JoVE Logo
发表
联系我们

登录

需要订阅 JoVE 才能查看此. 登录或开始免费试用。

本文内容

  • 摘要
  • 摘要
  • 引言
  • 研究方案
  • 结果
  • 讨论
  • 披露声明
  • 致谢
  • 材料
  • 参考文献
  • 转载和许可

摘要

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.

摘要

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.

引言

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。

Access restricted. Please log in or start a trial to view this content.

研究方案

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。

  1. 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 。
  2. 准备0.48中号碳酸氢钠原液（200倍浓度）。将25ml添加到上述溶液中。的NaHCO 3的最终浓度为2.4毫摩尔。
  3. 制备0.4M的溴化钠储备液（500X浓度）。加入10ml其添加到上述溶液中。溴化钠的最终浓度为0.8毫摩尔。补至5升蒸馏水。
  4. 过滤用0.45μm的膜过滤器的溶液。在使用前用无菌水两次稀释（V / V）。
    注意:可以使Brujewicz人工海水的2倍浓度作为原液。

2.一般培养条件

  1. 培养实验室饲养或在20和30℃之间的无菌100毫升烧杯野生捕获轮虫。较高的温度导致寿命较短，加速繁殖。使用25℃下的实验的方便性。轮虫密度或人工海水量是不是在这里一个大问题。
  2. 文化饮食微藻在人工海水中11。见斯内尔等人 （2014年）的详细信息11。通常情况下，使用四爿tetrathele（〜2×10 5个细胞/ ml 27,28），T。 suecica（〜6×10 5个细胞/ ml 25,29），和微绿球藻 （〜7×10 6个细胞/ ml 30,31），由于饮食藻（种类，培养康迪特离子，生化成分）显著影响轮虫臂尾轮虫的存活时间，用同一地段微藻各实验组。
  3. 喂养偶尔改变介质（ 如 ，喂，每2天，每周接种）保持在分批培养轮虫的股票群体。许多新生儿和成人轴承鸡蛋2-3个可以在轮虫是在最佳条件下进行观察。
    注:另一种方式来获得实验轮虫是孵化休眠卵。从休眠卵孵出轮虫被认为是很好地同步，并且他们的寿命是不从来自amictic鸡蛋11孵出轮虫显著不同。然而，从休眠卵轮虫早于从amictic鸡蛋开始再现。因此，需要对它们的繁殖性状测量慎用。

3.同步轮虫通过预培养

  1. 选择从ST单轮虫玉珠人口和培养在2.1-2.3如上所述建立一个亚群将被用于实验。通常培养两周。
  2. 收集来自亚群蛋承载轮虫（收集将用于实验的个体的双号）。培养它们作为一个单一的队列（密度:〜50个/毫升），在新鲜的介质下自由采食在2.1-2.3中描述的6孔培养板中。控制人口密度是重要的，因为条件培养基影响轮虫臂 32,33的生殖生理。
  3. 转移从成人的第一个卵孵化到新制备培养基新生儿。重复此过程在2-3代。
  4. 使用用于测量生存时间的特定时间段（ 例如 ，<3小时）内画影线的新生儿。为了避免可能偏向于选择单一的个体，虽然很不可能的，用几个不知疲倦重复性检查侧亚群。
    注:由于轮虫繁殖通常只无性这种情况下，确保无男性是整个实验过程中存在。男性比新生儿小，典型的移动比女性更快。混交雌体具有amictic女性在一定条件下29种不同的寿命。

4.生存时间的测量

  1. 在塑料板代替新生儿（通常24或48孔板，每孔含有1ml人工海水）。
  2. 在24小时的时间间隔，如果在较高的温度（30℃以上）传送轮虫到新制备的培养基，或在12小时的时间间隔。记录的后代的数量和每一个人无论是死是活。记录轮虫死时电晕的纤毛运动完全停止。
    注意:轮虫经常附着到孔的侧壁。水的轻柔吹打有助于找到他们。如果轮虫找不到或ArË小心被移液破坏，记录他们为"封杀"，而不是"死"。
  3. 新生儿取出实验时，轮虫正在积极重现。新生儿快速增长，这是有时很难从实验轮虫区分。

5.数据分析

  1. 创建Kaplan-Meier生存曲线（ 图1和2）通过在Y轴和时间在X轴上标绘的累积存活率。这是存活数据的最常见的代表。使用的存活时间34统计比较的非参数数秩检验（也称为曼特尔-考克斯测试）。对数秩检验也被包括在其他标准统计软件包如JMP和R.
    注:请不要使用学生t检验或后面的参数多重比较方差分析的分析，因为正态分布通常不是由生存数据35满足。此外，这些方法具DS不采取截尾的个人考虑。如果没有删数据曼 - 惠特尼U检验都可以使用。

Access restricted. Please log in or start a trial to view this content.

结果


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...

Access restricted. Please log in or start a trial to view this content.

讨论

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?...

Access restricted. Please log in or start a trial to view this content.

披露声明

The authors have nothing to disclose.

