Name: measurement of survival time in brachionus rotifers: synchronization of maternal conditions
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Description: Watch this Scientific Journal Video about Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions at JoVE.com

Wir danken George Jarvis, Martha Bock und Bette Hecox-Lea, Marine Biological Laboratory, für ihre Hilfe bei der Verfilmung.

1. Herstellung von Medien Hinweis: Verwenden Sie Halb verdünnt Brujewicz künstlichem Meerwasser Salzgehalt 16,5 ppt (PSU). Andere künstliche seawaters werden auch häufig verwendet , um Kultur Brachionus rotifers 25,26. <ol><li> Hinzufügen 454 mM NaCl, 26 mM MgCl 2, 27 mM MgSO 4, 10 mM KCl, und 10 mM CaCl 2 bis 4,5 L destilliertem Wasser (Endvolumen 5 wird L sein). Alternativ dazu können Sie entsalztem Verdünnungswasser anstelle von destilliertem Wasser. In CaCl 2 , nachdem alle anderen Salze aufzulösen. </li><li> Bereiten Sie die 0,48 M NaHCO 3 - Stammlösung (200x Konzentration). 25 ml davon zu der obigen Lösung. Die Endkonzentration von NaHCO 3 beträgt 2,4 mM. </li><li> Bereiten Sie die 0,4 M NaBr-Stammlösung (500x Konzentration). 10 ml davon zu der obigen Lösung. Die Endkonzentration von NaBr 0,8 mM. Machen Sie bis zu 5 L mit destilliertem Wasser. </li><li> Filtern der Lösung mit einem 0,45 um-Membranfilter.Verdünnen zweimal mit sterilem Wasser vor der Verwendung (v / v). Hinweis: Es ist möglich, eine 2x Konzentration von Brujewicz künstlichem Meerwasser als Stammlösung zu machen. </li></ol> 2. Allgemeine Kulturbedingungen <ol><li> Kultur im Labor erhöht oder Wild gefangen Rotatorien in einem sterilen 100-ml-Becher zwischen 20 und 30 ° C. Höhere Temperaturen führen zu kürzeren Lebensdauer und eine beschleunigte Wiedergabe. Verwenden 25 ° C für die Bequemlichkeit der Experimente. Die rotifer Dichte oder Volumen von künstlichem Meerwasser ist kein großes Problem hier. </li><li> Kultur Nahrungs Mikroalge Spezies in künstlichem Meerwasser 11. Siehe Snell et al. (2014) für weitere Einzelheiten 11. Typischerweise verwenden Tetraselmis tetrathele (~ 2 x 10 5 Zellen / ml 27,28), T. suecica (~ 6 x 10 5 Zellen / ml 25,29) und Nannochloropsis oculata (~ 7 x 10 6 Zellen / ml 30,31). Da Diätalgen (Arten, Kultur conditIonen, biochemische Zusammensetzungen) , beeinflusst maßgeblich die Überlebenszeit von Brachionus Rotatorien, die gleiche Menge Mikroalge in allen Versuchsgruppen verwenden. </li><li> Halten Sie eine Stammpopulation von Rotatorien in einer Batch - Kultur durch die Fütterung und wechselnden Medien gelegentlich (zB füttern alle 2 Tage und impfen jede Woche). Viele Neugeborene und Erwachsene 2-3 Eier tragen, können beobachtet werden, wenn Rotatorien unter optimalen Bedingungen sind. Hinweis: Eine alternative Möglichkeit, experimentelle Rotatorien zu erhalten, ist Dauereier auszubrüten. Rotifers aus ruhenden Eier ausgebrütet werden als gut synchronisiert werden, und ihre Lebensdauer ist nicht signifikant verschieden von der aus amictic Eier 11 schraffiert Rotatorien. Allerdings rotifers aus ruhenden Eier beginnen Reproduktion früher als die aus amictic Eier. Daher ist Vorsicht für Messungen ihrer Reproduktionsmerkmale erforderlich. </li></ol> 3. Synchronisation von Rotifers von Pre-Kultur <ol><li> Wählen Sie einen einzelnen rotifer aus dem stock Bevölkerung und Kultur wie in 2,1-2,3 beschrieben eine Subpopulation zu etablieren, die für Experimente verwendet werden. Typischerweise Kultur für zwei Wochen. </li><li> Sammeln