作者热烈感谢安德森, j. Adamczyk, e. 伦, 我 Carstensen, Giansiracusa b. 莫特, 霍尔斯特和 ar. 史迪威, 讨论设计和执行以及在实地的帮助。

下面描述的协议遵循美国农业部农业研究服务机构的动物保育指南. 
 1. 完全评估 注意: 要确定 1) 所有蜂房部件 (包括盖子、盖子、盒、底板、框架和 2) 的重量, 需要进行充分的评估。所有蜂巢的重量将被总结和减去的总蜂房重量时, 所有的蜜蜂都存在, 以产生所有的成年蜜蜂的重量。在今后的评价中, 可以保留和使用在很大程度上不变的蜂巢部件的重量数据, 如盖子、底板和箱 (es), 从而加速评价。在研究开始或临近的时候进行全面的评估。木制零件在含水量方面可能会发生变化 24 , 因此在长周期后建议重复完整的评估。在每个评估中称量蜂巢的一部分, 例如盖子, 以估计其他蜂巢部分的变化。在评估过程中, 如果其他蜂箱在附近, 并且抢劫压力是一个关注的问题, 请在工作人员和蜂房周围放置一个屏蔽的前庭. 
<ol> <li> 对所有配置单元部分进行权衡。使用精度为1克或更低的刻度. 
	<ol> <li> 将盖子、内盖、皇后排除、糖浆送纸器和配置单元顶部的任何其他部件进行权衡. </li>
		<li> 使用刮刀或蜂房工具, 取出并称量任何食物或治疗材料, 如蛋白质补充剂或油脂馅饼, 搁在车架的顶端。必要时替换. </li>
		<li> 放置一个空的配置单元框 (以下是 #34; 临时框和 #34;) 配置单元旁边作为帧的过渡位置。将临时箱子放在平坦的表面上, 如盖子或底板上, 以防蜜蜂, 特别是皇后从盒子里掉到地上. </li>
		<li> 将帧快速而柔和地移动, 最好是在两个或三组中保留蜂簇的部分, 以使帧的顺序与原始框中的相同。一旦所有的帧都被移动, 摇晃任何剩余的蜜蜂到框架在临时框和权衡蜂房框. </li>
		<li> 如果箱子是最低的箱子, 在摇晃蜂入临时箱子之后称底部板、入口减速器和其他蜂房零件。编号帧与一个不可磨灭的标记之前移动它们, 使他们可以被替换为原来的方向. </li>
		<li> 重新安装底板、入口减速机、育雏箱等设备。将临时框中的帧从原来的顺序和方向移回原来的巢盒, 再次注意避免蜜蜂的不必要的干扰. </li>
	</ol> </li> <li> 从框的一端移除框架, 并以可视方式检查框架的两侧是否存在女王。如果女王被发现, 把她轻轻地放在已经被评估的盒子的一部分, 以避免进一步的干扰。如果可能的话, 把工蜂轻轻地摇动到框间, 而不是放在上面. </li>
	<li> 将整个框架的两侧与数码相机进行权衡, 使其尽可能接近框架, 这样就可以很容易地区分出有盖的巢和蜂蜜细胞, 但在足够的距离, 使整个框架包括在内。拍摄框架, 而另一名工作人员持有. 
	<ol> <li> 使用 #39; 框架支架和 #39; (任何夹具, 保持框架直立, 而在规模和不3月的梳子) 的规模。称量蜂房内的糖浆喂食器. 
		注意: 照片中的帧的大小将用于将曲面区域从相对于绝对测量的转换. </li>
	</ol> </li> <li> 在配置单元框中替换其原始位置和方向的框架。在重新安装前一帧之前删除下一帧, 以提供更多的空间来安全地摇动盒子内的蜜蜂。检查女王的存在, 晃动, 以消除蜜蜂, 重量, 照片, 并更换每个后续帧, 直到所有的框架已经处理. </li>
	<li> 如果配置单元有两个或更多的框, 请首先从最低的框中重并拍摄帧. 
	<ol> <li> 依次移除上部框或框, 将其堆叠在配置单元的旁边, 使其与原始顺序相反。一旦对底部框进行了评估, 请将下一个框放在底部框的顶部, 将临时框放在剩余的计算框的顶部, 然后将帧从第二个框移动到临时框, 如上所示。将帧移回其原始框, 以其原始方向. 
		注: 首先检查下盒, 摇动的蜜蜂将落入蜂巢箱中, 不再被打扰. </li>
	</ol> </li> <li> 在最近一段时间的不活动期间获取总蜂房重量, 如在评估当天蜜蜂在飞行之前的晚上或清晨。这个重量代表整个殖民地, 包括觅食. </li>
	<li> 计算成年蜂质量通过对所有蜂房零件 (木器、帧、蜂饲料等) 的权重求和, 然后从和 #34 中减去该总和; 总蜂房重量和 #34; 前一步获得的值。两者之间的区别是对成年蜂质量的估计. 
	注: 在进行全面评估后, 可在部分基础上进行后续评估, 假设无蜂巢部件 (如盒和底板) 的重量没有明显变化。部分评估花费的时间更少. </li>
	<li> 若要使用单个框对配置单元进行部分计算, 请删除、拍摄、称量和替换帧, 而不将帧移动到临时框中。在具有多个框的配置单元中, 按照上面所述的步骤操作 (步骤 1.5), 但不称量配置单元框. </li>
</ol> 2。在刻度上安装配置单元 注意: 本研究中使用的蜂巢秤是室外电子台秤, 最大容量至少100公斤, 在5000中一个部件的负载单元精度与温度补偿, 以及平底锅大小460毫米 x 610 毫米。这些刻度与 24 VDC 指示器和12位记录连接。指示器输出可以是数字或模拟;这里使用的缩放系统在 mA 中输出, 并且需要校准曲线将数据转换为 kg. 
<ol> <li> 在一个牢固的水平曲面上安装刻度. 
	注: 使用混凝土或陶瓷块或窑干木片将鳞片从地面上提升, 将使水垢和电气连接保持与土壤或水接触. </li>
	<li> 用塑料、布、纸板或其他材料覆盖秤上的顶部, 以防过度反射和加热蜂巢入口. </li>
	<li> 确保靠近蜂箱的入口在不同的方向上, 以减少蜂群间的漂移. </li>
	<li> 在安装后定期校准配置单元秤, 特别是在移动刻度时. 
	<ol> <li> 将刻度校准权重放在称重盘上, 并记录刻度输出。确保总重量超过规模最大容量的 50%. 
		注: 如果刻度输出为电压或电流单位, 而不是质量单位, 则使用校准曲线的斜率和截距 (平移和缩放输出的总重量之间的关系) 将比例输出转换为质量单位. </li>
	</ol> </li> </ol> 3。温度传感器的安装 注意: 本研究中使用的温度传感器有两种类型: 1) 热电偶连接到直接连接到电池供电的记录 (这可能是放在内部或外部的电缆蜂房, 如果电缆足够长);和 2) 带有集成传感器和数据采集的小型电池供电设备, 需要从配置单元中删除数据才能下载. 
<ol> <li> 获取可在帧之间安装的温度传感器, 对内部的高湿度条件有抵抗力配置单元, 并具有足够的电池电量和内存 (或可通过电缆或无线连接访问). </li>
	<li> 将可能受蜡或蜂胶影响的传感器放置在保护性容器中, 如组织嵌入盒式 (小的、一次性的塑料盒、带扣盖和通风槽)。保护容器减少蜡和蜂胶的积聚, 可以堵塞传感器访问端口的传感器或其他影响数据下载. </li>
	<li> 将传感器放在盒中心顶部的下方, 以便提供有关育雏的信息 (见下文). 