致谢

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。

Access restricted. Please log in or start a trial to view this content.

材料

NameCompanyCatalog NumberComments
Sodium chlorideWako190-13921
Magnesium chlorideWako136-03995
Magnesium sulfateWako131-00427
Potassium chlorideWako168-22111
Calcium chlorideWako035-00455
Sodium bicarbonateWako199-05985
Sodium bromideWako190-01515
Membrane filter (0.45 µm pore size)MilliporeHAWP04700
Culture plate, 6-well, non-treatedThomas Scientific6902D01Flat bottom
Culture plate, 48-well, non-treatedThomas Scientific6902D07Flat bottom
Tetraselmis, LivingCarolina Biological Supply Company152610
PRISM 6GraphPad SoftwareVersion 6.0d

参考文献

  1. Wallace, R. L., Snell, T. W., Ricci, C., Nogrady, T. Rotifera Vol.1: Biology, ecology and systematics. , 2nd edn, SPB Academic Publishing. (2006).
  2. Segers, H. Annotated checklist of the rotifers (Phylum Rotifera), with notes on nomenclature, taxonomy and distribution. , Magnolia Press. Auckland. (2007).
  3. Mark Welch, D. B., Meselson, M. Evidence for the evolution of bdelloid rotifers without sexual reproduction or genetic exchange. Science. 288 (5469), 1211-1215 (2000).
  4. Suga, K., Mark Welch, D., Tanaka, Y., Sakakura, Y., Hagiwara, A. Analysis of expressed sequence tags of the cyclically parthenogenetic rotifer Brachionus plicatilis. PLoS ONE. 2, e671(2007).
  5. Denekamp, N. Y., et al. Discovering genes associated with dormancy in the monogonont rotifer Brachionus plicatilis. BMC Genomics. 10, 108(2009).
  6. Lee, J. -S., et al. Sequence analysis of genomic DNA (680 Mb) by GS-FLX-Titanium sequencer in the monogonont rotifer, Brachionus ibericus. Hydrobiologia. 662 (1), 65-75 (2010).
  7. Flot, J. -F., et al. Genomic evidence for ameiotic evolution in the bdelloid rotifer Adineta vaga. Nature. 500 (7463), 453-457 (2013).
  8. Dunn, C. W., et al. Broad phylogenomic sampling improves resolution of the animal tree of life. Nature. 452 (7188), 745-749 (2008).
  9. Yoshinaga, T., Kaneko, G., Kinoshita, S., Tsukamoto, K., Watabe, S. The molecular mechanisms of life history alterations in a rotifer: a novel approach in population dynamics. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 136 (4), 715-722 (2003).
  10. Dahms, H. -U., Hagiwara, A., Lee, J. -S. Ecotoxicology, ecophysiology, and mechanistic studies with rotifers. Aquat. Toxicol. 101 (1), 1-12 (2011).
  11. Snell, T. W. Rotifers as models for the biology of aging. Int. Rev. Hydrobiol. 99 (1-2), 84-95 (2014).
  12. Snell, T. W., Johnston, R. K., Gribble, K. E., Mark Welch, D. B. Rotifers as experimental tools for investigating aging. Invertebr. Reprod. Dev. 59, Suppl 1. 5-10 (2015).
  13. Kaneko, G., et al. Molecular characterization of Mn-superoxide dismutase and gene expression studies in dietary restricted Brachionus plicatilis rotifers. Hydrobiologia. 546, 117-123 (2005).
  14. Yoshinaga, T., et al. Insulin-like growth factor signaling pathway involved in regulating longevity of rotifers. Hydrobiologia. 546, 347-352 (2005).
  15. Ozaki, Y., Kaneko, G., Yanagawa, Y., Watabe, S. Calorie restriction in the rotifer Brachionus plicatilis enhances hypoxia tolerance in association with the increased mRNA levels of glycolytic enzymes. Hydrobiologia. 649 (1), 267-277 (2010).
  16. Kailasam, M., et al. Effects of calorie restriction on the expression of manganese superoxide dismutase and catalase under oxidative stress conditions in the rotifer Brachionus plicatilis. Fish. Sci. 77 (3), 403-409 (2011).
  17. Garcìa-Garcìa, G., Sarma, S., Núñez-Orti, A. R., Nandini, S. Effects of the mixture of two endocrine disruptors (ethinylestradiol and levonorgestrel) on selected ecological endpoints of Anuraeopsis fissa and Brachionus calyciflorus (Rotifera). Int. Rev. Hydrobiol. 99 (1-2), 166-172 (2014).
  18. Yang, J., Mu, Y., Dong, S., Jiang, Q., Yang, J. Changes in the expression of four heat shock proteins during the aging process in Brachionus calyciflorus (rotifera). Cell Stress Chaperones. 19 (1), 33-52 (2014).
  19. Han, J., et al. Sublethal gamma irradiation affects reproductive impairment and elevates antioxidant enzyme and DNA repair activities in the monogonont rotifer Brachionus koreanus. Aquat. Toxicol. 155, 101-109 (2014).