egg tragenden Rotatorien vom Subpopulation (eine doppelte Anzahl von Personen zu sammeln, die für das Experiment verwendet wird). Kultur als eine einzige Kohorte (Dichte: ~ 50 Personen / ml) in einer 6-Well - Kulturplatte wie in 2,1-2,3 in einem frischen Medien unter Ad - libitum - Fütterung beschrieben. Controlling Bevölkerungsdichte ist wichtig , weil das konditionierte Medium 32,33 Fortpflanzungsphysiologie von Brachionus rotifers beeinflusst. </li><li> Übertragen ausgebrütet Neugeborenen von der ersten Eier &quot;Erwachsene neu vorbereitet Kulturmedien. Wiederholen Sie diese Prozedur über 2-3 Generationen. </li><li> Verwenden innerhalb eines bestimmten Zeitraums schraffierten Neugeborenen (zB &lt;3 h) , um die Überlebenszeit gemessen wird . Um zu vermeiden, eine mögliche Verzerrung des einzelnen Individuums bei der Auswahl, wenn auch sehr unwahrscheinlich, überprüfen Reproduzierbarkeit unter Verwendung mehrerer independent Subpopulationen. Hinweis: Da Rotatorien der Regel nur ungeschlechtlich unter dieser Bedingung zu reproduzieren, sicherzustellen, dass keine Männchen während des gesamten Experiments vorhanden sind. Die Männchen sind kleiner als Neugeborene und in der Regel bewegen sich schneller als Frauen. Mictic Weibchen haben unterschiedliche Lebensdauern von amictic Weibchen unter bestimmten Bedingungen 29. </li></ol> 4. Messungen der Überlebenszeit <ol><li> Ort Neugeborenen in Kunststoffplatten (typischerweise 24- oder 48-Well-Platten, wobei jede Vertiefung mit 1 ml künstlichem Meerwasser). </li><li> Nach 24 h Intervallen, übertragen Sie die Rotatorien neu vorbereitet Kulturmedien oder in 12 Stunden Intervallen, wenn bei höherer Temperatur (30 ° C oder höher). Notieren Sie die Anzahl der Nachkommen und ob jeder einzelne ist tot oder lebendig. Notieren Sie sich die rotifer als tot, wenn Zilien Bewegung der Korona vollständig zum Stillstand gekommen ist. Anmerkung: Rotifers befestigen häufig an den Seitenwänden der Vertiefungen. Vorsichtiges Pipettieren des Wassers hilft, sie zu finden. Wenn rotifers nicht gefunden oder are versehentlich durch Pipettieren beschädigt, sie aufzunehmen als &quot;zensiert&quot;, nicht als &quot;tot&quot;. </li><li> Entfernen Neugeborenen, wenn experimentelle rotifers aktiv reproduzieren sind. Neugeborenen wachsen schnell und es ist manchmal schwierig, sie aus experimentellen Rotatorien zu unterscheiden. </li></ol> 5. Datenanalyse <ol><li> Erstellen Sie die Kaplan-Meier - Überlebenskurve (Abbildungen 1 und 2) durch kumulative Überlebensrate auf der Y - Achse und die Zeit auf der X - Achse aufgetragen ist . Dies ist die häufigste Darstellung der Überlebensdaten. Verwenden Sie den nicht-parametrischer Log-Rank - Test auf statistische Vergleich der Überlebenszeit 34 (auch Mantel-Cox - Test genannt). Die Log-Rank-Test wird auch in anderen statistischen Standardpakete wie JMP und R. enthalten Hinweis: da Normalverteilung 35 durch das Überleben Daten erfüllt in der Regel nicht nicht Student-t-Test oder Varianzanalyse (ANOVA) , gefolgt von parametrischer mehrere Vergleich verwenden. Auch sind diese methods nehmen nicht zensiert Personen berücksichtigt. Mann-Whitney U-Test kann verwendet werden, wenn es keine zensierten Daten ist. </li></ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+parental+age+effects+on+the+evolution+of+aging.">The role of parental age effects on the evolution of aging.</a> Evolution. 56 (5), 927-935 (2002).</li></ol>

The authors have nothing to disclose.

