作者感谢克拉克和蒂姆克罗提供蜂筑巢芦苇, 梅瑞狄斯 Nesbitt 和莫莉比德韦尔协助在实验室, Drs. 卡梅伦·柯里, Christelle Guédot, 特里格里斯沃尔德, 迈克尔 Branstetter 和三匿名评论家为他们有用的评论, 改进了手稿。这项工作得到了农业部-农业研究服务划拨资金 (目前研究信息系统 #3655-21220-001)、威斯康星农业、贸易和消费者保护部 (#197199)、国家科学基金会 (根据格兰特号DEB-1442148), 能源部大湖生物能源研究中心 (能源部的科学 DE-FC02-07ER64494)。

1. 为杀菌剂暴露实验准备丙环唑解决方案
<ol><li>通过在无菌水中溶解适当数量的商业购买的丙环唑 14.3%, 准备0.1x 杀菌剂解决方案的一天的实验。确保只有新配制的杀菌剂溶液用于所有治疗。</li>
	<li>添加23µL 的0.1x 杀菌剂溶液每克花粉规定, 以获得丙环唑的最大浓度以前报告的蜂收集花粉24 (0.361 PPM 或µg 活性成分 g-1的花粉)。</li>
</ol>2. 从蜂芦苇中收获卵和寄主花粉的规定
<ol><li>使用消毒手术刀, 解剖新鲜堵塞的蜂筑巢芦苇, 分裂成两个部分沿芦苇的长度, 以暴露各自的房间。 注: 每个巢可能包含在8到14个房间和一个单独蛋在房间之内。</li>
	<li>根据先前公布的准则77, 目视检查芦苇以识别含有雄性卵的腔室。使用灭菌弯曲针去除每个花粉供应连同伴生的蛋从巢芦苇, 并且安置在干净的重量小船。</li>
	<li>用干净的细漆刷将鸡蛋从规定中轻轻地分开, 用标准的实验室平衡记录花粉供应和卵子的新鲜重量。计算男性花粉规定的平均重量。</li>
	<li>执行随后的步骤, 以最小的延迟, 以减少伤害的机会, 从暴露在过量的温度和脱水。</li>
</ol>3. 准备寄主植物花粉的规定
<ol><li>目视检查母系收集的寄主植物花粉从巢室挖掘, 以确保没有寄生虫存在78。为了减少任何潜在的产妇偏见, 将花粉的规定组合成一个不育的培养皿中的单一质量, 并使用消毒针混合良好。</li>
	<li>将组合质量分为新的花粉规定, 确保每一个重组的规定的重量大约等于一个自然分配的男性提供的平均重量 (平均值, 0.35, 0.01 克, N = 42)。 注: 由于蜂 sp。将较小的花粉规定分配给雄性后代, 这会导致雄性幼虫的体重降低77。为了避免因性别差异而引起的任何这种偏见, 在实验中只使用雄性卵子。</li>
</ol>4. 准备非寄主植物花粉供应
<ol><li>粉碎商业购买蜂蜜蜂收集花粉使用一个标准的实验室球磨机的细粉。</li>
	<li>根据母系花粉的含水量 (~ 20%), 利用适当体积的40% 种灭菌糖溶液79 , 将花粉粉水合, 并将其彻底混合, 形成面团状的稠度。</li>
	<li>分成单独的花粉质量, 每一个体重约相当于一个自然分配的男性规定的平均重量。 注: 母系收集的花粉的含水量可以通过比较30随机选择的男性腔80中花粉的新鲜和干重来进行标准化。为了获得干重, 花粉的规定应冷冻干燥的冻干机 (1.5 Pa 为72小时)。</li>
</ol>5. 制备多井细胞培养板
<ol><li>用蒸压锡杯 (5 x 9 厘米) 的无菌48井培养板的线个体井。使用无菌钳, 轻轻地把胶囊的顶端边缘, 以便它可以容纳花粉供应。</li>
	<li>使用基于治疗组的无菌工具, 在锡杯内放置单一质量的寄主或非宿主花粉供应。 注意: 为了避免交叉污染, 使用单独的钢板治疗和控制组。</li>
</ol>6. 添加杀菌剂
<ol><li>用不育的木棍在花粉质量中放置一个中央凹陷。使用新的棍子为每个花粉供应。</li>
	<li>添加适当数量的杀菌剂溶液 (用于治疗), 或无菌水 (用于控制) 进入抑郁症。用无菌钳捏住抑郁症的开口, 以尽量减少杀菌剂/无菌水与卵子之间的表面接触。</li>
	<li>确保实验设计的阶乘设置与示意图表示形式 (图 4) 中所描述的对齐。</li>
</ol><img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57876/57876fig4.jpg"> 图4。实验装置的示意图表示法.该实验表示一个完全交叉的 2 x 2 阶乘设置。因子1代表杀菌剂暴露, 包括2级: (i) 无杀菌剂 (n = 10) 和 (ii) 杀菌剂 (n = 10)。因子2代表花粉源, 包括2层: (i) 寄主花粉 (n = 8) 和 (ii) 非寄主花粉 (n = 8)。<a href="/files/ftp_upload/57876/57876fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
7. 后方和观察幼虫
<ol><li>使用干净的细漆刷将随机选择的雄性卵放在花粉供应的顶端表面。一旦鸡蛋被放置在所有的规定, 更换细胞培养板的盖子, 确保它与标签胶带在角落。</li>
	<li>把井板放在干净的托盘上, 用深色的布盖住, 以阻挡直接光线的接触。在托盘内放置6个含30毫升不育水的井板, 以防止脱水。在室温下将孵化盘放在孵化器内不受干扰。</li>
	<li>每天在解剖显微镜下观察井板, 而不去除井板盖。通过检查运动来确保幼虫活着。如果检测不到运动, 丢弃含有死幼虫和剩余花粉的锡杯。允许所有幸存的幼虫在井板内不受干扰地发育, 直到到达 prepupal 阶段。</li>
	<li>从锡杯中取出幼虫, 一旦到达 prepupal 阶段41。用刷子清洁丝绸茧里的大便。用解剖显微镜仔细切开丝茧, 用橡皮钳提取预蛹。</li>
	<li>轻轻地处理预蛹, 确保工具不会刺穿柔软的身体。记录预蛹 (prepupal 生物量) 的新鲜重量和从卵到 prepupal 阶段 (幼虫发育时间) 的发育时间。 注: 任何死幼虫应立即丢弃, 以防止不需要的微生物生长在尸体和剩余的花粉供应。这减少了感染的风险, 其余的健康幼虫。</li>
</ol>

