אנו מודים ג&#39;ורג ג&#39;רוויס, מרתה בוק, ובט היקוקס-לאה, במעבדה לביולוגיה ימית, על עזרתם הצילומים.

1. הכנת Media הערה: השתמש חצי מדולל Brujewicz מי ים מלאכותי של ppt המליחה 16.5 (PSU). Seawaters המלאכותית אחרת גם משמשת לעתים קרובות כדי rotifers תרבות Brachionus 25,26. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להוסיף 454 מ&quot;מ NaCl, 26 מ&quot;מ MgCl 2, 27 mM MgSO 4, 10 מ&quot;מ KCl, ו -10 מ&quot;מ CaCl 2 כדי 4.5 ליטר של מים מזוקקים (נפח סופי יהיה 5 L). לחלופין, להשתמש במי דילול יונים במקום מים מזוקקים. להוסיף CaCl 2 לאחר המסת כל המלחים האחר. </li><li style=";text-align:right;direction:rtl"> הכן את פתרון 3 מניות 0.48 M NaHCO (ריכוז 200x). הוסף 25 מ&quot;ל של אותו הפתרון לעיל. הריכוז הסופי של NaHCO 3 הוא 2.4 מ&quot;מ. </li><li style=";text-align:right;direction:rtl"> כן פתרון המניות 0.4 M NaBr (ריכוז 500x). הוסף 10 מ&quot;ל של אותו הפתרון לעיל. הריכוז הסופי של NaBr הוא 0.8 מ&quot;מ. הפוך עד 5 ליטר עם מים מזוקקים. </li><li style=";text-align:right;direction:rtl"> סנן את הפתרון עם מסנן הממברנה 0.45 מיקרומטר.לדלל אותו פעמיים עם מים סטריליים לפני השימוש (V / V). הערה: אפשר להכין ריכוז 2x של מי ים מלאכותי Brujewicz כפתרון מניות. </li></ol> 2. תנאי תרבות כלליות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> תרבות הגדלים במעבדה או rotifers בר-שנתפסו בכוס 100 מ&quot;ל סטרילי בין ג &#39;20 ו -30 מעלות. טמפרטורות גבוהות לגרום תוחלת חיים קצרות יותר ורבייה מואצת. השתמש 25 ° C לנוחיות ניסויים. צפיפות rotifer או הנפח של מי ים מלאכותי אינה בעיה גדולה כאן. </li><li style=";text-align:right;direction:rtl"> מיני תרבות microalgae תזונתיים במי ים מלאכותיים 11. ראה ואח סנל. (2014) לפרטים 11. בדרך כלל, להשתמש Tetraselmis tetrathele (~ 2 x 10 5 תאים / מ&quot;ל 27,28), צ &#39; suecica (~ 6 x 10 5 תאים / מ&quot;ל 25,29), ו Nannochloropsis oculata (~ 7 x 10 6 תאים / מ&quot;ל 30,31). מאז אצות דיאטה (מינים, קונדיט תרבותיונים, קומפוזיציות ביוכימי) משפיע זמן ההישרדות משמעותית של rotifers Brachionus, להשתמש באותו הרבה microalga בכל קבוצות הניסוי. </li><li style=";text-align:right;direction:rtl"> שמור אוכלוסיית מניות של rotifers בתרבות תצווה על ידי האכלה והשינוי בתקשורת מדי פעם (למשל, להאכיל כל 2 ימים לחסן כל שבוע). רבים ילודים ומבוגרים נושאות 2-3 ביצים ניתן להבחין כאשר rotifers הם בתנאים אופטימליים. הערה: דרך חלופית להשיג rotifers ניסיוני היא לבקוע ביצים נחות. Rotifers בקע מן הביצים נחות נחשב להיות מסונכרנים היטב, ואת תוחלת החיים שלהם אינה שונה באופן מהותי מזו של rotifers בקע מן הביצים amictic 11. עם זאת, rotifers מביצים נח להתחיל רבייה מוקדם יותר מאלו מביצים amictic. לכן, זהירות נדרשת למדידות של תכונות הרבייה שלהם. </li></ol> סנכרון 3. rotifers ידי תרבות קדם <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בחר rotifer אחת מן stאוכלוסייה ותרבות Ock כמתוארת 2.1-2.3 להקים אוכלוסיית משנה אשר תשמש עבור ניסויים. בדרך כלל התרבות במשך שבועיים. </li><li style=";text-align:right;direction:rtl"> אסוף rotifers נושאות הביצית מן האוכלוסייה משנה (לאסוף מספר כפול של אנשים אשר ישמשו לצורך הניסוי). תרבות בם עוקבה יחידה (צפיפות: ~ 50 אנשים / מיליליטר) בצלחת תרבות 6 היטב כמתואר 2.1-2.3 בתוך תקשורת טריה תחת האכלה כרצונך. שליטת צפיפות אוכלוסייה חשובה כי המדיום המותנה משפיע פיזיולוגית רבייה של rotifers Brachionus 32,33. </li><li style=";text-align:right;direction:rtl"> עבר בילודים בקעו מן הביצים הראשונות של המבוגרים למדיה ותרבות מוכנים חדש. חזור על הליך זה על פני 2-3 דורות. </li><li style=";text-align:right;direction:rtl"> השתמש בילודים בקעו בתוך פרק זמן מסוים (למשל, &lt;3 שעות) למדידת זמן ההישרדות. כדי למנוע הטיות אפשריות בבחירת האדם היחיד, אם כי מאוד לא סביר, לבדוק שחזור באמצעות מספר indeתת-אוכלוסיות משתלשלות. הערה: כפי rotifers בדרך כלל לשחזר רק asexually תחת תנאי זה, להבטיח ללא זכרים נוכחים לאורך כל הניסוי. הזכרים קטנים יותר בילודים ובדרך כלל לנוע מהר יותר מאשר נקבות. לנקבות יש Mictic: אורך חיים שונים נקבות amictic בתנאים מסוימים 29. </li></ol> 4. מדידות של הישרדות זמן <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בילודים מקום בצלחות פלסטיק (בדרך כלל 24 או צלחות 48-היטב, עם כל 1 מ&quot;ל המכיל גם של מי ים מלאכותיים). </li><li style=";text-align:right;direction:rtl"> בגיל 24 מרווחי hr, להעביר את rotifers למדיה ותרבות המוכנות חדש או ב 12 מרווחי hr אם בטמפרטורה גבוהה (30 ° C ומעלה). רשום את מספר הצאצאים והאם כל אדם חי או מת. רשום את rotifer כמת כאשר תנועת ריסים של העטרה הפסיקה לחלוטין. הערה: rotifers לעתים קרובות לצרף הקירות הצדדיים של בארות. pipetting העדין של המים מסייע למצוא אותם. אם rotifers לא נמצא או arדואר ניזוק באופן מקרי על ידי pipetting, להקליט אותם, כמו &quot;צנזורה&quot;, לא כמו &quot;מת&quot;. </li><li style=";text-align:right;direction:rtl"> סור בילודים כאשר rotifers הניסיון להתרבות באופן פעיל. ילודים לצמוח במהירות וזה לפעמים קשה להבדיל ביניהם לבין rotifers הניסיון. </li></ol> ניתוח 5. נתונים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> צור את עקומת ההישרדות קפלן-מאייר (איורים 1 ו -2) על ידי התוויית שיעור ההישרדות המצטברת על ציר Y וזמן על ציר X. זהו הייצוג הנפוץ ביותר של נתוני הישרדות. השתמש בבדיקת הלא פרמטרית log-rank (נקרא גם מבחן מנטל-קוקס) עבור השוואה סטטיסטית של זמן ההישרדות 34. מבחן log-rank כלול גם חבילות סטטיסטיות מקובלות אחרות כגון JMP ור &#39; הערה: אל תשתמש מבחן t או הניתוח של סטודנטים שונים (ANOVA) ואחריו השוואה מרובה פרמטרית כי התפלגות נורמלית בדרך כלל לא פגשה נתוני הישרדות 35. כמו כן, אלה methods לא לוקח אנשים מצונזרים בחשבון. מאן-ויטני U הבדיקה יכולה לשמש אם אין נתונים מצונזרים. </li></ol>

