Name: measurement of survival time in brachionus rotifers: synchronization of maternal conditions
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私たちは、撮影中の彼らの助けのために、ジョージ・ジャービス、マーサボック、およびベティヒーコックス-リー、海洋生物学研究所に感謝しています。

メディアの調製注意：使用して、塩分16.5 PPT（PSU）の半希釈Brujewicz人工海水を。他の人工海水にも頻繁に文化Brachionusのワムシ25,26に使用されています。 <ol><li> 454のNaCl、26のMgCl 2、27mMのMgSO 4を、10のKCl、および10mMのCaCl 2（最終容量は5 Lになります）の蒸留水4.5 Lに追加します。また、脱イオン希釈水の代わりに蒸留水を使用しています。すべての他の塩を溶解させた後のCaCl 2を追加します。 </li><li> 0.48 MのNaHCO 3ストック溶液（200倍濃度）を準備します。上記の溶液に25mlのを追加します。 NaHCO 3を最終濃度が2.4 mmです。 </li><li> 0.4 MのNaBr原液（500X濃度）を準備します。上記の溶液に10mlのを追加します。 NaBrの最終濃度は0.8 mmです。蒸留水で5 Lにメイクアップ。 </li><li> 0.45μmのメンブレンフィルターで溶液をフィルタリングします。（v / v）を使用前に滅菌水で2回希釈します。 注：原液としてBrujewicz人工海水の2倍の濃度とすることができます。 </li></ol> 2.一般的な培養条件<ol><li>培養実験室で上昇または20と30℃の間の滅菌100ミリリットルのビーカー内の野生捕獲さワムシ。温度が高いほど短い寿命と加速再生につながります。実験の便宜のために25°Cを使用してください。ワムシ密度または人工海水の量は、ここで大きな問題ではありません。 </li><li>人工海水11中の文化食物微細藻類種。詳細は11のためにスネルら。（2014）を参照してください。一般的に、Tetraselmisのtetrathele（〜2×10 5細胞/ mlの27,28）、Tを使用ダイエット藻類（種、培養コンディットので、suecica（〜6×10 5細胞/ mlの25,29）、およびNannochloropsisのoculata（〜7×10 6細胞/ mlの30,31）。イオン、生化学的組成物）は大幅にすべての実験群では、微細藻類の同一ロットを使用し、Brachionusのワムシの生存期間に影響を与えます。 </li><li>供給し、（ 例えば 、2日毎に供給し、毎週接種）時折メディアを変更することにより、バッチ培養におけるワムシの株式集団をキープ。ワムシは、最適な条件である場合、多くの新生児2-3卵を有する成人を観察することができます。 注意：実験的なワムシを取得する別の方法は、安静時の卵を孵化することです。休んで卵から孵化しワムシはよく同期させることが考えられ、その寿 ​​命は倍数性単為生殖の卵11から孵化ワムシのそれとは大きく異なっていないされています。しかし、安静時の卵からのワムシは倍数性単為生殖の卵からのものよりも早く再生を開始します。このように、注意が彼らの繁殖形質の測定のために必要とされます。 </li></ol>前培養によるワムシの3同期<ol><li>目から単一のワムシを選択実験に使用される亜集団を確立するために、2.1〜2.3で説明したように、人口培養OCK。典型的には、2週間の培養。 </li><li> （実験に使用される個人の二重数を収集する）サブ集団から卵ベアリングワムシを集めます。単一のコホートとしての文化、それらを（密度：〜50人/ ml）を不断給餌下で新鮮な培地に2.1から2.3に記載されているように6ウェル培養プレートインチ馴化培地はBrachionusのワムシ32,33の生殖生理学に影響を与えるため、人口密度を制御することが重要です。 </li><li>新たに用意した培養培地に大人の最初の卵から孵化した新生児を転送します。 2-3世代を超えて、この手順を繰り返します。 </li><li>生存時間を測定するための一定期間（ 例えば 、&lt;3 ​​時間）以内に孵化した新生児を使用してください。単一の個人の選択の可能性バイアスを避けるために、非常にありそうにないものの、いくつかのINDEを使用して再現性を確認しますペンダントのサブ集団。 注：ワムシは、通常、この条件の下でのみ無性再現するように、何人の男性が、実験を通して存在しないことを確認します。男性は新生児よりも小さく、一般的に速い女性より移動します。 Mictic女性が一定の条件の29の下で倍数性単為生殖の雌は異なる寿命を持っています。 </li></ol>生存期間の4測定<ol><li>プラスチックプレートに入れ新生児（人工海水の各ウェルを含む1ミリリットルで、通常、24または48ウェルプレート、）。 </li><li> 24時間の間隔で、より高い温度（上記30℃以上）であれば、新たに調製した培養培地に、または12時間の間隔でワムシを転送します。子孫の数を記録し、各個人が死んでいるか生きているかどうか。コロナの繊毛の動きが完全に停止したときに死んだとしてワムシを記録します。 注：ワムシは、多くの場合、ウェルの側壁に付着します。水の穏やかなピペッティングは、それらを見つけるのに役立ちます。ワムシが見つかり又はArされていない場合電子誤ってピペット操作によって損傷を受け、ない「死んだ」として、「検閲」としてそれらを記録。 </li><li>実験ワムシが積極的に再現しているときに新生児を削除します。新生児は急速に成長し、実験ワムシと区別することが困難な場合があります。 </li></ol> 5.データ解析<ol><li> X軸上のY軸と時間の累積生存率をプロットしたカプラン・マイヤー生存曲線（ 図1及び2）を作成します 。これは、生存データの最も一般的な表現です。生存期間34の統計的比較のために（もマンテル-コックスのテストと呼ばれる）ノンパラメトリックログランク検定を使用してください。ログランク検定はまた、JMPおよびR.などの他の標準的な統計パッケージに含まれています 注意：正規分布は、通常、生存データ35によって満たされていないため、スチューデントのt検定またはパラメトリック多重比較に続いて分散分析（ANOVA）を使用しないでください。また、これらのメトDSのアカウントに検閲個人を取ることはありません。何打ち切りデータが存在しない場合はマンホイットニーU検定を使用することができます。 </li></ol>