	注: 用传感器将一根短金属丝连接到容器上, 将导线钉在车架顶部, 这样容器就会挂在一边。直接放置在巢体上的传感器可能会干扰育雏和梳子的维护, 而放置在靠近外部的框架上的传感器, 或放置在内盖或盖子上, 可能会受到外部条件的影响, 特别是在凉爽天气下. </li>
</ol> 4。糖浆处理的制备 注意: 使用分析级吡虫啉对每种喂养的日进行处理解决方案。吡虫啉是高度水溶性的, 因此容易在糖浆中加入;有些杀虫剂的水溶性很低, 应采用其他方法。使用 1 L 塑料瓶的1公斤处理液的配方如下: <ol> <li> 混合控制 (无杀虫剂) 蔗糖溶液 1:1 w: w ( 如 500 g sucrose:500 毫升蒸馏水)。将蔗糖溶于蒸馏水中, 在热板上加热到不超过60和 #176 的大搅拌烧瓶;
	<li> 对于包括吡虫啉在内的解决方案, 将蔗糖溶液与上述混合, 但保留100毫升 (因此和 #34; 短和 #34;), 以允许各自的吡虫啉和 #34 的增加量; 穗和 #34; 解决方案. 
	<ol> <li> 在400毫升蒸馏水中溶解500克糖, 允许添加100毫升的穗, 以达到1公斤的治疗方案。为方便在外地, 转移900克和 #34; 短期和 #34; 糖溶液到另一个瓶子, 然后添加穗到每个单独的瓶子. </li>
	</ol> </li> <li> 在分析天平上测量1.0 毫克杀虫剂, 并在100毫升蒸馏水中使用无热量的搅拌棒将其溶解, 以此来准备 10 ppm 的吡虫啉库存解决方案. 
	注意: 为了避免静电的问题, 将吡虫啉称量为小的、反应的塑料贮器, 并将这些容器直接放在溶液中。在吡虫啉溶解后, 搅拌溶液并取出贮器. </li>
	<li> 对于 5 ppb 溶液, 将0.5 毫升的库存溶液混合成99.5 毫升的蒸馏水, 达到100毫升的峰值溶液。加入900克的短蔗糖溶液, 达到1公斤 5 ppb 糖浆。对于 20 ppb 溶液, 混合2.0 毫升的库存溶液成98.0 毫升的蒸馏水, 以产生100毫升的峰值解决方案。对于 100 ppb 溶液混合, 10.0 毫升的库存溶液成90.0 毫升蒸馏水的穗解决方案. </li>
</ol> 5。蜂蜂箱的制备及处理方法的应用 <ol> <li> 评估在应用治疗之前和之后确定成年蜂和育雏种群的菌落。在治疗前确定基线农药水平的样品, 如蜂蜜、蜜蜂面包、蜜蜂或蜡等. 
	<ol> <li> 若要取样蜡, 请打开50毫升离心管, 选择一节空梳, 然后沿梳子刮开管口, 直到收集到所需的蜡量。避免碰到管子里的蜡。更换管子上的盖子, 并给管子贴上标签。替换配置单元中的帧. </li>
		<li> 要取样蜂蜜或花蜜, 打开离心管, 然后按管口对含有蜂蜜或花蜜的梳子部分进行按压。允许物料流入管内, 而不是刮管, 以减少样品中的蜡量。更换管子上的盖子, 并给管子贴上标签。替换配置单元中的帧. </li>
		<li> 要取样蜂面包, 请选择一个含有蜂面包的框架, 并使用干净的金属或塑料刮刀去除蜂面包的几个细胞的内容, 并将材料放在离心管中。替换配置单元中的帧. 
		注: 收集足够的材料通常需要几分钟每帧。典型的农药分析样品至少有3克物质;在每个蜂房中取样蜂蜜、蜡和蜂面包至少2或3不同的点, 并将这些子组合到每个蜂房中. </li>
	</ol> </li> <li> 在评估后不久 (如次日), 删除包含蜂蜜和花蜜的任何 broodless 帧, 并将这些帧替换为基础或空绘制梳, 以方便糖浆存储. 
	注: 目的是增加空贮容量和对花蜜的需求。如果将治疗方法应用在蛋白质补充剂中, 可能不需要去除食物的骨架. </li>
	<li> 确保配置单元具有进纸器。在预处理评估后尽快将糖浆 (步骤 4.4) 倒入喂料器中, 尽可能快地进行处理。小心不要把任何糖浆洒在蜂巢外, 其他蜂群的蜜蜂可能会发现它. 
	注: 内部馈线可能最有效地减少抢劫;一些杀虫剂影响成年蜂活动和被处理的蜂房可能不太警惕保护馈线从抢夺蜂 9 . </li>
</ol> 6。蜂巢架照片分析 注意: 帧照片是在每个配置单元评估过程中进行的。可以使用以下协议从照片中提取信息. 
<ol> <li> 激活图像分析软件 (如 ImageJ), 它允许使用跟踪工具在照片上选择形状并测量所选形状的区域. </li>
	<li> 检索帧的数字照片。选择和 #39; 文件和 #39; 单击并 #39; 打开和 #39;。导航到保存照片的文件夹, 选择照片进行分析。图像现在将在 ImageJ 程序中打开, 并且 ImageJ 工具栏将处于活动状态. </li>
	<li> 从工具栏中选择 #39;P olygon 和 #39; 工具。使用鼠标点击每个角落, 以确定的框架范围内的梳子覆盖。当到达起始角时, 形状将一起对齐. </li>
	<li> 一旦使用多边形工具定义了梳子区域, 请单击 "#39"; 分析和 #39; 选择和 #39; 测量和 #39;; 新的对话框将随测量一起弹出。使用列和 #39; 区域和 #39; 测量所绘制的定义区域中的像素. 
	注意: 第一次使用该程序时, 定义要报告的预期结果。
	<ol> <li> 单击并 #39; 结果和 #39; 在 "结果" 对话框中, 选择并 #39; 设置测量和 #39;。检查区域, 并取消选中所有其他项. </li>
	</ol> </li> <li> 定义覆盖的巢的区域, 选择和 #39; 手绘和 #39; 工具。按住鼠标上的按钮, 并在起始点处进行跟踪。到达起点时, 松开鼠标按钮;该形状将捕捉到位. 
	<ol> <li> 单击和 #39; 分析和 #39; 选择和 #39; 测量和 #39;。如果育雏模式不是固体, 由于疾病等因素, 使用和 #39; 多点和 #39; 选择工具来计算单元格的个数。将单元格计数直接转换为 cm 2 以便于比较;通常大约有4.01 单元格/cm 2 20 。
	</ol> </li> <li> 将跟踪的育雏形状的值除以框架内部的值, 以获得覆盖范围为上限的育雏的百分比帧覆盖率. </li>
	<li> 测量代表性帧的内部以获得一个标准, 以便将育雏曲面区域转换为 cm 2 . 
	注意: 典型的兰斯特罗思深框架的表面面积约为880厘米 2 20 。例如, 如果20% 的内部兰斯特罗思深框架被发现覆盖的育雏, 然后表面 area 将是 0.20 x 880 cm 2 = 176 cm 2 。
	<li> 对配置单元的育雏曲面区域求和。要将此量值转换为质量, 请将曲面面积乘以0.77 克/cm 2 20 。
	<li> 计算蜂蜜、上限花蜜和储存花粉的表面积, 例如, 使用步骤6.3 至6.6 所述的方法. 
	注意: 如果花蜜和花粉被视为和 #34; 食品商店和 #34; 这将把一个框架的组成部分减少到两个 (食品商店和育雏)。一个典型的兰斯特罗思框架与塑料基础和空画梳子重约 556 g 20 , 因此计算食物商店的重量, 总结的重量的框架与空的拉长的梳子和巢质量的重量, 并且减去那重量从观察到的框架的重量的问题. </li>
</ol>

