作者 (s) 感谢威斯康星大学生物技术中心 DNA 测序设施, 提供放大和测序设施和服务, 凯特琳卡尔森, 珍妮弗诀窍, 杰克奥托, 和最大 Haase 提供技术援助分子分析。这项工作得到了农业部-农业研究处拨款资助 (目前的研究信息系统 #3655-21220-001)。国家科学基金会提供进一步支助 (赠款 No。DEB-1442148), 美国能源部大湖生物能源研究中心 (能源部 DE-FC02-07ER64494), 和美国农业部国家食品和农业研究所 (孵化项目 1003258)。C.T.H. 是一个皮尤学者在生物医学科学和阿尔弗雷德克劳斯·特普费尔教员研究员, 支持的皮尤慈善信托基金和亚历山大·冯洪堡基金会分别。

1. 使用田间网箱试验检查杀菌剂照射对蜂群成功的影响 <ol> <li> 在燕麦种植场中设置了十网笼。在每个笼子周围挖一条沟, 把网笼的四条边挖到地上, 以确保蜜蜂不能逃脱。将笼子与盆栽, 开花的植物, 是已知的吸引蜜蜂 ( 如 荞麦, 琉璃, alyssum, 宇宙和向日葵) ( 图 2 )。
	<li> 用一个单托盘 (36 厘米 x 42 厘米) 的盛开三叶草补充笼子。集群花卉资源在笼子的一个角落里, 占用一个空间大约 2.5 m x 1 m. 植物剩余的笼子区域由燕麦. </li>
	<li> 随机分配大黄蜂蜂群, 每个包含工人和一个女王, 治疗 (杀菌剂存在/缺席, N = 5 殖民地每治疗, 10 殖民地共计), 然后安置他们在一个领域笼子 ( n = 1/笼子) 29 天 (6月23日-2014年7月21日). </li>
	<li> 将菌落盒定向到菌落和 #39; s 开口指向南方, 为蜜蜂提供最佳的航行条件。用糖水囊来补贴蜂群, 放在蜂巢盒子里, 以补充花蜜的可用性. </li>
	<li> 在研究期间 (0 和 13), 使用手持杀虫剂喷雾器, 在五个杀菌剂处理笼中应用 chlorothalonil-based 杀菌剂在田间相关水平 (20 克/升) 到开花植物。将花朵均匀地涂上, 这样就不会有进一步的液体附着在花表面 ( 图 3 ). </li>
	<li> 在字段保持架研究结束时, 将 B. 凤仙花 从笼子中手工取出, 通过放置-20 和 #176 冷却蜂房; C 型冷冻机20分钟 </li>
	<li> 使用无菌钳清除蜜蜂并记录幼虫、蛹、成年雌性的数量 ( i . e . 觅食) 和成年男性。使用分析天平记录母亲皇后、幼虫、蛹、成年雌性 ( 即 觅食) 和成年雄性的干重. </li>
</ol> 2。使用实验室 在体内 试验中检测杀菌剂暴露对花粉中微生物群落的影响 <ol> <li> 用标准的方法将商业购买的花粉粉碎为细粉实验室球磨机。通过浸泡70% 乙醇, 使花粉在紫外线照射下一夜之间蒸发, 从而杀菌。在 general-purpose 琼脂培养基上通过电镀0.5 毫克来验证花粉的不育性. 
	<ol> <li> 对干燥、灭菌的花粉, 使用灭菌管, 添加田间相关剂量的杀菌剂: 环在 14.3%; 嘧22.9% 用于治疗 (0.74 和 #181; l 和0.65 和 #181; 分别/天/蜂房)。用消毒的木棍拌匀. </li>
	</ol> </li> <li> 在室温下保持的干净、卫生的实验室中放置6个实验性荨麻疹 (n = 3 个用于控制和治疗). 台式。每一天体重4.27 克的花粉 34 , 35 与杀菌剂混合 (用于治疗) 或无菌水 (用于控制) 在一个罩内使用标准的无菌技术. 
	<ol> <li> 使用包含蜂巢的纸板盒侧面提供的陷阱门, 介绍蜂房内的花粉。每周用消毒的糖溶液补充蜂房。继续喂养制度四周. </li>
	</ol> </li> <li> 在实验室研究的结论中, 通过放置-20 和 #176 来冷却荨麻疹; C 型冷冻机20分钟, 用灭菌钳和铲子在不育的储藏管中, 用消毒的镊子将花粉的规定刮掉。存储在-80 和 #176; c. 在实验开始和结束时清点和记录工人和王后母亲的体重 (步骤 1.7). </li>
	<li> 使用商业上可用的 dna 隔离套件从花粉提供样品中分离出 dna (详见材料表). 
	<ol> <li> 添加0.25 克花粉-提供给萃取管, 短暂的漩涡混合. </li>
		<li> 在去离子蒸馏水中制作200毫克/毫升溶菌酶溶液, 足够50和 #181; L/样品。用力摇动, 完全形成解决方案. </li>
		<li> 添加50和 #181; 溶菌酶溶液中的萃取管与样品在其中, 并混合好几次。在水浴中孵育试管10分钟, 在37和 #176; C。如果沉淀形成, 热溶液到60和 #176; C 直到溶解在用途之前. </li>
		<li> 添加70和 #181; L 对萃取管进行水裂解处理, 在平板涡垫上用胶带将其水平固定, 在室温下为三十年代 1万 x g 离心管10分钟. </li>
		<li> 将上清液转移到干净的2毫升收集管。添加250和 #181, L 蛋白质沉淀溶液, 和涡流 5 s. 在4和 #176; C 在冰浴中孵育5分钟. 
		注: 400 至 #181; 升至 500 #181; 上清。上清液可能还含有一些微粒. </li>
		<li> 在室温下离心管的温度为1分钟, 在 1万 x g, 并转移到600和 #181; 升上清到一个干净的2毫升收集管。添加200和 #181; 水抑制剂去除溶液, 涡旋, 并孵育在4和 #176; C 为5分钟离心管在室温下为1分钟, 在 1万 x g. 转移到750和 #181; 升上清成干净的2毫升收集管. </li>
