我们要感谢Julia Fine博士在协议创建过程中的想法和讨论，以及Cassondra Vernier博士在整个编辑过程中的有用评论。这些材料为部分由粮食和农业研究基金会根据赠款ID 549025支持的项目做出了贡献。

1. 大规模蜜蜂提取选项1：幼虫自我清除
<ol><li>把一只蜜蜂女王关在一个空的、拉长的Langstroth框架上，把她送回她的殖民地。让蜂王在这个框架上产卵24小时。<ol>
<li>24小时后检查框架，以确保大多数梳状细胞含有新产卵。根据蜂王和蚁群的不同，卵有时在最初的24小时内产得不是很好。如果发生这种情况，请再留出24小时，并根据需要调整时间。</li>
	</ol></li>
	<li>在24小时产卵期后，释放蜂王。清楚地标记框架并将其返回殖民地。</li>
	<li>在将蚁后笼养满后正好192小时（8天）（假设在前24小时内产卵正常;8天标志着化蛹前的点），从菌落中取出标记的框架。刷掉所有成年蜜蜂，并将框架转移到与蜂巢内部条件（34°C和50%相对湿度（RH））相匹配的培养箱中。<ol>
<li>检查以确保框架的大部分都充满了5龄 幼虫，这些幼虫可以通过它们大而白色的C形身体紧紧地压在梳状细胞的底部边缘来识别。也可能有一些细胞已经被蜡盖覆盖，特别是在框架中心附近。</li>
	</ol></li>
	<li>准备与幼虫填充框架的高度和宽度相匹配的容器，通过用肥皂和水彻底清洁内表面和外表面，然后用漂白剂溶液，最后用乙醇来接收5龄 幼虫。在继续操作之前，请彻底干燥容器。<ol>
<li>在容器底部铺上几层纸巾。然后添加几层重叠的较薄、吸水的清洁湿巾（例如，精致的任务雨刷）或滤纸。材料必须具有吸水性。避免重叠顶层，以提高未来幼虫转移的便利性。</li>
	</ol></li>
	<li>将框架面朝下（焦点幼虫朝下）放在容器顶部，以便幼虫可以落在清洁湿巾层上。<ol>
<li>用一块帐篷状铝箔或其他覆盖物覆盖容器和框架，以保持水分并将设置返回到培养箱。离开设置过夜。幼虫的天然觅食倾向会导致它们爬出细胞并落在下面的填充表面上。</li>
	</ol></li>
	<li>使用 1.4 中详述的相同步骤彻底清洁单独的传输托盘，以准备这些托盘。让托盘干燥，并用清洁湿巾将底部分层。托盘不需要匹配任何特定尺寸，但较浅的托盘将允许更容易的幼虫操作。<ol>
<li>开始将幼虫从容器中移出容器中的顶层，然后将幼虫轻轻倒入托盘上，从而将幼虫从容器转移到托盘上。幼虫应该在一夜之间落入容器中，形成几个粘稠的团块（图1A）。</li>
		<li>使用钝的软镊子分离幼虫，并将它们铺在托盘的表面上。它们不需要均匀分布，可以彼此靠近，但不应该接触。参见 图1B ，了解分离幼虫的视觉描述。</li>
		<li>借此机会清除任何受损（变色）或低于平均水平大小的幼虫。这些在成熟过程中更容易死亡，并可能在整个托盘中带来感染/真菌生长。</li>
	</ol></li>
	<li>用帐篷铝箔盖住托盘以保持水分，并将设置返回到培养箱。</li>
	<li>每天检查幼虫并去除任何变色的（深棕色或黑色）。 注意：幼虫/蛹前期会排便到清洁湿巾上，表现为小的棕色斑块。它们还可能生产少量的白色，细长的织带，作为其封盖过程的一部分。这些情况都不需要更换清洁湿巾，只要它们是吸水性的。</li>
	<li>让幼虫化蛹并成熟到白眼阶段，这可以通过它们的一般形状与成年蜜蜂的形状相匹配来识别，同时它们的眼睛和身体的大部分其余部分仍然缺乏色素沉着。这应该发生在女王笼养后的14至15天之间。蛹现在已准备好进行病毒注射。 图1C，1D 为白眼蛹的例子。</li>
</ol>2. 大量蜜蜂提取选项2：手动切除蛹
注：虽然选项2（蛹切除）是一种可行的蜜蜂提取方法，但与选项1（幼虫自我清除）相比，它也有几个缺点。选项2的劳动强度要高得多，在蛹龄时更难控制，并且通常对蜜蜂本身的压力更大。尽可能建议使用选项 1。
<ol><li>从合适的蜂群中选择一帧在白眼阶段或附近含有蛹的带盖蜜蜂的育雏（参见1.9的描述）。从位于框架中心和边缘附近的梳状细胞中取出封盖，以确认是否存在适当的发育阶段。</li>
	<li>将框架转移到设置为34°C和50%相对湿度的培养箱中。未立即使用时，始终将框架放回培养箱。</li>
	<li>准备并清洁与 1.6 中描述的转移托盘完全相同的传输托盘。</li>