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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sublethal+effects+of+imidacloprid+on+honey+bee+colony+growth+and+activity+at+three+sites+in+the+U.S.">Sublethal effects of imidacloprid on honey bee colony growth and activity at three sites in the U.S.</a> PLoS ONE. 11 (12), e0168603 (2016).</li><li>Johnson, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Within-nest+temporal+polyethism+in+the+honey+bee.">Within-nest temporal polyethism in the honey bee.</a> Behav. Ecol. Sociobiol. 62 (5), 777-784 (2008).</li><li>Moritz, R. F. A., Southwick, E. 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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hypothetical+homeotherm:+The+honey+bee+hive.">A hypothetical homeotherm: The honey bee hive.</a> Comp. Biochem. Physiol. 40A, 935-944 (1971).</li><li>Stabentheiner, A., Kovac, H., Brodschneider, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Honeybee+colony+thermoregulation+-+regulatory+mechanisms+and+contribution+of+individuals+in+dependence+on+age,+location+and+thermal+stress.">Honeybee colony thermoregulation - regulatory mechanisms and contribution of individuals in dependence on age, location and thermal stress.</a> PLoS ONE. 5 (1), e8967 (2010).</li><li>Delaplane, K. S., van der Steen, J., Guzman-Novoa, E., Dietemann, V., Ellis, J. 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G., Mercadier, G., Holst, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Within-day+variation+in+continuous+hive+weight+data+as+a+measure+of+honey+bee+colony+activity.">Within-day variation in continuous hive weight data as a measure of honey bee colony activity.</a> Apidologie. 39, 694-707 (2008).</li><li>Meikle, W. G., Weiss, M., Stilwell, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+colony+phenology+using+within-day+variability+in+continuous+weight+and+temperature+of+honey+bee+hives.">Monitoring colony phenology using within-day variability in continuous weight and temperature of honey bee hives.</a> Apidologie. 47, 1-14 (2016).</li><li>Meikle, W. G., Holst, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+continuous+monitoring+of+honey+bee+colonies.">Application of continuous monitoring of honey bee colonies.</a> Apidologie. 46, 10-22 (2015).</li><li>De Smet, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+indicator+gene+expression+profiles,+colony+dynamics+and+tissue+development+of+honey+bees+exposed+to+sub-lethal+doses+of+imidacloprid+in+laboratory+and+field+experiments.">Stress indicator gene expression profiles, colony dynamics and tissue development of honey bees exposed to sub-lethal doses of imidacloprid in laboratory and field experiments.</a> PLoS ONE. 12 (2), e0171529 (2017).</li><li>Danka, R. G., Rinderer, T. E., Hellmich, R. A., Collins, A. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The thermology of wintering honey bee colonies. , (1971).</li><li>Szabo, T. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermology+of+wintering+honey-bee+colonies+in+4-colony+packs.">Thermology of wintering honey-bee colonies in 4-colony packs.</a> Am. Bee J. 189, 554-555 (1989).</li><li>Human, H., Nicolson, S. W., Dietemann, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+honeybees,+Apis+mellifera+scutellata,+regulate+humidity+in+their+nest?.">Do honeybees, Apis mellifera scutellata, regulate humidity in their nest?.</a> Naturwissenschaften. 93, 397-401 (2006).</li></ol>

