这份出版物得到了教育部的资助 (PR 奖 #P031C160025 17, 项目标题: 84.031C) 到科罗拉多州立大学-普韦布洛 (CSU-普韦布洛) 社区, 以建立积极的干细胞参与 (C 基地)。我们感谢目前的动物学和 Elsevier, 提供使用前几份论文中发表的代表性成果的权利, 以及朱庇特的编辑们为我们提供了发表本议定书的机会。我们还要感谢 C 基地计划, 布莱恩凯文·范德·艾格 Heuvel 博士 (c 基地和生物学系, csu-普韦布洛), csu-普韦布洛生物系, 托马斯格拉齐亚诺, 伯纳德 Possidente 博士 (生物学系, 斯基德莫尔学院) 和克莱尔博士拉莫斯(科罗拉多州立大学生物系) 给予他们的支持和帮助。

下面的协议是一种实验性的协议, 用于后方物种在果蝇属的污染培养基, 当口服摄取毒素是适当的;其他形式的暴露是可能的使用果蝇模型11,15,16,26。本协议中描述的方法以前是由. 19和彼得森等。23,24,25。
1. 在研究实验室建立果蝇种群
<ol><li>建立一个由环境控制的孵化器 (或小房间), 通过设置恒温器, 光照: 暗循环和湿度, 取决于测试物种的喜好, 为果蝇的种群提供住房。 注意: 首选的环境条件将因该研究所选择的物种的本土生态学而异。例如, 黑腹是原产于撒哈拉以南非洲27并且通常保持在25°c, 12:12 光: 黑暗的周期, 并且大约60% 湿气16,18,19,20 ,21,22,23,24,25,28,29,30. 另一方面,蒙大拿州的范围遍及加拿大和美国中西部, 这是一个更寒冷的地区;因此,蒙大拿通常保持在19–20°c, 有时是24小时的光政权, 以模拟条件在交配季节31。有关不同种类果蝇的地理范围的更详细的描述, 请参见果蝇形态模式网站32。</li>
	<li>
获得一个首选的果蝇品种和/或自交系从一个股票中心 (见资料表), 另一研究实验室应要求, 或收集野生, 基因变量的人口从外地。 注: 以下步骤解释了收集野生的、基因变量的果蝇种群以维持在研究实验室中的方法。这些方法已从马尔可夫和 O ' 格雷迪33和沃纳和 Jaenike34 , 以收集最广泛的物种, 一次, 而不是针对特定物种与一个诱饵来源。<ol>
<li>在冷藏库中冻结半打成熟的香蕉过夜, 在设置诱饵陷阱之前解冻。</li>
		<li>在瓶子的前面切一个 u 形的狭缝, 让苍蝇在饵瓶中被捕获, 而不是逃跑, 准备多部的塑料瓶。用瓶盖盖上塑料瓶, 这样苍蝇就不会通过盖子逃走。</li>
		<li>把解冻香蕉放到瓶子的底部, 这样瓶子的底部就含有大约一英寸的香蕉。把一大块成熟的西红柿放在瓶子里。将酵母浆 (从啤酒酿造过程中残留的酵母) 添加到瓶子底部的香蕉上, 这样香蕉就能浸泡在酵母浆中。</li>
		<li>添加木杆 (在直立垂直位置) 的瓶子, 使苍蝇有一个干净的基板走在远离酵母浆和香蕉。 图 1说明了这些方法的最终产品。</li>
		<li>一夜之间在树上挂饵瓶, 每24小时检查一次口吸出的瓶子, 并单独放置女性在小瓶中, 以创建 iso 女性线。 注意: 可以创建多雌性线, 但是, 只有当每个物种的雌性可以清楚地辨认。另外,果蝇在种类之内占领不同的生态学利基并且将有不同的膳食要求取决于那些适当位置 (Werner 并且 Jaenike34);参见沃纳和 Jaenike34的饮食建议和食物食谱。</li>
		<li>检查解剖显微镜下的成人 F1子代, 以确定收集的果蝇的种类 (见马尔可夫和 O ' 格雷迪33和沃纳和 Jaenike34 , 以协助鉴定各种物种).</li>
	</ol>
</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57450/57450fig1.jpg"> 图 1: 用于收集野外果蝇野生种群的陷阱和诱饵的图案表示.(a)在科罗拉多州的一个当地实地地点设置了飞行陷阱。(B)更密切地了解这一实地地点设置的飞行陷阱。<a href="/files/ftp_upload/57450/57450fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<ol start="3"><li>保持在环境控制的孵化器或室内恒温, 光照: 暗循环和湿度的 iso 女性或多女性线。要做到这一点, 房子苍蝇在瓶子或瓶子的首选培养基, 并允许妊娠雌性产卵的培养基。监测小瓶的幼虫和蛹的存在。 注:果蝇内蝇属不同的生态龛位, 将有不同的饮食要求和环境非生物偏好取决于这些利基33,34。在埃尔金和米勒28, 谢弗等地可以找到环境偏好和饮食建议 (以及进一步的畜牧指导)。29, 公牛和英勇的30, 马尔可夫和 O ' 格雷迪33, 和沃纳和 Jaenike34。如果使用野生捕获的物种, 可以在孵化器中模拟当地环境条件, 直到能够识别出物种。</li>
	<li>经常将库存转移到新鲜的培养基上, 丢弃旧瓶子, 保持健康的线条, 避免螨感染。 注意: 转移的频率将取决于物种的生命周期。例如, 每2周将果蝇移植到新鲜培养基中。有关在实验室中维护线路的详细信息,请参阅兰德等。16, 埃尔金和米勒28, 谢弗等。29, 公牛和英勇30, 格林斯潘35和科学教育数据库36。</li>
</ol>2. 受污染介质中的后果蝇
注: 如果测试果蝇在实验室中第一次或与新的污染物 (s), 确定致命剂量 (见卡洛斯·卡斯塔涅达等。37和马西等。38的方法) 和 LD50 (见卡洛斯·卡斯塔涅达等.37和埃金斯等。39为方法) 首先。然后, 运行剂量响应曲线, 以确定所需的表型输出的生物相关浓度;看到了吗？19和周等。40为方法。
<ol><li>根据污染物的化学成分, 在所需浓度 (s) 的情况下, 准备受污染介质的库存溶液。 注: 例如, 准备 PbAc 的库存解决方案: 通过将污染物添加到蒸馏水 (dH20) 中, 准备醋酸铅 (PbAc) 介质的库存溶液, 直到用污染物达到所需浓度的介质。例如, 1000 µM PbAc 的库存解决方案, 可以通过将 PbAc 添加到 dH20 中, 直到达到1000µM PbAc。此外, 稀释库存解决方案 (如1000 µM PbAc 解决方案) 到所需浓度 (如500µM PbAc), 并保持这些解决方案作为库存。</li>