作者没有什么可透露的。

在实验室条件下, 在其天然筑巢芦苇外饲养蜜蜂, 可以测试与幼虫体质有关的多个假说。如果不明因素继续导致蜜蜂死亡, 使用体外实验进行的风险评估研究可以帮助确定潜在的威胁, 并告知该物种丰富的野生传粉者群的管理做法12 ,38,49,76,81,82。
一般而言, 发育时间较短, prepupal 生物量高, 与幼虫适应性较高有关。在所有的治疗中, 幼虫发育的持续时间与 prepupal 生物量呈负相关。然而, 四组幼虫发育期和 prepupal 生物量之间存在显著差异。没有得到任何杀菌剂和允许食用寄主花粉的幼虫的幼虫发育时间最短, prepupal 生物量最高。相比之下, 那些食用杀菌剂处理的非寄主花粉的幼虫, 花了最长的时间完成幼虫的发育, 并具有最低的 prepupal 生物量。然而, 幼虫存活率的趋势不那么清楚, 只有在寄主花粉上饲养的杀菌剂治疗组才会发现死亡率。解构杀菌剂暴露和花粉源的主要作用和交互作用表明: (i.) 食用杀菌剂处理花粉的主要作用对 prepupal 生物量有显著的负面影响, 但幼虫发育期不明显。虽然先前的研究表明, 在较高浓度的成人蜂急性口服毒性,这些结果表明, 口服接触丙环唑低浓度可以影响健身通过减少 prepupal 生物质23.(ii) 食用非寄主花粉的主要效果对幼虫的健康有不良影响。这些结果与先前发表的研究表明花粉质量 (即存在毒素, 保护化合物, 缺乏必需营养素) 和蜜蜂生理学上的差异可以限制蜜蜂利用非寄主花粉68.也有可能, 缺乏有益的微生物群通常从寄主花粉和/或蜂作物获得可能加剧这种效果。(iii) 杀菌剂暴露与花粉源对幼体的适应性没有显著的交互作用。在杀菌剂治疗和未治疗组中, 与寄主花粉饲养的幼虫相比, 食用非寄主花粉的幼虫显著降低了 prepupal 生物量和较长的幼虫发育时间。无论花粉来源如何, 幼虫食用杀菌剂处理的规定与未经处理的花粉饲养的幼虫相比, prepupal 生物量显著降低。除显著的主要影响外, 这两个因素的交互作用符合添加剂模型, 即杀菌剂暴露和花粉类型对幼虫适应性的负面影响是不协同的。蜜蜂健康的各种决定因素相互作用引起的不确定性 (这里详述), 限制了授粉管理战略的有效性。通过帮助预测多种危险因素的交互作用, 类似实验室实验的结果可以帮助规避蜜蜂保护工作中长期存在的挑战。
该协议中有几个关键步骤可以影响实验的结果。只要有可能, 最好使用无农药残留的有机花粉。使用未知来源的花粉会增加各种农用化学品的污染风险, 这会混淆实验结果。重要的是要获得新的堵塞蜂筑巢芦苇, 以便只有孵化鸡蛋或非常年轻的幼虫被用于研究。这保证幼虫几乎完全地被饲养在意欲的花粉治疗类型。嵌套芦苇应使用浅切口进行解剖, 否则花粉的规定和卵在收割过程中可能会受损。一旦鸡蛋被删除, 他们必须轻轻地处理, 以防止损坏和保留在重量船在凉爽的黑暗卫生环境 (如, 生物安全柜), 直到他们转移。此保持时间应保持在最低限度 (< 30 分钟), 以确保鸡蛋的质量不会受到损害。所有程序都应在生物安全柜内进行, 以确保清洁的工作环境和减少污染的机会。为确保其功效, 只使用新鲜配制的杀菌剂溶液进行治疗。鉴于花粉是疏水性的, 应将杀菌剂溶液/不育水引入花粉中的抑郁症。这就最大限度地通过该规定渗透液体的数量。然而, 重要的是, 抑郁症不会刺穿整个供应的深度, 因为它会导致损失的解决方案坚持胶囊地板。个别的治疗和控制应在单独的油井板, 以减少交叉污染的机会, 从挥发性化合物和/或花粉传播的微生物群。折叠胶带应贴在板的边缘, 以允许足够的空气间隙一旦盖子到位。在日常观察中, 盘子应该轻轻地处理, 以尽量减少对幼虫的干扰。观察应在显微镜下进行, 最小光强度, 不应删除盖子, 除非丢弃死幼虫。在意外的普遍死亡情况下, 必须对幼虫及其花粉的规定进行目视检查, 以检查是否有感染和出没的迹象。含有受损复制的井板应立即丢弃, 工作区消毒, 并进行消毒, 以防止感染蔓延。
尽管有广泛的应用, 这种方法还是有一定的局限性。例如, 尽管使用有机花粉是最好的做法, 但在温室内饲养植物以产生足够数量的纯未被污染的花粉, 在后勤上是令人望而却步的。在这种情况下, 可以使用野生收集的花粉, 只要它被筛查是否存在农药残留物。另一种减少使用野生收集花粉的危险的策略是从不太可能被喷洒的来源获得花粉 (例如, 远离农业农场的原始未受干扰的地区)。本研究所用的寄主花粉是从位于犹他州 Kaysville 附近瓦萨奇山脉山麓的天然林地和草原上的筑巢芦苇中获得的。考虑到这个地区是由野生的, 非托管的天然森林, 远离任何商业农业地区, 和这些蜜蜂不飞到蜜蜂在觅食83,84,85, 这是极不可能他们收集的花粉将被喷洒。因此, 这里收集到的花粉中的杀虫剂残留物很可能是微不足道的。在这样的野生景观中觅食, 成年雌性不太可能遇到受污染的花粉, 减少了幼虫间暴露的风险。在这项研究中使用的商业购买蜜蜂花粉是从威斯康星州和密歇根州北部的自然森林地区收集的。销售为人的消耗量, 公开地可利用的信息和与供应商的个人通信表明蜂巢没有被化学治疗, 并且花粉以它的自然, 原始的形式出售, 不用任何修改86。因此, 合理地假设在商业上购买的蜜蜂花粉中的污染物负荷是极小的。对于没有野生天然植被的非托管区域获得花粉的研究, 最好从花粉化学分析中获得直接的经验证据, 以确保在风险化验中使用的花粉是无污染的。另一个限制涉及人工饲养环境引入的发明。尽管做了最好的努力, 在一个天然嵌套芦苇 (例如, 水分, 氧气浓度, 三维的个体腔结构) 中复制精确的微环境是没有逻辑上可行的, 这可能会影响幼虫适应未知的程度。为了 defensibly 模拟自然饮食的特点, 必须在体外饮食操作研究之前获得巢状芦苇的初步数据。虽然这项研究中使用的非宿主花粉是从俄勒冈葡萄缺乏或稀有87的地区获得的, 但在商业性购买的花粉中可能有寄主花粉的痕迹, 可能影响结果。这种技术的另一个缺点是在实验期间处理压力可能对蜜蜂产生不良影响。最后, 虽然在自然界63遇到孵化卵是常见的, 但在实验室条件下, 很难确定孵化失败是否是由于处理压力、实验性治疗或自然原因造成的。由于这些因素可能会给研究带来未知程度的偏见, 所以在解释所获得的结果时必须谨慎使用。
通过控制可以明显偏倚实验结果的因素 (例如, 产妇觅食效率75, 性别特异性变异77, 以及膳食花粉68,72), 所述议定书提供对以前发布的技术76的实质性改进, 并为假设测试提供了更严格的框架。例如, 使用体外饲养的蜜蜂允许对 xenobiotic 治疗的性别特定反应进行调查, 否则将对研究49、88腔巢蜜蜂的野生种群有挑战性。操控和测试多种相互作用因素的潜力 (例如饮食质量和数量、与饮食相关的微生物群、接触到协同杀虫剂), 可以为蜜蜂健康的关键决定因素提供宝贵的见解。该协议的灵活性可以方便地进行修改 (例如, 使用不同大小的油井板来容纳不同尺寸的幼虫, 改变饮食的种类和数量), 使之适于与其他几种单独的蜜蜂和黄蜂一起使用。76. 虽然这项研究根据在喂养阶段的发展和生存状况评估了体能, 但昆虫可以孵化, 直到出现, 才能获得关于出现率、食物对身体转换和出现后长寿的额外数据。49. 这一信息有助于评估治疗在毒性化验中的亚致死效应。随着对花粉菌群功能的兴趣日益浓厚, 基于实验室的研究可以根据花粉微生物群的71识别敏感抗性蜂类。通过对蜂巢储存花粉中的微生物群进行排序, 可以探索不同蜂组的杀菌剂敏感性差异。这可以帮助确定花粉菌群在赋予不同程度的抗 xenobiotic 压力的作用。未来的研究还可能有助于确定寄主和非寄主花粉的天然微生物群之间的差异, 这可能是导致选择蜂种中 oligolectic 行为的一个潜在因素。
幼虫孤蜂的体外饲养可以帮助控制野外体验的自然变异, 从而界定个体和相互作用因素在影响蜜蜂健康方面的角色。这种可访问和廉价的技术扩展了昆虫学家的工具箱, 允许对多个参数进行操作, 这可以很容易地修改以解决特定的研究目标。如果从体外实验中得到的证据可以帮助确定高危人群, 对蜜蜂保护策略的影响将是巨大的。