Das aktuelle Protokoll beschreibt ein Verfahren zur in Brachionus Rotatorien die Überlebenszeit gemessen wird . Der entscheidende Schritt ist die Synchronisation von rotifer Bedingungen über mehrere Generationen. Bei experimentellen rotifers gut synchronisiert sind, ist eine typische Typ - I - Überlebenskurve mit sehr wenig frühe Sterblichkeit beobachtet , wie 18,24,37,38 in mehreren früheren Studien berichtet. Die Standardabweichungen der Überlebenszeit werden deshalb kleiner im Vergleich zu schlecht synchronisierten Rotatorien, in hoher statistischer Aussagekraft zur Folge hat. Die Synchronisation wird auch die Reproduzierbarkeit der Überlebenszeitmessungen zu erhöhen erwartet - weil Mütter unter optimalen Bedingungen kultiviert werden, die aktuelle Protokoll Offsets möglich schädlichen Auswirkungen der mütterlichen Generationen. Wenn frühe Sterblichkeit noch nach sorgfältiger Synchronisation beobachtet wird, sollten Sie neu vorbereitet Kulturmedien verwenden, eine andere Menge Fütterung Algen, oder eine neu gegründete experimentelle Kohorte (dhStart von Protokoll 3.1). Eine Einschränkung dieses Protokolls besteht darin, dass die gut synchronisiert Rotatorien sind potentiell überempfindlich. Zum Beispiel beim Screening von Chemikalien , die Lebensdauer verlängern, einige durch dieses Protokoll gescreent Chemikalien versagen kann erhebliche Auswirkungen auf die Lebensdauern von schlecht synchronisierten Rotatorien (zB Personen aus wilden und Batch-kultivierten Populationen) zu erkennen. Somit sollten die Ergebnisse dieser Versuche mit Vorsicht interpretiert werden. Die Wirkung des mütterlichen Alters auf die Nachkommen , die Überlebenszeit wurde auch in anderen wirbellosen Modelle einschließlich der Fruchtfliege Drosophila melanogaster und dem Nematoden Caenorhabditis elegans 39,40 berichtet. Obwohl es zeitraubend in dieser langlebigen Modelle ist, wäre die Synchronisationsprozedur über mehrere Generationen, die für diese Tiere in der Messung der Überlebenszeit experimentellen Schwankungen zu verringern.