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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=Light-dependent+transcriptional+events+during+resting+egg+hatching+of+the+rotifer+Brachionus+manjavacas.">Light-dependent transcriptional events during resting egg hatching of the rotifer Brachionus manjavacas.</a> Mar. Genomics. 20, 25-31 (2015).</li><li>Gribble, K. E., Welch, D. B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life-span+extension+by+caloric+restriction+is+determined+by+type+and+level+of+food+reduction+and+by+reproductive+mode+in+Brachionus+manjavacas+(Rotifera).">Life-span extension by caloric restriction is determined by type and level of food reduction and by reproductive mode in Brachionus manjavacas (Rotifera).</a> J. Gerontol. A Biol. Sci. Med. Sci. 68 (4), 349-358 (2013).</li><li>Kaneko, G., Kinoshita, S., Yoshinaga, T., Tsukamoto, K., 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=Changes+in+expression+patterns+of+stress+protein+genes+during+population+growth+of+the+rotifer+Brachionus+plicatilis.">Changes in expression patterns of stress protein genes during population growth of the rotifer Brachionus plicatilis.</a> Fish. Sci. 68 (6), 1317-1323 (2002).</li><li>Kim, H. J., Sawada, C., Hagiwara, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavior+and+reproduction+of+the+rotifer+Brachionus+plicatilis+species+complex+under+different+light+wavelengths+and+intensities.">Behavior and reproduction of the rotifer Brachionus plicatilis species complex under different light wavelengths and intensities.</a> Int. Rev. Hydrobiol. 99 (1-2), 151-156 (2014).</li><li>Yoshinaga, T., Hagiwara, A., Tsukamoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+conditioned+media+on+the+asexual+reproduction+of+the+monogonont+rotifer+Brachionus+plicatilis+O.+F.+Müller.">Effect of conditioned media on the asexual reproduction of the monogonont rotifer Brachionus plicatilis O. F. 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.

