Somos gratos a George Jarvis, Martha Bock, e Bette Hecox-Lea, Laboratório de Biologia Marinha, por sua ajuda na filmagem.

1. Preparação de Mídia Nota: Use meio diluída Brujewicz água do mar artificial da salinidade 16,5 ppt (PSU). Outros seawaters artificiais também são frequentemente usados ​​para cultura Brachionus rotíferos 25,26. <ol><li> Adicionar NaCl a 454 mM, 26 mM de MgCl2, 27 mM de MgSO4, 10 mM de KCl e 10 mM de CaCl 2 a 4,5 L de água destilada (volume final será de 5 L). Em alternativa, usar água de diluição desionizada em vez de água destilada. Adicionar CaCl2 depois de se dissolverem todos os outros sais. </li><li> Prepare o M NaHCO solução 0,48 3 estoque (concentração de 200x). Adicionar 25 ml de solução para o acima. A concentração final de NaHCO 3 é de 2,4 mm. </li><li> Preparar a solução stock M NaBr 0,4 (concentração 500x). Adicionar 10 ml da solução para o acima. A concentração final de NaBr é de 0,8 mm. Perfaz-se 5 L com água destilada. </li><li> Filtra-se a solução com um filtro de membrana de 0,45 um.Diluir duas vezes com água estéril antes da sua utilização (v / v). Nota: É possível fazer uma concentração 2x de Brujewicz água do mar artificial como uma solução estoque. </li></ol> 2. Condições Gerais Cultura <ol><li> laboratório-raise Cultura ou rotíferos selvagens-capturado em um copo de 100 ml estéril entre 20 e 30 ° C. As temperaturas mais elevadas resultam em expectativa de vida mais curtos e reprodução acelerada. Usar 25 ° C para a conveniência dos experimentos. A densidade de rotíferos ou o volume de água do mar artificial não é um grande problema aqui. </li><li> Cultura espécies de microalgas alimentares em água do mar artificial 11. Veja Snell et al. (2014) para mais detalhes 11. Tipicamente, usar Tetraselmis tetrathele (~ 2 x 10 5 células / ml) 27,28, T. suecica (~ 6 x 10 5 células / mL), 25,29 e Nannochloropsis oculata (~ 7 X 10 6 células / ml 30,31). Uma vez que dieta algas (espécies, Condit culturaíons, composições bioquímicas) influencia significativamente o tempo de sobrevivência de rotíferos Brachionus, use o mesmo lote de microalga em todos os grupos experimentais. </li><li> Manter uma população de ações de rotíferos em uma cultura em lote através da alimentação e mudando media ocasionalmente (por exemplo, alimentar a cada 2 dias e inocular todas as semanas). Muitos recém-nascidos e adultos portadores de 2-3 ovos pode ser observado quando rotíferos estão em condições óptimas. Nota: Uma forma alternativa de obter rotíferos experimentais é para chocar ovos de repouso. Rotíferos eclodidos de ovos de repouso são considerados para ser bem sincronizados, e o seu tempo de vida não é significativamente diferente da de rotíferos eclodidos de ovos amictic 11. No entanto, rotíferos de ovos que descansam iniciar a reprodução mais cedo do que os de ovos amictic. Assim, é necessária cautela para medições de suas características reprodutivas. </li></ol> 3. Sincronização de rotíferos pelo Pré-cultura <ol><li> Selecione um único rotíferos do stpopulação e cultura ock como descrito no 2,1-2,3 para estabelecer uma sub-população que vai ser utilizado para experiências. Normalmente