	<li>从培养箱中取出框架，并设置在光源下方的倾斜支架上。用水润湿一小叠纸巾。</li>
	<li>用乙醇清洁一对钝硬镊子。使用干净的镊子，从含有白眼蛹的细胞中取出封盖，注意在此过程中不要损坏蛹。</li>
	<li>逐个轻轻地从梳状细胞中切除蛹。如果有足够的空间，使用镊子尖端抓住胸部和腹部周围的蛹是最安全的。然而，如果没有，蛹也可以通过抓住头部并慢慢摆动它来去除，然后抓住身体。<ol>
<li>用湿纸巾覆盖框架中未立即接触的部分，以保持水分。在切除过程中根据需要移除和更换。</li>
	</ol>
</li>
	<li>将切除的蛹沿着转移托盘中的清洁湿巾间隔，确保它们都没有接触。任何毁容或变色的蛹都应丢弃。蛹现在已准备好进行病毒注射。<ol>
<li>丢弃与湿巾接触后释放液体的蛹;他们很可能被刺穿了。有时，蛹在去除后1小时内会在与镊子接触点附近表现出小的黑色素化黑斑。这应该不会影响生存。如果出现大片的黑色素化，应丢弃受影响的蛹。</li>
	</ol>
</li>
</ol>3. 蛹病毒注射
注意：如果首次执行此方案（即，没有先前的病毒接种物），首先使用来自疑似感染的菌落的成虫，蛹或幼虫提取和浓缩颗粒。如步骤5所述测量所得接种中的病毒滴度，并确定要进一步传播的颗粒。
<ol><li>在开始使用蜜蜂病毒之前，使用漂白水和乙醇对所有工作台面进行消毒。在整个过程中应佩戴丁腈手套。</li>
	<li>制备能够注射〜1μL量的流体的进样器装置。 注意：一种廉价但有效的方法是使用柔性环氧树脂或其他液体粘合剂将30 G皮下注射针头连接到100μL多分配器尖端（参见 材料表）的尖端，从而创建手工制作的设备。确保针帽的边缘紧紧地密封在多功能分配器尖端上，并允许设备干燥。参见 图2 ，了解示例喷油器装置的直观描述。</li>
	<li>通过在15 mL锥形离心管中将所需类型的病毒颗粒与灭菌的1x PBS（磷酸盐缓冲盐水）混合来制备病毒注射稀释溶液。所需的总量将取决于需要注射的蛹的数量，每个蛹需要注射1μL（例如，500蛹= 5μL病毒颗粒+ 495μL PBS）。</li>
	<li>将注射器设备连接到手动多移液器上，并通过从单独的烧杯中抽取100μL水并将其分配为1μL剂量来测试功效。确保分配的每个量相等，必要时更换注射器设备。清除喷油器中残留的水。</li>
	<li>从培养箱中取出步骤1或步骤2中产生的蛹托盘，并取下铝箔覆盖物。</li>
	<li>用乙醇清洁一对钝硬镊子。沿着胸部轻轻抓住单个蛹，施加足够的压力以迫使内部液体进入腹部。这应该使tergite的划分更加明显。</li>
	<li>使用多移液管抽取100μL病毒颗粒溶液，将针头插入第三和第四腹部三次剁咽之间，然后注射1μL病毒溶液。对托盘上铺设的每个蛹重复，注意避免重复注射。在此过程中损坏的任何蛹都应丢弃。 注意：所有与病毒制备相关的材料均被指定为生物危害物质，应按照机构指南进行高压灭菌和处置。针头也应按照机构指南进行处理。</li>
	<li>用铝箔覆盖注射蛹的托盘并返回培养箱。让病毒颗粒在蛹内繁殖3-5天。<ol>
<li>对蛹进行日常检查，并清除任何死亡或腐烂的标本，以防止细菌或真菌积聚。小的黑色素点可能出现在腹部的注射部位，但不应影响生存。</li>
	</ol>
</li>
	<li>将蛹取样到50 mL锥形离心管中并涡旋以均质化内容物，并注意按菌落源将蛹采样到管中，以减少非目标病毒的污染。确保没有整个蛹残留，并将管子转移到-80°C的冰箱中，直到准备好病毒颗粒沉淀和浓缩。</li>
</ol>4. 病毒颗粒浓度
注意：此协议尚未经过包膜病毒恢复测试。
<ol><li>使用前高压灭菌所有材料和容器。在整个过程中应佩戴丁腈手套，实验室外套和护目镜。在开始之前，使用漂白水，乙醇和RNase灭活溶液对所有工作表面进行灭菌。<ol>
<li>准备1x TES（Tris-EDTA-盐）缓冲液：混合10mM Tris-HCL pH 7.5，2mM EDTA和150mM NaCl。通过高压灭菌灭菌。</li>
	</ol></li>
	<li>解冻均质化的蛹并转移到离心机瓶中。加入大约三个体积的1x PBS（例如，将一整管50 mL的蛹加入150 mL的1x PBS），并将体积与最满的瓶子的体积相等。</li>
	<li>首先用手混合，然后在室温下置于轨道振荡器上10分钟。</li>