	<li>准备介质, 遵循制造商的指导方针作为控制介质。根据制造商的指导方针, 准备其他介质;不过, 为弶镀 ' p20 制备的污染溶液补充剂。 注: 例如, 如果使用瞬时果蝇培养基, 添加大约一茶匙即时培养基到塑料瓶。向培养基中添加大约5–5.5 毫升 dH20。洒几粒活面包酵母, 准备控制培养基。准备实验介质, 补充20 µM 的库存溶液 (如 500 PbAc)。</li>
	<li>
将繁殖存活成熟的雄性和雌性从种群中转移到控制和实验培养基中。 注: 羽化的时间后, 生殖成熟度是不同的果蝇物种41。<ol>
<li>用占主导地位的手轻轻地敲打一小瓶的股票。确保苍蝇自动移动到瓶子的底部。另一方面, 在轻拍瓶子的同时取下瓶子的瓶盖, 在瓶子顶端放一小瓶控制或污染的培养基。把瓶子放在一起, 翻转过来, 轻轻敲击, 这样苍蝇就会自动转移到控制或污染介质的新鲜瓶子里。趁苍蝇还在敲瓶子的时候, 把瓶子盖上。</li>
		<li>重复与更多的瓶子, 确保标准化的苍蝇在每个瓶子。 注:通过单转移或麻醉进行转移的成年人总数将取决于用于避免过度拥挤的小瓶的大小。</li>
		<li>在标准环境条件下孵化成人 (即孵化器), 允许成年人在培养基中交配和产卵 24–96 h。</li>
		<li>在 24–96 h 之后, 在停尸房里丢弃成年人 (一个装满矿物油的瓶子, 用一个紧的漏斗) 留下受精卵 (后来成为实验对象), 以成熟的测试。把瓶子放进孵化器, 让卵发育。</li>
		<li>通过寻找从培养基中产生的幼虫来监测流浪龄幼虫的小瓶。</li>
	</ol>
</li>
</ol>3. 在不同的发展阶段收集实验对象
注: 实验对象可在任何发育阶段收集, 放置在盲编码的15毫升锥形管中, 并进行测试以积累。污染物积累的测试方法将取决于所研究的污染物。例如, PbAc 的积累可以用电感耦合等离子体质谱 (ICP-MS)42进行测试。此外, 实验对象可以收集在任何发展阶段, 以测试各种表型效应的污染物。图 2说明了果蝇生命周期43。图 3说明了暴露的实验协议和收集的不同发展阶段。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57450/57450fig2.jpg"> 图 2: 对果蝇生命周期的概念性概述 (最常用的果蝇模型系统).果蝇生命周期的阶段为: 1) 蛋, 2) 第一龄幼虫, 3) 二龄幼虫, 4) 第三龄幼虫, 5) 流浪第三龄幼虫, 6) 白眼蛹, 7) 红眼蛹, 8) 新 eclosed 成人, 9) 成熟成人。<a href="/files/ftp_upload/57450/57450fig2large.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/57450/57450fig3.jpg"> 图 3: 在父母 (f0) 和随后世代 (f1和以后) 对被污染的媒介的果蝇的口头暴露方法的概念概述.(A)暴露的世代在发展过程中的口腔接触方法。(B)测试将污染物转移到后代的方法 (F1到所需的世代)。这个数字已经从彼得森等。24<a href="/files/ftp_upload/57450/57450fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<ol><li>
收集流浪-第三龄幼虫
	<ol>
<li>启动监控瓶时, 灯打开的孵化器, 因为幼虫将出现从培养基和向上移动的瓶子一侧在 h 后, 灯打开在孵化器。在这个 h 中, 用木棍或镊子小心地从瓶子两侧移走流浪的第三龄幼虫。 注: 可供采集的幼虫数量将取决于 "2.3.4" 中产卵的数量。</li>
		<li>要去除幼虫中多余的培养基, 将幼虫放在一个小烧杯中, 用 dh2o 将 dh2O 倒入烧杯中, 将幼虫放在一个微妙的任务雨刷上。使用一个微妙的任务雨刷, 轻轻地去除多余的 dH2O 从幼虫。</li>
		<li>在环境控制的孵化器中维持实验种群。</li>
	</ol>
</li>
	<li>
收集新 eclosed 的成年人
	<ol>
<li>通过观察小瓶两侧的蛹的颜色来监测羽化的瓶子。 注意: 蛹在发育过程中会变暗。发育时间, 特别是预羽化, 取决于所测试的物种。</li>
		<li>当第一个成年人开始 eclose 的时候, 把这些成年人扔进一个含矿物油的太平间里。</li>
		<li>第二天早晨, 当在孵化器中打开灯时, 倾倒并丢弃任何可能在夜间或 morningbefore 灯亮起 eclosed 的未知年龄 (或童贞) 的成年人。</li>
		<li>大约4小时后, 麻醉任何成人出现作为新 eclosed 的成年人与2枪的瓶子。在解剖显微镜下将成人放置在 CO2板上。性成人通过寻找性别梳子的男性和 ovipositors 在女性的前肢。 注: 羽化必须在6小时内收集, 以避免交配, 但其他物种可能有较长的发育时间 (因此, 不需要在这个时间范围内收集)。</li>
		<li>用木棍在 CO2板上分离成人。用木棍轻轻地将成年人转移到与性别相关的群体中, 以此来匹配先前存在的历史。</li>
	</ol>
</li>
	<li>
收集成年后羽化
	<ol>
<li>允许成年人留在培养基匹配前羽化暴露从卵阶段到预期年龄后羽化在一个环境控制的孵化器。</li>
		<li>在测试前, 将成年人单独转移到控制培养基上24小时, 以允许成年人从身体上培养过量的污染介质。</li>
	</ol>
</li>
</ol>4. 后方实验对象测试几代或跨代暴露的影响。
<ol><li>后方父母世代 (ak a P0或 F0代), 转移成人从存货人口到控制和实验性媒介跟随步骤在 "2.1" 到 "2.3" 和 "3.1" 到 "3.3"。</li>
	<li>成人繁殖成熟时 (见 Pitnick等.41), 单独转移 (如2.3.1 所述) 一小瓶雄性到一瓶新的控制或实验培养基。单独转移一小瓶女性到现在包含男性的新鲜的小瓶。允许成年人在培养基中交配产卵24-96 小时, 然后将成年人丢弃在含有矿物油的太平间里, 重新孵化瓶子, 让后代发育。</li>
	<li>根据所需的世代数重复步骤4.1 到4.2。</li>
</ol>