鉴于它们作为昆虫传粉者的主导群体1, 全球蜜蜂种群的损失对粮食安全和生态系统稳定性构成威胁2、3、4、5、6 ,7。管理和野生蜜蜂种群的下降趋势归因于几个共同的危险因素, 包括生境分裂、新出现的寄生虫和病原体、遗传多样性的丧失以及入侵物种的引进3 ,4,7,8,9,10,11,12。特别是, 杀虫剂的使用急剧增加 (例如, neonicotinoids) 与蜜蜂13、14、15之间的有害影响直接相关。一些研究表明, neonicotinoids 和麦角甾醇生物合成抑制 (EBI) 杀菌剂之间的协同作用可能导致高死亡率横跨多蜂种类16,17,18,19,20,21,22. 然而, 长期以来被认为是 "蜜蜂安全" 的杀菌剂继续喷洒在开花作物上, 但没有对23进行过多的审查。觅食蜜蜂已被记录下来, 例行带回花粉负荷污染的杀菌剂残留物24,25,26。这种杀菌剂 ladenpollen 的消费量会导致幼虫蜂27、28、29、30之间的高死亡率, 以及成年蜜蜂中的一组亚致死效应16,31,32,33,34. 最近的一项研究表明, 杀菌剂可能会通过改变蜂巢储存的花粉中的微生物群落来造成蜜蜂的损失, 从而扰乱蜜蜂和花粉传播的微生物之间的关键共生35。
虽然孤蜂对几种野生和农业植物的授粉至关重要,36、37、38, 但这种不同的传粉群体在农药监测研究中受到的关注较少。一个成年的独居女性的巢包含5-10 个封闭的巢室, 每个都有一个有限质量的母系收集的花粉和花蜜, 和一个单一的鸡蛋39。孵化后, 幼虫依赖于分配的花粉提供, 和相关的花粉传播的微生物群获得足够的营养40,41。因为他们缺乏社会生活方式的好处, 孤独的蜜蜂可能更容易受到杀虫剂的照射42。例如, 虽然在喷洒后的社会蜜蜂的赤字可能会得到工人和新出现的巢的一些扩大, 一个单一的成年单身女性的死亡结束所有的生殖活动43。这种易感性的差异突出表明需要在生态研究中纳入不同的蜜蜂分类, 以确保对被管理和野生蜜蜂有足够的保护。然而, 除了少数研究, 对杀菌剂暴露的影响的调查主要集中在社会蜜蜂18,23,32,44,45 ,46,47,48,49。
属于蜂属的孤蜂 (图 1) 已被全世界用作多种重要水果和坚果作物的有效传粉者, 39,50,51,53,53. 与其他被管理的授粉小组 24,54,55,56,57,58, 成年蜂蜜蜂通常暴露于杀菌剂喷洒在开花作物44。在最近喷洒的农作物上觅食的成年雌性可能会收集和储存他们的巢室与杀菌剂的花粉, 后来形成了唯一的饮食发育幼虫。食用受污染的花粉的规定随后可将幼虫暴露于杀菌剂残留物42。在 oligolectic 物种中, 接触的风险可能较高, 仅在少数几个密切相关的寄主植物上觅食59、60、61。例如, 某些 megachilid 蜜蜂似乎优先饲料低品质的菊科花粉, 作为减少寄生62的一种手段。然而, 杀菌剂对 oligolectic 孤蜂幼虫适应性的影响程度尚未得到实证量化。本研究的目的是制定一个协议, 以检验杀菌剂暴露和花粉源对体外饲养孤蜂的适应性的主要和交互作用。为了调查, ribifloris lato (广义) 的卵可以在商业上获得 (材料表)。这个人口是理想的, 因为它作为一个土生土长的授粉的重要性, 并且它强烈偏爱花蜜丰富的十大功劳 aquifolium (俄勒冈葡萄) 发现在区域53,63,64(图 2)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57876/57876fig1.jpg"> 图1。一张成人蜂 ribifloris的高分辨率照片。照片信贷, 美国农业部研究昆虫学家吉姆. 甘蔗,<a href="/files/ftp_upload/57876/Figure_1.pdf" target="_blank">请点击这里查看这个数字的更大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57876/57876fig2.jpg"> 图2。芦苇蜂 ribifloris (广义)的嵌套芦苇, 在前景中嵌套雌性. 芦苇的室隔断和端子插头是由咀嚼叶构造的。照片信用 NativeBees.com 先生,<a href="/files/ftp_upload/57876/57876fig2large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
本研究的第一个目的是评估食用杀菌剂处理花粉对幼虫体质的影响 (以发育时间和 prepupal 生物量计算)。虽然接触到普遍应用的杀菌剂丙环唑已与成年蜜蜂之间的死亡率增加23,24,32,44,45, 54、55、56、57、58、65、66、67, 对蜜蜂幼虫的影响较小知道。本研究的第二个目的是评估食用非寄主花粉对幼虫体质的影响。以往的研究表明, oligolectic 蜜蜂的幼虫在被迫食用非寄主花粉68时未能发育。这种结果可能归因于蜜蜂生理学69、花粉生物化学70和与天然花粉有关的有益菌群的变化 (71)。本研究的第三个目的是评价杀菌剂处理和日粮花粉对幼体健康的交互作用。
众所周知, 许多生物学特性包括产妇体型、调配率、觅食策略和花粉量72、73、74、75都影响了孤蜂幼虫的适应性。这些因素可以在芦苇之间引入显著的变异性, 这在评估幼虫的健康状况时, 对发展防御性实验设计构成了挑战。此外, 鉴于幼虫的发育发生在密封的筑巢芦苇内, 这种变异对后代的影响很难想象和量化而不使用非致命技术 (图 3)。为了克服这一挑战, 本研究中的所有假说都是使用饲养在巢状芦苇外的幼虫来测试的。实验设计代表一个完全交叉的 2 x 2 阶乘设置, 每个因素由2级组成;因子 1: 杀菌剂暴露 (杀菌剂;无杀菌剂);因子 2: 花粉源 (寄主花粉, 非寄主花粉)。在受控实验室条件下, 蜜蜂在无菌多井细胞培养板中由卵向 prepupal 阶段培养。每个井都有一个标准的花粉供应量和一个鸡蛋。孵化后, 幼虫在井内分配的花粉上进食, 完成幼虫发育, 并启动蛹。过去的研究表明, 在这种人工饲养环境中所饲养的蜜蜂中, 无法解释的死亡率低于野外49、76所遇到的。在体外饲养的蜜蜂的使用比基于田间的研究有几个优点: 1) 它最大限度地减少自然变异性和不受控制因素的混杂效应, 通常与实地研究相关;2) 它允许在治疗组中同时测试每个感兴趣因素的多层次操作;3) 复制的次数可以预先确定, 每个复制的实验因素可以单独操作;4) 幼虫响应变量可在不干扰相邻幼虫的情况下, 独立地进行可视化和记录;5) 可以修改该协议, 以适应涉及多个因素和响应变量的更复杂的实验设计。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57876/57876fig3.jpg"> 图3。蜂 ribifloris (广义)天然嵌套芦苇中的含量.关闭(a)解剖的芦苇, 显示个别的房间, 花粉的规定和分区, (B)新鲜收获的花粉规定, 和相关的鸡蛋 (用黑色圆圈表示)。<a href="/files/ftp_upload/57876/57876fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