	<li>在4°C下以15，000 ×g 离心5分钟以除去细胞碎片。如果残留细胞碎片，请根据需要重复此步骤。<ol>
<li>如果上清液表面漂浮着大球脂肪，则在继续之前通过干酪布过滤到单独的无菌离心机瓶中。</li>
	</ol></li>
	<li>用0.3体积的24∶1氯仿：异戊醇溶液（例如，190mL上清液+ 57mL氯仿：异戊醇）提取上清液，并通过倒置混合。在4°C下以21，000 ×g 离心20分钟。 注意：避免直接接触皮肤或眼睛上的氯仿。始终穿戴适当的个人防护用品。所有氯仿废物均应按照体制准则进行处置。</li>
	<li>通过在冰浴或4°C冷室中将上清液倾析到单独的无菌500mL烧杯中，从每个瓶子中回收水相。注意避免上清液被氯仿污染，因为这会使纯化过程更加困难;最好失去一点点水相。</li>
	<li>加入不含RNase的水，使每个烧杯的体积达到200 mL。将烧杯放在磁力搅拌板上，放入中等大小的搅拌棒中。将板设置为在中低设置下搅拌。</li>
	<li>在恒定的温和搅拌下，向每个烧杯中缓慢加入NaCl，使每个烧杯达到2.3%的最终浓度（例如，每200mL上清液4.6g NaCl）。向每个烧杯中加入聚乙二醇8000（PEG）至终浓度为7%（例如，每200毫升上清液14克PEG）。</li>
	<li>用铝箔覆盖烧杯，并继续在冰上或冷室中以中低速搅拌1-5小时以溶解PEG。搅拌时间越长，PEG溶解得越彻底。</li>
	<li>关闭搅拌板。将覆盖的烧杯在冰上或冷藏室中孵育1-3天，以使病毒颗粒和蛋白质沉淀。孵育时间越长，沉淀的颗粒就越多。</li>
	<li>将每个烧杯的内容物转移到单独的干净离心机瓶中，并在4°C下以15，000 ×g 离心30分钟以回收PEG颗粒沉淀。弃去上清液。</li>
	<li>小心地从瓶子的侧面刮下PEG颗粒，并通过缓慢向颗粒中添加少量TES（每100只原始蜜蜂约10 mL）将其重悬于清洁烧杯内的最小体积的1x TES缓冲液中。</li>
	<li>将悬浮的沉淀通过18G针头至少十次，然后等分到2mL离心管中。将所有试管保持在冰上，直到准备好4.14。</li>
	<li>在4°C下以13，000 ×g 离心2mL管15分钟以除去额外的PEG。使用1，000μL移液器将上清液转移到另一个2 mL离心管中，并重复离心步骤以确保完全去除所有PEG。</li>
	<li>使用离心过滤装置（100 kDa截止）通过几轮在室温（RT）下以14，000 ×g 离心10分钟，将上清液中的剩余颗粒浓缩到新的离心管中，每次10分钟，直到达到原始浓度的约五分之一（10 mL颗粒-TES溶液至约2 mL浓缩颗粒）。过滤单元有不同的尺寸;选择最适合正在处理的样品数量。适合 15 mL 大小的锥形管的装置通常是最合适的。</li>
	<li>通过通过26 G皮下注射针头重悬浓缩颗粒，并在室温下以14，000 ×g 离心5分钟，以进行最后一轮PEG去除。如果液体仍然浑浊，重复此步骤，直到除去所有 PEG 沉淀物。</li>
	<li>将粘稠的上清液等分到所需量，并储存在-80°C直至准备使用。</li>
</ol>5. 病毒RNA提取和定量
<ol><li>使用任何适当的RNA提取方法（例如，TRIzol RNA提取试剂，然后进行DNA酶处理）从整个蜜蜂或浓缩的病毒颗粒中提取RNA。</li>
	<li>通过RT-qPCR量化步骤5.1中产生的接种物中的病毒滴度，优选使用不依赖于宿主基因表达18的基于标准曲线的方法，尽管其他方法也可以进行估计。</li>
</ol>6. 病毒喂养生物测定
<ol><li>在框架收集步骤（6.2）之前或在24小时蜜蜂出苗期间（6.3）准备生物测定的所有必要材料。<ol>
<li>准备干净的笼子或其他能够容纳生物测定所需数量的蜜蜂的围栏（例如，尺寸为10.16 cm x 10.16 cm x 7.62 cm的丙烯酸盒笼），方法是用适当尺寸的离心管堵塞所有进料器孔（图3）。确定并随机分配笼子之间的处理方法，并标记每个处理方式，以方便将来参考。</li>
		<li>通过标记单个中型称重船及其相应的笼子和处理来准备接种托盘。</li>
		<li>通过将适量的蔗糖与去离子水混合（例如，每1升水300g蔗糖用于30%蔗糖溶液）来制备进料溶液，确保在加入蔗糖之前和之后对水进行灭菌。无菌蔗糖溶液可以在4°C冰箱中储存数周，但如果出现任何混浊，应丢弃。</li>