作者没有什么可透露的。

果蝇作为一个强大的模型, 为一系列的生物过程, 由于广泛的保护基因和通路之间的人和人类 13,14。由于同样的原因, 它是一个强大的医学模型,果蝇已经成为一个合适的模型系统来研究人类污染对一系列毒理学端点的影响。几个实验室成功地使用了D.黑腹作为模型系统研究一系列化合物, 包括重金属11,16,18-25,37 ,38,39,40,44,45, 乙醇46, 纳米粒子26,47, 杀虫剂48和溶剂49。尽管最近努力利用果蝇作为毒理学模型, 它作为一个模型系统来回答无数毒理学问题仍处于起步阶段。然而, 鉴于它广泛用作医学相关端点的模型, 以及它在生态学50和进化研究17中的应用, 它作为毒理学模型系统的潜力是巨大的。
在此, 我们提出了在受污染培养基上培育果蝇属中各种物种的方法, 以测试各种毒理学端点。虽然其他形式的暴露是可能的使用果蝇作为模型 (如吸入和皮肤接触), 本议定书的重点是对污染物的口头消费, 这是必要的污染物, 自然会被摄取 (比如通过食物链)。这些方法可以适应多种果蝇种类和污染物的使用。野生的, 基因变量的果蝇种群也可以收集在田间和维持在研究实验室。有许多选择的陷阱和诱饵, 可以使用, 取决于物种的食物偏好;有关字段集合的字段指南, 请参阅马尔可夫和 O ' 格雷迪33和沃纳和 Jaenike34。此外, 这些方法可以改变, 以确定发展暴露在不同的关键发展时期的影响, 并允许长期多代测试污染物暴露。
这些方法的关键步骤包括: (1) 在环境控制条件下维持飞行种群, (2) 避免飞行种群过度拥挤, (3) 根据化学性质稀释试验污染物, (4) 选择测试污染物的生物相关浓度。在环境调节的孵化器 (或一间小房间) 内维持库存, 可确保在不同条件下的额外变化不会混淆结果。此外, 以往的行为季节性变化已被发现51和几个果蝇物种进入滞育在冬季52。第二, 幼虫过度拥挤对发育30、成人体型30和长寿53有长远影响。此外, 污染物的稀释是确保果蝇在生物上可以积累污染物的重要步骤。例如, PbAc 溶解在 dH2O23,24,25, 而其他化学品可能需要溶解在盐水或乙醇。选择与生物相关的污染物浓度会影响结果的方向;例如 , 低剂量的 PbAc 增加了与雄配的平均数雌性 ( 20 分钟内 ) , 而较高的剂量显示雌配19的平均数量显著下降。为了确定测试污染物的生物相关浓度, 读者应考虑运行初步研究, 以确定致命剂量和 LD50, 以确定适当剂量, 以执行剂量反应曲线。通过执行剂量响应曲线来测试某一特定端点上的浓度范围, 读者可以确定对个人或人群来说, 对其进行进一步测试的剂量是 "有益的" 或 "危险的"。
该协议提供了一个途径来确定: 1) 组织的多重生物水平对健康和毒理学终点的相互作用;2) 发展和紧急因素的作用;3) 生态重要端点;4) 医学上-重要端点;5) 多重压力源如何相互作用产生结果;和 6) 长期暴露的影响, 超越世代。为了说明本议定书的有效性, 提供了证据表明, 在整个发展过程中暴露的个人积累了 Pb (表 1)23,24。此外, 具有代表性的结果表明, 该协议可用于测试暴露在生态重要端点 (例如, 发育铅暴露对交配选择的影响24) 的影响。此外, 其他人已经测试了污染物对组织的多重生物水平的影响 (包括生理18,21, 遗传20,22和表型-水平19,23,24,25), 医学上-重要端点 18,20,21,22,23, 和长期多代效应23,24 ,25,54。此外, 初步数据表明, 发育铅暴露诱导跨代遗传效应的生育力在54。这项议定书的一个重要限制是, 使用这项议定书与果蝇处于萌芽阶段。因此, 有有限的出版物18,19,20,21,22,23,24,25解决《议定书》对补充毒理学问题的潜力, 例如发展和紧急因素的作用、额外的生态重要终点、多重压力源以及暴露的演变影响。
使用此协议, 读者可以测试自然摄取的污染物, 使用生物相关的方法。连续液体喂养, 由苏亚雷斯和al开发。55是一种替代的口服方法, 特别是对农药的接触。然而, 连续液体喂养适合成人摄取液体污染物, 不适用于个人可能暴露前羽化的污染物。这一点尤其重要, 因为在开发中可能存在暴露的关键时期。先前的研究显示, 铅暴露的关键时期为23。因此,果蝇应该在整个开发过程中暴露出来, 以避免果蝇在测试之前有可能积极消除污染物, 直到确定关键时期。
总之, 我们提供了一种将果蝇口头暴露于污染物的协议。使用这个协议和模型系统, 毒理学家可以转向伦理和非侵入性的方法, 以动物测试, 同时纳入一个更全面的方法, 以了解污染物的影响8。