Rotifers sind mikroskopisch kleine kosmopolitische Zooplankton (&lt;1 mm), die die phylum Rotatorier 1 darstellen. Sie haben eine einfache Körperplan von ca. 1.000 somatischen Zellen besteht, sowie eine charakteristische radähnlichen ciliary Vorrichtung die corona genannt, die für die Fortbewegung und Fütterung verwendet wird. Die meisten rotifers gehören zu den Klassen Monogononta oder Bdelloida, die etwa 1600 und 500 Arten enthalten jeweils 2. Monogononta rotifers haben in der Regel sowohl sexuelle und asexuelle Reproduktionsphasen (zyklische Parthenogenese), während Bdelloid Rotatorien durch obligatorische Parthenogenese 3 reproduzieren. Es ist somit möglich, genetisch identische rotifer Individuen zu erhalten, die in Experimenten eine hohe Reproduzierbarkeit gewährleistet. Darüber hinaus haben sie mehrere andere Vorteile als Modellorganismen, wie beispielsweise eine kurze Lebensdauer, Einfachheit der Kultur, die Verfügbarkeit von genomischen und transkriptomischen Sequenzdaten 4-7 und eine einzigartige phylogenetische Position entfernt von a8 rthropods und Nematoden. Rotifers versprechen daher invertebrate Modelle in ökologische, toxikologische und Alterungsuntersuchungen 12.09. Die Überlebenszeit unter der Exposition gegenüber Umweltstress oder Chemikalien ist ein häufig gemessene Parameter in diesen Forschungsfeldern 13-19. Allerdings ist Vorsicht geboten, wenn die Überlebenszeit von Rotatorien Messung, weil sie anfällig gegenüber Umweltbedingungen ihrer Mütter ist. Und zwar in der Monogononta rotifer Brachionus Manjavacas, weiblichen Nachkommen von im Alter von Mütter haben eine kürzere Lebensdauer als die von jungen Mütter; jedoch mütterliche Kalorienrestriktion (CR) kompensiert teilweise die schädlichen Auswirkungen von fortgeschrittenen Alter der Mutter 20. In B. plicatilis, mütterliche CR bietet Nachkommen Langlebigkeit, Beständigkeit lange Überlebenszeit unter Hunger und hohen oxidativen Stress verbunden mit erhöhter Expression von antioxidativen Enzymen 21,22. Das Alter der Mutter WirkungAuch in Bdelloid Rotatorien 23 beobachtet. Daher sollten die Bedingungen der experimentellen Rotatorien sorgfältig über mehrere Generationen vor der Messung der Überlebenszeit synchronisiert werden. Hier bieten wir ein Protokoll für die Messung der Überlebenszeit in Brachionus rotifers nach der Synchronisation von Kulturbedingungen über mehrere Generationen hinweg. Intermittierende Fasten (IF), eine Variation von CR wo rotifers periodisch zugeführt werden, um die Wirkung der Synchronisation aufgrund der bekannten Wirkung von IF auf Langlebigkeit 22,24 offenbaren angewendet.

<table><tbody><tr><td>Sodium chloride</td><td>Wako</td><td>190-13921</td><td></td></tr><tr><td>Magnesium chloride</td><td>Wako</td><td>136-03995</td><td></td></tr><tr><td>Magnesium sulfate</td><td>Wako</td><td>131-00427</td><td></td></tr><tr><td>Potassium chloride</td><td>Wako</td><td>168-22111</td><td></td></tr><tr><td>Calcium chloride</td><td>Wako</td><td>035-00455</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Wako</td><td>199-05985</td><td></td></tr><tr><td>Sodium bromide</td><td>Wako</td><td>190-01515</td><td></td></tr><tr><td>Membrane filter (0.45 &amp;micro;m pore size)</td><td>Millipore</td><td>HAWP04700</td><td></td></tr><tr><td>Culture plate, 6-well, non-treated</td><td>Thomas Scientific</td><td>6902D01</td><td>Flat bottom</td></tr><tr><td>Culture plate, 48-well, non-treated</td><td>Thomas Scientific</td><td>6902D07</td><td>Flat bottom</td></tr><tr><td>Tetraselmis, Living</td><td>Carolina Biological Supply Company</td><td>152610</td><td></td></tr><tr><td>PRISM 6</td><td>GraphPad Software</td><td>Version 6.0d</td><td></td></tr></tbody></table>

measurement of survival time in brachionus rotifers: synchronization of maternal conditions

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.