cultura durante duas semanas. </li><li> Recolha rotíferos portadores de ovos da sub-população (a cobrar o dobro do número de indivíduos que serão utilizados para o experimento). Cultura-los como uma única coorte (densidade: ~ 50 indivíduos / mL) em uma placa de cultura de 6 poços, como descrito no 2,1-2,3 no estado fresco de comunicação sob alimentação ad libitum. Controlando a densidade de população é importante porque o meio condicionado afecta a fisiologia reprodutiva de rotíferos Brachionus 32,33. </li><li> Transferir recém-nascidos nascidos de primeiros ovos dos adultos para meios de cultura recém-preparado. Repita este procedimento ao longo de 2-3 gerações. </li><li> Use neonatos nascidos dentro de um determinado período de tempo (por exemplo, &lt;3 h) para medir o tempo de sobrevivência. Para evitar um possível viés na seleção do único indivíduo, embora muito improvável, verificar a reprodutibilidade usando vários independentes sub-populações. Nota: Como rotíferos normalmente se reproduzem assexuadamente sob esta condição, garantir que não haja homens estão presentes durante todo o experimento. Os machos são menores do que recém-nascidos e, normalmente, se mover mais rápido do que as fêmeas. Fêmeas Mictic têm diferentes tempos de vida das fêmeas amictic sob certas condições 29. </li></ol> 4. Medições de Tempo de Sobrevivência <ol><li> neonatos lugar em placas de plástico (tipicamente de 24 ou 48 poços placas, com cada uma das cavidades contendo 1 ml de água do mar artificial). </li><li> Em intervalos de 24 horas, transferir os rotíferos aos meios de cultura recém-preparados ou em intervalos de 12 horas, se a temperatura mais elevada (acima de 30 ° C ou). Grave o número de filhos e se cada indivíduo está vivo ou morto. Grave o rotíferos como morto quando o movimento dos cílios da corona parou completamente. Nota: Rotíferos muitas vezes atribuem às paredes laterais das cavidades. pipetagem suave da água ajuda a encontrá-los. Se rotíferos não são encontrados ou are acidentalmente danificado pelo pipetagem, gravá-las como &quot;censurado&quot;, não como &quot;morto&quot;. </li><li> Remover neonatos quando rotíferos experimentais estão reproduzindo ativamente. Os recém-nascidos crescem rapidamente e às vezes é difícil distingui-los dos rotíferos experimentais. </li></ol> Análise 5. Os dados <ol><li> Criar a curva de sobrevivência de Kaplan-Meier (Figuras 1 e 2) através da representação gráfica taxa de sobrevivência cumulativa no eixo Y e o tempo no eixo x. Esta é a representação mais comum de dados de sobrevivência. Use o teste de log-rank não paramétrico (também chamado de teste de Mantel-Cox) para comparação estatística de tempo de sobrevivência 34. O teste de log-rank também está incluído em outros pacotes estatísticos padrão, tais como JMP e R. Nota: Não use t-teste ou análise de variância (ANOVA), seguido de comparação múltipla paramétrico Student porque distribuição normal geralmente não é encontrado por dados de sobrevivência 35. Além disso, estes metods não levam indivíduos censurado em conta. Mann-Whitney U Test pode ser usada se não houver dados censurados. </li></ol>