		<li> 添加1200和 #181; 升水溶液到上清, 和涡流 5 s. 加载约675和 #181; l 上清到一个旋转过滤器, 离心在 1万 x g, 在室温下1分钟。放弃流. </li>
		<li> 重复2.4.7 两次. 
		注: 所处理的每个样品总共需要三负载. </li>
		<li> 添加500和 #181; L 乙醇, 在室温下离心三十年代在 1万 x g. 放弃流动, 并再次离心在室温下1分钟, 在 1万 x g. 在干净的2毫升收集管中放置旋转滤镜. </li>
		<li> 添加100和 #181; 洗脱缓冲液到滤膜中心。离心机在室温下为三十年代在 1万 x g. 丢弃旋转滤镜。在-20 和 #176 之间存储收集的 dna; c 和-80 和 #176; c </li> </ol> </li> <li> 使用分离的 dna 进行测序. 
	<ol> <li> 通过荧光分析对 2 ng/和 #181 的分离 DNA 进行量化和规范化;
		<ol> <li> 为每个提取的样品准备一式三份的反应, 比较 28S (植物) 的相对数量, 分析其 (真菌) 和每个花粉的 16S (细菌) 成分--提供样本 36 。确保每一个反应包含 10 ng 的总 DNA, 2x 的不对称的菁染料为主混合, 和2.5 和 #181; L 每个向前和反向底漆对28焦/28 b 37 用于植物和 ITS1/ITS5.8R 真菌 DNA 的 38.</li>
		</ol> </li> <li> 使用下列参数放大 DNA: 2 分钟 pre-denaturation 在50和 #176; c, 2 分钟初变性在95和 #176; c, 40 周期 (十五年代在98和 #176; c, 十五年代在58和 #176; c, 六十年代在72和 #176; c), 后跟一个熔体曲线. </li>
		<li> 使用面向 16S rRNA V3/V4 变量区域和其/5.8s rRNA 间隔区域的下一代测序库准备2步嵌套的 PCR 协议。
		<ol> <li> 将12.5 和 #181 添加到每个底漆和2x 主组合的 5 pmol。对每个区域分别作出反应 (16S 或其; 请参见 表 1 )。修改区域特定的引物, 如前面所描述的 39 , 40 , 以向特定于基因的序列添加排序器特定的适配器悬垂核苷酸序列. </li>
			<li> 使用以下参数执行初始放大: 3 分钟初变性在95和 #176; c, 25 周期 (三十年代在95和 #176; c, 三十年代在55和 #176; c, 三十年代在72和 #176; c), 5 min 最后的延长在72和 #176; c. </li>
			<li> 在初始放大后, 通过电泳移动验证库大小和数量, 并使用1x 体积固相可逆 imm 进行清洗obilization 珠去除残留底漆和反应试剂。池16S 和它的增定量地为每个示例创建一个增池. </li>
			<li> 使用以下引物添加排序器特定的适配器和示例特定的双索引 (请参见 表 1 )。增加2.5 和 #181; L 放大 DNA 到 5 pmol 的每个底漆和2x 主混合。使用以下参数执行库放大: 3 分钟初变性在95和 #176; c, 8 周期 (三十年代在95和 #176; c, 三十年代在55和 #176; c, 三十年代在72和 #176; c), 5 min 最后的引伸在72和 #176; c. </li>
		</ol> </li> <li> 遵循 PCR 方法, 使用 1 x 体积的固相可逆固定化微珠清洁成品库。使用电泳移动和荧光分别评估成品库的质量和数量。在排序前将库标准化到2和 #181; M 和池. </li>
		<li> 在与次级 PCR 中添加的适配器类似的平台上执行下一代测序, 其长度适当以覆盖整个增 41 . </li>
	</ol> </li> <li> 序列注释和微生物成分分析。
	<ol> <li> 将对所有顺序库的对端排序数据 (R1 和 R2) 合并为单个重叠。在合并 R1 和 R2 文件之后, 将生成每个库的单个 fasta 文件. 
		注意: 此步骤和下面描述的过程 (除非另有说明) 在 Mothur 版本 1.38.0 38 中执行. </li>
		<li> 筛选每个 fasta 文件以删除不明确的基、意外的长序列和长物. 
		注: 筛选的参数和序列去除 maxambig = 0, maxlength = 600, 和 maxhomop = 8 为两个基因。删除相同的序列, 但为所有库分别保留一个 contingence 表 (又称 Mothur 中的计数表). </li>
		<li> 将基因16S 库的唯一序列与席尔瓦数据库版本123对齐, 并将其库与其数据库进行联合, 以使其不被合并为 42 . 
		注意: 序列必须从王国水平到属层的注释。
		<ol> <li> 随后, 使用 diff=5 对具有函数前的群集的群集对齐序列, 并删除合体. </li>
		</ol> </li> <li> 根据席尔瓦 (用于基因 16S) 的方法对序列进行分类, 并将其 (用于基因的) 分类文件 (截止值为 80%) 联合使用. 
		<li> 加载到 R 44 在 Mothur 的分类过程中生成的 contingence 表 (每个基因一个)。在每个分类学层面上, 获取每个图书馆的相对丰度, 进一步分析微生物群落的变化. 
		注意: 在每个分类学层面上, 当相对丰度小于2% 时, 所有的实验重复都将分类组合并在一起. </li>
	</ol>
	</li>
</ol>

