ونحن ممتنون لجورج جارفيس، مارثا بوك، وبات Hecox-ليا، المختبر البيولوجي البحري، لمساعدتهم في التصوير.

1. إعداد وسائل الإعلام ملاحظة: استخدام مياه البحر الاصطناعي نصف المخفف Brujewicz الملوحة 16.5 جزء في الألف (PSU). وكثيرا ما تستخدم أيضا مياه البحر الاصطناعية الأخرى لثقافة الدوارات Brachionus 25،26. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إضافة 454 ملي مول كلوريد الصوديوم، و 26 ملي MgCl 2، 27 ملي MgSO 4، 10 ملي بوكل، و 10 ملي CaCl 2-4،5 لتر من الماء المقطر (الحجم النهائي سيكون 5 لتر). بدلا من ذلك، واستخدام الماء منزوع الأيونات تخفيف بدلا من الماء المقطر. إضافة CaCl 2 بعد حل جميع الأملاح الأخرى. </li><li style=";text-align:right;direction:rtl"> إعداد M NaHCO حل 3 الأوراق المالية 0.48 (تركيز 200X). إضافة 25 مل منه إلى الحل السالف الذكر. التركيز النهائي من NaHCO 3 هو 2.4 ملم. </li><li style=";text-align:right;direction:rtl"> إعداد M NaBr حل الأوراق المالية 0.4 (تركيز 500X). إضافة 10 مل منه إلى الحل السالف الذكر. التركيز النهائي من NaBr هو 0.8 ملم. ما يصل إلى 5 لتر مع الماء المقطر. </li><li style=";text-align:right;direction:rtl"> تصفية حل مع فلتر الغشاء 0.45 ميكرون.تمييع مرتين مع الماء المعقم قبل الاستخدام (ت / ت). ملاحظة: من الممكن أن تجعل تركيز 2X من Brujewicz مياه البحر الاصطناعي كحل الأسهم. </li></ol> 2. شروط الثقافة العامة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ثقافة أثار مختبر أو الدوارات البرية المأسورة في العقيمة كوب 100 مل بين 20 و 30 درجة مئوية. ارتفاع درجات الحرارة يؤدي إلى أقصر عمرا وتسارع الاستنساخ. استخدام 25 درجة مئوية لمدة راحة من التجارب. كثافة حيدة الخلية والروتيفر أو حجم مياه البحر الاصطناعي ليست قضية كبيرة هنا. </li><li style=";text-align:right;direction:rtl"> ثقافة أنواع الطحالب الدقيقة الغذائية في مياه البحر الاصطناعي (11). انظر سنيل وآخرون (2014) لمزيد من التفاصيل 11. عادة، استخدم جنس (Tetraselmis) tetrathele (~ 2 × 10 5 خلية / مل 27،28)، T. suecica (~ 6 × 10 5 خلية / مل 25،29)، وNannochloropsis oculata (= 7 × 10 6 خلية / مل 30،31). ومنذ الحمية الطحالب (الأنواع، كونديت الثقافةالأيونات، التراكيب الكيميائية الحيوية) يؤثر بشكل كبير وقت بقاء الدوارات Brachionus، استخدم نفس الكثير من microalga في كل المجموعات التجريبية. </li><li style=";text-align:right;direction:rtl"> الحفاظ على السكان الأسهم من الدوارات في ثقافة دفعة عن طريق تغذية وتغيير وسائل الإعلام في بعض الأحيان (على سبيل المثال، وإطعام كل أيام 2 و تطعيم كل أسبوع). العديد من حديثي الولادة والبالغين تحمل 2-3 البيض ويمكن ملاحظة عند الدوارات هي في أفضل الظروف. ملاحظة: طريقة بديلة للحصول على الدوارات التجريبية هي أن يفقس البيض يستريح. تعتبر الدوارات التي تحاك من البيض يستريح لتكون متزامنة بشكل جيد، وحياتها لا يختلف كثيرا عن أن من الدوارات التي تحاك من البيض amictic 11. ومع ذلك، الدوارات من البيض يستريح تبدأ التكاثر في وقت سابق من تلك التي من البيض amictic. وبالتالي، لا بد من الحذر لقياس الصفات الإنجاب. </li></ol> 3. تزامن الروتيفيرا من قبل الثقافة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> حدد حيدة الخلية والروتيفر واحد من شارعسكان اوك والثقافة كما هو موضح في 2،1-2،3 لإقامة شبه سكان التي سيتم استخدامها