		<li>准备喂料管，方法是用6.1.3中产生的进料溶液部分填充15 mL离心管，将它们倒置，并用拇指钉在管尖周围戳1-2个孔。也可以使用在火焰上加热的18G皮下注射针头熔化孔。确保喂料器管盖拧得非常紧，因为松动的盖子会导致缓慢泄漏，从而在一夜之间淹死笼子居民。 注意：可以根据需要调整喂料溶液和喂料管体积，以满足实验需求。</li>
		<li>通过在大桶的边缘轻轻涂上植物油或类似的油腻物质，为新出现的蜜蜂准备一个可接受的集合。分别，使用相同的包衣方法制备几个小杯子。</li>
	</ol></li>
	<li>选择并移除来自至少三个独立蜂巢的濒临灭绝的蜜蜂育雏框架。适当老化的蜜蜂类似于梳状细胞盖下的完全色素沉着的成虫。持续出现的一个明确迹象是观察细胞居民慢慢地咀嚼和/或最近清空的细胞，沿着蜡盖带有特征性的锯齿状咀嚼痕迹。 注意：所需的确切帧数将取决于实验的大小，每帧的新兴育雏数量以及一年中的时间。标准的Langstroth深框架每侧包含约3，000个梳状细胞;一个包含大部分有盖蛹的框架，观察到一些新兴的可以在24小时内轻松产生400 +蜜蜂。根据需要在整个季节进行调整。</li>
	<li>在将框架放入出苗箱并将其转移到与蜂巢内部条件（34°C和50%RH）相匹配的培养箱之前，刷掉所有成年蜜蜂。让蜜蜂出现24小时。</li>
	<li>从培养箱中取出出料盒，将所有新出现的蜜蜂刷入收集桶中。确保从出苗箱中取出所有蜜蜂，以防止在任何后续刷牙中包含错误老化的蜜蜂。任何能够飞行的蜜蜂>24小时前出现，应排除在生物测定之外。</li>
	<li>通过手工在收集桶中轻轻混合蜜蜂（它们在这个年龄不能刺痛）来产生新出现的蜜蜂的均质化混合物，以尽量减少来自任何单个菌落的蜂巢遗传效应。对于特定应用，为菌落源安排可能是可取的。</li>
	<li>数出35只蜜蜂，将每只蜜蜂放入同一个涂有油脂的杯子中，然后将内容物转移到丙烯酸笼中。或者，将较小的蜜蜂分成几个涂有油脂的杯子（例如，5个杯子，每个杯子7只蜜蜂）可能更容易，以避免错误计数错误。使用的蜜蜂数量可以根据需求和应用而变化。</li>
	<li>将蜜蜂的笼子转移到培养箱（34°C和50%RH），确保遵循6.1.1中创建的随机放置顺序，以尽量减少任何潜在的微气候影响。</li>
	<li>在开始之前，用漂白水、乙醇和 RNase 灭活溶液清洁所有表面和移液器，为病毒工作做好准备。确保在处理病毒颗粒时戴上丁腈手套。</li>
	<li>通过在无菌容器中解冻出适当量的浓缩病毒颗粒（步骤4.17）并与足够的蔗糖溶液（步骤6.1.3）充分混合来制备所需浓度的病毒接种物。对于35只蜜蜂的笼子，每个需要600μL接种物。如果所需病毒浓度等于或低于0.1%，建议连续稀释。<ol>
<li>例如，涉及40个笼子的实验都需要1%的病毒接种物，将需要24 mL病毒溶液，这可以通过将240μL浓缩病毒颗粒与23.76 mL蔗糖溶液组合来创建。建议在初始体积中包括大约15%的超额，因为粘性液体预计会造成一些损失。</li>
	</ol></li>
	<li>将按处理类型分类的6.1.2中制备的接种物托盘布置好，并将600μL适当接种物的移液器移液到每个托盘上。<ol>
<li>小心地将接种物托盘插入相应的笼子中，注意不要意外释放任何蜜蜂。借此机会扫描蜜蜂并替换任何可能在转移过程中死亡的蜜蜂。</li>
	</ol></li>
	<li>在取出阻挡顶部喂料器孔的离心管之前，允许完全消耗接种管（约12-14小时），插入6.1.4中制备的适当进料管。这有助于确保笼子里的蜜蜂在整个种群中均匀地共享接种物。随意 提供蔗 糖溶液，并且在整个实验过程中应根据需要重新填充试管。</li>
	<li>在每个实验的前72小时内，以12小时的间隔记录每个笼子内的死亡率，然后将记录转移到24小时的间隔。从笼子里移走死蜜蜂，通过把笼门往上滑动，让一对镊子伸进去舀出死蜜蜂，增加未来计数的便利性。确保在笼子之间的酒精灯上对镊子进行消毒，以防止病毒交叉污染。</li>
	<li>用于病毒滴度测量的蜜蜂样本（5.1-5.2），方法是在每个笼子内随意选择活标本并将其放入干冰上的离心管中。通常，三只蜜蜂足以在任何给定的时间点进行病毒滴度测量，而不会使笼子减少人口。</li>
	<li>根据需要继续进行常规死亡率测量。</li>
</ol>