每年, 化工行业引进约1000种新化学品1、2;然而, 在2、3分销之前, 仅有很小一部分化学物质对环境的影响进行了测试。虽然大规模的灾难是罕见的, 致死和长期接触大量的污染物在人类和野生动物4,5普遍存在。生态和环境毒理学的历史焦点是测试致死性、单一化学暴露、急性暴露和暴露的生理效应, 作为衡量污染物对生存6的影响的一种手段,7,8,9,10. 虽然对动物试验采取了伦理和非侵入性的做法, 但目前的做法受到限制, 因为发展、紧急性质和外部因素 (如人口水平和生态系统级相互作用) 在斡旋生态学重要端点8。因此, 有必要在不牺牲野生动物和/或脊椎动物的实验室中纳入更全面的方法。
无脊椎动物模型系统, 如果蝇, 是一个有吸引力的替代方案, 以解决对毒性测试的更全面的方法的需要。在一个世纪前的11, 它最初被发展为人类相关基因研究的无脊椎动物模型系统。由于以下几个原因, 黑腹现在已被广泛用作脊椎动物模型的替代品: 1) 保护的基因和通路之间的黑腹和人类;2) 与脊椎动物模型相比短世代时间;3) 维护成本低廉;4) 易于生成大样本尺寸;和 5) 过多的表型和生态相关的端点可用于测试11,12,13,14,15,16,17.
几个实验室11,15,16,17,18,19, 20,21,22,23,24,25现在使用D.黑腹作为一种脊椎动物模型的毒性试验, 以了解污染对人类的影响。当地野生果蝇可以被利用, 以及作为野生动物 (和人类) 的毒性模型, 以回答生态, 行为上和进化相关的问题, 在多个生物层次的组织。利用果蝇属中的物种作为模型, 几个可测量的端点是可能的11,15,16,18,19,20 ,21,22,23,24,25。另外, 使用果蝇模型, 毒理学家可以: 1) 道德上的链接效应在组织的多生物水平;2) 纳入紧急因素和发展的作用;3) 研究生态学上重要端点 (除医学上重要端点);4) 同时测试多个压力源;5) 和测试的长期多代 (如进化和跨代) 的影响的压力。因此, 利用果蝇作为模型系统, 可以采取多种方法, 不限于研究机械方法与自交系的近亲在实验室 。
本文介绍了果蝇的饲养和采集方法, 以回答各种毒理学问题。更具体地说, 我们描述的方法 1) 饲养果蝇的中掺有一个或多个污染物;2) 在整个发育过程中收集果蝇(如流浪的第三龄幼虫、蛹病例、新 eclosed 成年人和成年成年人);3) 在受污染的培养基中饲养果蝇, 以测试代际和跨代的传播, 以及长期毒物暴露对进化的影响。使用此协议, 以前的作者18,19,20,21,22,23,24,25报告了发育铅 (Pb2 +) 暴露的不同生理、遗传和行为效应。这项议定书使毒理学家能够使用一种更整体的毒理学方法, 这对于了解污染物是人类和野生动物在日益污染的环境中的危险因素是必不可少的。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Carolina Biological Instant Drosophila Medium Formula 4-24</td>
			<td>Carolina Biological</td>
			<td>173204</td>
			<td></td>
		</tr>
		<tr>
			<td>Drosophila vials, Narrow (PS), Polystyrene, Superbulk, 1000 vials/unit</td>
			<td>Genessee Scientific</td>
			<td>32-116SB</td>
			<td>Used to store flies</td>
		</tr>
		<tr>
			<td>Flugs Closures for vials and bottles, Narrow plastic vials</td>
			<td>Genessee Scientific</td>
			<td>49-102</td>
			<td>Used to store flies</td>
		</tr>
		<tr>
			<td>Cardboard trays, trays only, narrow</td>
			<td>Genessee Scientific</td>
			<td>32-124</td>
			<td>Used to organize populations of flies</td>
		</tr>
		<tr>
			<td>Cardboard trays, dividers only, narrow</td>
			<td>Genessee Scientific</td>
			<td>32-126</td>
			<td>Used to organize populations of flies</td>
		</tr>
		<tr>
			<td>Thermo Scientific&#160;Nalgene Square Wide-Mouth HDPE Bottles with Closure</td>
			<td>Fischer Scientific</td>
			<td>03-312D</td>
			<td>Useful for storage of contaminants</td>
		</tr>
		<tr>
			<td>Thermo Scientific&#160;Nalgene Color-Coded LDPE Wash Bottles</td>
			<td>Fischer Scientific</td>
			<td>03-409-17C</td>
			<td>Useful for storage of contaminants</td>
		</tr>
		<tr>
			<td>Eppendorf&#160;Repeater M4 Manual Handheld Pipette Dispenser</td>
			<td>Fischer Scientific</td>
			<td>14-287-150</td>
			<td>Used to prepare medium</td>
		</tr>
		<tr>
			<td>Combitips Advanced Pipetter Tips - Standard, Eppendorf Quality Tips</td>
			<td>Fischer Scientific</td>
			<td>13-683-708</td>
			<td>Used to prepare medium</td>
		</tr>
		<tr>
			<td>Flypad, Standard Size (8.1 X 11.6cm)</td>
			<td>Genessee Scientific</td>
			<td>59-114</td>
			<td>Used to anesthetize flies</td>
		</tr>
		<tr>
			<td>Flystuff foot valve</td>
			<td>Genessee Scientific</td>
			<td>59-121</td>
			<td>Used to anesthetize flies</td>
		</tr>
		<tr>
			<td>Tubing, green (1 continguous foot/unit)</td>
			<td>Genessee Scientific</td>
			<td>59-124G</td>
			<td>Used to anesthetize flies</td>
		</tr>
		<tr>
			<td>Mineral Oil, Light, White, High Purity Grade, 500 mL HDPE Bottle</td>
			<td>VWR</td>
			<td>97064-130</td>
			<td>Used to make a morgue</td>
		</tr>
		<tr>
			<td>Glass Erlenmeyer Flask Set - 3 Sizes - 50, 150 and 250ml, Karter Scientific 214U2</td>
			<td>Walmart</td>
			<td>Not applicable</td>
			<td>Used to make a morgue</td>
		</tr>
		<tr>
			<td>BGSET5 Glass Beaker Set Of 5</td>
			<td>Walmart</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inbred or wildtype line of Drosophila</td>
			<td>Bloomington Drosophila Stock Center at Indiana University</td>
			<td></td>
			<td>https://bdsc.indiana.edu</td>
		</tr>
		<tr>
			<td>Wild popultions of Drosophila</td>
			<td>UC San Diego Drosophila Stock Center</td>
			<td></td>
			<td>https://stockcenter.ucsd.edu/info/welcome.php</td>
		</tr>
	</tbody>
</table>

experimental protocol for using drosophila as an invertebrate model system for toxicity testing in the laboratory

紧急性质和外部因素 (特别是人口水平和生态系统层面的相互作用) 在生态重要的端点的调解中起着重要作用, 尽管它们在毒理学研究中很少被考虑。作为毒物的行为、神经和遗传影响的毒理学模型, 腹腹已经成为了一个典型。更重要的是,果蝇属中的物种可以作为一种综合框架方法的模型系统, 在毒物学研究中纳入紧急性质并回答生态相关问题。本文的目的是提供一个协议, 以揭露果蝇属物种的污染物, 作为一个模型系统的一系列表型产出和生态相关的问题。更具体地说, 本议定书可用于 1) 将组织的多个生物层次联系起来, 了解毒物对个人和人口水平的影响;2) 测试毒物在发育暴露的不同阶段的影响;3) 检验污染物的几代和演变影响;4) 同时测试多种污染物和压力源。