Abbildung 1 zeigt repräsentative Überlebenskurven von schlecht synchronisierten Populationen (von zwei Wiederholungen). In diesem Experiment rotifers waren entweder gefüttert jeden Tag [ad libitum (AL) Gruppe] oder jeden zweiten Tag (IF - Gruppe). Das mediane Überleben betrug 13 und 18 Tage in der AL und IF-Gruppe. Obwohl es bekannt ist, dass, wenn die Lebensdauer der rotifer erstreckt, scheiterte dieser Versuch einen statistisch signifikanten Unterschied zwischen den Lebensdauern der AL zu erkennen und IF-Gruppen. Empirisch unzureichende Synchronisation Ergebnisse in der frühen Sterblichkeit und allmählichen Rückgang der Überlebensrate wie in diesem Experiment beobachtet. Schäden an Rotatorien durch unsachgemäße Behandlung oder niedrige Wasserqualität für die Subpopulation verursacht tendenziell ähnliche Ergebnisse zu erzielen. Wenn rotifer Bedingungen sind optimal und gut synchronisiert, ist frühe Sterblichkeit kaum beobachtet und accordingly Rotatorien neigen dazu , in einer synchronisierten Art und Weise während der späteren Phase des Experiments (Abbildung 2) sterben. Das mediane Überleben betrug 13 und 20 Tage in der AL und IF-Gruppe. Obwohl weniger Tiere als das Experiment in Figur 1 verwendet wurden, war der Unterschied in der Lebensdauer zwischen diesen Gruppen statistisch signifikant. Dies ist die repräsentative Ergebnisse aus mehr als fünf Experimenten , die vorher 22 veröffentlicht wurden. <img alt="Abbildung 1" src="/files/ftp_upload/54126/54126fig1.jpg" /> Abb . 1: Kaplan-Meier - Kurven für schlecht synchronisierte Personen zu intermittierenden Fastens (IF) unterworfen , um die AL - Gruppe wurde ad libitum während des gesamten Experiments eingespeist, während die ZF - Gruppe jeden zweiten Tag gefüttert wurde. N = 11 und N = 12 für das AL und IF-Gruppen jeweils (N bezieht sich auf die Anzahl der einzelnen in dem Experiment verwendet). Das Experiment wurde PERFORmed bei 25 ° C. Kein signifikanter Unterschied in der Lebensdauer festgestellt wurde , wenn Log-Rank - Test verwendet wurde (P = 0,1207). Allerdings ist diese Daten schwer zu interpretieren , da Log-Rank - Test nicht verwendet werden sollte , zwei sich kreuzende Überlebenskurven zu vergleichen , obwohl der Test bekannt ist robust 36 zu sein. Keine etablierten Methoden sind zur Zeit für die Überquerung Überlebenskurven mit zensierten Daten verfügbar. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54126/54126fig1large.jpg" target="_blank">Bitte hier klicken</a> , um <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54126/54126fig1large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> <img alt="Figur 2" src="/files/ftp_upload/54126/54126fig2.jpg" /> Abbildung 2:. Kaplan-Meier - Kurven für synchronisierte Individuen ausgesetzt IF Die rotifer Kohorte, erhalten durch Vorkultur wurde der gleichen IF Zeitplan unterworfen (jeden zweiten Tag gefüttert). N = 6 und N = 8 für AL und IF-Gruppe. Der Versuch wurde bei 25 ° C durchgeführt wird. Log-Rank - Test, p = 0,0057. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54126/54126fig2large.jpg" target="_blank">Bitte hier klicken</a> , um <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54126/54126fig2large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a>

Watch this Scientific Journal Video about Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions at JoVE.com

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

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.

Die Messung der Überlebensdauer in<em&gt; Brachionus</em&gt; Rotifers: Synchronisation von Maternal Bedingungen

Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short ...

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Universidad San Sebastian

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Biology

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

8.4K Aufrufe.  The University of Tokyo. 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.

Serie: Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus allowing a single investigator to screen more treatments or conditions over the same amount of time without loss of data quality. The method requires common equipment found in most laboratories working with C. elegans and is thus simple to adopt. The approach centers on assaying independent samples of a population at each observation point, rather than a single sample over time as with traditional longitudinal methods. Scoring entails adding liquid to the wells of a multi-well plate, which stimulates C. elegans to move and facilitates quantifying changes in healthspan. Other major benefits of the Replica Set method include reduced exposure of agar surfaces to airborne contaminants (e.g. mold or fungus), minimal handling of animals, and robustness to sporadic mis-scoring (such as calling an animal as dead when it is still alive). To appropriately analyze and visualize the data from a Replica Set style experiment, a custom software tool was also developed. Current capabilities of the software include plotting of survival curves for both Replica Set and traditional (Kaplan-Meier) experiments, as well as statistical analysis for Replica Set. The protocols provided here describe the traditional experimental approach and the Replica Set method, as well as an overview of the corresponding data analysis.