作者没有什么可透露的。

对杀菌剂对蜜蜂健康的影响的调查仍然是害虫管理战略的一个理解方面。我们的研究目的是通过一套互补的技术来弥合这一知识鸿沟, 这一方法明确地隔离了推动蜜蜂衰落的潜在因素。下面详细介绍这些实验的规划、基本原理和渲染。
重要的是要确保不允许蜜蜂逃脱笼实验的网格, 因为这将损害人口学分析。同样重要的是, 人工巢有足够的绝缘, 以防止雨水和阳光直射。应注意不要将杀菌剂直接喷洒在巢上。应向菌落提供糖水囊, 以补充采集的花蜜, 防止脱水。
对于以实验室为基础的喂养试验, 在准备花粉餐时应保持严格的无菌条件, 以防止污染。商业购买的花粉必须是粉状和紫外线灭菌之前使用。必须保持蜂房卫生, 以防止外来寄生虫的侵扰, 如茶水间蛾和蟑螂。液体膀胱应辅以无菌糖溶液, 以防止脱水。进行前处理普查时, 应注意不要过度搬运蜜蜂的压力, 并将它们长期留在转为外。
由于 dna 的产量取决于起始材料的年龄和类型, 只有新鲜或 well-frozen 的样本必须用于 dna 提取和扩增。dna 提取样品的重量不应超过 0.25 gm, 因为这将降低 dna 的产量。应使用凝胶电泳对 DNA 的产率进行量化。前期电泳定量的孤立 DNA 是至关重要的, 然后再进行 qPCR 反应, 以确保有效放大47。
对于基因分析, 可以通过限制注释序列的数量 (通过放宽序列之间可容忍差异的分数) 和减少分类群的数量来获得对微生物动态的更全面的分析, 如以及合并那些相对丰度相当低 (和 #60; 2%)。
保守的样本大小 (N = 5 用于杀菌剂治疗和未处理的笼型研究单元) 可以夸大数据的方差, 这将在将来的研究中使用更大的复制来最小化。为了给结果增加更大的分辨率和统计力, 蜂群将在杀菌剂暴露前后 censused 和称量。长时间运行笼子试验将允许生产新的皇后, 这可以作为一个额外的反应变量。随着对当前协议的这些修改, 未来的研究将提供更多关于蜂巢种群动态的细节。
花粉的疏水性使其有效地与水混合。粉碎和筛选花粉颗粒, 以细粉, 助于混合过程。花粉粉的数量可以调整, 以达到预期的一致性。应注意彻底混合液体 (水或杀菌剂), 以达到均匀分布的活性成分, 甚至交付到觅食。
由于过量的 dna 可以抑制 PCR 反应, 建议样品不应超过0.25 克. 由于这种剪切 dna, 降低了产量, 应避免过量的涡流。低的 DNA 产量也可能是由于长期暴露于裂解缓冲液的结果。细菌引物应从 16S rRNA 的 V7-V9 区优选, 以尽量减少叶绿体 DNA 的非特异性放大48。另外, 其他基因的测序, 如18S 核糖体 RNA 基因, 可以更好地了解花粉中的微生物多样性, 以及对杀菌剂利用的种群变化。
考虑到使用 Mothur 处理的库的数量, 计算时间可能会相当高。在比较序列时移除单身者 (如果计算资源有限, 在嵌合体过滤之前删除它们) 可以大大减少计算操作的分类单元所需的时间。
笼子实验限制了蜜蜂的自然飞行范围 (相对于允许它们广泛地在整个景观中觅食), 而且不清楚这些限制对反应变量的影响程度。虽然控制和治疗笼经验相同范围的生物和非生化变量, 它是在后勤上不可能控制的微环境在每个笼子。
在社区内的微生物相互作用的真正范围是太复杂和复杂的复制通过实验试验。虽然我们的数据可能记录花粉中总微生物多样性的一个子集, 但它是第一次洞察细菌和真菌在存在/没有杀菌剂的情况下的交互作用。
使用可选数据库对齐序列49或基于花粉细菌多样性预测功能代谢状态50可以为花粉微生物群提供更广阔的视野。最后, 为了更好地表征微生物群落在杀菌剂应用后的代谢和功能动态, 需要对花粉微生物群进行全基因组测序。
使用商业购买的蜂箱的笼子实验提供了一个最好的框架来测量 semi-controlled 设置中的响应变量。对蜜蜂的自然生态 (觅食效率、社会结构、子代保育) 进行最小化改造, 笼型试验使人工环境和手工搬运在实验室过程中的发明和压力降到最低。实验.
据我们所知, 目前还没有进行研究, 以评估杀菌剂对蜂花粉中微生物群落动态的影响。接触杀菌剂可以消除一个或多个敏感物种, 破坏真菌和细菌之间的生态休战。这项研究的目的是利用田间和实验室试验作为一个坩埚来进行这些相互作用, 以证明对居民微生物物种的灭绝会破坏花粉微生物群的生态平衡, 进而损害蜜蜂的健康。
基于文化的技术, 如稀释和电镀标准介质捕获只在特定环境中的微生物区的一小部分。这可能导致微生物多样性的严重不足, 而 unculturable 的微生物可能会被发现51。为了研究微生物的广度, 科学家必须依靠与文化无关的技术, 这是更具包容性和全面性的, 例如基因分析和下一代测序。从这些强大的分子技术, 这项研究的目的是获得更大的分辨率进入花粉微生物群比传统的文化技术仅提供。
这项研究的结果揭示了大黄蜂群中接触杀菌剂的工人数量的巨大损失。为了使大黄蜂群体获得成功, 工人们需要提供足够的资源和照顾母皇后, 特别是对发育中的幼虫。最终, 该殖民地的目标是最大限度地获取资源 (花粉和花蜜), 这样尽可能多的女儿皇后可以在夏末时生产。健康的女儿-皇后是一个群体的健身措施。然后, 大量削减工人, 有效地削弱了殖民地的能力, 生产皇后下一年。在这项研究中, 不仅工人人数明显减少, 但从杀菌剂治疗的蜂箱的母皇后提出了较低的生物量, 推测是由于粮食供应不足的觅食。鉴于花粉中微生物相互作用的复杂性, 很难辨别出真菌和细菌之间的确切交互作用。然而, 在这里所描述的复制和重复实验的结果, 将提供重要的洞察力的转变共生之间的两个微生物组 (无论是竞争性的, 互惠, 或共生), 以响应异的压力,及其对花粉微生物的下游影响。
通过使用强大的分子工具, 可以更好地解决花粉微生物群的生态复杂性。初步的理解揭示了大量的自然发生的细菌和真菌, 共同贡献, 以维持群体的健康。特别是, 从花粉中分离出来的酵母被认为具有杀菌和发酵的特性, 这两者对幼虫的发育都是至关重要的。这些生态协会暗示了蜜蜂和它们的花粉微生物之间的高度互惠互利。因此, 花粉的规定, 是一个由关键功能作用的微生物群落的培养所带来的一种突现的多样性效应。在没有这些关键共生的情况下, 花粉的供应似乎受到了未知程度的损害。继续朝这个方向努力, 很可能解释这些微生物的功能多样性, 并揭露它们作为蜜蜂共生的作用。
最近的研究已经揭穿了杀菌剂对蜜蜂无害的概念19,24,52,53,54。本研究的目的是分离驱杀真菌对蜜蜂的影响机制, 从而揭示杀菌剂使用与蜂衰的因果关系。假设中心围绕的概念, 蜜蜂微生物共生正在改变显着的杀菌剂残留的花粉。最终, 这项工作将看法如何在农业和城市环境中查看和使用杀菌剂。鉴于目前的全球授粉危机, 拟议的研究产生了新的关于本地蜜蜂生态学的知识, 因为它与农业生产实践有关。杀菌剂仍然是最后一个 agrichemical 组, 可以有效地免除限制在开花期间对农作物的应用的规定。因此, 管理和野生蜜蜂都经常接触到杀菌剂。越来越多的文献表明, 虽然杀菌剂对蜜蜂没有致命的接触, 在高剂量, 暴露是相当有害的, 导致更高的幼虫死亡率21和改变觅食行为。预计这项工作将阐明杀菌剂对传粉者的危害机制。此外, 这项研究将形成更明智的虫害管理政策的基础, 并向种植者通报最佳管理做法。这项工作还将有助于为改进杀虫剂喷洒提供指导方针, 这可能影响农药的法律和法规。更广泛地讲, 这些发现将与任何有管理的生态系统有关, 其中开花植物通过收集花粉的昆虫来访问。随着全球蜜蜂专用授粉服务的估计值非常高55,56, 如果这项工作能够减少蜜蜂数量的减少, 潜在的影响将是巨大的。