<table>
	<tbody>
		<tr>
			<td>eggs of O. ribifloris sensu lato (s.l.)</td>
			<td></td>
			<td></td>
			<td>Kaysville, Davis County, Utah, USA</td>
		</tr>
		<tr>
			<td>Osmia reeds</td>
			<td>Nativebees.com</td>
			<td>NA</td>
			<td>Freshly plugged reeds</td>
		</tr>
		<tr>
			<td>Dissection set</td>
			<td>VWR</td>
			<td>89259-964</td>
			<td>Sterilize before use</td>
		</tr>
		<tr>
			<td>Long Nose Pliers</td>
			<td>Husky</td>
			<td>1006</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well culture plates</td>
			<td>VWR</td>
			<td>10062-892</td>
			<td>Sterile sealed</td>
		</tr>
		<tr>
			<td>48 well culture plates</td>
			<td>VWR</td>
			<td>10062-898</td>
			<td>Sterile sealed</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>VWR</td>
			<td>25373100</td>
			<td>Sterile sealed</td>
		</tr>
		<tr>
			<td>Square Weighing Boats</td>
			<td>VWR</td>
			<td>10770-448</td>
			<td></td>
		</tr>
		<tr>
			<td>Camel Hair Brush</td>
			<td>Bioquip</td>
			<td>1153A</td>
			<td></td>
		</tr>
		<tr>
			<td>Tin capsules</td>
			<td>EA Consumables</td>
			<td>D1021</td>
			<td>Sterilize before use</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>VWR</td>
			<td>470302-808</td>
			<td></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>Honey bee pollen</td>
			<td>Bee energised</td>
			<td>897098001244</td>
			<td>Untreated, natural, raw pollen</td>
		</tr>
		<tr>
			<td>Microbalance</td>
			<td>VWR</td>
			<td>10204-990</td>
			<td></td>
		</tr>
		<tr>
			<td>Pulverisette</td>
			<td>LAB SYNERGY INC.</td>
			<td>30334913</td>
			<td></td>
		</tr>
		<tr>
			<td>Wooden sticks</td>
			<td>VWR</td>
			<td>470146908</td>
			<td>Sterilize before use</td>
		</tr>
		<tr>
			<td>Sealing tape</td>
			<td>VWR</td>
			<td>89097-912</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>VWR</td>
			<td>89403-384</td>
			<td></td>
		</tr>
		<tr>
			<td>Planting tray</td>
			<td>VWR</td>
			<td>470150-632</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>VWR</td>
			<td>BDH1158-4LP</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tube</td>
			<td>VWR</td>
			<td>21008936</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsyringe</td>
			<td>Cole-Palmer</td>
			<td>UX-07940-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Rubber tweezer</td>
			<td>Amazon</td>
			<td>B0135HWPN4</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe needles</td>
			<td>VWR</td>
			<td>89219-334</td>
			<td></td>
		</tr>
		<tr>
			<td>Freeze drier</td>
			<td>Labcono</td>
			<td>LFZ-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Statistical software</td>
			<td>SPSS</td>
			<td>Version 21.0</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro rearing of solitary bees: a tool for assessing larval risk factors