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

Here we describe the Replica Set method, an approach to quantitatively measure C. elegans lifespan/survival and healthspan in a high-throughput and robust manner, thus allowing screening of many conditions without sacrificing data quality. This protocol details the strategy and provides a software tool for analysis of Replica Set data.

Die Replica-Set-Methode: Eine Hochdurchsatz-Ansatz zu quantitativ Messen Caenorhabditis Elegans Lebensdauer

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus ...

Forschung

Genetik

Resurrection of Dormant Daphnia magna: Protocol and Applications

Long-term studies enable the identification of eco-evolutionary processes that occur over extended time periods. In addition, they provide key empirical data that may be used in predictive modelling to forecast evolutionary responses of natural ecosystems to future environmental changes. However, excluding a few exceptional cases, long-term studies are scarce because of logistic difficulties associated with accessing temporal samples. Temporal dynamics are frequently studied in the laboratory or in controlled mesocosm experiments with exceptional studies that reconstruct the evolution of natural populations in the wild.
Here, a standard operating procedure (SOP) is provided to revive or resurrect dormant Daphnia magna, a widespread zooplankton keystone species in aquatic ecosystems, to dramatically advance the state-of-the-art longitudinal data collection in natural systems. The field of Resurrection Ecology was defined in 1999 by Kerfoot and co-workers, even though the first attempts at hatching diapausing zooplankton eggs date back to the late 1980s. Since Kerfoot&#39;s seminal paper, the methodology of resurrecting zooplankton species has been increasingly frequently applied, though propagated among laboratories only via direct knowledge transfer. Here, an SOP is described that provides a step-by-step protocol on the practice of resurrecting dormant Daphnia magna eggs.
Two key studies are provided in which the fitness response of resurrected Daphnia magna populations to warming is measured, capitalizing on the ability to study historical and modern populations in the same settings. Finally, the application of next generation sequencing technologies to revived or still dormant stages is discussed. These technologies provide unprecedented power in dissecting the processes and mechanisms of evolution if applied to populations that have experienced changes in selection pressure over time.

Resurrection of Dormant Daphnia magna: Protocol and Applications

Long-term studies are essential to understanding the process of evolution and the mechanisms of adaptation. Generally, these studies require commitments beyond the life-time of researchers. Here, a powerful method is described that dramatically advances state-of-the-art data collection to generate longitudinal data in natural systems.

Auferstehung des ruhenden Daphnia Magna: Protokoll und Anwendungen

Long-term studies enable the identification of eco-evolutionary processes that occur over extended time periods. In addition, they provide key empirical ...

Umwelt

Measuring Replicative Life Span in the Budding Yeast

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice 1. Due to the lack of easily quantified age-associated phenotypes, aging in yeast has been assayed almost exclusively by measuring the life span of cells in different contexts, with two different life span paradigms in common usage 2. Chronological life span refers to the length of time that a mother cell can survive in a non-dividing, quiescence-like state, and is proposed to serve as a model for aging of post-mitotic cells in multicellular eukaryotes. Replicative life span, in contrast, refers the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Here we present a generalized protocol for measuring the replicative life span of budding yeast mother cells. The goal of the replicative life span assay is to determine how many times each mother cell buds. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160X), such as the Zeiss Axioscope 40 or another comparable model. Physical separation of daughter cells from mother cells is achieved using a manual micromanipulator equipped with a fiber-optic needle. Typical laboratory yeast strains produce 20-30 daughter cells per mother and one life span experiment requires 2-3 weeks.

In this article we present a general protocol for measuring the replicative life span of yeast mother cells.

Mess-replikative Lebensspanne in der Hefe

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding ...