作者没有什么可透露的。

在这里，我们概述了详细介绍病毒扩增和接种原液制备过程的每个步骤的方法，包括幼虫收集和病毒繁殖，提取和浓缩，以及以笼中喂养实验的形式进行病毒处理。这些方法允许生产半纯病毒颗粒（图4），其有效性可以通过对成人致命的病毒的剂量反应死亡率测试来一致地量化（图5）。在确认感染能力和/或病理学后，生成的颗粒可用于生物测定，以?...

蜜蜂（Apis mellifera）在现代全球农业景观中发挥着关键作用，但目前正遭受生物和非生物胁迫因素的结合，包括农药暴露，饲料不良，寄生虫和病原体1，2。最受关注的最重要病原体之一是病毒，其中许多是由另一个主要的蜜蜂压力源，寄生的Varroa螨（Varroa destructor）传播的。这些病毒可以在蜜蜂中引起一系列负面影响，包括育雏存活率降低，发育缺陷和瘫痪，从而导致越冬前后蜂巢完全崩溃3，4，5。尽管在用于对抗病毒感染的技术开发方面取得了可喜的进步6，7，8，9，但许多病毒在蜜蜂或蜂群中传播，传播和相互作用的动态仍然知之甚少5，10.了解蜜蜂和病毒相互作用的基本生物学及其与其他环境因素的关系对于开发有效的病毒管理技术至关重要。
然而，研究蜜蜂 - 病毒相互作用带来了挑战，许多已知和未知因素使该过程复杂化。这些包括与饮食11，12，农药暴露13和蜜蜂遗传背景14，15的相互作用。即使仅关注病毒感染，并发症也很常见，因为蜜蜂种群，无论是管理的还是野生的，总是有一定程度的背景病毒感染，尽管通常没有表现出急性症状16，17，并且病毒合并感染的影响尚不清楚18。这使得对蜜蜂病毒影响的研究难以解开。
许多蜜蜂病毒研究已经使用间接病毒感染来寻找与其他应激源的相互作用，观察背景感染如何随着其他治疗而变化12，19，20，21。虽然这种方法已经成功地确定了重要的效果，特别是发现农药或饮食治疗如何影响病毒水平和复制，但用已知含量和浓度的病毒治疗进行接种对于病毒感染动态的实验测试至关重要。即便如此，将实验性治疗与背景感染分开也可能带来挑战。在实地研究中，研究人员已经区分了变形翅膀病毒（DWV）的菌株，为病毒从蜜蜂传播到大黄蜂22提供证据，但仅使用这种方法很难在蜜蜂中。病毒传染性克隆是一种强大的工具，不仅用于跟踪感染23，24，25 ，还用于蜜蜂病毒的反向遗传学研究和病毒 - 宿主相互作用研究26，27，28。然而，在大多数情况下，感染性克隆仍然需要满足细胞内的感染周期以产生颗粒。这种颗粒优选作为实验治疗的接种，因为它们的感染性高于裸病毒RNA，并且用包裹的基因组接种模仿自然感染。
生产纯的、未受污染的蜜蜂病毒接种（野生型病毒株或来自传染性克隆的病毒株）也带来了挑战。这些主要是由于难以获得可靠，连续复制，无病毒的蜜蜂细胞系来产生纯病毒株29，30。虽然已经产生了一些细胞系，但这些系统仍然不完美;尽管如此，仍有希望产生29个可行的细胞系，这将允许更好地控制病毒的产生和研究。在这样的生产线变得广泛可用之前，大多数病毒生产方案将继续依赖于使用 体内 病毒生产和纯化18，31，32，33，34。这些方法涉及鉴定和纯化感兴趣的病毒颗粒（或产生传染性克隆），并使用它们来感染蜜蜂，通常为蛹。将蛹注入靶病毒然后处死，并提取和纯化进一步的颗粒。然而，由于没有蜜蜂一开始就没有病毒，因此任何此类浓缩物中其他病毒的痕迹总是存在一定程度的污染，因此，在选择背景感染可能性较低的蜜蜂时必须格外小心。此外，用于从梳状细胞中除去蛹的方法用于这些方案33 是非常劳动密集型的，并且可以在蜜蜂中诱导压力，通过这些方法限制生产18，32。在这里，我们报告了一种替代方法，该方法允许大规模去除幼虫，只需很少的劳动和对蜜蜂的机械应力。
一旦获得蛹并注射起始病毒接种物，必须将它们孵育以提供病毒复制时间。随后，产生的病毒颗粒可以被加工成可用于感染实验蜜蜂的形式。有几种简单的方法可以实现这一点，包括使用由病毒感染的蜜蜂产生的粗匀浆35，36 或血淋巴作为感染源37。这些方法是有效的，但从背景基质中引入未知变量的机会更大（例如，死蜂匀浆中的其他因素）。此外，如果实验需要在短时间内给予大剂量的已知剂量的病毒，则希望浓缩颗粒。因此，为了更好地控制，最好使用允许一定程度的病毒颗粒纯化和浓缩的方法。通常，一系列的沉淀和离心步骤将导致除去几乎所有可能的非目标病毒物质33。
在生产这种浓缩的接种物后，定量病毒滴度（qPCR）并用 体内 生物测定法表征它是有益的，以测试其活力和导致死亡的能力，以及证实感染后获得的病毒滴度增加。这可以通过注射实验（进入蛹或成虫）或喂养实验（进入幼虫或成虫）来实现。虽然所有这些方法都是可能的，但在笼子里喂养成群的成年蜜蜂通常是最快和最简单的。笼式测定法还广泛用于测试蜜蜂的各种其他处理方法，包括农药毒性38，卵巢发育39和营养对行为的影响40，41 ，因此，可以形成将病毒感染与其他因素42联系起来的实验的良好基础。
在这里，我们描述了一种在不使用昂贵的超速离心机的情况下生产大量半纯，高度富集的病毒颗粒的可靠方法，包括一种去除蛹的方法，以减少蜜蜂的劳动和机械应力，以及用于测试病毒感染和影响的高可重复性，高通量生物测定。通过严格控制病毒接种的纯度，研究人员能够减少蜜蜂病毒反应相对于其他病毒接种方法的变化。此外，生物测定可以在扩展到现场现实设置之前，使用高度可重复的实验单元在小组级别筛选病毒效应，这需要管理更多的劳动密集型。结合这两种方法，这两种方法为研究提供了必要的工具，可以帮助提高我们对蜜蜂 - 病毒生理相互作用的整体理解。