管理和野生蜜蜂物种正经历着广泛的下降, 对自然和农业系统的主要影响1。尽管共同努力, 以了解这一问题的原因, 驱动蜂蜜蜂下降的因素仍然没有很好地理解2,3,4。对于某些种类的野生, 本地蜜蜂, 情况已经变得可怕5,6。如果蜜蜂种群在与工业农业相交时不能持续, 其种群数量将继续下降, 而需要传粉者的作物 (全球总产量的 35%7) 将会减少收成。
虽然许多潜在的因素, 如农药暴露, 疾病和栖息地损失1,4,8,9,10都牵连到蜜蜂的衰落,在农业系统内部或附近, 这些压力源对本地蜜蜂健康的交互作用的了解相对较少。许多目前的研究工作继续侧重于杀虫剂, (例如, 烟碱11,12), 尽管过去的研究表明, 杀菌剂也可能通过损害记忆形成而在蜜蜂减少中发挥作用,嗅觉接收13, 巢识别14, 酶活性和代谢功能15,16,17。在全球范围内, 杀菌剂继续应用于开花的农作物。最近的研究表明, 蜜蜂通常会将杀菌剂残留物带回蜂巢18, 事实上, 研究显示, 有很大比例的被测试的蜂箱含有杀菌剂残留物19,20。进一步的工作表明, 杀菌剂残留与蜜蜂幼虫死亡率的高比率21,22,23和在殖民地内存在 "被埋葬的花粉", 虽然无毒,缺乏微生物活性, 营养受损24。尽管杀菌剂长期以来被认为是 "蜜蜂安全" 的事实, 但现在有证据表明, 单独接触杀菌剂可能会在本地大黄蜂物种中造成严重的蜂群损失,熊凤仙花25。
为了建立杀菌剂暴露与群体死亡率之间的因果关系, 需要确定这些化学品的作案手法。如土壤26, 沉积物27和水生环境28, 通过瞄准真菌, 杀菌剂最有可能改变真菌丰度和花粉中的多样性-规定, 从而调用一个主要的社区转移,可能强烈偏爱细菌。没有真菌竞争者或拮抗剂, 某些致病细菌可以增殖相对不受控制, 促进花粉变质的规定。过去的研究表明, 微生物, 特别是酵母菌和丝状真菌, 作为营养共生的蜜蜂29,30,31, 防止寄生虫和病原体32 ,33, 并提供花粉存储的长期保存。因此, 杀菌剂可能会通过破坏微生物群落来间接地伤害幼蜂, 因为它们需要提供这些服务和/或增加对机会性病原体和寄生虫的易感性12。随着对粮食生产的需求日益增加, 世界各地的农作物每年都在开花时喷洒杀菌剂, 这突出了需要了解这种杀菌剂引起的影响的严重性。
到目前为止, 与本地蜜蜂微生物生态学有关的主要知识鸿沟可以由以下问题来表示: 在何种程度上杀菌剂改变蜂花粉中的微生物群落-规定？什么是消耗花粉与一个深刻改变的微生物群落的下游影响？与这些生态学上相关的问题, 实验开发了以显露的主要目标 1) 仅杀菌剂残余可能导致严重殖民地衰落在本地蜂种;2) 花粉中微生物群落的程度--规定被杀菌剂改变, 3) 蜜蜂的健康受到严重改变的微生物群落的影响。实验目标的定义是通过结合实验室和实地实验来解决上述问题。本研究利用先进的基因和分子技术和传统的野外观察方法, 将杀菌剂对蜜蜂健康的潜在影响结合起来。
这项研究的第一个目的是证明单独的杀菌剂暴露会在本地蜜蜂物种中造成巨大的群体损失。一项涉及大型田间网箱的研究被用来调查杀菌剂暴露对熊凤仙花群体生长的影响, 这是一个无处不在的、丰富的美国本土蜜蜂 (图 1、图 2、图 3)。据推测, 与医护蜂房相比, 杀菌剂治疗的蜂箱会呈现较低的适应性和不典型的人口学。从这个实验中获得的数据证实了这一假说, 证明花粉中的杀菌剂残留物可能是造成本地大黄蜂物种严重群体损失的唯一原因25。本研究的第二个目的是探讨花粉微生物对杀菌剂暴露的反应。据推测, 花粉中微生物的群落组成--暴露在杀菌剂中的成分会与未经处理的花粉不同。虽然真菌的丰度和多样性预计会显著下降, 但细菌和/或单一的优势真菌物种在没有其他竞争真菌的情况下可能会无节制地生长。通过一系列的体内试验, 将利用基因分析微生物群落组成的这些变化。

<table>
	<tbody>
		<tr>
			<td>Natupol Beehive</td>
			<td>Koppert</td>
			<td>USRESM1</td>
			<td>16 hives</td>
		</tr>
		<tr>
			<td>Propiconazole 14.3</td>
			<td>Quali-Ppro</td>
			<td>60207-90-1</td>
			<td>Propiconazole 14.3%</td>
		</tr>
		<tr>
			<td>Abound</td>
			<td>Syngenta</td>
			<td>4033540</td>
			<td>Azoxystrobin 22.9%</td>
		</tr>
		<tr>
			<td>Chlorothalonil</td>
			<td>Syngenta</td>
			<td>3452</td>
			<td>Fungicide used for trials</td>
		</tr>
		<tr>
			<td>Pollen granules</td>
			<td>Bee rescued</td>
			<td>B004D5650C</td>
			<td>3X 16oz bottles, pollen for trials</td>
		</tr>
		<tr>
			<td>Bacterial strains for inoculation</td>
			<td>Currie Lab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast strains for inoculation</td>
			<td>Hittinger lab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Primer pairs</td>
			<td>UW Biotech Center</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Isolation Kit</td>
			<td>Mo Bio</td>
			<td>12830-50</td>
			<td>Commercial DNA isolation kit</td>
		</tr>
		<tr>
			<td>Qubit dsDNA HS Assay Kit</td>
			<td>Thermo Fisher</td>
			<td>Q32851</td>
			<td>DNA quantification tool</td>
		</tr>
		<tr>
			<td>Select Master Mix for CFX</td>
			<td>Thermo Fisher</td>
			<td>4472952</td>
			<td>Used to perform real-time PCR using SYBR GreenER dye.</td>
		</tr>
		<tr>
			<td>Real-Time PCR Detection System</td>
			<td>Bio Rad</td>
			<td>1855196</td>
			<td>Instrument used for PCR amplification</td>
		</tr>
		<tr>
			<td>PCR Clean-Up Kit,</td>
			<td>Axygen</td>
			<td>10159-696</td>
			<td>Used for efficient removal of unincorporated dNTPs, salts and enzymes</td>
		</tr>
		<tr>
			<td>DNA 1000 Kit</td>
			<td>Agilent</td>
			<td>5067-1504</td>
			<td>Used for sizing and analysis of DNA fragments</td>
		</tr>
		<tr>
			<td>MiSeq Sequencer</td>
			<td>Illumina</td>
			<td></td>
			<td>Used for next-generation sequencing</td>
		</tr>
		<tr>
			<td>Assorted glassware (beaker, flasks, pipettes, test tubes, repietters)</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

empirical, metagenomic, and computational techniques illuminate the mechanisms by which fungicides compromise bee health