لإجراء التجارب. ثقافة عادة لمدة أسبوعين. </li><li style=";text-align:right;direction:rtl"> جمع الدوارات التي تحمل البيض من سكان الفرعي (جمع عدد مزدوج من الأفراد التي سيتم استخدامها لتجربة). الثقافة بأنها مجموعة نموذجية واحدة (الكثافة: ~ 50 فردا / مل) في لوحة الثقافة 6 جيدا كما هو موضح في 2،1-2،3 في وسائل الإعلام الجديدة تحت تغذية libitum الإعلانية. السيطرة على الكثافة السكانية مهم جدا لأنه وسيلة مكيفة يؤثر علم وظائف الأعضاء التناسلية من الدوارات Brachionus 32،33. </li><li style=";text-align:right;direction:rtl"> نقل حديثي الولادة تحاك من البيض الأولى الراشدين إلى وسائل الإعلام ثقافة أعد حديثا. كرر هذا الإجراء أكثر من 2-3 أجيال. </li><li style=";text-align:right;direction:rtl"> استخدام حديثي الولادة تحاك في غضون فترة زمنية معينة (على سبيل المثال، &lt;3 ساعة) لقياس الوقت البقاء على قيد الحياة. لتجنب التحيز المحتمل في اختيار فرد واحد، على الرغم من واردا جدا، والتحقق من استنساخ باستخدام العديد INDEمعلقة-الفرعية من السكان. ملاحظة: كما الدوارات عادة ما تتكاثر لاجنسيا فقط تحت هذا الشرط، ضمان عدم وجود الذكور موجودة في كافة مراحل التجربة. الذكور هم أصغر من حديثي الولادة وعادة ما تتحرك بسرعة أكبر من الإناث. الإناث Mictic لها أعمار مختلفة من الإناث amictic في ظل ظروف معينة (29). </li></ol> 4. قياسات الوقت البقاء على قيد الحياة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> مكان حديثي الولادة في لوحات بلاستيكية (عادة 24- أو 48-جيدا لوحات، مع كل منها يحتوي أيضا 1 مل من مياه البحر الاصطناعي). </li><li style=";text-align:right;direction:rtl"> في 24 فترات ساعة، ونقل الدوارات إلى وسائل الإعلام الثقافة أعدت حديثا أو في 12 ساعة فترات إذا في ارتفاع درجة الحرارة (30 درجة مئوية أو أعلى). تسجيل عدد من ذرية وإذا كان كل فرد هو حيا أو ميتا. تسجيل حيدة الخلية والروتيفر في عداد الموتى عند حركة أهداب من الهالة قد توقف تماما. ملاحظة: الروتيفيرا غالبا ما نعلق على الجدران الجانبية من الآبار. pipetting لطيف من الماء يساعد على العثور عليها. إذا لم يتم العثور على الدوارات أو عالبريد التالفة عن طريق الخطأ من قبل pipetting ل، تسجيلها باسم &quot;رقابة&quot;، وليس كما &quot;حيا&quot;. </li><li style=";text-align:right;direction:rtl"> إزالة حديثي الولادة عند الدوارات التجريبية تتوالد بنشاط. حديثي الولادة تنمو بسرعة وأنه من الصعب أحيانا التمييز بينها وبين الدوارات التجريبية. </li></ol> تحليل 5. البيانات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إنشاء بقاء منحنى كابلان ماير (الشكلان 1 و 2) من خلال التآمر معدل البقاء على قيد الحياة التراكمي على المحور Y والوقت على المحور العاشر. هذا هو التمثيل الأكثر شيوعا من بيانات البقاء على قيد الحياة. استخدام غير حدودي الاختبار سجل رتبة (وتسمى أيضا رف كوكس test) لمقارنة إحصائية من الوقت البقاء على قيد الحياة 34. كما يتم تضمين اختبار سجل رتبة في الحزم الإحصائية القياسية الأخرى مثل أحزاب اللقاء المشترك وR. ملاحظة: لا تستخدم ر الطالب اختبار أو تحليل التباين (ANOVA)، يليه مقارنة متعددة حدودي لأنه عادة ما يتم الوفاء التوزيع العادي عن طريق البيانات البقاء على قيد الحياة 35. أيضا، هذه methoس لا تأخذ الأفراد رقابة بعين الاعتبار. مان ويتني U اختبار يمكن أن تستخدم إذا كان هناك أية بيانات للرقابة. </li></ol>