作者没有相互竞争的金融利益。

正如农作物科学家需要精确准确的数据来评估不同处理方法对植物生长和产量的影响, 蜜蜂研究人员需要精确准确的数据来评估蜂群的生长和活动。这些类型的数据是特别重要的, 当治疗效果可能是微妙的和长期的, 如可能预计当蜜蜂接触到低浓度的农药。
连续的蜂巢重量数据包含了许多关于正在研究的殖民地的生长和活动的信息, 以及这些殖民地对外来干扰的反应, 例如从抢夺蜜蜂和加入糖糖浆到饲养者9。然而, 对连续的重量和温度数据的正确解释需要定期了解菌落的大小。当成年蜂人口的大小可以估计从每日振幅趋势小时数据在花蜜流程期间, 由于增加的觅食交通, 在花蜜流动外面关系不举行井20。同样, 由于觅食的成功、食物消耗和蜜蜂数量的增加和减少 (例如蜜蜂死亡), 蜂群重量的变化部分是菌落大小的函数。较小的殖民地可能是健康的, 但显示较低的增长率和较少觅食的质量, 例如, 仅仅由于一个较小的觅食人口。
确定蜂房内温度传感器的一致和信息固定位置对于产生可重现的结果至关重要, 特别是在越冬时。 以前的研究表明, 在巢 (下) 框中的中央框架的顶轨温度监测与周围环境 (外部) 温度的关系与蜂巢中的其他几个位置相比较少, 并且受蜂群本身的影响更小20. 在冬季, 蜂群蜂簇和周围环境之间的温差预计会很高, 因此蜂巢内不同位置之间的差异同样会比在温暖月份中更大。此外, 蜜蜂群集将移动到蜂巢中的不同位置以利用食物资源25,26。这里所描述的冬季研究表明, 水平位置的温度, 从顶部到顶端的外框, 在中间框架上的垂直差异是明显不同的。横向空气运动横跨框架是相对限制的, 而垂直运动沿一个框架是不, 因此, 垂直混合将是预期的更大。这些结果证实, 在中间框架的顶端轨上的传感器位置可能是关于温度影响从殖民地的信息。
这里描述的蜂巢评估解决了其中的一些问题。通过将蜂房部分的总和减去在不活动期间 (如夜间) 测量的总蜂房重量, 对成年蜂质量的估计与觅食蜂的数量无关, 因而对评估时间不敏感。记录数据从一个规模允许很少空间的主观性, 和帧照片提供一个永久性的记录, 所以对育雏或食物表面积的分析可以重新考虑, 如果有必要, 从而减少对培训的人进行分析的依赖。
此处描述的方法并非没有错误来源。蜂巢的部件需要尽可能的不受蜜蜂的影响, 任何重要的梳状物或其它被去除的物质都应该在丢弃之前权衡。木质成分的含水量会随着时间的推移而改变, 而空蜂巢可以在环境相对湿度24的每日变化中表现出可测量的每日重量波动。包含蜂群的蜂巢可能不会有太大的变化, 因为蜜蜂倾向于在蜂房27内保持相对恒定的湿度, 这将减弱环境影响。这些方法也依赖于快速的工作。一旦蜂巢被拆开, 温度和气体条件就会急剧变化, 凉爽的温度会影响育雏的健康14。 拆掉一个没有屏幕的蜂巢来防止蜜蜂从邻近的殖民地里出来, 可以促进抢劫, 特别是在花蜜稀少的时候。
这些规程的修改偶尔地是必要的。虽然研究人员有兴趣收集尽可能多的数据, 但在某些情况下, 如必须检查许多蜂箱, 当抢劫压力很高, 或者天气条件不理想时, 工人必须迅速行动, 放弃一些数据。如果粮食储备的数据不重要, 只含有蜂蜜和/或花粉的框架, 或只有蜡或粉底的框架, 则应称重但不能拍照。一些研究人员也对上限幼虫和卵的水平感兴趣, 它们经常在框架照片中被发现;然而, 为了避免抽样偏差, 研究者必须有信心在所有的照片中都能发现它们。经常仅盖帽的巢可以可靠地和一致地被辨认在拍摄的相片在领域情况下。关于治疗的应用, 一些蜂箱, 特别是那些喂食吡虫啉的高剂量的蜂巢, 可能会变得昏昏欲睡, 导致他们的糖浆消耗减少, 以及它们保护蜂巢的能力。如果一个蚁群在5-7 维后不能饮用糖浆, 那么糖浆应该被除去, 称量, 丢弃 (以避免因发酵而产生的污染), 而菌落则提供新鲜的糖浆。通过称量丢弃的糖浆, 研究人员将记录每个菌落消耗了多少, 从而可以计算总用量。最后, 在糖糖浆的应用是最有效的, 当有很少或没有替代花蜜来源。替代花蜜来源将稀释治疗到一个未知的程度。正如《议定书》一节所指出的, 研究人员应牢记, 吡虫啉等杀虫剂与降低活性水平有关, 包括蜂房保护, 这取决于浓度。减少的蜂群防御也可能导致更多的抢劫和可能的治疗混杂。
研究结果表明, 对蜂群的持续监测和内部温度是敏感的群体水平的行为, 即使在行为变化引起的非常低 (5 ppb) 浓度的神经毒性农药。将这些方法进一步应用到其他毒素和杀虫剂与其他行动模式, 如昆虫生长调节剂, 将增加我们的理解的影响, 田间实际剂量的蜜蜂蜂群。