种植者经常在开花时使用杀菌剂来保护农作物免受疾病的危害, 从而使蜜蜂暴露于杀菌剂残留物中。虽然被认为是 "蜜蜂安全", 但有越来越多的证据表明花粉中的杀菌剂残留物与蜜蜂的下降有关 (蜂蜜和蜜蜂的种类)。虽然这些机制仍然不为人知, 但研究人员推测, 蜜蜂微生物共生的参与。微生物在保存和/或处理花粉方面起着举足轻重的作用, 它们作为幼虫的营养。通过改变微生物群落, 杀菌剂可能破坏这些微生物介导的服务, 从而危害蜜蜂的健康。这份手稿描述了用于研究杀菌剂可能导致蜂群衰退的间接机制的协议。笼子试验揭露蜜蜂对杀菌剂处理过的花朵已经提供了第一个证据, 即杀菌剂造成了本地大黄蜂 (熊凤仙花) 的严重蜂群损失。利用田间相关剂量的杀菌剂, 进行了一系列的实验, 以提供更精细的描述微生物群落动态的杀菌剂暴露花粉。通过下一代测序和基因分析, 研究了花粉微生物群中真菌和细菌组合结构组成的变化。本文所开发的实验, 旨在提供一个机械的了解如何杀菌剂影响花粉的微生物-规定。最终, 这些发现应该揭示出杀菌剂可能导致蜂群衰退的间接途径。

野外网箱研究:
从笼型实验中获得的数据表明, 大黄蜂的蜂群对杀菌剂暴露有明显的反应。杀菌剂处理的蜂箱比控制单元 (43.2 ± 11.2, F19= 6.8, p = 0.03) (图 4) 产生的工人数量显著减少 (12.2 ± 3.8, 平均± SE)。此外, 杀菌剂处理的蜂箱的蜂生物量 (0.91 克± 0.15) 明显低于对照蜂房 (2.36 克± 0.55;F19 = 8.3, p = 0.02)。在母蜂中也观察到了这种在杀菌剂治疗的蜂箱中降低生物量的模式。在杀菌剂处理过的蜂巢中, 蜂王的生物量明显低于控制蜂巢的皇后 (0.27 克± 0.01, 0.14 克± 0.04);Z = 2.5, p = 0.01)。然而, 杀菌剂的暴露并没有影响到幼虫、蛹和雄性在治疗中的数量。离散生命阶段 (幼虫、蛹、工人和成年雄性) 的生物量和幼虫、蛹和工人的个体重量在杀菌剂治疗和控制荨麻疹上没有显示出任何差异。
实验室研究:
从实验室实验获得的数据表明, 在杀菌剂暴露之前, 控制和杀菌剂治疗的蜂箱有统计学可比的平均工人计数 (控制蜂巢 = 28.0 ± 3.1; 杀菌剂治疗的荨麻疹 = 31.67 ± 2.0; n = 3每个) 和皇后重量 (控制荨麻疹 = 0.77 克± 0.04; 杀菌剂治疗的荨麻疹 = 0.74 克± 0.01)。然而, 在研究结束时, 相对于杀菌剂处理的荨麻疹 (45.33 ± 3.8) (t4 = 3.89, p = 0.01) 相比, 在控制配置单元 (67.67 ± 4.3) 中, 工人计数显著提高。工人人口增加了 ~ 150% 在控制蜂箱比 ~ 45% 在杀菌剂被对待的蜂房。同样, 在控制荨麻疹 (0.76 克± 0.02) 中, 女王母亲的最终体重保持不变, 相比之下, 杀菌剂处理的蜂箱的减少 35% (0.49 克± 0.16)。两者结合在一起, 这些结果与先前发布的结果25一致, 并表明杀菌剂暴露受影响的群体适应性, 这是由较少的工作人员数量和降低的蚁后体重 (图 5) 证明的。
基因对花粉的分析表明, 从杀菌剂治疗和控制荨麻疹收集的微生物群落之间存在着明显的差异 (图 6)。在杀菌剂治疗的荨麻疹中, Streptomycetales、Enterobacteriales 的相对丰度下降 (#62; 95%)。有趣的是, 这两组都知道他们的抗真菌活性和在花粉保存30,45在大黄蜂蜂房环境中的作用。顺序 Rickettsiales 的细菌成员, 包括节肢动物的共同的病原体46, 显示了更大丰盈在杀菌剂被对待的荨麻疹。杀菌剂治疗的蜂箱有更低的真菌, 属于 Eurotiales 和 Sordariales 的命令, Capnodiales 和 Ascosphaerales 与控制荨麻疹的数量更高 (图 7)。正如所料, 对于细菌和真菌, 香农的多样性指数(H)和均匀度(E)在杀菌剂处理的蜂箱中比对照组低, 尽管这些差异没有统计学意义 (细菌: H控制配置单元 =1.25 ± 0.3, H杀菌剂处理的荨麻疹 = 0.82 ± 0.2, E控制配置单元 = 0.46 ± 0.1;E杀菌剂处理的荨麻疹 = 0.31 ± 0.1;真菌: H控制配置单元 =0.99 ± 0.3, H杀菌剂处理的蜂箱 = 0.72 ± 0.4, E控制配置单元 = 0.57 ± 0.2;E杀菌剂处理的荨麻疹 = 0.42 ± 0.2)。虽然详细说明了这种社区转移的代谢和功能的影响是超出了本研究的范围, 结合菌落计数和体重数据, 这些结果表明, 杀菌剂暴露可能影响群体健康下降扰乱蜜蜂和花粉微生物的共生关系。为了更好地评估花粉微生物群在维持健康蜂种群中的作用, 建议进一步研究影响幼虫存活率的特定微生物群体的作用。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/54631/54631fig1.jpg"> 
图 1: 在熊凤仙花嵌套中.高分辨率图像的工人倾向于育雏细胞。巢细胞在任何特定时间都可以含有几个卵和幼虫。开发幼虫饲料的花粉-规定定期引入会议厅。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54631/54631fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/54631/54631fig2.jpg"> 
图 2: 为蜜蜂聚居地建造的大型野战网箱.每个网笼 (N = 10) 都储存了一个商业购买的熊凤仙花蜂巢, 以及盛开的植物品种, 以吸引蜜蜂。在笼子的一个角落里摆放着鲜花, 剩下的地方用燕麦草覆盖。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54631/54631fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/54631/54631fig3.jpg"> 
图 3: 最近喷洒的向日葵上的杀菌剂残留物.在实验0和13天喷洒了田间相关剂量的杀菌剂。使用杀虫剂喷雾器, 花在黄昏/傍晚均匀涂上杀菌剂溶液, 以避免与觅食蜂直接接触。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54631/54631fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/54631/54631fig4.jpg"> 
图 4: 网箱试验中杀菌剂残留对黄蜂的影响在花粉中接触到杀菌剂残留物的大黄蜂菌落数量显著下降 (约简离子在成年女性丰盈) 在一个月的过程中。误差线表示± 1SE;p & #60; 0.05。此图是从 Bernauer et al.中修改的25.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54631/54631fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/54631/54631fig5.jpg"> 
图 5: 实验室实验中杀菌剂残留对蜜蜂的影响受杀菌剂处理的花粉感染的大黄蜂菌落数量显著下降 (成年工人数量减少), 在一个月的过程中, 蚁后的体重下降。误差线表示± 1SE;p = 0.03。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54631/54631fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/54631/54631fig6.jpg"> 
图 6: 基因分类的饼图真菌和细菌多样性在命令等级.基于 (a) 16S 和 (b) 的分析, 根据从控制和杀菌剂处理的蜂箱收集的花粉提供样本进行分类。控制蜂巢在微生物分布上表现出更高的多样性和均匀性。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54631/54631fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/54631/54631fig7.jpg"> 
图 7: 按顺序排列的百分比更改细菌和真菌的相对丰度对杀菌剂暴露的反应.基因微生物群落的分类使用 (a) 16S 和 (b) 其基础引物<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54631/54631fig7large.jpg" target="_blank">请点击这里查看这个数字的更大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>底漆对</td>
			<td>底漆类型</td>
			<td colspan="4">底漆顺序</td>
		</tr>
		<tr>
			<td rowspan="2">28焦/28 b</td>
			<td rowspan="2">植物</td>
			<td colspan="4">GGC GGT AAA 增值税</td>
		</tr>
		<tr>
			<td colspan="4">增值税 CCG AAG 动漫</td>
		</tr>
		<tr>
			<td rowspan="2">ITS1/ITS5。8</td>
			<td rowspan="2">真菌</td>
			<td colspan="4">GGT GAA CCT GCG 克</td>
		</tr>
		<tr>
			<td colspan="4">堵嘴 ATC 增值税 TGT TGA AAG TT</td>
		</tr>
		<tr>
			<td rowspan="2">16s 向前/反向</td>
			<td rowspan="2">嵌套细菌</td>
			<td colspan="4">5 "-ACACTCTTTCCCTACACGACGCTCTTCCGATCTCCTACGGGNGGCWGCAG-3'/</td>
		</tr>
		<tr>
			<td colspan="4">5 "-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGACTACHVGGGTATCTAATCC-3"</td>
		</tr>
		<tr>
			<td rowspan="2">ITS1F 底漆/ITS4</td>
			<td rowspan="2">嵌套真菌</td>
			<td colspan="4">5 "-ACACTCTTTCCCTACACGACGCTCTTCCGATCTTTCGTAGGTGAACCTGCGG-3"</td>
		</tr>
		<tr>
			<td colspan="4">5 "-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTCCTCCGCTTATTGATATGC-3"</td>
		</tr>
		<tr>
			<td rowspan="2"></td>
			<td rowspan="2">适配器底漆</td>
			<td colspan="4">5 '-AATGATACGGCGACCACCGAGATCTACAC [55555555] ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3 的</td>
		</tr>
		<tr>
			<td colspan="4">5 '-CAAGCAGAAGACGGCATACGAGAT [77777777] GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3 "</td>
		</tr>
	</tbody>
</table>
表 1: 用于 DNA 放大的底漆对和底漆序列的列表.请参见文本以查看引用。