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The authors have nothing to disclose.

O actual protocolo descreve um método para medir o tempo de sobrevivência em rotíferos Brachionus. O passo crítico é a sincronização das condições de rotíferos ao longo de várias gerações. Quando rotíferos experimentais são bem sincronizados, um tipo de curva típica I sobrevivência é observada com muito pouco mortalidade precoce como relatado em vários estudos anteriores 18,24,37,38. desvios-padrão de seu tempo de sobrevivência, portanto, tornam-se menores em comparação com rot...

Rotíferos são microscópicos cosmopolita zooplâncton (&lt;1 mm), que constituem o filo rotifera 1. Eles têm um plano de corpo simples composto por cerca de 1000 células somáticas, bem como um dispositivo de roda-ciliar como característica chamada coroa, que é usado para locomoção e de alimentação. A maioria dos rotíferos pertencem a classes monogononta ou bdelloidea, que contêm cerca de 1.600 e 500 espécies, respectivamente 2. Rotíferos monogononta geralmente têm duas fases reprodução sexuada e assexuada (partenogênese cíclico), enquanto rotíferos bdelloid reproduzir por partenogênese obrigatória 3. É, assim, possível a obtenção de indivíduos geneticamente idênticos rotíferos, o que assegura uma elevada reprodutibilidade em experiências. Além disso, eles têm várias outras vantagens como organismos modelo, tal como um tempo de vida curto, facilidade de cultura, a disponibilidade de dados de sequências genómicas e transcriptomic 4-7, e uma posição filogenética a partir de uma única distanterthropods 8 e nemátodos. Rotíferos são, portanto, prometendo modelos de invertebrados em ecológica, toxicológicas e envelhecimento estudos 9-12. O tempo de sobrevivência sob a exposição ao stress ambiental ou produtos químicos é um parâmetro frequentemente medida nestes campos de pesquisa 13-19. No entanto, é preciso ter cuidado quando se mede o tempo de sobrevivência de rotíferos porque é suscetível às condições ambientais de suas mães. Ou seja, no monogononta Manjavacas rotíferos Brachionus, descendentes do sexo feminino a partir de mães com idade têm uma vida útil mais curta do que os de mães jovens; no entanto, a restrição calórica materna (CR) compensa parcialmente os efeitos deletérios da avançada idade materna 20. Em B. plicatilis, CR materno proporciona longevidade prole, o tempo de sobrevivência muito tempo sob fome e de resistência ao estresse oxidativo alta associada com a expressão aumentada de enzimas antioxidantes 21,22. O efeito da idade maternaTambém tem sido observado em rotíferos bdelloid 23. Portanto, as condições de rotíferos experimentais devem ser cuidadosamente sincronizada ao longo de várias gerações antes das medições do tempo de sobrevivência. Aqui nós fornecemos um protocolo para a medição do tempo de sobrevivência em rotíferos Brachionus seguinte sincronização das condições de cultivo ao longo de várias gerações. Jejum intermitente (IF), uma variação do CR onde rotíferos são alimentados periodicamente, foi aplicado para revelar o efeito de sincronização devido aos efeitos conhecidos de SE na longevidade 22,24.

<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.

A Figura 1 mostra as curvas de sobrevida representativas de populações mal sincronizados (de duas repetições). Neste experimento, os rotíferos ou eram todos os dias Fed [ad libitum grupo (AL)] ou em dias alternados (IF grupo). A sobrevida média foi de 13 e 18 dias na AL e se os grupos, respectivamente. Embora seja bem sabido que se estende a vida útil do rotíferos, esta experiência não conseguiu detectar uma diferença estatisticamente significat...

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.

Measurement of Survival Time in<em&gt; Brachionus</em&gt; Rotifers: Sincronização de condições maternas

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

8.4K visualizações.  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.

Vídeo: 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.

O método de conjunto de réplica: Uma abordagem de alto rendimento para quantitativamente medida Caenorhabditis elegans expectativa de vida

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

Genética

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.

Ressurreição de dormentes Daphnia magna: protocolo e aplicações

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

Ambiente

Measuring Caenorhabditis elegans Life Span on Solid Media

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has emerged as a principal model used to study the biology of aging. Because virtually every biological subsystem undergoes functional decline with increasing age, life span is the primary endpoint of interest when considering total rate of aging. In nematodes, life span is typically defined as the number of days an animal remains responsive to external stimuli. Nematodes can be propagated either in liquid media or on solid media in plates, and techniques have been developed for measuring life span under both conditions. Here we present a generalized protocol for measuring life span of nematodes maintained on solid nematode growth media and fed a diet of UV-killed bacteria. These procedures can easily be adapted to assay life span under various common conditions, including a diet consisting of live bacteria, dietary restriction, and RNA interference.

Measuring Caenorhabditis elegans Life Span on Solid Media

In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.

Medição Caenorhabditis elegans Expectativa de Vida em meios sólidos

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

Biologia

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.

Pesquisando métodos de baixo custo para medir a expectativa de vida e a saúde em 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.

Mensuração da Viabilidade Embrionária e do Tamanho da Ninhada em Caenorhabditis elegans

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

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

Medição do Tempo de vida em<em&gt; Drosophila melanogaster</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

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.

Medição das taxas de consumo de oxigênio em intacta 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.

Padronizar um método não letal para caracterizar o estado reprodutivo e o desenvolvimento larval de mexilhões de água doce (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.

Medições de Respostas de Estresse Fisiológico em 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://jove.unicartagenaproxy.elogim.com/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.

Sincronização de nematóides: um método para obter populações de vermes em estágios idênticos de desenvolvimento

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

Measurement of Survival Time in

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