通过将果蝇口头暴露在整个开发过程中, 可以通过将果蝇暴露在不同的生物组织水平来测试各种毒理学问题。本节介绍在以前发表的论文23、24中使用本协议获得的代表性结果。特别是, 本议定书以前用于评估在同一世代的暴露和跨第一代后代23中铅 (Pb) 的积累、消除和封存;并研究了积累对交配选择24的意义。
表 1和图 4显示了使用此协议获得的具有代表性的结果, 用于确定 f0和 f1代中 Pb 的累积和消除。
表 1显示了在多个发育阶段 (徘徊的第三龄幼虫, 在不同的剂量: 0、10、40、50、75、100、250和500µM PbAc) 的样品中铅累积的代表性结果蛹病例, 新 eclosed 的成年人, 成熟的女性和男性) 在彼得森等。在不同发育阶段收集了23份样品, 冷冻-20 摄氏度, 用硝酸和过氧化氢处理, 用 ICP-MS23,42测定 Pb。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>剂量 (µM PbAc)</td>
			<td style="text-align: center;">幼虫</td>
			<td style="text-align: center;">蛹案例</td>
			<td style="text-align: center;">新 Eclosed 成人</td>
			<td style="text-align: center;">成熟的成年女性</td>
			<td style="text-align: center;">成熟成年男性</td>
		</tr>
<tr>
<td>0</td>
			<td style="text-align: center;">0.009 @ 0.001</td>
			<td style="text-align: center;">0.099 @ 0.02</td>
			<td style="text-align: center;">0.04 @ 0.005</td>
			<td style="text-align: center;">0.069 @ 0.031</td>
			<td style="text-align: center;">0.0015 @ 0.003</td>
		</tr>
<tr>
<td>10</td>
			<td style="text-align: center;">0.42 @ 0.025</td>
			<td style="text-align: center;">0.46 @ 0.03</td>
			<td style="text-align: center;">0.12 @ 0.009</td>
			<td style="text-align: center;">0.12 @ 0.012</td>
			<td style="text-align: center;">0.056 @ 0.008</td>
		</tr>
<tr>
<td>40</td>
			<td style="text-align: center;">7.5 @ 0.67</td>
			<td style="text-align: center;"></td>
			<td style="text-align: center;"></td>
			<td style="text-align: center;"></td>
			<td style="text-align: center;"></td>
		</tr>
<tr>
<td>50</td>
			<td style="text-align: center;">5.2 @ 0.71</td>
			<td style="text-align: center;">2.77 @ 0.30</td>
			<td style="text-align: center;">1.11 @ 0.14</td>
			<td style="text-align: center;"></td>
			<td style="text-align: center;"></td>
		</tr>
<tr>
<td>75</td>
			<td style="text-align: center;">14 @ 1.06</td>
			<td style="text-align: center;"></td>
			<td style="text-align: center;"></td>
			<td style="text-align: center;"></td>
			<td style="text-align: center;"></td>
		</tr>
<tr>
<td>100</td>
			<td style="text-align: center;">12 @ 0.95</td>
			<td style="text-align: center;">4.7 @ 0.38</td>
			<td style="text-align: center;">3.36 @ 0.58</td>
			<td style="text-align: center;">1.20 @ 0.63</td>
			<td style="text-align: center;">1.24 @ 0.46</td>
		</tr>
<tr>
<td>250</td>
			<td style="text-align: center;">89 @ 8.49</td>
			<td style="text-align: center;"></td>