最近人们对蜜蜂接触低浓度的农用化学品, 特别是杀虫剂的问题提出了关注。蜂在领域可能通过直接应用暴露在觅食在领域或甚而殖民地自己, 通过接触处理的表面, 由种子处理的尘土和由植物产品的消耗量例如花粉、花蜜和渗出物1,2,3,4,5. 急性毒性显然对蜜蜂有害, 但许多杀虫剂 (如吡虫啉等烟碱) 的试验昆虫 (LC50) 中的浓度导致50% 的死亡率远远高于蜜蜂面包中所观察到的。和蜡6 , 高于被视为 "字段实际" 范围7的内容。然而, 最近的工作表明, 致死接触农药对菌落级别8的影响很大, 即使浓度低至每亿 (ppb)9的5份。
蜜蜂的蜂群被称为 "超级", 因为许多与单个动物相关的功能, 包括食物采购、繁殖 (蜂蜜蜂群通过菌落裂变进行的行为) 和温度控制, 都是由成人工蜂组10,11,12,13,14。蜜蜂群落, 被认为是有机体, 提供一个独特的研究机会, 因为支持殖民地的结构可以拆卸, 检查, 并重新组装, 如果做仔细, 很少, 或没有副作用。养蜂人和研究人员利用这一点, 通过拆卸蜂箱来检查它们的存在和状态, 包括蜂王、害虫和病原体、食物储藏的充足性、育雏数量和其他因素15。蜂群有另一个重要的特性, 那就是, 与大多数 homeotherms 不同的是, 它们通常是静止的, 通过部署觅食蜂来获取资源, 这些蜜蜂可以在距离10公里的16处收集食物。因此, 蜂巢可以无限期地维持在电子秤上, 由此产生的连续重量数据已被证明可以提供蜂蜜储存、觅食活动、觅食成功、蜂拥、糖浆消耗、致死和致死的信息。农药的影响, 害虫侵扰和抢劫9,17,18,19,20。由于菌落的所有部分, 包括巢, 可以被访问, 温度, 湿度, CO2, 声音和其他因素的传感器可以安装在蜂房内, 并用于获取连续的数据, 通知了殖民地的健康和状态21。
估计成年蜜蜂和育雏人口的大小是有用的, 如果不是关键的解释许多类型的连续数据。视觉评估在野外研究中的应用非常迅速, 并且在22中有很多缺点。目视检查通常在白天进行, 因此不考虑觅食蜂, 它可以构成人口的很大一部分23。目视检查估计数的精确度和准确性也取决于检查员的培训和经验。客观性是另一个考虑因素, 特别是如果蜂房检查员知道哪些蜂箱在哪些治疗组;关于这种措施错误的分配的假设可能难以自圆其说。
蜂群的体重增加率, 觅食人口的大小, 和蜂房内的温度, 提供几个例子, 将被期望是数量上不同的大和小的殖民地, 所以测量和控制的群体大小往往大大在随机实验中促进了治疗效果的检测。为了研究目的, 制定了一项关于成年蜂的精确和客观的测量方法, 其原则是在不活动期间 (如夜间) 的总蜂房重量与所有蜂房部件的重量之和 (包括木器、蜡、蜂蜜和育雏) 是成年蜂总质量的重量。通过用帧照片称量帧和测量育雏质量, 在控制空框和蜡的重量之后, 可以通过从框架重量中减去巢体质量来估计食物资源。这些方法旨在提高现场目视评估所期望的精确度。

<table border="0" cellpadding="0" cellspacing="0" width="814">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:427px;">Langstroth hive equipment (box)</td>
			<td style="width:301px;">Mann Lake Ltd, Hackensack, MN</td>
			<td style="width:87px;">KD-700</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Langstroth hive equipment (frame)</td>
			<td>Mann Lake Ltd, Hackensack, MN</td>
			<td>WW-900</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Langstroth hive equipment (lid and cover)</td>
			<td>Mann Lake Ltd, Hackensack, MN</td>
			<td style="width:87px;">WW-302 
			&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Langstroth hive equipment (base)</td>
			<td>Mann Lake Ltd, Hackensack, MN</td>
			<td>WW-316</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Langstroth hive equipment (internal feeder)</td>
			<td>Mann Lake Ltd, Hackensack, MN</td>
			<td>FD-505</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cordovan Italian queens and bee packages</td>
			<td>C.F. Koehnen &#38; Sons, Glenn, CA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scale, bench (100 kg max. capacity): model B-2418&#160;</td>
			<td>TEKFA, Copenhagen, Denmark</td>
			<td>discontinued</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scale, bench (100 kg max. capacity): Diamond Series</td>
			<td>Avery Weigh-Tronix , Fairmont, MN</td>
			<td>1824-200</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imidacloprid, analytical-grade CAS # 138261-41-3</td>
			<td>Sigma-Aldrich, St. Louis, MO 63103</td>
			<td align="right">37894</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Electronic scale, precision (precision = 0.1 mg): Adventurer Pro 260</td>
			<td>Ohaus,&#160; Parsippany, NJ</td>
			<td>AV264C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Electronic scale, portable (15 kg max. cap.): Ranger Count 3000</td>
			<td>Ohaus, Parsippany, NJ</td>
			<td>RC31P15</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thermocouple probe: TMC6-HD</td>
			<td>Onset Computer Corp., Bourne, MA</td>
			<td>TMC6-HD</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Datalogger, 12-bit: Hobo U-12</td>
			<td>Onset Computer Corp., Bourne, MA</td>
			<td>U12-012</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Temperature and r.h. datalogger: iButton Hygrochron</td>
			<td>Baulkham Hills, NSW 2153, Australia</td>
			<td>DS1923</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Temperature datalogger: iButton Thermochron</td>
			<td>Baulkham Hills, NSW 2153, Australia</td>
			<td>DS1922L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nalgene plastic bottle</td>
			<td>Thermo Scientific, Rochester, NY</td>
			<td>2104-0032</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue embedding cassette</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>B1000731WH</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Digital camera: Pentax K-01</td>
			<td>Ricoh Imaging Co., Ltd.</td>
			<td align="right">15241</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ImageJ version 1.47 software</td>
			<td>W. Rasband, National Institutes of Health, USA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge tubes, 50 ml</td>
			<td>Fisher Scientific, Asheville, NC</td>
			<td>14-959-49A</td>
		</tr>
	</tbody>
</table>

monitoring colony-level effects of sublethal pesticide exposure on honey bees

致死杀虫剂暴露对蜜蜂蜂群的影响可能是显著的, 但在野外使用标准目视评估方法难以发现。在这里, 我们描述的方法, 以衡量的数量, 成年蜜蜂, 育雏和食物资源, 通过权衡蜂巢和蜂房的部分, 通过拍摄帧, 并安装在规模和内部传感器的蜂箱。这些周期性评估的数据, 然后结合运行平均和每日趋势数据的蜂房重量和内部蜂房温度。所得到的数据集已被用于检测吡虫啉在糖浆中的菌落级效应, 其含量低至每亿5份。这些方法是客观的, 需要很少的训练, 并以传感器输出和照片的形式提供永久记录。