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<li>Gribble, K. E., Welch, D. B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Life-span+extension+by+caloric+restriction+is+determined+by+type+and+level+of+food+reduction+and+by+reproductive+mode+in+Brachionus+manjavacas+%28Rotifera%29%5BTitle%5D)+AND+%22J.+Gerontol.+A+Biol.+Sci.+Med.+Sci%22%5BJournal%5D)">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/pubmed?term=((Changes+in+expression+patterns+of+stress+protein+genes+during+population+growth+of+the+rotifer+Brachionus+plicatilis%5BTitle%5D)+AND+%22Fish.+Sci%22%5BJournal%5D)">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/pubmed?term=((Behavior+and+reproduction+of+the+rotifer+Brachionus+plicatilis+species+complex+under+different+light+wavelengths+and+intensities%5BTitle%5D)+AND+%22Int.+Rev.+Hydrobiol%22%5BJournal%5D)">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/pubmed?term=((Effect+of+conditioned+media+on+the+asexual+reproduction+of+the+monogonont+rotifer+Brachionus+plicatilis+O.+F.+M%C3%BCller%5BTitle%5D)+AND+%22Hydrobiologia%22%5BJournal%5D)">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/pubmed?term=((A+novel+growth-promoting+protein+in+the+conditioned+media+from+the+rotifer+Brachionus+plicatilis+at+an+early+exponential+growth+phase%5BTitle%5D)+AND+%22Hydrobiologia%22%5BJournal%5D)">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>GraphPad Statistics Guide. , Available from:
 <a target="_blank" href="http://www.graphpad.com/guides/prism/6/statistics/">http://www.graphpad.com/guides/prism/6/statistics/</a> (2015).</li>
<li>Collet, D. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aCollet%2C+D+%22Modelling+Survival+Data+in+Medical+Research%22">Modelling Survival Data in Medical Research</a>. , 2nd edn, Chapman & Hall/CRC. 151-193 (1993).</li>
<li>Bouliotis, G., Billingham, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Crossing+survival+curves%3A+alternatives+to+the+log-rank+test%5BTitle%5D)+AND+%22Trials%22%5BJournal%5D)">Crossing survival curves: alternatives to the log-rank test.</a> Trials. 12, Suppl 1. A137(2011).</li>
<li>Yang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Changes+in+expression+of+manganese+superoxide+dismutase%2C+copper+and+zinc+superoxide+dismutase+and+catalase+in+Brachionus+calyciflorus+during+the+aging+process%5BTitle%5D)+AND+%22PloS+ONE%22%5BJournal%5D)">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/pubmed?term=((Joint+inhibition+of+TOR+and+JNK+pathways+interacts+to+extend+the+lifespan+of+Brachionus+manjavacas+%28Rotifera%29%5BTitle%5D)+AND+%22Exp.+Gerontol%22%5BJournal%5D)">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/pubmed?term=((Aging+in+nematode+Caenorhabditis-elegans+-+major+biological+and+environmental-factors+influencing+life-span%5BTitle%5D)+AND+%22Mech.+Ageing+Dev%22%5BJournal%5D)">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/pubmed?term=((The+role+of+parental+age+effects+on+the+evolution+of+aging%5BTitle%5D)+AND+%22Evolution%22%5BJournal%5D)">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.

يصف البروتوكول الحالي وسيلة لقياس الوقت البقاء على قيد الحياة في الدوارات Brachionus. الخطوة الحاسمة هي تزامن الظروف حيدة الخلية والروتيفر على مدى عدة أجيال. عندما تتم مزامنة الدوارات التجريبية بشكل جيد، لوحظ نوع نموذجي منحنى أنا البقاء على قيد الحياة مع القليل جدا ...