Watch this Scientific Journal Video about Empirical, Metagenomic, and Computational Techniques Illuminate the Mechanisms by which Fungicides Compromise Bee Health at JoVE.com

经验、宏基因组和计算技术阐明了杀菌剂损害蜜蜂健康的机制环境 |视频

Empirical, Metagenomic, and Computational Techniques Illuminate the Mechanisms by which Fungicides Compromise Bee Health

黄蜂蜂巢内的微生物联合体丰富和保存蜂幼虫的花粉。使用下一代测序, 连同实验室和实地实验, 这份手稿描述的协议用于测试的假说, 杀菌剂残留改变花粉微生物群, 和群体人口统计, 最终导致殖民地损失.

经验、基因和计算技术阐明了杀菌剂危害蜜蜂健康的机制

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered ...

empirical-metagenomic-computational-techniques-illuminate-mechanisms

Nanyang Technological University

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9.6K 次观看.  USDA-ARS. 黄蜂蜂巢内的微生物联合体丰富和保存蜂幼虫的花粉。使用下一代测序, 连同实验室和实地实验, 这份手稿描述的协议用于测试的假说, 杀菌剂残留改变花粉微生物群, 和群体人口统计, 最终导致殖民地损失.

视频: Empirical, Metagenomic, and Computational Techniques Illuminate the Mechanisms by which Fungicides Compromise Bee Health

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa1,2, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol 3 . Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high&#8208;resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography4,5. Moreover, OpNS can be a high&#8208;throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples 6. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.

More than half of proteins are small proteins (molecular mass &#60;200 kDa) that are challenging for both electron microscope imaging and three-dimensional reconstructions. Optimized negative staining is a robust and high-throughput protocol to obtain high contrast and relatively high resolution (~1 nm) images of small asymmetric proteins or complexes under different physiological conditions.

优化负染:通过电子显微镜检查小和不对称蛋白结构的高通量协议

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of ...

科研

环境科学

Spotting Cheetahs: Identifying Individuals by Their Footprints

The cheetah (Acinonyx jubatus) is Africa's most endangered large felid and listed as Vulnerable with a declining population trend by the IUCN1. It ranges widely over sub-Saharan Africa and in parts of the Middle East. Cheetah conservationists face two major challenges, conflict with landowners over the killing of domestic livestock, and concern over range contraction. Understanding of the latter remains particularly poor2. Namibia is believed to support the largest number of cheetahs of any range country, around 30%, but estimates range from 2,9053 to 13,5204. The disparity is likely a result of the different techniques used in monitoring.
Current techniques, including invasive tagging with VHF or satellite/GPS collars, can be costly and unreliable. The footprint identification technique5 is a new tool accessible to both field scientists and also citizens with smartphones, who could potentially augment data collection. The footprint identification technique analyzes digital images of footprints captured according to a standardized protocol. Images are optimized and measured in data visualization software. Measurements of distances, angles, and areas of the footprint images are analyzed using a robust cross-validated pairwise discriminant analysis based on a customized model. The final output is in the form of a Ward's cluster dendrogram. A user-friendly graphic user interface (GUI) allows the user immediate access and clear interpretation of classification results.
The footprint identification technique algorithms are species specific because each species has a unique anatomy. The technique runs in a data visualization software, using its own scripting language (jsl) that can be customized for the footprint anatomy of any species. An initial classification algorithm is built from a training database of footprints from that species, collected from individuals of known identity. An algorithm derived from a cheetah of known identity is then able to classify free-ranging cheetahs of unknown identity. The footprint identification technique predicts individual cheetah identity with an accuracy of &gt;90%.