			<td style="text-align: center;"></td>
			<td style="text-align: center;">25.06 @ 4.72</td>
			<td style="text-align: center;">5.46 @ 0.75</td>
		</tr>
<tr>
<td>500</td>
			<td style="text-align: center;">271 @ 41.01</td>
			<td style="text-align: center;">334.30 @ 39.43</td>
			<td style="text-align: center;">8.44 @ 0.84</td>
			<td style="text-align: center;">46.50 @ 14.72</td>
			<td style="text-align: center;">14.43 @ 1.83</td>
		</tr>
</tbody></table>表 1: 平均铅负荷 (ng/个人) 测试 在从卵期到试验阶段, 口服铅后, 在发育过程中发生黑腹。手段 (ng/飞行) 的标准误差显示 (n = 8 幼虫, n = 3 控制饲养的成人, n = 3 Pb 饲养的成人)。野生型黑腹黑腹养殖控制或含铅培养基 (0, 10, 40, 50, 75, 100, 250 或500µM PbAc) 从卵阶段到不同的发展阶段。采用 ICP-42 -质谱法收集和试验了铅积累的样品. 这张桌子已经从彼得森和al修改过了。23
在图 4中, 父母世代 (f0) 接触到 Pb 从卵子阶段到成年, 交配在控制培养基中, 第一代后代 (f1) 在控制培养基中饲养直到成年24。检测铅积累和清除的方法类似于彼得森等。23. 这项实验的结果表明, 父母的接触不会传给第一代成年后代24。因此, 使用此协议, 可以测试不同进化尺度下的自适应响应, 以及 F0暴露的跨代效应。在彼得森和al也发现了类似的结果。23
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57450/57450fig4.jpg"> 图 4:Pb 积累在 ( A) 父母 (f0) 和 (B) 未曝光的后代 (f1).在 ( a)和(b)中的条形显示平均值 (ng/成人) ±SEM. 在 (a) 和 (b) 栏上方显示的样本大小。= p < 0.001。(A) 0名成人经口头接触250µM PbAc, 使用本议定书从卵子阶段到 5 d 岁后羽化, 并收集6天后羽化 (24 小时净化) 后, 使用 ICP-MS.42(B) 进行 Pb 堆积试验。0例成人在控制培养基治疗中交配。从卵期到成年 (使用本协议) 在控制培养基中饲养未暴露的 F1子代, 并采用 ICP-MS 进行铅积累试验。在 (B): "cf + cm"= f1成人与父母在控制媒介饲养, "CF + PbM" = f1个成人与父亲在含铅的媒介饲养, "PbF + CM" = f1个成人与母亲在含铅的媒介饲养, "PbF + PbM" = f1成人与父母在含铅培养基饲养。这个数字已经从彼得森等。24<a href="/files/ftp_upload/57450/57450fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a> 
表 1和图 4所示的结果表明,果蝇在不同剂量、发育阶段和使用本议定书的进化尺度上都能很容易地积累 Pb。因此, 这表明协议的有效性, 揭露D.
在图 5中, 这里描述的协议被彼得森等使用。24测试发育铅暴露对交配偏好的影响。实验对象从卵期到成年, 在控制或含铅培养基从卵阶段到成年期, 并在24小时净化后测试交配首选。彼得森等。24发现铅暴露的女性优先交配与铅暴露的男性, 当考虑到一个控制或铅暴露的男性的选择。这些结果是执行《议定书》以检查表单输出的一个典型例子。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57450/57450fig5.jpg"> 图 5:男性和女性的交配偏好在250µM PbAc 从卵子阶段到成年.酒吧在 (A), (B) 和 (C) 显示平均百分比 (%) 交配成功 (在60分钟) 的电子显微镜. ** = p < 0.001. * = p < 0.05。实验对象 (A), (B) 和 (C) 被暴露在控制或含铅培养基 (250 µM PbAc) 从卵子阶段到成熟成年和测试的差异, 配偶选择。(A)女性偏爱控制或 Pb 饲养的雄性 (即两种选择试验)。样品大小是: N = 126 控制饲养的女性和 137 Pb 饲养的女性。(B)男性偏爱控制和铅饲养的雌性 (即两种选择试验)。样品大小是: N = 59 控制饲养的男性和 N = 64 Pb 饲养的男性。(C)男性和女性的交配偏好, 当单独配对一个伴侣的任何接触 (即无选择测试)。在 (C): "CF + CM" = 一个控制饲养的女性配对与一个控制饲养的男性 (N = 85 对), "CF + PbM" = 一个控制饲养的女性配对与一个铅饲养男性 (n = 79 对), "PbF + CM" = 一 Pb 饲养的女性配对与一个控制饲养的男性 (N = 91 对), "PbF +PbM "= 一 pb 饲养的女性 + 一 pb 饲养的男性 (N = 98 对)。这个数字已经从彼得森等。24.<a href="/files/ftp_upload/57450/57450fig5large.jpg" target="_blank">请点击这里查看这个数字的更大版本.</a>