  20. Gribble, K. E., Jarvis, G., Bock, M., Mark Welch, D. B. Maternal caloric restriction partially rescues the deleterious effects of advanced maternal age on offspring. Aging Cell. 13 (4), 623-630 (2014).
  21. Yoshinaga, T., Hagiwara, A., Tsukamoto, K. Effect of periodical starvation on the survival of offspring in the rotifer Brachionus plicatilis. Fish. Sci. 67 (2), 373-374 (2001).
  22. Kaneko, G., et al. Calorie restriction-induced maternal longevity is transmitted to their daughters in a rotifer. Funct. Ecol. 25 (1), 209-216 (2011).
  23. Lansing, A. I. A transmissible, cumulative, and reversible factor in aging. J. Gerontol. 2 (3), 228-239 (1947).
  24. Yoshinaga, T., Hagiwara, A., Tsukamoto, K. Effect of periodical starvation on the life history of Brachionus plicatilis O. F. Müller (Rotifera): a possible strategy for population stability. J. Exp. Mar. Biol. Ecol. 253 (2), 253-260 (2000).
  25. Gribble, K. E., Kaido, O., Jarvis, G., Mark Welch, D. B. Patterns of intraspecific variability in the response to caloric restriction. Exp. Gerontol. 51, 28-37 (2014).
  26. Snell, T. W., Johnston, R. K. Glycerol extends lifespan of Brachionus manjavacas (Rotifera) and protects against stressors. Exp. Gerontol. 57, 47-56 (2014).
  27. Kim, H. -J., Hagiwara, A. Effect of female aging on the morphology and hatchability of resting eggs in the rotifer Brachionus plicatilis Müller. Hydrobiologia. 662 (1), 107-111 (2011).
  28. Kim, H. -J., et al. Light-dependent transcriptional events during resting egg hatching of the rotifer Brachionus manjavacas. Mar. Genomics. 20, 25-31 (2015).
  29. Gribble, K. E., Welch, D. B. M. Life-span extension by caloric restriction is determined by type and level of food reduction and by reproductive mode in Brachionus manjavacas (Rotifera). J. Gerontol. A Biol. Sci. Med. Sci. 68 (4), 349-358 (2013).
  30. Kaneko, G., Kinoshita, S., Yoshinaga, T., Tsukamoto, K., Watabe, S. Changes in expression patterns of stress protein genes during population growth of the rotifer Brachionus plicatilis. Fish. Sci. 68 (6), 1317-1323 (2002).
  31. Kim, H. J., Sawada, C., Hagiwara, A. Behavior and reproduction of the rotifer Brachionus plicatilis species complex under different light wavelengths and intensities. Int. Rev. Hydrobiol. 99 (1-2), 151-156 (2014).
  32. Yoshinaga, T., Hagiwara, A., Tsukamoto, K. Effect of conditioned media on the asexual reproduction of the monogonont rotifer Brachionus plicatilis O. F. Müller. Hydrobiologia. 412, 103-110 (1999).
  33. Ohmori, F., Kaneko, G., Saito, T., Watabe, S. A novel growth-promoting protein in the conditioned media from the rotifer Brachionus plicatilis at an early exponential growth phase. Hydrobiologia. 667 (1), 101-117 (2011).
  34. GraphPad Statistics Guide. , Available from: http://www.graphpad.com/guides/prism/6/statistics/ (2015).
  35. Collet, D. Modelling Survival Data in Medical Research. , 2nd edn, Chapman & Hall/CRC. 151-193 (1993).
  36. Bouliotis, G., Billingham, L. Crossing survival curves: alternatives to the log-rank test. Trials. 12, Suppl 1. A137(2011).
  37. Yang, J., et al. Changes in expression of manganese superoxide dismutase, copper and zinc superoxide dismutase and catalase in Brachionus calyciflorus during the aging process. PloS ONE. 8 (2), e57186(2013).
  38. Snell, T. W., Johnston, R. K., Rabeneck, B., Zipperer, C., Teat, S. Joint inhibition of TOR and JNK pathways interacts to extend the lifespan of Brachionus manjavacas (Rotifera). Exp. Gerontol. 52, 55-69 (2014).
  39. Klass, M. R. Aging in nematode Caenorhabditis-elegans - major biological and environmental-factors influencing life-span. Mech. Ageing Dev. 6 (6), 413-429 (1977).
  40. Priest, N. K., Mackowiak, B., Promislow, D. E. L. The role of parental age effects on the evolution of aging. Evolution. 56 (5), 927-935 (2002).

Access restricted. Please log in or start a trial to view this content.

转载和许可

请求许可使用此 JoVE 文章的文本或图形

请求许可

探索更多文章

113 Monogononta

This article has been published

Video Coming Soon

JoVE Logo

政策

使用条款

隐私

联系我们

向图书馆推荐

JoVE 新闻简报

科研

教育

发表

图书馆员

关于 JoVE

版权所属 © 2025 MyJoVE 公司版权所有，本公司不涉及任何医疗业务和医疗服务。