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Müller.</a> Hydrobiologia. 412, 103-110 (1999).</li><li>Ohmori, F., Kaneko, G., Saito, T., Watabe, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+growth-promoting+protein+in+the+conditioned+media+from+the+rotifer+Brachionus+plicatilis+at+an+early+exponential+growth+phase.">A novel growth-promoting protein in the conditioned media from the rotifer Brachionus plicatilis at an early exponential growth phase.</a> Hydrobiologia. 667 (1), 101-117 (2011).</li><li>Collet, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Modelling Survival Data in Medical Research. , 151-193 (1993).</li><li>Bouliotis, G., Billingham, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crossing+survival+curves:+alternatives+to+the+log-rank+test.">Crossing survival curves: alternatives to the log-rank test.</a> Trials. 12, A137 (2011).</li><li>Yang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+expression+of+manganese+superoxide+dismutase,+copper+and+zinc+superoxide+dismutase+and+catalase+in+Brachionus+calyciflorus+during+the+aging+process.">Changes in expression of manganese superoxide dismutase, copper and zinc superoxide dismutase and catalase in Brachionus calyciflorus during the aging process.</a> PloS ONE. 8 (2), e57186 (2013).</li><li>Snell, T. W., Johnston, R. K., Rabeneck, B., Zipperer, C., Teat, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Joint+inhibition+of+TOR+and+JNK+pathways+interacts+to+extend+the+lifespan+of+Brachionus+manjavacas+(Rotifera).">Joint inhibition of TOR and JNK pathways interacts to extend the lifespan of Brachionus manjavacas (Rotifera).</a> Exp. Gerontol. 52, 55-69 (2014).</li><li>Klass, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+in+nematode+Caenorhabditis-elegans+-+major+biological+and+environmental-factors+influencing+life-span.">Aging in nematode Caenorhabditis-elegans - major biological and environmental-factors influencing life-span.</a> Mech. Ageing Dev. 6 (6), 413-429 (1977).</li><li>Priest, N. K., Mackowiak, B., Promislow, D. E. 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.