虽然孤蜂为野生和被管理的农作物提供重要的授粉服务, 但在农药管制研究中, 这种富含物种的群体已基本被忽视。如果喷雾发生在或靠近寄主植物的情况下, 当蜜蜂收集花粉来供应巢时, 接触杀菌剂残留物的风险可能会特别高。对于从一组植物中摄取花粉的蜂物种 (oligolecty), 不能利用非寄主植物的花粉可以增加其与杀菌剂相关毒性的危险因素。本手稿描述了用于成功后方 oligolectic 梅森蜜蜂,蜂 ribifloris严格 lato, 从鸡蛋到 prepupal 阶段在细胞培养板的标准化实验室条件下的协议。在体外饲养的蜜蜂随后被用来研究杀菌剂暴露和花粉来源对蜜蜂健康的影响。以 2 x 2 全交叉因子设计为基础, 研究了杀菌剂暴露和花粉源对幼虫体质的主要影响和交互作用, prepupal 生物量、幼虫发育时间和存活率进行量化。这种技术的一个主要优点是, 使用体外饲养的蜜蜂可以减少自然背景的变异性, 同时也能同时操作多个实验参数。描述的协议提供了一个通用的工具, 假设测试涉及一系列影响蜜蜂健康的因素。为了使保护工作取得重大的、持久的成功, 对导致蜜蜂下降的生理和环境因素的复杂相互作用的这种洞察将被证明是至关重要的。

幼虫的体质是使用三指标 (i) 幼虫发育时间, (ii) prepupal 生物量, (iii) 存活率。利用杀菌剂暴露 (两个层次: 无杀菌剂、杀菌剂) 和花粉源 (两个层次: 寄主花粉、非寄主花粉) 作为独立变量, 以幼虫发育时间为相关变量, 进行了双向方差分析。杀菌剂暴露的主要效果 (F128 = 1.24, P = 0.28) 在杀菌剂治疗 (平均 SE) (28.14 ±1.98 d, N = 14) 和未处理 (25.39 @ 1.65 d, n = 18) 组之间是非显著的。花粉源的主要效应然而, 表明在寄主花粉的发育时间 (20.00 0.50 d, n = 16) 和非寄主花粉 (33.19 ±0.81 d, n = 16) (F128 = 179.83 ) 之间的显著差异;P < 0.001)。Bonferroni 修正后的特别比较表明, 在寄主 (p = 0.57) 和非宿主 (p = 0.32) 花粉中, 杀菌剂治疗和未治疗组的幼虫发育时间没有显著差异。然而, 与非寄主花粉相比, 在寄主花粉上饲养的幼虫发育时间明显短于杀菌剂治疗 (p < 0.001) 和未经处理的 (p < 0.001) 花粉。相互作用作用 (杀菌剂曝光 x 花粉来源) 不是重要的 (F128 = 0.09, P = 0.77)。用 prepupal 生物量作为相关变量进行了重复分析。杀菌剂接触的主要效果表明, 杀菌剂处理 (0.123 ±0.01 g, n = 14) 和未治疗 (0.149, 0.01 克, n = 18) 组之间有显著差异 (F128 = 4.66, P = 0.04)。花粉源 (F128 = 56.30, P < 0.001) 的主要效果表明, 在寄主花粉 (0.170 @ 0.01 克, N = 16) 和非寄主花粉 (0.105 ±0.01 g, n = 16) 上饲养的幼虫之间有显著的差异。.Bonferroni 纠正后的比较表明, prepupal 生物量在寄主 (p = 0.22) 和非宿主 (p = 0.08) 花粉的杀菌剂治疗和未治疗组之间没有显著差异。然而, 在寄主花粉饲养的幼虫中, prepupal 生物量明显高于非寄主花粉 (p < 0.001) 和未经处理的 (p < 0.001) 花粉。相互作用作用 (杀菌剂曝光 x 花粉来源) 不是重要的 (F128 = 0.132, P = 0.72)。图 5和图 6是上述分析所得结果的图形表示。独立样本t检验表明花粉源对幼虫存活率的显著影响 (N = 18, t9 =-2.45, P =0.04).
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57876/57876fig5.jpg"> 图5。条形图显示了基于幼虫发育时间和 prepupal 生物量的幼虫适应性指标.幼虫体质指标是基于(A)和(B)花粉源的聚类;和 ( C)和(D)杀菌剂的暴露。(意思是 1 SE)。P < 0.001<a href="/files/ftp_upload/57876/57876fig5large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57876/57876fig6.jpg"> 图6。幼虫健身指标的交互图。杀菌剂暴露和花粉源对(A)幼虫发育时间和(B) prepupal 生物量的交互作用。(意思是 1 SE)。<a href="/files/ftp_upload/57876/57876fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
利用 Pearsons 相关性研究了幼虫发育时间与 prepupal 生物量的关系 (图 7)。在所有治疗组 (r =-0.83, P < 0.001, N = 32) 和整个杀菌剂治疗 (无杀菌剂: r =-0.76, p < 0.001, N = 18, 有显著的负相关;杀菌剂: r =-0.92, P < 0.001, N = 14)。在非寄主花粉 (r =-0.64, P < 0.01, N = 16) 上饲养的幼虫有显著的负相关, 但在寄主花粉 (r =-0.01, p =) 上没有观察到这种关系。0.98, N = 16)。
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/57876/57876fig7.jpg"> 图7。幼虫发育时间与 prepupal 生物量的关系.皮尔逊生长时间与 prepupal 生物量的相关性(A)所有治疗组 (P < 0.001) (B)花粉源 (寄主花粉: p = 0.98, 非寄主花粉: p < 0.01);(C)杀菌剂暴露 (无杀菌剂:磷< 0.001, 杀菌剂: p < 0.001)。对于面板(B)和(C),趋势线与图图例中的符号颜色匹配。<a href="/files/ftp_upload/57876/57876fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Video 1" src="/files/ftp_upload/57876/57876video1.jpg"> 动画图1。多井板单井中ribifloris的第五级幼虫龄。幼虫被注意到已经开始纺丝绸茧准备蛹。<a href="/files/ftp_upload/57876/1.mov" target="_blank">请单击此处查看此视频。(右键单击可下载.</a>