The cheetah (Acinonyx jubatus) is an iconic, endangered species, but conservation efforts are challenged by habitat shrinkage and conflict with commercial farmers. The footprint identification technique, a robust, accurate and cost-effective image classification system, is a new approach to monitoring cheetahs.

通过他们的足迹识别个体:合模猎豹

The cheetah (Acinonyx jubatus) is Africa's most endangered large felid and listed as Vulnerable with a declining population trend by the IUCN1. It ranges ...

Evaluating the Effect of Environmental Chemicals on Honey Bee Development from the Individual to Colony Level

The presence of pesticides in the beekeeping environment is one of the most serious problems that impacts the life of a honey bee. Pesticides can be brought back to the beehive after the bees have foraged on flowers that have been sprayed with pesticides. Pesticide contaminated food can be exchanged between workers which then feed larvae and therefore can potentially affect the development of honey bees. Thus, residual pesticides in the environment can become a chronic damaging factor to honey bee populations and gradually lead to colony collapse. In the presented protocol, honey bee feeding methods are described and applied to either an individual honey bee or to a colony. Here, the insect growth regulator (IGR) pyriproxyfen (PPN), which is widely used to control pest insects and is harmful to the development of honey bee larvae and pupae, is used as the pesticide. The presenting procedure can be applied to other potentially harmful chemicals or honeybee pathogens for further studies.

Herein we present a method to feed pesticide contaminated food to both an individual honey bee and a beehive colony. The procedure evaluates the pesticide effect on individual honey bees by in vivo feeding of basic larval diet and also on the natural condition of beehive colony.

从个体到群体度评价环境化学物质对蜜蜂发展的影响

The presence of pesticides in the beekeeping environment is one of the most serious problems that impacts the life of a honey bee. Pesticides can be ...

Nanopore DNA Sequencing for Metagenomic Soil Analysis

This article describes the steps for construction of a DNA library from soil, preparation and use of the nanopore flow cell, and analysis of the DNA sequences identified using computer software. Nanopore DNA sequencing is a flexible technique that allows for rapid microbial genome sequencing to identify bacterial and viral species, to characterize bacterial strains, and to detect genetic mutations that confer resistance to antibiotics. The advantages of nanopore sequencing (NS) for life sciences include its low complexity, reduced cost, and rapid real-time sequencing of purified genomic DNA, PCR amplicons, cDNA samples, or RNA. NS is an example of "strand sequencing" which involves sequencing DNA by guiding a single stranded DNA molecule through a nanopore that is inserted into a synthetic polymer membrane. The membrane has an electrical current applied across it, so as the individual bases pass through the nanopore the electrical current is disrupted to varying degrees by the four nucleotide bases. The identification of each nucleotide occurs by detecting the characteristic modulation of the electrical current by the different bases as they pass through the nanopore. The NS system consists of a handheld, USB powered portable device and a disposable flow cell that contains a nanopore array. The portable device plugs into a standard laptop computer that reads and records the DNA sequence using computer software.

Nanopore technology for sequencing biomolecules has wide applications in the life sciences, including identification of pathogens, food safety monitoring, genomic analysis, metagenomic environmental monitoring, and characterization of bacterial antibiotic resistance. In this article, the procedure for metagenomic soil DNA sequencing for species identification using the nanopore sequencing technology is demonstrated.

基因土壤分析的纳米 DNA 测序

This article describes the steps for construction of a DNA library from soil, preparation and use of the nanopore flow cell, and analysis of the DNA ...

RGB and Spectral Root Imaging for Plant Phenotyping and Physiological Research: Experimental Setup and Imaging Protocols

Better understanding of plant root dynamics is essential to improve resource use efficiency of agricultural systems and increase the resistance of crop cultivars against environmental stresses. An experimental protocol is presented for RGB and hyperspectral imaging of root systems. The approach uses rhizoboxes where plants grow in natural soil over a longer time to observe fully developed root systems. Experimental settings are exemplified for assessing rhizobox plants under water stress and studying the role of roots. An RGB imaging setup is described for cheap and quick quantification of root development over time. Hyperspectral imaging improves root segmentation from the soil background compared to RGB color based thresholding. The particular strength of hyperspectral imaging is the acquisition of chemometric information on the root-soil system for functional understanding. This is demonstrated with high resolution water content mapping. Spectral imaging however is more complex in image acquisition, processing and analysis compared to the RGB approach. A combination of both methods can optimize a comprehensive assessment of the root system. Application examples integrating root and aboveground traits are given for the context of plant phenotyping and plant physiological research. Further improvement of root imaging can be obtained by optimizing RGB image quality with better illumination using different light sources and by extension of image analysis methods to infer on root zone properties from spectral data.

An experimental protocol is presented for assessment of soil grown plant root systems with RGB and hyperspectral imaging. Combination of RGB image time series with chemometric information from hyperspectral scans optimizes insights into plant root dynamics.

RGB 和光谱根成像植物表型和生理研究: 实验装置和成像协议

Better understanding of plant root dynamics is essential to improve resource use efficiency of agricultural systems and increase the resistance of crop ...

Techniques for Investigating the Anatomy of the Ant Visual System

This article outlines a suite of techniques in light microscopy (LM) and electron microscopy (EM) which can be used to study the internal and external eye anatomy of insects. These include traditional histological techniques optimized for work on ant eyes and adapted to work in concert with other techniques such as transmission electron microscopy (TEM) and scanning electron microscopy (SEM). These techniques, although vastly useful, can be difficult for the novice microscopist, so great emphasis has been placed in this article on troubleshooting and optimization for different specimens. We provide information on imaging techniques for the entire specimen (photo-microscopy and SEM) and discuss their advantages and disadvantages. We highlight the technique used in determining lens diameters for the entire eye and discuss new techniques for improvement. Lastly, we discuss techniques involved in preparing samples for LM and TEM, sectioning, staining, and imaging these samples. We discuss the hurdles that one might come across when preparing samples and how best to navigate around them.

This article outlines a suite of techniques in light and electron microscopy to study the internal and external eye anatomy of insects. These include several traditional techniques optimized for work on ant eyes, detailed troubleshooting, and suggestions for optimization for different specimens and regions of interest.

蚂蚁视觉系统解剖学研究方法

This article outlines a suite of techniques in light microscopy (LM) and electron microscopy (EM) which can be used to study the internal and external eye ...