現在のプロトコルはBrachionusのワムシの生存時間を測定するための方法が記載されています。重要なステップは、数世代にわたるワムシ条件の同期です。実験ワムシはよく同期化されるとき、典型的なタイプIの生存曲線は、いくつかの以前の研究18,24,37,38で報告されたように、非常に小さな初期の死亡率が観察されます。彼らの生存時間の標準偏差が高いため、統計的パワーで、その結果、不十分同期ワムシに比べて小さくなります。同期化はまた、生存時間の測定の再現性を高めることが期待される - 母親は最適な条件下で培養されているため、現在のプロトコルは、母体の世代の可能性有害な影響を相殺します。早期の死亡率は依然として慎重な同期の後に観察されている場合は、新たに用意した培地を使用することを検討して、 すなわち供給藻類、または新たに確立された実験コホートの他の多く（、）プロトコル3.1から開始します。 このプロトコルの制限がよく同期ワムシが潜在的に過剰に敏感であるということです。例えば、寿命を延長する化学物質のスクリーニング時に、このプロトコルによりスクリーニングいくつかの化学物質が不十分同期ワムシ（ 例えば 、野生およびバッチ培養集団からの個人）の寿命に大きな影響を検出できないことがあります。したがって、このような実験の結果は慎重に解釈されるべきです。 子孫の生存時間に母性年齢の影響もフルーツフライキイロショウジョウバエや線虫線虫（Caenorhabditis elegans）39,40を含む他の無脊椎動物のモデルで報告されています。それはより多くの時間がかかり、これらの長寿命モデルであるが、これらの動物は生存時間の測定における実験的変動を減少させるために、数世代にわたって同期手順は有用であろう。

ワムシは門ワムシ1を構成する微細な国際的な動物プランクトン（&lt;1ミリメートル）です。彼らは、約1,000体細胞から成るシンプルなボディープランだけでなく、特徴的な車輪状の毛様体装置が移動し、給餌のために使用されているコロナと呼ばれています。ほとんどのワムシはそれぞれ2、約1600と500種を含むクラスMonogonontaまたはヒルガタワムシ綱に属します。 bdelloidワムシが義務付け単為生殖3によって再生するMonogonontワムシは、一般的に、両方の性的および無性生殖相（循環的単為生殖を）持っています。実験において高い再現性を保証する、遺伝的に同一のワムシの個体を得ることができます。さらに、それらは、短い寿命、培養の容易さ、ゲノム及びトランスクリプトーム配列データ4-7の可用性、およびから離れユニークな系統発生位置などのモデル生物のようないくつかの他の利点を有しますrthropodsと線虫8。ワムシは、したがって、生態系における無脊椎動物のモデルを有望な毒物学、および研究9-12を老化されています。 環境ストレスや化学物質への曝露下での生存時間は、これらの研究分野13-19で頻繁に測定されたパラメータです。ワムシの生存時間を測定する場合、それは母親の環境条件に敏感であるためしかし、注意が必要です。すなわち、monogonontワムシBrachionus manjavacasで、高齢者の母親からの女性の子孫は、若い母親からのものよりも短い寿命を持っています。しかし、母体のカロリー制限（CR）は、部分的に高齢出産20の有害な影響を相殺します。 B.でplicatilis、母体のCRは、子孫の長寿、飢餓の下で長い生存期間、および抗酸化酵素21,22の発現増強に伴う高い酸化ストレス耐性を提供します。母体の年齢効果またbdelloidワムシ23で観察されています。したがって、実験ワムシの条件は慎重に生存時間の測定の前に数世代にわたって同期する必要があります。 ここでは、数世代にわたって培養条件の同期以下のBrachionusのワムシでの生存時間を測定するためのプロトコルを提供します。間欠絶食（IF）、ワムシは、定期的に供給されるCRの変化は、原因寿命22,24にIFのよく知られた効果のために、同期の影響を明らかにするために適用しました。

<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.