Watch this Scientific Journal Video about Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory at JoVE.com

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

在本文中, 我们提供了一个详细的协议, 以暴露在果蝇属物种的污染物, 目的是研究暴露对一系列表型产出的影响, 在不同的发展阶段和超过一个世代。

利用果蝇作为无脊椎动物毒性试验模型系统的实验协议

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ...

experimental-protocol-for-using-drosophila-as-an-invertebrate-model

Nanyang Technological University

Research

JoVE Journal

Biology

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

12.6K 次观看.  Colorado State University-Pueblo. 在本文中, 我们提供了一个详细的协议, 以暴露在果蝇属物种的污染物, 目的是研究暴露对一系列表型产出的影响, 在不同的发展阶段和超过一个世代。

视频: Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

Testing Nicotine Tolerance in Aphids Using an Artificial Diet Experiment

Plants may upregulate the production of many different seconday metabolites in response to insect feeding. One of these metabolites, nicotine, is well know to have insecticidal properties. One response of tobacco plants to herbivory, or being gnawed upon by insects, is to increase the production of this neurotoxic alkaloid. Here, we will demonstrate how to set up an experiment to address this question of whether a tobacco-adapted strain of the green peach aphid, Myzus persicae, can tolerate higher levels of nicotine than the a strain of this insect that does not infest tobacco in the field.

In this video and assay is demonstrated that tests the tolerance of nicotine to two types of aphids one that infests tobacco plants in the field and one that does not.

使用人工饲料实验测试蚜虫的尼古丁公差

Plants may upregulate the production of many different seconday metabolites in response to insect feeding. One of these metabolites, nicotine, is well ...