<table>
	<tbody>
		<tr>
			<td>10% bleach solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>24:1 chloroform:isoamyl alcohol</td>
			<td>SigmaAldrich</td>
			<td>C0549</td>
			<td></td>
		</tr>
		<tr>
			<td>70% ethanol solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cages for bioassay</td>
			<td></td>
			<td></td>
			<td>Dependent on experimental setup</td>
		</tr>
		<tr>
			<td>Combitips Advanced 0.1 mL</td>
			<td>Eppendorf</td>
			<td>30089405</td>
			<td>Optional (if no injector appartus is available)</td>
		</tr>
		<tr>
			<td>Containers for larval self-removal</td>
			<td></td>
			<td></td>
			<td>Should measure roughly 19&#34; x 9-1/8&#34; (Langstroth deep frame dimensions)</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td></td>
			<td></td>
			<td>Blunt, soft forceps for larval separationl; blunt, hard forceps for pupal excision</td>
		</tr>
		<tr>
			<td>Fume hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td></td>
			<td></td>
			<td>Capable of maintaining 34 &#186;C and 50% relative humidity</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Fisher Scientific</td>
			<td>06-666</td>
			<td>Any absorbent wipe will work</td>
		</tr>
		<tr>
			<td>Medium-sized weight boats</td>
			<td></td>
			<td></td>
			<td>Serve as inoculum trays</td>
		</tr>
		<tr>
			<td>Microcon-100kDa with Biomax membrane</td>
			<td>MilliporeSigma</td>
			<td>MPE100025</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrile gloves</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>SigmaAldrich</td>
			<td>P5119</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene glycol 8000 (PEG)</td>
			<td>SigmaAldrich</td>
			<td>1546605</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated benchtop centrifuge</td>
			<td></td>
			<td></td>
			<td>Capable of 15,000 x g</td>
		</tr>
		<tr>
			<td>Refrigerated centrifuge</td>
			<td></td>
			<td></td>
			<td>Capable of 21,000 x g</td>
		</tr>
		<tr>
			<td>Repeater M4 Multipipette</td>
			<td>Eppendorf</td>
			<td>4982000322</td>
			<td>Optional (if no injector appartus is available)</td>
		</tr>
		<tr>
			<td>RNAse Away</td>
			<td>ThermoFisher</td>
			<td>7000TS1</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse-free water</td>
			<td>SigmaAldrich</td>
			<td>W4502</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TES</td>
			<td>SigmaAldrich</td>
			<td>T1375</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of virus-enriched inoculum for oral infection of honey bees (apis mellifera)

蜜蜂在世界各地具有重要的生态和农业重要性，但也受到各种压力的影响，这些压力对蜜蜂健康产生负面影响，包括暴露于病毒病原体。这种病毒可以引起各种各样的破坏性影响，并且由于多种因素使得难以将实验性治疗的影响与先前存在的背景感染区分开来，因此通常很难进行研究。在这里，我们提出了一种大规模生产大量病毒颗粒的方法以及高通量生物测定法，以测试病毒感染和效果。由于目前缺乏连续的无病毒蜜蜂细胞系，因此使用蜜蜂蛹 在体内 扩增病毒颗粒，使用最小压力的方法从蜂巢中大量提取病毒颗粒。然后，这些病毒颗粒可用于蜜蜂笼生物测定，以测试接种的活力，以及各种其他病毒感染动力学，包括与营养，杀虫剂和其他病原体的相互作用。使用这种颗粒的一个主要优点是，与目前的替代品相比，它大大减少了在后续实验中引入未知变量的机会，例如通过受感染的蜜蜂血淋巴或匀浆物感染，尽管在采购蜜蜂时仍应小心，以尽量减少背景病毒污染。笼式测定不能替代在菌落水平上测试病毒感染效应的大规模，现场现实实验，而是作为建立基线病毒反应的方法，与半纯病毒颗粒相结合，可以作为检查蜜蜂 - 病毒生理相互作用各个维度的重要工具。

成功遵循蛹注射和病毒提取的方案（图1）应产生大量病毒颗粒。然而，在多个时间点对来自各种菌落的蛹进行取样和注射，可以最大限度地提高获得低污染目标病毒的机会。病毒在蜜蜂中相互复制和相互作用的动态尚不清楚;再加上预先存在的感染的可能性，不能保证注射的（所需的）病毒会成为任何给定蛹中的优势，即使蛹是从同一原始菌落取样的。 图4 显示...

Watch this Scientific Journal Video about Preparation of Virus-Enriched Inoculum for Oral Infection of Honey Bees Apis mellifera at JoVE.com

Preparation of Virus-Enriched Inoculum for Oral Infection of Honey Bees Apis mellifera

在这里，我们描述了两种方案：第一种是繁殖，提取，纯化和量化大量蜜蜂非包膜病毒颗粒，包括去除蜜蜂蛹的方法，其次是使用高度可重复的高通量笼式生物测定来测试病毒感染的影响。

一种富含病毒的接种物用于蜜蜂口腔感染（Apis mellifera）的制备

Honey bees are of great ecological and agricultural importance around the world but are also subject to a variety of pressures that negatively affect bee ...

preparation-virus-enriched-inoculum-for-oral-infection-honey-bees

Research

JoVE Journal

Biology

Preparation of Virus-Enriched Inoculum for Oral Infection of Honey Bees (Apis mellifera)

2.8K 次观看.  University of Illinois. 在这里，我们描述了两种方案：第一种是繁殖，提取，纯化和量化大量蜜蜂非包膜病毒颗粒，包括去除蜜蜂蛹的方法，其次是使用高度可重复的高通量笼式生物测定来测试病毒感染的影响。

视频: Preparation of Virus-Enriched Inoculum for Oral Infection of Honey Bees Apis mellifera

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 ...

科研

生物学

Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging, Cell Injury Measurements, and Adenoviral Infection

The pancreatic acinar cell is the main parenchymal cell of the exocrine pancreas and plays a primary role in the secretion of pancreatic enzymes into the pancreatic duct. It is also the site for the initiation of pancreatitis. Here we describe how acinar cells are isolated from whole pancreas tissue and intracellular calcium signals are measured. In addition, we describe the techniques of transfecting these cells with adenoviral constructs, and subsequently measuring the leakage of lactate dehydrogenase, a marker of cell injury, during conditions that induce acinar cell injury in vitro. These techniques provide a powerful tool to characterize acinar cell physiology and pathology.