蜜蜂群体生长和物候, 测量使用定期蜂房检查与连续重量监测, 显示显着较低的巢在接触到吡虫啉 100 ppb。 蜂房检查显示, 成年蜂的质量不受致死浓度为5和 100 ppb 的影响, 但对育雏帧照片的分析显示, 100 ppb 处理中的育雏量明显较低 (图 1)。连续蜂房重量数据显示不同浓度的吡虫啉群体间的菌落生长率有所不同。连续的蜂房重量数据分为两部分:25 h 运行平均数据, 这是有关的群体生长和觅食的成功, 以及每小时的原始数据和25小时的平均运行时间的差异, 每小时趋势数据。趋势数据的每日振幅与觅食活动有关19,20 (图 2)。虽然在某种程度上, 称蜂箱有时可能检测到平均重量差异, 但连续称重提供了趋势数据, 以获取有关群体行为的信息。
在典型的商用蜂巢中, 中间框架的顶轨是温度传感器的有效位置。 在殖民地的成年工蜂产生和维持高温 (33-36°C) 特别是在育雏14和集群本身是移动的优势, 以利用食品商店在蜂房的不同部分25,26。为了减少热量损失, 群集避免与蜂巢的外部部分 (如侧面或底部) 有较大的接触面, 因此从盒子的顶部中心到簇的距离很少大。与蜂巢中的其他位置相比, 巢盒的顶部中心已被显示出降低了温度的变异性, 例如在夏季和秋季条件下的外部框架的顶部和第二个盒子 ("super") 的顶部20。在冬季的情况下, 环境温度和星团之间的差异将是最大的, 同一地点被发现具有最高的温度和最低的变异性与环境条件相比, 表明群集本身的最大贡献 (图 3和4;表 1)。温度制度的差异不能归因于成年蜂质量的差异 (表 2)。
在100 ppb 治疗中, 与 5 ppb 治疗相比, 正弦曲线与连续温度数据相适应的振幅明显高于对照组, 而治疗组差异无统计学意义9. 振幅与温度的变异性成正比, 因此较高的振幅表示蜂巢中那个点的温度变高。相对于 100 ppb 治疗组 (图 5), 较低的振幅反映了 5 ppb 和对照组的更高的育雏产量。这些结果, 结合蜂巢检查和连续蜂房重量数据, 证实了 100 ppb 吡虫啉对蜂群水平的影响。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56355/56355fig1.jpg"> 
图 1:成年蜂和育雏措施。蜂房检查数据从 2014年5月, 到 2015年3月, 为菌落暴露在含糖浆 0 (控制), 5 和 100 ppb 吡虫啉附近图森, 亚利桑那州.(A) 平均 (+ SEM) 成年蜂总质量;(B) 平均 (+ SEM) 密封的巢面面积。灰色地带表示治疗期 (每蜂房6周处理的蔗糖糖浆每周4-6 公斤)。每治疗组四菌落;一个殖民地在 100 ppb 在冬天死了。8月没有记录成年蜂的质量。此处的数据以前已发布, 并在此处使用权限9。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56355/56355fig1large.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/56355/56355fig2.jpg"> 
图 2:蜜蜂蜂房重量数据。连续重量数据从6月到 2014年12月, 为蜂蜜蜂殖民地暴露了糖浆包含 0, 5 和 100 ppb 吡虫啉在图森附近, AZ.(A) 总蜂房重量 (± SEM);(B) 正弦曲线的振幅与同一期间的趋势权重数据相匹配。灰色地带表示治疗期 (每蜂房6周处理的蔗糖糖浆每周4-6 公斤)。每治疗组四菌落;一个殖民地在 100 ppb 在冬天死了。此处的数据以前已发布, 并在此处使用权限9。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56355/56355fig2large.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/56355/56355fig3.jpg"> 
图 3:在不同位置的蜂房内温度。每天平均 (± SEM) 温度 (° c) 的3传感器位置在蜜蜂蜂巢内.(a) 4 数据蜂箱保存在高海拔地点 (MLEM: 2412 米);和 (B) 从2013年12月至2014年2月在亚利桑那州图森市附近保存的3蜂箱 (SRER: 719 米)。蜂箱是木制兰斯特罗思深盒 (43.65 升容量) 装有木制内盖和金属衬里的伸缩盖。热电偶温度探头连接到车架的顶部, 并将一个集成的记录/温度传感器放在中间框架的底部轨上。这些数据表明, 与其他传感器位置相比, 中间帧顶部的传感器位置始终高于周围环境, 从而使蜜蜂菌落对温度管理有了信息。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56355/56355fig3large.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/56355/56355fig4.jpg"> 
图 4:在不同位置的单元内温度可变性正弦 c 的日均 (± SEM) 振幅urves 适合每小时趋势温度数据的3传感器位置在蜜蜂蜂巢。(A) 在高海拔地点保存的4蜂箱 (MLEM: 2412 米);和 (B) 从2013年12月至2014年2月在亚利桑那州图森市附近保存的3蜂箱 (SRER: 719 米)。蜂箱是木制兰斯特罗思深盒 (43.65 升容量) 装有木制内盖和金属衬里的伸缩盖。热电偶温度探头连接到车架的顶部, 并将一个集成的记录/温度传感器放在中间框架的底部轨上。这些数据显示, 中间帧顶部的传感器位置在可变性上一直较低 (这里测量的是正弦曲线的振幅, 适合每小时趋势数据), 而不是与其他传感器位置相比, 因此信息温度管理由蜂殖民地。环境温度 (实心黑线) 的刻度在每个图的右侧。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56355/56355fig4large.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/56355/56355fig5.jpg"> 
图 5:在蜂房内温度为被对待的殖民地.连续温度数据从6月到 2014年12月, 为蜂蜜蜂殖民地暴露了糖浆包含 0, 5 和 100 ppb 吡虫啉在图森附近, AZ. (A) 平均蜂房温度 (± SEM);(B) 正弦曲线的振幅与同一期间的趋势温度数据相适应。灰色地带表示治疗期 (每蜂房6周处理的蔗糖糖浆每周4-6 公斤)。每治疗组四菌落;一个殖民地在 100 ppb 在冬天死了。此处的数据以前已发布, 并在此处使用权限 (米克尔et al.2016a).<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56355/56355fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>比较</td>
			<td>t 检验</td>
			<td>P</td>
			<td>网站</td>
			<td>平均差异 (° c)</td>
		</tr>
		<tr>
			<td>pos 2-pos 1</td>
			<td>2.05</td>
			<td>0.0485</td>
			<td>mlem</td>
			<td>1.00 ±0.25</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>SRER</td>
			<td>4.38 ±0.12</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pos 2-pos 3</td>
			<td>0.84</td>
			<td>1</td>
			<td>mlem</td>
			<td>2.05 ±0.21</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>SRER</td>
			<td>0.12 ±0.11</td>
		</tr>
	</tbody>
</table>
表 1:蜜蜂蜂房内不同传感器位置的平均温差和后点比较.位置 1: 顶部外框;位置 2: 顶部中间框;位置 3: 底部中间框。4数据蜂箱被保存在一个高海拔地点 (MLEM: 2412 m) 和3蜂箱保存在一个较低海拔地点 (SRER: 719 米) 附近图森, 亚利桑那州从2013年12月至2014年2月。蜂箱是木制兰斯特罗思深盒 (43.65 升容量) 装有木制内盖和金属衬里的伸缩盖。热电偶温度探头连接到1和2位置, 并将一个集成的记录/温度传感器连接到位置3。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>组</td>
			<td>日期</td>
			<td>平均 (± SEM) 成年蜂质量 (g)</td>
			<td>平均 (± SEM) 育雏表面积 (cm2)</td>
		</tr>
		<tr>
			<td>mlem</td>
			<td>2013年11月18日</td>
			<td>2119±412</td>
			<td>1372±396</td>
		</tr>
		<tr>
			<td>SRER</td>
			<td>2013年11月15日</td>
			<td>2270±312</td>
			<td>53 30</td>
		</tr>
		<tr>
			<td>mlem</td>
			<td>2014年2月13日</td>
			<td>2171±105</td>
			<td>0</td>
		</tr>
		<tr>
			<td>SRER</td>
			<td>2014年2月11日</td>
			<td>2027±487</td>
			<td>867±79</td>
		</tr>
	</tbody>
</table>
表 2:平均成年蜂质量和育雏质量, 估计使用这里描述的协议, 为四个蜂箱保存在一个高海拔地点 (MLEM: 2412 m); 和三个蜂箱保存在一个较低海拔地点 (SRER: 719 m) 附近图森, 亚利桑那州从2013年12月至 2014年2月.蜂箱是木制兰斯特罗思深盒 (43.65 升容量) 装有木制内盖和金属衬里的伸缩盖。热电偶温度探头连接到1和2位置, 并将一个集成的记录/温度传感器连接到位置3。