Biologie

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many historical and contemporary studies have identified thousands of lifespan-altering paradigms, including genetic mutations, transgenic gene expression, and hormesis, a beneficial, low-grade exposure to stress. With its many advantages, including a short lifespan, easy and low-cost maintenance, and fully sequenced genome with homology to almost two-thirds of all human genes, C. elegans has quickly been adopted as an outstanding model for stress and aging biology. Here, several standardized methods are surveyed for measuring lifespan and healthspan that can be easily adapted into almost any research environment, especially those with limited equipment and funds. The incredible utility of C. elegans is featured, highlighting the capacity to perform powerful genetic analyses in aging biology without the necessity of extensive infrastructure. Finally, the limitations of each analysis and alternative approaches are discussed for consideration.

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

Caenorhabditis elegans serve as an excellent model system with robust and low-cost methods for surveying healthspan, lifespan, and resilience to stress.

Erhebung kostengünstiger Methoden zur Messung von Lebensdauer und Gesundheitserwartung bei Caenorhabditis elegans

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many ...

Measuring Embryonic Viability and Brood Size in Caenorhabditis elegans

Caenorhabditis elegans is an excellent model organism for the study of meiosis, fertilization, and embryonic development. C. elegans exist as self-fertilizing hermaphrodites, which produce large broods of progeny-when males are present, they can produce even larger broods of cross progeny. Errors in meiosis, fertilization, and embryogenesis can be rapidly assessed as phenotypes of sterility, reduced fertility, or embryonic lethality. This article describes a method to determine embryonic viability and brood size in C. elegans. We demonstrate how to set up this assay by picking a worm onto an individual Modified Youngren's, Only Bacto-peptone (MYOB) plate, establish the appropriate timeframe to count viable progeny and non-viable embryos, and explain how to accurately count live worm specimens. This technique can be used to determine viability in self-fertilizing hermaphrodites as well as cross-fertilization by mating pairs. These relatively simple experiments are easily adoptable for new researchers, such as undergraduate students and first-year graduate students.

Measuring Embryonic Viability and Brood Size in Caenorhabditis elegans

Here, we present a general method to determine the embryonic viability and total number of embryos produced (brood) using the model organism C. elegans.

Messung der Embryonalviabilität und Brutgröße bei Caenorhabditis elegans

Caenorhabditis elegans is an excellent model organism for the study of meiosis, fertilization, and embryonic development. C. elegans exist as ...

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. To improve the mating efficiency of B. Xylophilus and to increase the chances of mating observation, virgin adults were cultured and used for the investigation. Eggs were obtained by keeping the nematodes in water and allowing the females to lay eggs for 10 min. The second-stage juveniles (J2) were synchronized by incubating the eggs for 24 h at 25 &#176;C in the dark, and the early J4 were obtained by culturing the J2 with grey mold, Botrytis cinerea, for another 52 h. At this time point, most J4 nematodes could be clearly distinguished as being male or female using their genital morphology. The male and female J4 were collected and cultured separately in two different Petri dishes for 24 h to get virgin adult nematodes. A virgin male and a virgin female were paired in a drop of water in the well of a concave slide. The mating behavior was filmed with a video recorder under a stereomicroscope. The whole period of the mating process was 82.8 &#177;3.91 min (mean &#177;SE) and could be divided into 4 different phases: searching, contacting, copulating, and lingering. The mean minutes of duration were 21.8 &#177; 2.0, 28.0 &#177; 1.9, 25.8 &#177; 0.7 and 7.2 &#177; 0.5, respectively. Eleven sub-behaviors were described: cruising, approaching, encountering, touching, hooping, locating, attaching, ejaculating, separating, quiescence, and roaming. Interestingly, obvious intra-sexual competition was observed when one female was grouped with 3 males or one male with 3 females. This protocol is useful and valuable, not only in investigating the mating behavior of B. xylophilus, but also in acting as a reference for ethological studies of other nematodes.

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

A protocol for investigating the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, is presented. Behavioral features of B. xylophilus are described in the mating process.

Beobachtung und Quantifizierung der Paarungsverhalten in der Pinewood Nematode,<em&gt; Bursaphelenchus xylophilus</em

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation&#160;of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile Caenorhabditis elegans.