הפרוטוקול הנוכחי מתאר שיטה למדידת זמן ההישרדות rotifers Brachionus. השלב הקריטי הוא סנכרון תנאי rotifer במשך כמה דורות. כאשר rotifers ניסיוני מסונכרנים היטב, עקומת ההישרדות לי סוג טיפוסי הוא ציין עם תמותה מוקדמת מעט מאוד כפי שדווח בכמה מחקרים קודמים 18,24,37,38. סטיות התקן של ז?...

Rotifers הוא זואופלנקטון מיקרוסקופי קוסמופוליטית (&lt;1 מ&quot;מ) המהווה את המערכה Rotifera 1. יש להם תוכנית פשוטה הגוף מורכב כ -1,000 תאים סומטיים כמו גם מנגנון ריסי גלגל דמוי מאפיין שנקרא קורונה, אשר משמש לתנועה והאכלה. רוב rotifers שייכים כיתות Monogononta או Bdelloidea, המכילים כ -1,600 ו -500 מינים, בהתאמה 2. Rotifers Monogonont יש בדרך כלל שני שלבי רבייה מיניים מינית (בשכפול הגנטי מחזורי), בעוד rotifers bdelloid לשכפל בשכפול גנטי 3 חובה. בסך הכול אפשר להשיג אנשי rotifer זהים גנטי, אשר מבטיח שחזור גבוה בניסויים. בנוסף, יש להם כמה יתרונות אחרים כמו אורגניזמים מודל, כגון תוחלת חיים קצרה, קלות תרבות, זמינות של נתוני רצף גנומי transcriptomic 4-7, ו עמדת פילוגנטי ייחודית רחוקה מנקודהrthropods נמטודות 8. Rotifers ולכן מבטיח מודלים חסרי חוליות ב אקולוגית, טוקסיקולוגית, והזדקנות מחקרים 9-12. זמן ההישרדות תחת חשיפה ללחץ או כימיקלים סביבתיים הוא פרמטר נמדד לעתים קרובות בתחומי מחקר אלה 13-19. עם זאת, זהירות נדרשת כאשר מודד את זמן ההישרדות של rotifers כי זה רגיש לתנאי סביבה של האמהות שלהם. כלומר, בתוך manjavacas monogonont rotifer Brachionus, צאצא נקבה מאמהות בגילאי יש תוחלת חיים קצרה יותר מאשר אלו של אמהות צעירות; עם זאת, הגבלת קלוריות אימהיות (CR) מקזזת בחלקה את ההשפעות המזיקות של גיל האם מתקדם 20. ב B. plicatilis, אימהית CR מספק לאריכות הצאצאים, זמן ההישרדות הארוך תחת רעב והתנגדות סטרס חמצוני הגבוהות הכרוכות עם ביטוי מוגבר של אנזימים נוגדי חמצון 21,22. השפעת גיל האםגם נצפה rotifers bdelloid 23. לכן, תנאי rotifers ניסיוני צריכים להיות מתואמים היטב במשך כמה דורות לפני המדידות של זמן הישרדות. כאן אנו מספקים פרוטוקול מדידת זמן הישרדות rotifers Brachionus הבא סנכרון של תנאי תרבות במשך כמה דורות. צום לסירוגין (IF), וריאציה של CR איפה rotifers ניזונים מעת לעת, יושמה כדי לחשוף את השפעת הסנכרון בשל ההשפעות הידועות של IF על אריכות ימים 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.

איור 1 מציג עקומות הישרדות נציג של אוכלוסיות מסונכרנות היטב (מתוך שני משכפל). בניסוי זה, rotifers היו או היומיום הפד [כרצונך (AL) הקבוצה] או בכל יום אחר (IF קבוצה). חציון ההישרדות עמד על 13 ו -18 ימים באזור אל ואם קבוצות, בהתאמה. למרות זאת ידוע היט...

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.

מדידת הישרדות שעה<em&gt; Brachionus</em&gt; Rotifers: סנכרון של תנאי האימהי

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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Research

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Biology

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

8.4K Views.  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.

Video: 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.

השיטה קבע עותק משוכפל: בגישה תפוקה גבוהה באופן כמותי מידה Caenorhabditis elegans תוחלת החיים

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

Genetics

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.

תחיית המתים של רדום דפניה מגנה: פרוטוקול ויישומים

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

Environment

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.

מדידת Caenorhabditis elegans חיים טווח על מדיה מוצק

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

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.

סקר שיטות בעלות נמוכה למדידת תוחלת החיים ותוחלת הבריאות ב - 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.

מדידת כדאיות עוברית וגודל הברוד ב- 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.

מדידה של תוחלת חיים ב<em&gt; דרוזופילה 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.

מדידה של קצב הצריכה של חמצן בשלם 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.

סטנדרטיזציה של שיטה שאינה קטלנית לאפיון מעמד הרבייה והתפתחות הזחל של מולים מים מתוקים (ביובלויה: 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.

מדידות התגובות הפיזיולוגיות של הלחץ בג. אלגיה

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/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.

סנכרון נמטודות: שיטה להשגת אוכלוסיות של תולעים בשלבי התפתחות זהים

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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