Isolation of Mandibular Gland Reservoir Contents from Bornean 'Exploding Ants' (Formicidae) for Volatilome Analysis by GC-MS and MetaboliteDetector

The aim of this manuscript is to present a protocol describing the metabolomic analysis of Bornean &#39;exploding ants&#39; belonging to the Colobopsis cylindrica (COCY) group. For this purpose, the model species C. explodens is used. Ants belonging to the minor worker caste possess distinctive hypertrophied mandibular glands (MGs). In territorial combat, they use the viscous contents of their enlarged mandibular gland reservoirs (MGRs) to kill rival arthropods in characteristic suicidal &#39;explosions&#39; by voluntary rupture of the gastral integument (autothysis). We show the dissection of worker ants of this species for the isolation of the gastral portion of the wax-like MGR contents as well as listing the necessary steps required for solvent-extraction of the therein contained volatile compounds with subsequent gas chromatography-mass spectrometry (GC-MS) analysis and putative identification of metabolites contained in the extract. The dissection procedure is performed under cooled conditions and without the use of any dissection buffer solution to minimize the changes in the chemical composition of the MGR contents. After solvent-based extraction of volatile metabolites contained therein, the necessary steps for analyzing the samples via liquid-injection-GC-MS are presented. Lastly, data processing and putative metabolite identification with the use of the open-source software MetaboliteDetector is shown. With this approach, the profiling and identification of volatile metabolites in MGRs of ants belonging to the COCY group via GC-MS and the MetaboliteDetector software become possible.

Isolation of Mandibular Gland Reservoir Contents from Bornean 'Exploding Ants' Formicidae for Volatilome Analysis by GC-MS and MetaboliteDetector

Minor workers of the ant species Colobopsis explodens are dissected to isolate the wax-like content stored in their hypertrophied mandibular gland reservoirs for subsequent solvent-extraction and analysis by gas chromatography-mass spectrometry. The&#160;annotation and identification of volatile constituents using the open-source software MetaboliteDetector is also described.

婆罗洲 "爆炸蚂蚁" (蚁科昆虫) 对下颌腺储层含量的分离--gc-ms 和 MetaboliteDetector Volatilome 分析

The aim of this manuscript is to present a protocol describing the metabolomic analysis of Bornean &#39;exploding ants&#39; belonging to the Colobopsis ...

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ocean temperatures and ocean acidification, corals are increasingly under threat. To tackle these challenges, advances in cell and molecular biology have proven to be crucial for diagnosing the health of corals. Modifying some of the techniques commonly used in human medicine could greatly improve researchers&#39; ability to treat and save corals. To address this, a protocol for in situ hybridization used primarily in human medicine and evolutionary developmental biology has been adapted for use in adult corals under stress.
The purpose of this method is to visualize the spatial expression of an RNA probe in adult coral tissue that has been embedded in paraffin and sectioned onto glass slides. This method focuses on removal of the paraffin and rehydration of the sample, pretreatment of the sample to ensure permeability of the sample, pre-hybridization incubation, hybridization of the RNA probe, and visualization of the RNA probe. This is a powerful method when using non-model organisms to discover where specific genes are expressed, and the protocol can be easily adapted for other non-model organisms. However, the method is limited in that it is primarily qualitative, because expression intensity can vary depending on the amount of time used during the visualization step and the concentration of the probe. Furthermore, patience is required, as this protocol can take up to 5 days (and in many cases, longer) depending on the probe being used. Finally, non-specific background staining is common, but this limitation can be overcome.

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

The goal of this protocol is to perform in situ hybridization on adult coral samples that have been embedded in paraffin and sectioned onto glass slides. This is a qualitative method used to visualize the spatial expression of an RNA anti-sense probe in paraffin-embedded tissues.

就地石蜡镶嵌成人珊瑚样品的杂交技术

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ...

Necropsy-based Wild Fish Health Assessment

Anthropogenic influences from increased nutrients and chemical contaminants, to habitat alterations and climate change, can have significant effects on fish populations. Adverse effects monitoring, utilizing biomarkers from the organismal to the molecular level, can be used to assess the cumulative effects on fishes and other organisms. Fish health has been used worldwide as an indicator of aquatic ecosystem health. The necropsy-based fish health assessment provides data on visible abnormalities and lesions, parasites, condition and organosomatic indices. These can be compared by site, season and sex, as well as temporally, to document change over time. Severity ratings can be assigned to various observations to calculate a fish health index for more quantitative assessment. A drawback of the necropsy-based assessment is that it is based on visual observations and condition factors, which are not as sensitive as tissue and subcellular biomarkers for sublethal effects. Additionally, it is rarely possible to identify causes or risk factors associated with observed abnormalities. So, for instance a raised lesion or "tumor" on the fins, lips or body surface may be a neoplasm. However, it could also be a response to a parasite, chronic inflammation or hyperplasia of normal cells in response to an irritant. Conversely, neoplasms, certain parasites, other infectious agents and many tissue changes are not visible and so may be underestimated. However, during the necropsy-based assessment, blood (plasma), tissues for histopathology (microscopic pathology), genomics and other molecular analyses, and otoliths for aging can be collected. These downstream analyses, together with geospatial analyses, habitat assessments, water quality and contaminant analyses can all be important in comprehensive ecosystem evaluations.

The health of wild fishes can be used as an indicator of aquatic ecosystem health. Necropsy-based fish health assessments provide documentation of visible lesions or abnormalities, data used to calculate condition indices as well as the opportunity to collect tissues for microscopic evaluation, gene expression and other more in-depth analyses.

基于尸检的野生鱼类健康评估

Anthropogenic influences from increased nutrients and chemical contaminants, to habitat alterations and climate change, can have significant effects on ...

Measuring Hypopharyngeal Gland Acinus Size in Honey Bee (Apis mellifera) Workers

The nurse hypopharyngeal glands produce the protein fraction of the worker and royal jelly that is fed to developing larvae and queens. These paired glands that are located in the head of the bee are highly sensitive to the quantity and quality of pollen and pollen substitutes that the nurse bee consumes. The glands get smaller when nurses are fed deficient diets and are large when they are fed complete diets. Because nurse hypopharyngeal gland size is a robust indicator of nurse nutrition, it is essential that those studying honey bee nutrition know how to measure these glands. Here, we provide detailed methods for dissecting, staining, imaging, and measuring nurse bee hypopharyngeal glands. We present comparisons of unstained and stained tissue and data that were used to study the impact of pollen on gland size. This method has been used to test how diet impacts hypopharyngeal gland size but has further use for understanding the role of these glands in hive health.

Measuring Hypopharyngeal Gland Acinus Size in Honey Bee Apis mellifera Workers

Hypopharyngeal gland acinus size is a robust measure of nurse honey bee nutrition. Here, we provide a detailed protocol for dissecting, staining, imaging, and measuring nurse bee hypopharyngeal gland acini.

蜂蜜蜂 (api 蜜蜂) 中下咽腺性腺泡大小的测定

The nurse hypopharyngeal glands produce the protein fraction of the worker and royal jelly that is fed to developing larvae and queens. These paired ...