الدوارات هي المجهري العوالق الحيوانية عالمية (&lt;1 مم) التي تشكل الأسرة في اللغات الدوارات 1. لديهم خطة هيئة بسيطة تتألف من حوالي 1000 الخلايا الجسدية فضلا عن جهاز الهدبية عجلة تشبه مميزة تسمى الهالة، والذي يستخدم لتنقل والتغذية. معظم الدوارات تنتمي إلى الطبقات Monogononta أو Bdelloidea، والتي تحتوي على حوالي 1600 و 500 نوعا، على التوالي 2. يكون الدوارات Monogonont عموما مرحلتي الإنجابية الجنسية واللاجنسية (التوالد العذري الدوري)، في حين تتكاثر الدوارات bdelloid من التوالد العذري اجبة 3. ومن الممكن بالتالي الحصول على الأفراد حيدة الخلية والروتيفر متطابقة وراثيا، والتي تضمن استنساخ عالية في التجارب. وبالإضافة إلى ذلك، لديهم العديد من المزايا الأخرى مثل الكائنات النموذج، مثل عمر قصير، وسهولة الثقافة، وتوافر الجينومية وtranscriptomic البيانات تسلسل 4-7، وموقف النشوء والتطور الفريد من بعيدrthropods والديدان الخيطية 8. ولذلك الدوارات واعدة نماذج اللافقارية في الايكولوجية، السمية، والدراسات 9-12 الشيخوخة. الوقت البقاء على قيد الحياة تحت التعرض للإجهاد البيئي أو المواد الكيميائية غير معلمة قياس في كثير من الأحيان في هذه المجالات البحثية 13-19. ومع ذلك، هناك حاجة إلى الحذر عند قياس الوقت البقاء على قيد الحياة من الدوارات لأنها عرضة للظروف البيئية أمهاتهم. وهي، في monogonont manjavacas حيدة الخلية والروتيفر Brachionus، المواليد الإناث من الأمهات الذين تتراوح أعمارهم بين لها عمر أقصر من تلك التي من الأمهات الشابات. ومع ذلك، وتقييد السعرات الحرارية الأمهات (CR) تعوض جزئيا من الآثار الضارة للمتقدم عمر الأم 20. في B. plicatilis، توفر CR الأمهات طول العمر ذرية، وقت بقاء طويل تحت التجويع، وارتفاع الأكسدة المقاومة الضغط المرتبطة مع تعزيز التعبير عن الانزيمات المضادة للأكسدة 21،22. تأثير عمر الأمكما لوحظ في الدوارات bdelloid 23. ولذلك، فإن ظروف الدوارات التجريبية يجب أن تكون متزامنة بعناية على مدى عدة أجيال قبل أن قياسات الوقت البقاء على قيد الحياة. نحن هنا نقدم بروتوكول لقياس الوقت البقاء على قيد الحياة في الدوارات Brachionus التالية تزامن الظروف والثقافة على مدى عدة أجيال. الصوم المتقطع (IF)، والاختلاف من CR حيث يتم تغذية الدوارات بشكل دوري، تم تطبيق للكشف عن تأثير التزامن بسبب الآثار المعروفة للIF على طول العمر 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.

ويبين الشكل 1 منحنيات البقاء على قيد الحياة تمثيلية من السكان سيئة متزامنة (من أصل اثنين مكررات). في هذه التجربة، كانت الدوارات إما كل يوم بتغذية [libitum الإعلانية مجموعة (AL)] أو مرة كل يومين (IF المجموعة). كان بقاء متوسط ​​13 و 18 يوما في سو...

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.

قياس الوقت البقاء على قيد الحياة في<em&gt; Brachionus</em&gt; الروتيفيرا: التزامن من شروط صحة الأم

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

Research

JoVE Journal

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.

طريقة تعيين النسخة متماثلة: نهج الفائق "قياس كمي" ايليجانس كاينورهابديتيس عمر

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.

"القيامة نائمة" Daphnia magna: البروتوكول والتطبيقات

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

Environment

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.

قياس تنسخي سبان الحياة في الخميرة الناشيء.

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

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.

طرق المسح منخفضة التكلفة لقياس العمر الافتراضي والمدى الصحي في 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.

قياس الصلاحية الجنينية وحجم الحضنة في 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.

قياس عمر في<em&gt; ذبابة الفاكهة الدروسوفيلا</em

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

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.

المراقبة والقياس الكمي للسلوك التزاوج في غابة الصنوبر نيماتودا،<em&gt; Bursaphelenchus xylophilus</em

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

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

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

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

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

قياس معدلات استهلاك الأوكسجين في سليمة ايليجانس كاينورهابديتيس

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

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.

قياسات استجابات الإجهاد الفسيولوجي في C. Elegans

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

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

Source: Sarasija, S. and Norman, K. R. <a href="https://www.jove.com/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.

تزامن الديدان الخيطية: طريقة للحصول على مجموعات الديدان في مراحل متطابقة من التطور

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

قياس الوقت البقاء على قيد الحياة في

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