Watch this Scientific Journal Video about In Vitro Rearing of Solitary Bees: A Tool for Assessing Larval Risk Factors at JoVE.com

In Vitro Rearing of Solitary Bees: A Tool for Assessing Larval Risk Factors

体外孤蜂饲养: 一种评估幼虫危险因素的工具

在开花植物上喷洒杀菌剂可以使孤蜂接触到高浓度的花粉传播杀菌剂残留物。本研究以实验为基础, 结合体外饲养蜂幼虫, 探讨了寄主和非寄主植物对杀菌剂处理花粉的影响。

Although solitary bees provide crucial pollination services for wild and managed crops, this species-rich group has been largely overlooked in pesticide ...

in-vitro-rearing-solitary-bees-tool-for-assessing-larval-risk

Nanyang Technological University

Research

JoVE Journal

Environment

In Vitro Rearing of Solitary Bees: A Tool for Assessing Larval Risk Factors

8.1K 次观看.  University of Wisconsin-Madison. 在开花植物上喷洒杀菌剂可以使孤蜂接触到高浓度的花粉传播杀菌剂残留物。本研究以实验为基础, 结合体外饲养蜂幼虫, 探讨了寄主和非寄主植物对杀菌剂处理花粉的影响。

视频: In Vitro Rearing of Solitary Bees: A Tool for Assessing Larval Risk Factors

A Push-pull Protocol to Reduce Colonization of Bird Nest Boxes by Honey Bees

Introduction of the invasive Africanized honey bee (AHB) into the Neotropics is a serious problem for many cavity nesting birds, specifically parrots. These bees select cavities that are suitable nest sites for birds, resulting in competition. The difficulty of removing bees and their defensive behavior makes a prevention protocol necessary. Here, we describe a push-pull integrated pest management protocol to deter bees from inhabiting bird boxes by applying a bird safe insecticide, permethrin, to repel bees from nest boxes, while simultaneously attracting them to pheromone-baited swarm traps. Shown here is an example experiment using Barn Owl nest boxes. This protocol successfully reduced colonization of Barn Owl nest boxes by Africanized honey bees. This protocol is flexible, allowing adjustments to accommodate a wide range of bird species and habitats. This protocol could benefit conservation efforts where AHB are located.

Preventing colonization of bird nest boxes by invasive Africanized honey bees is important for conservation efforts of nest-site limited birds. We provide an integrated pest management approach to &#34;push&#34; bees away from nest boxes with a repellant insecticide, permethrin, and &#34;pull&#34; them toward pheromone baited swarm traps.

推挽议定书蜜蜂会影响家禽的殖民化巢箱

Introduction of the invasive Africanized honey bee (AHB) into the Neotropics is a serious problem for many cavity nesting birds, specifically parrots. ...

科研

环境科学

Reliable Method for Assessing Seed Germination, Dormancy, and Mortality under Field Conditions

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester fabric and glue can be folded and glued to construct a strip of compartments that house seeds and identifying information, while allowing contact with soil leachate, water, microorganisms, and ambient temperature. Strips may be constructed with a wide range of compartment numbers and sizes and allow the researcher to house a variety of genotypes within a single species, different species, or seeds that have experienced different treatments. As opposed to individual seed packets, strips are more easily retrieved as a unit. While replicate packets can be included within a strip, different strips can act as blocks or can be retrieved at different times for observation of seed behavior over time. We used a high temperature glue gun to delineate compartments and sealed the strips once the seed and tags identifying block and removal times were inserted. The seed strips were then buried in the field at the desired depth, with the location marked for later removal. Burrowing animal predators were effectively excluded by use of a covering of metal mesh hardware cloth on the soil surface. After the selected time interval for burial, strips were dug up and seeds were assessed for germination, dormancy and mortality. While clearly dead seeds can often be distinguished from ungerminated living ones by eye, dormant seeds were conclusively identified using a standard Tetrazolium chloride colorimetric test for seed viability.

Here we present a protocol for assessing seed survivorship, germination and dormancy under field conditions using buried, labeled seed strips and tetrazolium chloride (TZ) viability testing.

对于在野外条件下评估种子萌发，休眠和死亡率可靠的方法

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester ...

Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been hindered partly due to a lack of standardized methods and availability of robust, user-friendly technology. In this study, commercially available, division-arrested cell lines were employed to quantitatively screen for endocrine activity of chemicals present in water samples of interest to environmental quality professionals. A single, standardized protocol that included comprehensive quality assurance/quality control (QA/QC) checks was developed for Estrogen and Glucocorticoid Receptor activity (ER and GR, respectively) using a cell-based Fluorescence Resonance Energy Transfer (FRET) assay. Samples of treated municipal wastewater effluent and surface water from freshwater systems in California (USA), were extracted using solid phase extraction and analyzed for endocrine activity using the standardized protocol. Background and dose-response for endpoint-specific reference chemicals met QA/QC guidelines deemed necessary for reliable measurement. The bioassay screening response for surface water samples was largely not detectable. In contrast, effluent samples from secondary treatment plants had the highest measurable activity, with estimated bioassay equivalent concentrations (BEQs) up to 392 ng dexamethasone/L for GR and 17 ng 17&#946;-estradiol/L for ER. The bioassay response for a tertiary effluent sample was lower than that measured for secondary effluents, indicating a lower residual of endocrine active chemicals after advanced treatment. This protocol showed that in vitro transactivation bioassays that utilize commercially available, division-arrested cell &#34;kits&#34;, can be adapted to screen for endocrine activity in water.

Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

A protocol to screen for endocrine activity in organic extracts of water samples, including treated wastewater effluent and surface (receiving) water, was adapted using commercially available division-arrested ("freeze and thaw") in vitro transactivation bioassays.

在用水筛查内分泌活动市售的<em&gt;体外</em&gt;式激活生物测定

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been ...

Video Tracking Protocol to Screen Deterrent Chemistries for Honey Bees

The European honey bee, Apis mellifera L., is an economically and agriculturally important pollinator that generates billions of dollars annually. Honey bee colony numbers have been declining in the United States and many European countries since 1947. A number of factors play a role in this decline, including the unintentional exposure of honey bees to pesticides. The development of new methods and regulations are warranted to reduce pesticide exposures to these pollinators. One approach is the use of repellent chemistries that deter honey bees from a recently pesticide-treated crop. Here, we describe a protocol to discern the deterrence of honey bees exposed to select repellent chemistries. Honey bee foragers are collected and starved overnight in an incubator 15 h prior to testing. Individual honey bees are placed into Petri dishes that have either a sugar-agarose cube (control treatment) or sugar-agarose-compound cube (repellent treatment) placed into the middle of the dish. The Petri dish serves as the arena that is placed under a camera in a light box to record the honey bee locomotor activities using video tracking software. A total of 8 control and 8 repellent treatments were analyzed for a 10 min period with each treatment was duplicated with new honey bees. Here, we demonstrate that honey bees are deterred from the sugar-agarose cubes with a compound treatment whereas honey bees are attracted to the sugar-agarose cubes without an added compound.

The loss of honey bee colonies presents a challenge to crop pollination services. Current pollinator protection practices warrant an alternative approach to minimize the contact of honey bees to harmful pesticides using repellent chemistries. Here, we provide detailed methods for a visual tracking protocol to screen deterrents for bees.

用于蜂蜜蜜蜂筛选化学成分的视频跟踪协议

The European honey bee, Apis mellifera L., is an economically and agriculturally important pollinator that generates billions of dollars annually. Honey ...

Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron (Oxy)Hydroxides, Trace Elements, and Bacteria

Fate and speciation of trace elements (TEs), such as arsenic (As) and mercury (Hg), in aquifers are closely related to physio-chemical conditions, such as redox potential (Eh) and pH, but also to microbial activities that can play a direct or indirect role on speciation and/or mobility. Indeed, some bacteria can directly oxidize As(III) to As(V) or reduce As(V) to As(III). Likewise, bacteria are strongly involved in Hg cycling, either through its methylation, forming the neurotoxin monomethyl mercury, or through its reduction to elemental Hg&#176;. The fates of both As and Hg are also strongly linked to soil or aquifer composition; indeed, As and Hg can bind to organic compounds or (oxy)hydroxides, which will influence their mobility. In turn, bacterial activities such as iron (oxy)hydroxide reduction or organic matter mineralization can indirectly influence As and Hg sequestration. The presence of sulfate/sulfide can also strongly impact these particular elements through the formation of complexes such as thio-arsenates with As or metacinnabar with Hg.
Consequently, many important questions have been raised on the fate and speciation of As and Hg in the environment and how to limit their toxicity. However, due to their reactivity towards aquifer components, it is difficult to clearly dissociate the biogeochemical processes that occur and their different impacts on the fate of these TE.
To do so, we developed an original, experimental, column setup that mimics an aquifer with As- or Hg-iron-oxide rich areas versus iron depleted areas, enabling a better understanding of TE biogeochemistry in anoxic conditions. The following protocol gives step by step instructions for the column set-up either for As or Hg, as well as an example with As under iron and sulfate reducing conditions.

Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron OxyHydroxides, Trace Elements, and Bacteria

Fate and speciation of arsenic and mercury in aquifers are closely related to physio-chemical conditions and microbial activity. Here, we present an original experimental column setup that mimics an aquifer and enables a better understanding of trace element biogeochemistry in anoxic conditions. Two examples are presented, combining geochemical and microbiological approaches.

用于研究铁 (氧) 氢氧化物、微量元素和细菌之间厌氧生物地球化学相互作用的实验柱装置

Fate and speciation of trace elements (TEs), such as arsenic (As) and mercury (Hg), in aquifers are closely related to physio-chemical conditions, such as ...

Rearing and Long-Term Maintenance of Eristalis tenax Hoverflies for Research Studies

With an estimated 6000 species worldwide, hoverflies are ecologically important as alternative pollinators to domesticated honeybees. However, they are also a useful scientific model to study motion vision and flight dynamics in a controlled laboratory setting. As the larvae develop in organically polluted water, they are useful models for investigating investment in microbial immunity. While large scale commercial breeding for agriculture already occurs, there are no standardized protocols for maintaining captive populations for scientific studies. This is important as commercial captive breeding programs focusing on mass output during peak pollination periods may fail to provide a population that is consistent, stable and robust throughout the year, as is often needed for other research purposes. Therefore, a method to establish, maintain and refresh a captive research population is required. Here, we describe the utilization of an artificial hibernation cycle, in addition to the nutritional and housing requirements, for long term maintenance of Eristalis tenax. Using these methods, we have significantly increased the health and longevity of captive populations of E. tenax compared to previous reports. We furthermore discuss small scale rearing methods and options for optimizing yields and manipulating population demographics.

Rearing and Long-Term Maintenance of Eristalis tenax Hoverflies for Research Studies

The overall goal of these procedures is to establish, maintain and refresh a captive population of Eristalis tenax in a research setting.

研究性学习的Eristalis 管Hoverflies 的饲养和长期维护

With an estimated 6000 species worldwide, hoverflies are ecologically important as alternative pollinators to domesticated honeybees. However, they are ...

