genes simultaneously in honey bees using double-stranded RNA (dsRNA) injections. It also presents a protocol of proboscis extension response (PER) assay for measuring gustatory perception.	
	RNAi-mediated gene knockdown is an effective technique downregulating target gene expression. This technique is usually used for single gene manipulation, but it has limitations to detect interactions and joint effects between genes. In the first part of this video, we present two strategies to simultaneously knock down two genes (called double gene knockdown). We show both strategies are able to effectively suppress two genes, vitellogenin (vg) and ultraspiracle (usp), which are in a regulatory feedback loop. This double gene knockdown approach can be used to dissect interrelationships between genes and can be readily applied in different insect species.	
	The second part of this video is a demonstration of proboscis extension response (PER) assay in honey bees after the treatment of double gene knockdown. The PER assay is a standard test for measuring gustatory perception in honey bees, which is a key predictor for how fast a honey bee's behavioral maturation is. Greater gustatory perception of nest bees indicates increased behavioral development which is often associated with an earlier age at onset of foraging and foraging specialization in pollen. In addition, PER assay can be applied to identify metabolic states of satiation or hunger in honey bees. Finally, PER assay combined with pairing different odor stimuli for conditioning the bees is also widely used for learning and memory studies in honey bees.

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees Apis mellifera

In this protocol, we describe two strategies that simultaneously suppress two genes (double gene knockdown) in honey bees. Then we present how to use the proboscis extension response (PER) assay to study the effect of double gene knockdown on honey bee gustatory perception.

RNAi介导的双基因敲除和味觉感知测量蜜蜂（<em&gt;蜜蜂</em&gt;）

This video demonstrates novel techniques of RNA  interference (RNAi) which downregulate two genes simultaneously in honey bees  using double-stranded RNA ...

神经科学

Culturing and Genetically Manipulating Entomopathogenic Nematodes

Entomopathogenic nematodes in the genera Heterorhabditis and Steinernema are obligate parasites of insects that live in the soil. The main characteristic of their life cycle is the mutualistic association with the bacteria Photorhabdus and Xenorhabdus, respectively. The nematode parasites are able to locate and enter suitable insect hosts, subvert the insect immune response, and multiply efficiently to produce the next generation that will actively hunt new insect prey to infect. Due to the properties of their life cycle, entomopathogenic nematodes are popular biological control agents, which are used in combination with insecticides to control destructive agricultural insect pests. Simultaneously, these parasitic nematodes represent a research tool to analyze nematode pathogenicity and host anti-nematode responses. This research is aided by the recent development of genetic techniques and transcriptomic approaches for understanding the role of nematode secreted molecules during infection. Here, a detailed protocol on maintaining entomopathogenic nematodes and using a gene knockdown procedure is provided. These methodologies further promote the functional characterization of entomopathogenic nematode infection factors.

Entomopathogenic nematodes live in symbiosis with bacteria and together they successfully infect insects by undermining their innate immune system. To promote research on the genetic basis of nematode infection, methods for maintaining and genetically manipulating entomopathogenic nematodes are described.

培养和遗传操纵昆虫致病线虫

Entomopathogenic nematodes in the genera Heterorhabditis and Steinernema are obligate parasites of insects that live in the soil. The main characteristic ...

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four&#160;recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

Nonlytic insect cell expression systems are underutilized for production, cellular trafficking/localization, and recombinant protein functional analysis. Here, we describe methods to generate expression vectors and subsequent transient protein expression in commercially available lepidopteran cell lines. The co-localization of Bemisia tabaci aquaporins with subcellular fluorescent marker proteins is also presented.

瞬时表达和重组蛋白的细胞定位在培养的昆虫细胞

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and ...

生物学

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, chikungunya and Zika. Wolbachia-infected mosquitoes can be released into the field to either reduce population sizes through incompatible matings or to transform populations with mosquitoes that are refractory to virus transmission. For these strategies to succeed, the mosquitoes released into the field from the laboratory must be competitive with native mosquitoes. However, maintaining mosquitoes in the laboratory can result in inbreeding, genetic drift and laboratory adaptation which can reduce their fitness in the field and may confound the results of experiments. To test the suitability of different Wolbachia infections for deployment in the field, it is necessary to maintain mosquitoes in a controlled laboratory environment across multiple generations. We describe a simple protocol for maintaining Ae. aegypti mosquitoes in the laboratory, which is suitable for both Wolbachia-infected and wild-type mosquitoes. The methods minimize laboratory adaptation and implement outcrossing to increase the relevance of experiments to field mosquitoes. Additionally, colonies are maintained under optimal conditions to maximize their fitness for open field releases.

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes infected with Wolbachia are being released into natural populations to suppress the transmission of arboviruses. We describe methods to rear Ae. aegypti with Wolbachia infections in the laboratory for experiments and field release, taking precautions to minimize laboratory adaptation and selection.

维护伊蚊蚊蚊子感染沃尔巴克

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, ...

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, host-symbiont interactions, speciation, and venom synthesis. Despite the availability of several molecular tools, including CRISPR/Cas9, functional genetic studies are still limited in this organism. The major limitation of applying CRISPR/Cas9 technology in N. vitripennis stems from the challenges of embryonic microinjections. Injections of embryos are particularly difficult in this organism and in general in many parasitoid wasps, due to small embryo size and the requirement of a host pupa for embryonic development. To address these challenges, Cas9 ribonucleoprotein complex delivery into female&#160;ovaries by adult injection, rather than embryonic microinjection, was optimized, resulting in both somatic and heritable germline edits. The injection procedures were optimized in pupae and female wasps using either ReMOT Control (Receptor-Mediated Ovary Transduction of Cargo) or BAPC (Branched Amphiphilic Peptide Capsules). These methods are shown to be effective alternatives to embryo injection, enabling site-specific and heritable germline mutations.

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

Here, we describe methods for efficient pupal and adult injections in Nasonia vitripennis as accessible alternatives to embryo microinjection, enabling functional analysis of genes of interest using either RNA-silencing via RNA interference (RNAi) or gene knockout via CRISPR/Cas9 genome editing.

纳索尼亚维特里彭尼斯的 Rnai 和克里斯普基因编辑的普帕尔和成人注射

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, ...

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

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此视频中的章节

核酸的传递通过显微注射胚胎在世界范围内的农业害虫，

饲养并在隐藏甲壳虫双链RNA介导的基因敲除，