図1は不十分同期集団（2つの複製のうち）の代表的な生存曲線を示しています。この実験では、ワムシは、供給された日常の[ 自由摂取 （AL）グループ]または隔日（グループIF）のいずれかでした。生存期間中央値はそれぞれ、ALおよびIFグループで13と18日でした。よくIFは、ワムシの寿命を延長することが知られているが、この実験では、ALとIFグループの寿命の間に統計的に有意な差を検出することができませんでした。経験的には、早期の死亡率および生存率の緩やかな下落が不十分で、同期の結果は、この実験で観察しました。サブ集団についての不適切な処理または低水質に起因するワムシへの損傷は、同様の結果が得られる傾向にあります。 ワムシの条件が最適とも同期されている場合には、早期の死亡率はほとんど見られず、accordinされますGlyをワムシは、実験の後の段階（ 図2）中に同期の方法で死ぬ傾向にあります。生存期間中央値はそれぞれ、ALおよびIFグループで13と20日でした。少数の動物が図1の実験よりも、使用されたが、これらの群の間の寿命の差は、統計学的に有意でした。これは、以前に22公開されている以上の5回の実験からの代表的な結果です。 <img alt="図1" src="/files/ftp_upload/54126/54126fig1.jpg" /> 図1：IFグループは一日おきに供給したのに対し、 断続的な断食（IF）にかけ不十分同期個人用のカプラン・マイヤー曲線は ALグループは、実験を通して自由に供給しました。 N =それぞれ11およびN = ALとIFグループ12（Nは実験に用いた個体の数を指します）。実験はperforました25℃でのmed。ログランク検定は（P = 0.1207）を使用した場合、寿命に有意差は検出されませんでした。試験はロバスト36であることが知られているが、ログランク検定は、2つの交差する生存曲線を比較するために使用すべきではないため、このデータは解釈が困難です。いいえ確立された方法は、現在、打ち切りデータで生存曲線を横切るために用意されていません。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54126/54126fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図2" src="/files/ftp_upload/54126/54126fig2.jpg" /> 図2：IF にさらさ同期個人用のカプラン・マイヤー曲線は、前培養によって得られたワムシコホートは、（一日おきに供給された）同じIFスケジュールに供しました。 N = 6とALのためのN = 8とグループIF、それぞれ。実験は25℃で行いました。ログ-ランク検定、P = 0.0057。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54126/54126fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</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.

生存時間の測定で<em&gt; Brachionus</em&gt;ワムシ：母性条件の同期

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

measurement-survival-time-brachionus-rotifers-synchronization

Universidad San Sebastian

Research

JoVE Journal

Biology

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

8.4K ビュー.  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.

ビデオ: 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.

レプリカの設定方法: 定量的測定線虫の寿命を高スループット方法

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

遺伝学

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.

休眠復活オオミジンコ: プロトコルおよびアプリケーション

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

環境学

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.

出芽酵母の複製寿命の測定

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

生物学

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.

Caenorhabditis elegansの寿命と健康寿命を測定するための低コストの方法の調査

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.

カエノラブディティス・エレガンスにおける胚の生存率とひなサイズの測定

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.

パインウッドセンチュウの交配挙動の観察と定量化、<em&gt;マツノザイセンチュウ</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.

そのまま線虫の酸素消費量の測定

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.

淡水ムール貝の生殖状態と幼虫発達を特徴付けるための非致死的方法の標準化(ビバルビア:ユニオンダ)

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.

エレガンスにおける生理的ストレス応答の測定

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.

線虫の同期:同一の発生段階にある線虫の集団を取得する方法

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

生存時間の測定で

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