科研

生物学

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

编制果蝇实时成像的胚胎使用悬滴协议

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

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 ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

A Protocol for Using Gene Set Enrichment Analysis to Identify the Appropriate Animal Model for Translational Research

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison techniques resulted in contradictory conclusions regarding the relevance of animal models for translational research. A major reason for the discrepancies between different gene expression analyses is the arbitrary filtering of differentially expressed genes. Furthermore, the comparison of single genes between different species and platforms often is limited by technical variance, leading to misinterpretation of the con/discordance between data from human and animal models. Thus, standardized approaches for systematic data analysis are needed. To overcome subjective gene filtering and ineffective gene-to-gene comparisons, we recently demonstrated that gene set enrichment analysis (GSEA) has the potential to avoid these problems. Therefore, we developed a standardized protocol for the use of GSEA to distinguish between appropriate and inappropriate animal models for translational research. This protocol is not suitable to predict how to design new model systems a-priori, as it requires existing experimental omics data. However, the protocol describes how to interpret existing data in a standardized manner in order to select the most suitable animal model, thus avoiding unnecessary animal experiments and misleading translational studies.

We provide a standardized protocol for the use of gene set enrichment analysis of transcriptomic data to identify an ideal mouse model for translational research. 
This protocol can be used with DNA microarray and RNA sequencing data and can further be extended to other omics data if data are available.

一个协议来使用基因集富集分析以确定合适的动物模型转化型研究

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison ...

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

用基团特异 rDNA 引物对水生无脊椎动物进行遗传肠道含量分析的实验室规程

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...

Establishment of an Extracellular Acidic pH Culture System

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

胞外酸性 pH 培养体系的建立

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...

A Protocol for Laboratory Housing of Turquoise Killifish (Nothobranchius furzeri)

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of reference fish model systems, such as zebrafish and medaka. In recent years, an emerging fish &#8211; the turquoise killifish (Nothobranchius furzeri) &#8211; has been adopted by a growing number of research groups in the fields of biology of aging and ecology. With a captive life span of 4 - 8 months, this species is the shortest-lived vertebrate raised in captivity and allows the scientific community to test &#8211; in a short time &#8211; experimental interventions that can lead to alterations of the aging rate and life expectancy. Given the unique biology of this species, characterized by embryonic diapause, explosive sexual maturation, marked morphological and behavioral sexual dimorphism - and their relatively short adult life span -&#160;ad hoc husbandry practices are in urgent demand. This protocol reports a set of key husbandry measures that allow optimal turquoise killifish laboratory care, enabling the scientific community to adopt this species as a powerful laboratory animal model.

A Protocol for Laboratory Housing of Turquoise Killifish Nothobranchius furzeri

Laboratory housing of turquoise killifish can be scaled up&#160;to house and efficiently raise thousands of individual fish in a centralized water filtration system, employing the same infrastructure used for standard zebrafish facilities. Here we detail a list of standardized procedures that allow efficient killifish maintenance.

绿松石鳉鱼实验室外壳的协议 (Nothobranchiusfurzeri)

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of ...

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

新型杀虫化学对蚊子毒性测试规程

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...

An Air-liquid Interface Bronchial Epithelial Model for Realistic, Repeated Inhalation Exposure to Airborne Particles for Toxicity Testing

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic exposure conditions. This puts specific demands on the cell culture models. Many cell types are negatively affected by exposure to air (e.g., drying out) and only remain viable for a few days. This limits the exposure conditions that can be used in these models: usually relatively high concentrations are applied as a cloud (i.e., droplets containing particles, which settle down rapidly) within a short period of time. Such experimental conditions do not reflect realistic long-term exposure to low concentrations of particles. To overcome these limitations the use of a human bronchial epithelial cell line, Calu-3 was investigated. These cells can be cultured at ALI conditions for several weeks while retaining a healthy morphology and a stable monolayer with tight junctions. In addition, this bronchial model is suitable for testing the effects of repeated exposures to low, realistic concentrations of airborne particles using an ALI exposure system. This system uses a continuous airflow in contrast to other ALI exposure systems that use a single nebulization producing a cloud. Therefore, the continuous flow system is suitable for repeated and prolonged exposure to airborne particles while continuously monitoring the particle characteristics, exposure concentration, and delivered dose. Taken together, this bronchial model, in combination with the continuous flow exposure system, is able to mimic realistic, repeated inhalation exposure conditions that can be used for toxicity testing.

Described is the cell culture and exposure method of an in vitro bronchial model for realistic, repeated inhalation exposure to particles for toxicity testing.

用于真实、反复吸入空气颗粒以进行毒性测试的气液接口支气管上皮模型

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic ...

利用果蝇作为无脊椎动物毒性试验模型系统的实验协议

摘要

探索更多视频

此视频中的章节

使用人工饲料实验测试蚜虫的尼古丁公差

编制果蝇实时成像的胚胎使用悬滴协议

致命的甲壳类动物长期毒性试验卤虫franciscana

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

一个协议来使用基因集富集分析以确定合适的动物模型转化型研究

用基团特异 rDNA 引物对水生无脊椎动物进行遗传肠道含量分析的实验室规程

胞外酸性 pH 培养体系的建立

绿松石鳉鱼实验室外壳的协议 (Nothobranchiusfurzeri)

新型杀虫化学对蚊子毒性测试规程

用于真实、反复吸入空气颗粒以进行毒性测试的气液接口支气管上皮模型