Watch this Scientific Journal Video about Monitoring Colony-level Effects of Sublethal Pesticide Exposure on Honey Bees at JoVE.com

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

致死剂量的杀虫剂可能影响殖民地的方式是难以检测使用的只是周期性或视觉方法。方法通过称量蜂巢和蜂房部件、拍照帧和安装传感器, 来测量成年蜂的总质量、育雏和食物资源。数据分析也得到了解决。

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 ...

monitoring-colony-level-effects-sublethal-pesticide-exposure-on-honey

Nanyang Technological University

Research

JoVE Journal

Biology

9.0K 次观看.  Carl Hayden Bee Research Center. 致死剂量的杀虫剂可能影响殖民地的方式是难以检测使用的只是周期性或视觉方法。方法通过称量蜂巢和蜂房部件、拍照帧和安装传感器, 来测量成年蜂的总质量、育雏和食物资源。数据分析也得到了解决。

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

Monitoring Acupuncture Effects on Human Brain by fMRI

Functional MRI is used to study the effects of acupuncture on the BOLD response and the functional connectivity of the human brain.  Results demonstrate that acupuncture mobilizes a limbic-paralimbic-neocortical network and its anti-correlated sensorimotor/paralimbic network at multiple levels of the brain and that the hemodynamic response is influenced by the psychophysical response.  Physiological monitoring may be performed to explore the peripheral response of the autonomic nerve function. This video describes the studies performed at LI4 (hegu), ST36 (zusanli) and LV3 (taichong), classical acupoints that are commonly used for modulatory and pain-reducing actions. Some issues that require attention in the applications of fMRI to acupuncture investigation are noted.

FMRI and physiological monitoring is used to study the effects of Acupuncture on the central and peripheral nervous systems. Acupuncture mobilizes a limbic-paralimbic-neocortical network, with great overlap with the default mode network, to modulate neurological activity, possibly related to its autonomic effect in the peripheral nervous system.

通过功能磁共振成像监测针灸对人脑的影响

Functional MRI is used to study the effects of acupuncture on the BOLD response and the functional connectivity of the human brain.  Results demonstrate ...

科研

生物学

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and cardiovascular disease1-11. Traditional cell biological approaches to studying cellular function involve culturing cells on a rigid substratum (plastic dishes or glass coverslips) which cannot account for the effect of an elastic ECM or the variations in ECM stiffness between tissues. To model in vivo tissue compliance conditions in vitro, we and others use ECM-coated hydrogels. In our laboratory, the hydrogels are based on polyacrylamide which can mimic the range of tissue compliances seen biologically12. "Reactive" cover slips are generated by incubation with NaOH followed by addition of 3-APTMS. Glutaraldehyde is used to cross-link the 3-APTMS and the polyacrylamide gel. A solution of acrylamide (AC), bis-acrylamide (Bis-AC) and ammonium persulfate is used for the polymerization of the hydrogel. N-hydroxysuccinimide (NHS) is incorporated into the AC solution to crosslink ECM protein to the hydrogel. Following polymerization of the hydrogel, the gel surface is coated with an ECM protein of choice such as fibronectin, vitronectin, collagen, etc.
The stiffness of a hydrogel can be determined by rheology or atomic force microscopy (AFM) and adjusted by varying the percentage of AC and/or bis-AC in the solution12. In this manner, substratum stiffness can be matched to the stiffness of biological tissues which can also be quantified using rheology or AFM. Cells can then be seeded on these hydrogels and cultured based upon the experimental conditions required. Imaging of the cells and their recovery for molecular analysis is straightforward. For this article, we define soft substrata as those having elastic moduli (E) &lt;3000 Pascal and stiff substrata/tissues as those with E &gt;20,000 Pascal.

The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.