We describe a reproducible method of preparing mouse pancreatic acinar cells from a mouse for the purpose of examining acinar cell calcium signals and cellular injury with physiologically and pathologically relevant stimuli. A method for adenoviral infection of these cells is also provided.

制备胰腺腺泡细胞钙成像的目的，细胞损伤的测量，以及腺病毒感染

The pancreatic acinar  cell is the main parenchymal cell of the exocrine pancreas and plays a primary  role in the secretion of pancreatic enzymes into ...

Methods for Comparing Nutrients in Beebread Made by Africanized and European Honey Bees and the Effects on Hemolymph Protein Titers

Honey bees obtain nutrients from pollen they collect and store in the hive as beebread. We developed methods to control the pollen source that bees collect and convert to beebread by placing colonies in a specially constructed enclosed flight area. Methods were developed to analyze the protein and amino acid composition of the pollen and beebread. We also describe how consumption of the beebread was measured and methods used to determine adult worker bee hemolymph protein titers after feeding on beebread for 4, 7 and 11 days after emergence. Methods were applied to determine if genotype affects the conversion of pollen to beebread and the rate that bees consume and acquire protein from it. Two subspecies (European and Africanized honey bees; EHB and AHB respectively) were provided with the same pollen source. Based on the developed methods, beebread made by both subspecies had lower protein concentrations and pH values than the pollen. In general, amino acid concentrations in beebread made by either EHB or AHB were similar and occurred at higher levels in beebread than in pollen. Both AHB and EHB consumed significantly more of the beebread made by AHB than by EHB. Though EHB and AHB consumed similar amounts of each type of beebread, hemolymph protein concentrations in AHB were higher than in EHB. Differences in protein acquisition between AHB and EHB might reflect environmental adaptations related to the geographic region where each subspecies evolved. These differences could contribute to the successful establishment of AHB populations in the New World because of the effects on brood rearing and colony growth.

Here are methods to quantify nutrients in pollen before and after its conversion to beebread by two subspecies of honeybees. We describe techniques to measure beebread consumption and resulting protein titers in both subspecies.

对于在比较营养Beebread由非洲化和欧洲蜜蜂的方法和淋巴蛋白质滴度影响

Honey bees obtain nutrients from pollen they collect and store in the hive as beebread. We developed methods to control the pollen source that bees ...

Bone Conditioned Medium: Preparation and Bioassay

Autologous bone grafts are widely used in oral and maxillofacial surgery, orthopedics, and traumatology. Autologous bone grafts not only replace missing bone, they also support the complex process of bone regeneration. This favorable behavior of autografts is attributed to the three characteristics: osteoconductivity, osteogenicity, and osteoinductivity. However, there is another aspect: Bone grafts release a myriad of molecules, including growth factors, which can target mesenchymal cells involved in bone regeneration. The paracrine properties of bone grafts can be studied in vitro by the use of bone-conditioned medium (BCM). Here we present a protocol on how to prepare bone-conditioned medium from native pig cortical bone, and bone that underwent thermal processing or demineralization. Cells can be directly exposed to BCM or seeded onto biomaterials, such as collagen membranes, previously soaked with BCM. We give examples for in vitro bioassays with mesenchymal cells on the expression of TGF-&#946; regulated genes. The presented protocols should encourage to further reveal the paracrine effects of bone grafts during bone regeneration and open a path for translational research in the broad field of reconstructive surgery.

We describe here how to prepare bone-conditioned medium (BCM) and test its activity in vitro.

骨条件培养基的制备和生物测定

Autologous bone grafts are widely used in oral and maxillofacial surgery, orthopedics, and traumatology. Autologous bone grafts not only replace missing ...

Dissection and Observation of Honey Bee Dorsal Vessel for Studies of Cardiac Function

The European honey bee, Apis mellifera L., is a valuable agricultural and commercial resource noted for producing honey and providing crop pollination services, as well as an important model social insect used to study memory and learning, aging, and more. Here we describe a detailed protocol for the dissection of the dorsal abdominal wall of a bee in order to visualize its dorsal vessel, which serves the role of the heart in the insect. A successful dissection will expose a functional heart that, under the proper conditions, can maintain a steady heartbeat for an extended period of time. This allows the investigator to manipulate heart rate through the application of cardiomodulatory compounds to the dorsal vessel. By using either a digital microscope or a microscope equipped with a digital camera, the investigator can make video recordings of the dorsal vessel before and after treatment with test compounds. The videos can then be scored at a time convenient to the user in order to determine changes in heart rate, as well as changes in the pattern of heartbeats, following treatment. The advantages of this protocol are that it is relatively inexpensive to set up, easy to learn, requires little space or equipment, and takes very little time to conduct.

The abdominal dorsal vessel of the honey bee and other insects serves as the functional equivalent of the mammalian heart and plays an important role in nutrient transport, waste removal, immune function, and more. Here we describe a protocol for the visualization and pharmacological manipulation of bee heart rate.

解剖和蜜蜂背血管的观察心功能的研究

The European honey bee, Apis mellifera L., is a valuable agricultural and commercial resource noted for producing honey and providing crop pollination ...

Monitoring Colony-level Effects of Sublethal Pesticide Exposure on Honey Bees

The effects of sublethal pesticide exposure to honey bee colonies may be significant but difficult to detect in the field using standard visual assessment methods. Here we describe methods to measure the quantities of adult bees, brood, and food resources by weighing hives and hive parts, by photographing frames, and by installing hives on scales and with internal sensors. Data from these periodic evaluations are then combined with running average and daily detrended data on hive weight and internal hive temperature. The resulting datasets have been used to detect colony-level effects of imidacloprid applied in a sugar syrup as low as 5 parts per billion. The methods are objective, require little training, and provide permanent records in the form of sensor output and photographs.

