利用果蝇作为无脊椎动物毒性试验模型系统的实验协议
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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,<sup cl…
Representative Results
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…
Discussion
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,<…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Carolina Biological Instant Drosophila Medium Formula 4-24 | Carolina Biological | 173204 | |
| Drosophila vials, Narrow (PS), Polystyrene, Superbulk, 1000 vials/unit | Genessee Scientific | 32-116SB | Used to store flies |
| Flugs Closures for vials and bottles, Narrow plastic vials | Genessee Scientific | 49-102 | Used to store flies |
| Cardboard trays, trays only, narrow | Genessee Scientific | 32-124 | Used to organize populations of flies |
| Cardboard trays, dividers only, narrow | Genessee Scientific | 32-126 | Used to organize populations of flies |
| Thermo Scientific Nalgene Square Wide-Mouth HDPE Bottles with Closure | Fischer Scientific | 03-312D | Useful for storage of contaminants |
| Thermo Scientific Nalgene Color-Coded LDPE Wash Bottles | Fischer Scientific | 03-409-17C | Useful for storage of contaminants |
| Eppendorf Repeater M4 Manual Handheld Pipette Dispenser | Fischer Scientific | 14-287-150 | Used to prepare medium |
| Combitips Advanced Pipetter Tips – Standard, Eppendorf Quality Tips | Fischer Scientific | 13-683-708 | Used to prepare medium |
| Flypad, Standard Size (8.1 X 11.6cm) | Genessee Scientific | 59-114 | Used to anesthetize flies |
| Flystuff foot valve | Genessee Scientific | 59-121 | Used to anesthetize flies |
| Tubing, green (1 continguous foot/unit) | Genessee Scientific | 59-124G | Used to anesthetize flies |
| Mineral Oil, Light, White, High Purity Grade, 500 mL HDPE Bottle | VWR | 97064-130 | Used to make a morgue |
| Glass Erlenmeyer Flask Set – 3 Sizes – 50, 150 and 250ml, Karter Scientific 214U2 | Walmart | Not applicable | Used to make a morgue |
| BGSET5 Glass Beaker Set Of 5 | Walmart | ||
| Inbred or wildtype line of Drosophila | Bloomington Drosophila Stock Center at Indiana University | https://bdsc.indiana.edu | |
| Wild popultions of Drosophila | UC San Diego Drosophila Stock Center | https://stockcenter.ucsd.edu/info/welcome.php |
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Citazione di questo articolo
Peterson, E. K., Long, H. E. Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory. J. Vis. Exp. (137), e57450, doi:10.3791/57450 (2018).