使用丙烯酰胺水凝胶的细胞功能的研究矩阵刚度的影响

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

致命的甲壳类动物长期毒性试验卤虫franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, normal development or changes in environmental conditions2-5. Understanding the cellular signaling pathways that are involved in the response to hypoxia could provide new insight into treatment strategies for diverse human pathologies, from stroke to cancer. This goal has been impeded, at least in part, by technical difficulties associated with controlled hypoxic exposure in genetically amenable model organisms.
The nematode Caenorhabditis elegans is ideally suited as a model organism for the study of hypoxic response, as it is easy to culture and genetically manipulate. Moreover, it is possible to study cellular responses to specific hypoxic O2 concentrations without confounding effects since C. elegans obtain O2 (and other gasses) by diffusion, as opposed to a facilitated respiratory system6. Factors known to be involved in the response to hypoxia are conserved in C. elegans. The actual response to hypoxia depends on the specific concentration of O2 that is available. In C. elegans, exposure to moderate hypoxia elicits a transcriptional response mediated largely by hif-1, the highly-conserved hypoxia-inducible transcription factor6-9. C .elegans embryos require hif-1 to survive in 5,000-20,000 ppm O27,10. Hypoxia is a general term for "less than normal O2". Normoxia (normal O2) can also be difficult to define. We generally consider room air, which is 210,000 ppm O2 to be normoxia. However, it has been shown that C. elegans has a behavioral preference for O2 concentrations from 5-12% (50,000-120,000 ppm O2)11. In larvae and adults, hif-1 acts to prevent hypoxia-induced diapause in 5,000 ppm O212. However, hif-1 does not play a role in the response to lower concentrations of O2 (anoxia, operational definition &lt;10 ppm O2)13. In anoxia, C. elegans enters into a reversible state of suspended animation in which all microscopically observable activity ceases10. The fact that different physiological responses occur in different conditions highlights the importance of having experimental control over the hypoxic concentration of O2.
Here, we present a method for the construction and implementation of environmental chambers that produce reliable and reproducible hypoxic conditions with defined concentrations of O2. The continual flow method ensures rapid equilibration of the chamber and increases the stability of the system. Additionally, the transparency and accessibility of the chambers allow for direct visualization of animals being exposed to hypoxia. We further demonstrate an effective method of harvesting C. elegans samples rapidly after exposure to hypoxia, which is necessary to observe many of the rapidly-reversed changes that occur in hypoxia10,14. This method provides a basic foundation that can be easily modified for individual laboratory needs, including different model systems and a variety of gasses.

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

This paper details how to use continuous-flow hypoxia chambers to generate atmospheres with defined concentrations of O2 to understand biological responses to decreased O2. This system is easy to setup and maintain, and flexible enough to suit a wide range of O2 concentrations and model systems

创建定义的气体环境研究缺氧对<em&gt; C。线虫</em

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

测量细菌的影响<em&gt; C。线虫</em&gt;行为使用一个鸡蛋保留含量

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

Toxicological Assays for Testing Effects of an Epigenetic Drug on Development, Fecundity and Survivorship of Malaria Mosquitoes

Insecticidal resistance poses a major problem for malaria control programs. Mosquitoes adapt to a wide range of changes in the environment quickly, making malaria control an omnipresent problem in tropical countries. The emergence of insecticide resistant populations warrants the exploration of novel drug target pathways and compounds for vector mosquito control. Epigenetic drugs are well established in cancer research, however not much is known about their effects on insects. This study provides a simple protocol for examining the toxicological effects of 3-Deazaneplanocin A (DZNep), an experimental epigenetic drug for cancer therapy, on the malaria vector, Anopheles gambiae. A concentration-dependent increase in mortality and decrease in size was observed in immature mosquitoes exposed to DZNep, whereas the compound reduced the fecundity of adult mosquitoes relative to control treatments. In addition, there was a drug-dependent decrease in S-adenosylhomocysteine (SAH) hydrolase activity in mosquitoes following exposure to DZNep relative to control treatments. These protocols provide the researcher with a simple, step-by-step procedure to assess multiple toxicological endpoints for an experimental drug and, in turn, demonstrate a unique multi-prong approach for exploring the toxicological effects of water-soluble epigenetic drugs or compounds of interest against vector mosquitoes and other insects.

A protocol is developed to examine the effects of an epigenetic drug DZNep on the development, fecundity and survivorship of mosquitoes. Here we describe procedures for the aqueous exposure of DZNep to immature mosquitoes and a blood-based exposure of DZNep to adult mosquitoes in addition to measuring SAH hydrolase inhibition.

毒理学化验一个后生药物对疟蚊的发育，繁殖和存活率测试效果

Insecticidal resistance poses a major problem for malaria control programs. Mosquitoes adapt to a wide range of changes in the environment quickly, making ...

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 ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

内皮细胞粘附性定量<em&gt;在体外</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic levels. Tight control over ER calcium is imperative for protein folding, modification and trafficking. Perturbations to ER calcium can result in the activation of the unfolded protein response, a three-prong ER stress response mechanism, and contribute to pathogenesis in a variety of diseases. The ability to monitor ER calcium alterations during disease onset and progression is important in principle, yet challenging in practice. Currently available methods for monitoring ER calcium, such as calcium-dependent fluorescent dyes and proteins, have provided insight into ER calcium dynamics in cells, however these tools are not well suited for in vivo studies. Our lab has demonstrated that a modification to the carboxy-terminus of Gaussia luciferase confers secretion of the reporter in response to ER calcium depletion. The methods for using a luciferase based, secreted ER calcium monitoring protein (SERCaMP) for in vitro and in vivo applications are described herein. This video highlights hepatic injections, pharmacological manipulation of GLuc-SERCaMP, blood collection and processing, and assay parameters for longitudinal monitoring of ER calcium.

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies. A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store. This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.

监测内质网钙稳态使用<em&gt; Gaussia</em&gt;荧光素酶SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

Bacteriocins are believed to play a key role in defining microbial diversity in different ecological niches. Here, we describe an efficient procedure to assess how bacteriocins affect gut microbiota composition in an animal model.

评估细菌素对小鼠肠道微生物的影响的方法

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...

蜜蜂致死农药暴露对蜂群水平的影响

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通过功能磁共振成像监测针灸对人脑的影响

使用丙烯酰胺水凝胶的细胞功能的研究矩阵刚度的影响

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创建定义的气体环境研究缺氧对

测量细菌的影响

毒理学化验一个后生药物对疟蚊的发育，繁殖和存活率测试效果

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

内皮细胞粘附性定量

监测内质网钙稳态使用

评估细菌素对小鼠肠道微生物的影响的方法