Messung von Sauerstoff Verbrauchsmengen in intakten Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...

Standardizing a Non-Lethal Method for Characterizing the Reproductive Status and Larval Development of Freshwater Mussels (Bivalvia: Unionida)

Actively monitoring the timing, development, and reproductive patterns of endangered species is critical when managing for population recovery. Freshwater mussels are among the most imperiled organisms in the world, but information about early larval (glochidial) development and brooding periods is still lacking for many species. Previous studies have focused on the complex life history stage when female mussels are ready to parasitize host fish, but few studies have focused on the brooding period and timing of larval development. The protocol described here allows researchers to non-lethally evaluate the state of gravidity for female mussels. The results of this study show that this method does not affect a female mussel&rsquo;s ability to stay gravid or become gravid again after sampling has been performed. The advantage of this method may permit its use on federally threatened or endangered species or other populations of high conservation concern. This protocol can be adapted for use on both preserved or live individuals and was tested on a variety of mussel species. The database provided is a repository for a breadth of information on timing of reproductive habits and will facilitate future freshwater mussel research, conservation, and recovery efforts.

Standardizing a Non-Lethal Method for Characterizing the Reproductive Status and Larval Development of Freshwater Mussels Bivalvia: Unionida

Freshwater mussel conservation is dependent on monitoring reproductive patterns and processes of species. This study standardizes a non-lethal protocol for sampling gill contents, characterizing larval development, and providing a digital repository for data collected. This protocol-database package will be an important tool for mussel researchers in recovery of imperiled species.

Standardisierung einer nicht-tödlichen Methode zur Charakterisierung des Fortpflanzungsstatus und der Larval-Entwicklung von Süßwassermuscheln (Bivalvia: Unionida)

Actively monitoring the timing, development, and reproductive patterns of endangered species is critical when managing for population recovery. Freshwater ...

Measurements of Physiological Stress Responses in C. Elegans

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and physiology. Thus, organisms have evolved targeted and specific stress responses dedicated to repair damage and maintain homeostasis. These mechanisms include the unfolded protein response of the endoplasmic reticulum (UPRER), the unfolded protein response of the mitochondria (UPRMT), the heat shock response (HSR), and the oxidative stress response (OxSR). The protocols presented here describe methods to detect and characterize the activation of these pathways and their physiological consequences in the nematode, C. elegans. First, the use of pathway-specific fluorescent transcriptional reporters is described for rapid cellular characterization, drug screening, or large-scale genetic screening (e.g., RNAi or mutant libraries). In addition, complementary, robust physiological assays are described, which can be used to directly assess sensitivity of animals to specific stressors, serving as functional validation of the transcriptional reporters. Together, these methods allow for rapid characterization of the cellular and physiological effects of internal and external proteotoxic perturbations.

Measurements of Physiological Stress Responses in C. Elegans

Here, we characterize cellular proteotoxic stress responses in the nematode C. elegans by measuring the activation of fluorescent transcriptional reporters and assaying sensitivity to physiological stress.

Messung physiologischer Stressreaktionen in C. Elegans

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and ...

Nematode Synchronization: A Method to Obtain Populations of Worms in Identical Stages of Development

Source: Sarasija, S. and Norman, K. R. <a href="https://www-jove-com.bdigitaluss.remotexs.co/video/59277/measurement-oxygen-consumption-rates-intact-caenorhabditis">Measurement of Oxygen Consumption Rates in Intact&#160;Caenorhabditis elegans</a>.&#160;J. Vis. Exp. (2019).
In this video, we show a method of producing a developmentally synchronized population of sterile adult C. elegans. The example protocol synchronizes worms to study oxygen consumption rates in live animals.

Nematoden-Synchronisation: Eine Methode, um Populationen von Würmern in identischen Entwicklungsstadien zu erhalten

Source: Sarasija, S. and Norman, K. R. Measurement of Oxygen Consumption Rates in Intact&#160;Caenorhabditis elegans.&#160;J. Vis. Exp. (2019).
In this ...

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