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Biologia

生存时间的测量<em>臂</em>轮虫：产妇条件同步

Published: July 22, 2016
doi:
10.3791/54126
, , , ,
1Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences,The University of Tokyo, 2School of Marine Biosciences, Department of Marine Biosciences,Kitasato University, 3Josephine Bay Paul Center for Comparative Molecular Biology and Evolution,Marine Biological Laboratory, 4School of Arts and Sciences,University of Houston-Victoria

Summary

Rotifers are microscopic zooplankton used as models in ecotoxicological and aging studies. Here we provide a protocol for powerful and reproducible measurement of survival time in Brachionus rotifers. Synchronization of culture conditions over several generations is of particular importance because maternal condition affects life history of offspring.

Abstract

Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short lifespan, ease of culture, and parthenogenesis that enables clonal culture. However, caution is required when measuring their survival time as it is affected by maternal age and maternal feeding conditions. Here we provide a protocol for powerful and reproducible measurement of the survival time in Brachionus rotifers following a careful synchronization of culture conditions over several generations. Empirically, poor synchronization results in early mortality and a gradual decrease in survival rate, thus resulting in weak statistical power. Indeed, under such conditions, calorie restriction (CR) failed to significantly extend the lifespan of B. plicatilis although CR-induced longevity has been demonstrated with well-synchronized rotifer samples in past and present studies. This protocol is probably useful for other invertebrate models, including the fruitfly Drosophila melanogaster and the nematode Caenorhabditis elegans, because maternal age effects have also been reported in these species.

Introduction

轮虫是微观世界性浮游动物（<1毫米）构成门轮虫1。它们具有约1,000的体细胞，以及一个特性轮状睫状装置构成的简单的身体计划称为电晕，其用于运动和进纸。大多数轮虫属于类Monogononta或玄武湖的轮虫，其中包含约1600和500种，分别为2。 Monogonont轮虫一般同时具有有性和无性繁殖阶段（周期性孤雌生殖），而蛭形轮虫繁殖通过强制性孤雌生殖3。因此，有可能获得基因完全相同轮虫个人，从而确保在实验高再现性。此外，它们具有其它几个优点模式生物，如短寿命，易于培养，基因组和转录组序列数据4-7的可用性，以及独特的系统位置远离一rthropods和线虫8。因此轮虫是有希望在生态毒理学无脊椎动物模型和老化研究9-12。

下暴露于环境压力或化学品的生存时间是在这些研究领域一个13-19经常测量参数。但是，衡量轮虫的存活时间的时候，因为很容易受到他们的母亲的环境条件是需要谨慎。也就是说，在monogonont 臂尾轮虫manjavacas，雌性后代从母亲年龄比那些年轻的妈妈寿命较短;然而，产妇热量限制（CR），部分抵消了先进的孕产妇20岁的有害影响。在B.轮虫 ，母亲CR提供后代长寿，饥饿状态下存活时间长，并与抗氧化酶21,22表达增强相关的高氧化应激性。产妇年龄效应也已在蛭形轮虫23观察到。因此，实验轮虫的条件应仔细的存活时间测量同步前几代。

在这里，我们提供了以下几代的培养条件同步的轮虫臂存活时间测量的协议。间歇禁食（IF），其中轮虫周期性馈送CR的变化，施加于揭示的同步的效果由于中频对长寿22,24众所周知的效果。

Protocol

1.媒体的制备注意：使用盐度16.5个百分点（PSU）的一半稀释Brujewicz人造海水。其他人工海水也经常用于培养轮虫臂 25,26。 加454毫摩尔NaCl，26毫的MgCl 2，27毫硫酸镁 ，10毫米氯化钾和10mM的CaCl 2到4.5升蒸馏水（最终体积为5升）。另外，使用去离子水稀释代替蒸馏水。溶解所有其他盐后添加氯化钙 。 准备0.48中号?…

Representative Results

图1显示了同步较差人群（出两个重复的）的代表生存曲线。在这个实验中，轮虫要么美联储日常[ 自由采食 （AL）组]或隔日（IF组）。中位生存期在美联和IF组13和18日，分别。虽然这是众所周知的，如果延伸轮虫的寿命，本实验未能检测到的AL和IF组的寿命之间存在统计学显著差异。根据经验，在早期死亡率和生存率下降逐渐同步不足的结果在这个…

Discussion

目前的协议描述了测量在臂轮虫的存活时间的方法。关键的步骤是轮虫条件几代同步。当实验轮虫以及同步的，一个典型的I型的生存曲线是用很少的早期死亡率观察到几个以前的研究报告18,24,37,38。相比较差同步轮虫的存活时间的标准偏差因此变得更小，从而导致高的统计力量。同步也有望增加存活时间测量的再现 – 因为母亲在最佳条件下培养时，当前协议抵消母体世代的可能的?…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

我们感谢乔治·贾维斯，玛莎博克和贝特Hecox – 李，海洋生物实验室，他们在拍戏的帮助。

Materials

Sodium chloride Wako 190-13921
Magnesium chloride Wako 136-03995
Magnesium sulfate Wako 131-00427
Potassium chloride Wako 168-22111
Calcium chloride Wako 035-00455
Sodium bicarbonate Wako 199-05985
Sodium bromide Wako 190-01515
Membrane filter (0.45 µm pore size) Millipore HAWP04700
Culture plate, 6-well, non-treated Thomas Scientific 6902D01 Flat bottom
Culture plate, 48-well, non-treated Thomas Scientific 6902D07 Flat bottom
Tetraselmis, Living Carolina Biological Supply Company 152610
PRISM 6 GraphPad Software Version 6.0d
DOWNLOAD MATERIALS LIST

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Tags

Brachionus RotifersSurvival TimeSynchronizationMaternal ConditionsAging ResearchArtificial SeawaterMicro AlgaeNeonateCohortCulture Medium
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Kaneko, G., Yoshinaga, T., Gribble, K. E., Welch, D. M., Ushio, H. Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions. J. Vis. Exp. (113), e54126, doi:10.3791/54126 (2016).
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