Sublethal doses of pesticides may affect colonies in ways that are difficult to detect using only periodic or visual methods. Methods to measure total adult bee mass, brood, and food resources by weighing hives and hive parts, photographing frames, and installing sensors, are provided. Data analysis is also addressed.

蜜蜂致死农药暴露对蜂群水平的影响

The effects of sublethal pesticide exposure to honey bee colonies may be significant but difficult to detect in the field using standard visual assessment ...

Micromanipulation Techniques Allowing Analysis of Morphogenetic Dynamics and Turnover of Cytoskeletal Regulators

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how fluorescent recovery after photobleaching (FRAP) and photoactivation techniques can be used to study the spatiotemporal dynamics of proteins in subcellular locations. We also show how these techniques enable straightforward determination of various parameters linked to actin cytoskeletal regulation and cell motility. Moreover, the microinjection of cells is additionally described as an alternative treatment (potentially preceding or complementing the aforementioned photomanipulation techniques) to trigger instantaneous effects of translocated proteins on cell morphology and function. Micromanipulation such as protein injection or local application of plasma membrane-permeable drugs or cytoskeletal inhibitors can serve as powerful tool to record immediate consequences of a given treatment on cell behavior at the single cell and subcellular level. This is exemplified here by immediate induction of lamellipodial cell edge protrusion by the injection of recombinant Rac1 protein, as established a quarter-century ago. In addition, we provide a protocol for determining the turnover of enhanced green fluorescent protein (EGFP)-VASP, an actin filament polymerase prominently accumulating at lamellipodial tips of B16-F1 cells, employing FRAP and including associated data analysis and curve fitting. We also present guidelines for estimating the rates of lamellipodial actin network polymerization, as exemplified by cells expressing EGFP-tagged &#946;-actin. Finally, instructions are given for how to investigate the rates of actin monomer mobility within the cell cytoplasm, followed by actin incorporation at sites of rapid filament assembly, such as the tips of protruding lamellipodia, using photoactivation approaches. None of these protocols is&#160;restricted to components or regulators of the actin cytoskeleton, but can easily be extended to explore in analogous fashion the spatiotemporal dynamics and function of proteins in various different subcellular structures or functional contexts.

We describe how micro- and photomanipulation techniques such as FRAP and photoactivation enable the determination of motility parameters and the spatiotemporal dynamics of proteins within migrating cells. Experimental readouts include subcellular dynamics and turnover of motility regulators or of the underlying actin cytoskeleton.

微操作系统技术允许分析骨架调节器的形态发生动力学和营业额

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how ...

Oral Bacterial Infection and Shedding in Drosophila melanogaster

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic infections, there has been a recent increase of interest in the mechanisms of gut immunocompetence to pathogens, which require methods to orally infect flies. Here we present a protocol to orally expose individual flies to an opportunistic bacterial pathogen (Pseudomonas aeruginosa) and a natural bacterial pathogen of D. melanogaster (Pseudomonas entomophila). The goal of this protocol is to provide a robust method to expose male and female flies to these pathogens. We provide representative results showing survival phenotypes, microbe loads, and bacterial shedding, which is relevant for the study of heterogeneity in pathogen transmission. Finally, we confirm that Dcy mutants (lacking the protective peritrophic matrix in the gut epithelium) and Relish mutants (lacking a functional immune deficiency (IMD) pathway), show increased susceptibility to bacterial oral infection. This protocol, therefore, describes a robust method to infect flies using the oral route of infection, which can be extended to the study of a variety genetic and environmental sources of variation in gut infection outcomes and bacterial transmission.

Oral Bacterial Infection and Shedding in Drosophila melanogaster

This protocol describes methods to orally expose and infect the fruit fly Drosophila melanogaster with bacterial pathogens, and to measure the number of infectious bacteria shed following gut infection. We further describe the effect of immune mutants on fly survival following oral bacterial infection.

果蝇的口腔细菌感染和脱落

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic ...

Assessing Agrochemical Risk to Mated Honey Bee Queens

Current risk assessment strategies for honey bees rely heavily upon laboratory tests performed on adult or immature worker bees, but these methods may not accurately capture the effects of agrochemical exposure on honey bee queens. As the sole producer of fertilized eggs inside a honeybee colony, the queen is arguably the most important single member of a functioning colony unit. Therefore, understanding how agrochemicals affect queen health and productivity should be considered a critical aspect of pesticide risk assessment. Here, an adapted method is presented to expose honey bee queens and worker queen attendants to agrochemical stressors administered through a worker diet, followed by tracking egg production in the laboratory and assessing first instar eclosion using a specialized cage, referred to as a Queen Monitoring Cage. To illustrate the method&#39;s intended use, results of an experiment in which worker queen attendants were fed diet containing sublethal doses of imidacloprid and effects on queens were monitored are described.

This protocol was developed to enhance the understanding of how agrochemicals affect honey bee (Apis mellifera) reproduction by establishing methods to expose honey bee queens and their worker caretakers to agrochemicals in a controlled, laboratory setting and carefully monitoring their relevant responses.

评估交配蜜蜂女王的农用化学品风险

Current risk assessment strategies for honey bees rely heavily upon laboratory tests performed on adult or immature worker bees, but these methods may not ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

一种用于快速鉴定根系分泌物中根细菌化学引诱剂的改进趋化性测定

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...