A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for assessing plant responses to chemicals and other substances of interest is presented. This system is highly efficient in obtaining homogeneous and healthy seedlings of the C3 and C4 model species Arabidopsis thaliana and Setaria viridis, respectively. The sterile cultivation avoids algae and microorganism contamination, which are known limiting factors for plant normal growth and development in hydroponics. In addition, this system is scalable, enabling the harvest of plant material on a large scale with minor mechanical damage, as well as the harvest of individual parts of a plant if desired. A detailed protocol demonstrating that this system has an easy and low-cost assembly, as it uses pipette racks as the main platform for growing plants, is provided. The feasibility of this system was validated using Arabidopsis seedlings to assess the effect of the drug AZD-8055, a chemical inhibitor of the target of rapamycin (TOR) kinase. TOR inhibition was efficiently detected as early as 30 min after an AZD-8055 treatment in roots and shoots. Furthermore, AZD-8055-treated plants displayed the expected starch-excess phenotype. We proposed this hydroponic system as an ideal method for plant researchers aiming to monitor the action of plant inducers or inhibitors, as well as to assess metabolic fluxes using isotope-labeling compounds which, in general, requires the use of expensive reagents.

A simple, versatile, and low-cost in vitro hydroponic system was successfully optimized, enabling large-scale experiments under sterile conditions. This system facilitates the application of chemicals in a solution and their efficient absorption by roots for molecular, biochemical, and physiological studies.

一种灵活的低成本水培系统在无菌条件下评价植物对小分子的响应

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for ...

Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and toxicological studies. Using automated digital tracking platforms, recent efforts in neuropharmacology are leveraging larval fish locomotor behaviors to identify potential therapeutic targets for novel small molecules. Similar to these efforts, research in the environmental sciences and comparative pharmacology and toxicology is examining various behaviors of fish models as diagnostic tools in tiered evaluation of contaminants and real-time monitoring of surface waters for contaminant threats. Whereas the zebrafish is a popular larval fish model in the biomedical sciences, the fathead minnow is a common larval fish model in ecotoxicology. Unfortunately, fathead minnow larvae have received considerably less attention in behavioral studies. Here, we develop and demonstrate a behavioral profile protocol using caffeine as a model neurostimulant. Though photomotor responses of fathead minnows were occasionally affected by caffeine, zebrafish&#160;were markedly more sensitive for photomotor and locomotor endpoints, which responded at environmentally relevant levels. Future studies are needed to understand comparative behavioral sensitivity differences among fish with age and time of day, and to determine whether similar behavioral effects would occur in nature and be indicative of adverse outcomes at the individual or population levels of biological organization.

Here, we present a protocol to examine larval zebrafish and fathead minnow locomotor activities and photomotor responses (PMR) using an automated tracking software. When incorporated in common toxicology bioassays, analyses of these behaviors provide a diagnostic tool to examine chemical bioactivity. This protocol is described using caffeine, a model neurostimulant.

检测幼虫行为反应剖面的实验协议: 在神经刺激咖啡因中的应用

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and ...

Assessing the Particulate Matter Removal Abilities of Tree Leaves

Based on the conventional cleaning methods (water cleaning (WC) + brush cleaning (BC)), this study evaluated the influence of ultrasonic cleaning (UC) on collecting various sized particulate matter (PM) retained on leaf surfaces. We further characterized the retention efficiency of leaves to various sized PM, which will help to assess the abilities of urban trees to remove PM from ambient air quantitatively. 
Taking three broadleaf tree species (Ginkgo biloba, Sophora japonica, and Salix babylonica) and two needleleaf tree species (Pinus tabuliformis and Sabina chinensis) as the research objects, leaf samples were collected 4 days (short PM retention period) and 14 days (long PM retention period) after the latest rainfall. PM retained on the leaf surfaces was collected by means of WC, BC, and UC in sequence. Then, retention efficiencies of leaves (AEleaf) to three types of the various sized PM, including easily removable PM (ERP), difficult-to-remove PM (DRP), and totally removable PM (TRP), were calculated. Only around 23%-45% of the total PM retained on leaves could be cleaned off and collected by WC. When the leaves were cleaned through WC+BC, the underestimation of the PM retention capacity of different tree species was in the range of 29%-46% for various sized PM. Almost all PM retained on leaves could be removed if UC was supplemented to WC+BC. 
In conclusion, if the UC was complemented after the conventional cleaning methods, more PM on leaf surfaces could be eluted and collected. The procedure developed in this study can be used for assessing the PM removal abilities of different tree species.

The ultrasonic cleaning method was applied to elute the particulate matter (PM) retained on leaf surfaces after PM was eluted by the conventional cleaning methods (water cleaning only or water cleaning plus brush cleaning). The methodology can help to improve the estimation accuracy for PM retention capacity of leaves.

树叶颗粒物去除能力的评价

Based on the conventional cleaning methods (water cleaning (WC) + brush cleaning (BC)), this study evaluated the influence of ultrasonic cleaning (UC) on ...

A New Portable In Vitro Exposure Cassette for Aerosol Sampling

This protocol introduces a new in vitro exposure system, capable of being worn, including its characterization and performance. Air-liquid interface (ALI) in vitro exposure systems are often large and bulky, making transport to the field and operation at the source of emission or within the breathing zone difficult. Through miniaturization of these systems, the lab can be brought to the field, expediting processing time and providing a more appropriate exposure method that does not alter the aerosol prior to contacting the cells. The Portable In vitro Exposure Cassette (PIVEC) adapts a 37 mm filter cassette to allow for in vitro toxicity testing outside of a traditional laboratory setting. The PIVEC was characterized using three sizes of copper nanoparticles to determine deposition efficiency based on gravimetric and particle number concentration analysis. Initial cytotoxicity experiments were performed with exposed lung cells to determine the ability of the system to deposit particles while maintaining cell viability. The PIVEC provides a similar or increased deposition efficiency when comparing to available perpendicular flow in vitro exposure devices. Despite the lower sample throughput, the small size gives some advantages to the current in vitro ALI exposure systems. These include the ability to be worn for personal monitoring, mobility from the laboratory to the source of emission, and the option to set-up multiple systems for spatial resolution while maintaining a lower user cost. The PIVEC is a system capable of collecting aerosols in the field and within the breathing zone onto an air-interfaced, in vitro model.

A New Portable In Vitro Exposure Cassette for Aerosol Sampling

Here, we present a protocol to perform portable cellular aerosol exposures and measure cellular response. The method uses cells, grown at the air-liquid interface, mimicking in vivo physiology. Cellular response to copper nanoparticle aerosols was observed as oxidative stress through reactive oxygen species generation and cytotoxicity as lactate dehydrogenase release.

一种用于气溶胶取样的新型便携式体外曝光盒

This protocol introduces a new in vitro exposure system, capable of being worn, including its characterization and performance. Air-liquid interface (ALI) ...