We are grateful to George Jarvis,&#160;Martha Bock, and Bette Hecox-Lea, Marine Biological Laboratory, for their help in filming.

1. Preparation of Media
Note: Use half-diluted Brujewicz artificial seawater of salinity 16.5 ppt (PSU). Other artificial seawaters are also frequently used to culture Brachionus rotifers 25,26.
<ol>
	<li>Add 454 mM NaCl, 26 mM MgCl2, 27 mM MgSO4, 10 mM KCl, and 10 mM CaCl2 to 4.5 L of distilled water (final volume will be 5 L). Alternatively, use deionized dilution water instead of distilled water. Add CaCl2 after dissolving al...

<ol>
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Sci. 77 (3), 403-409 (2011).</li><li>Garc&#236;a-Garc&#236;a, G., Sarma, S., N&#250;&#241;ez-Orti, A. R., Nandini, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+mixture+of+two+endocrine+disruptors+(ethinylestradiol+and+levonorgestrel)+on+selected+ecological+endpoints+of+Anuraeopsis+fissa+and+Brachionus+calyciflorus+(Rotifera).">Effects of the mixture of two endocrine disruptors (ethinylestradiol and levonorgestrel) on selected ecological endpoints of Anuraeopsis fissa and Brachionus calyciflorus (Rotifera).</a> Int. Rev. Hydrobiol. 99 (1-2), 166-172 (2014).</li><li>Yang, J., Mu, Y., Dong, S., Jiang, Q., Yang, J. <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+the+expression+of+four+heat+shock+proteins+during+the+aging+process+in+Brachionus+calyciflorus+(rotifera).">Changes in the expression of four heat shock proteins during the aging process in Brachionus calyciflorus (rotifera).</a> Cell Stress Chaperones. 19 (1), 33-52 (2014).</li><li>Han, 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=Sublethal+gamma+irradiation+affects+reproductive+impairment+and+elevates+antioxidant+enzyme+and+DNA+repair+activities+in+the+monogonont+rotifer+Brachionus+koreanus.">Sublethal gamma irradiation affects reproductive impairment and elevates antioxidant enzyme and DNA repair activities in the monogonont rotifer Brachionus koreanus.</a> Aquat. Toxicol. 155, 101-109 (2014).</li><li>Gribble, K. E., Jarvis, G., Bock, M., Mark Welch, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+caloric+restriction+partially+rescues+the+deleterious+effects+of+advanced+maternal+age+on+offspring.">Maternal caloric restriction partially rescues the deleterious effects of advanced maternal age on offspring.</a> Aging Cell. 13 (4), 623-630 (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+periodical+starvation+on+the+survival+of+offspring+in+the+rotifer+Brachionus+plicatilis.">Effect of periodical starvation on the survival of offspring in the rotifer Brachionus plicatilis.</a> Fish. Sci. 67 (2), 373-374 (2001).</li><li>Kaneko, G., 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=Calorie+restriction-induced+maternal+longevity+is+transmitted+to+their+daughters+in+a+rotifer.">Calorie restriction-induced maternal longevity is transmitted to their daughters in a rotifer.</a> Funct. Ecol. 25 (1), 209-216 (2011).</li><li>Lansing, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transmissible,+cumulative,+and+reversible+factor+in+aging.">A transmissible, cumulative, and reversible factor in aging.</a> J. Gerontol. 2 (3), 228-239 (1947).</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+periodical+starvation+on+the+life+history+of+Brachionus+plicatilis+O.+F.+M&#252;ller+(Rotifera):+a+possible+strategy+for+population+stability.">Effect of periodical starvation on the life history of Brachionus plicatilis O. F. M&#252;ller (Rotifera): a possible strategy for population stability.</a> J. Exp. Mar. Biol. Ecol. 253 (2), 253-260 (2000).</li><li>Gribble, K. E., Kaido, O., Jarvis, G., Mark Welch, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+intraspecific+variability+in+the+response+to+caloric+restriction.">Patterns of intraspecific variability in the response to caloric restriction.</a> Exp. Gerontol. 51, 28-37 (2014).</li><li>Snell, T. W., Johnston, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycerol+extends+lifespan+of+Brachionus+manjavacas+(Rotifera)+and+protects+against+stressors.">Glycerol extends lifespan of Brachionus manjavacas (Rotifera) and protects against stressors.</a> Exp. Gerontol. 57, 47-56 (2014).</li><li>Kim, H. -. J., 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=Effect+of+female+aging+on+the+morphology+and+hatchability+of+resting+eggs+in+the+rotifer+Brachionus+plicatilis+M&#252;ller.">Effect of female aging on the morphology and hatchability of resting eggs in the rotifer Brachionus plicatilis M&#252;ller.</a> Hydrobiologia. 662 (1), 107-111 (2011).</li><li>Kim, H. -. 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. 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The current protocol describes a method for measuring the survival time in Brachionus rotifers. The critical step is the synchronization of rotifer conditions over several generations. When experimental rotifers are well synchronized, a typical type I survival curve is observed with very little early mortality as reported in several previous studies 18,24,37,38. Standard deviations of their survival time therefore become smaller compared to poorly synchronized rotifers, resulting in high statistical p...

Rotifers are microscopic cosmopolitan zooplankton (&#60;1 mm) that constitute the phylum Rotifera 1. They have a simple body plan composed of approximately 1,000 somatic cells as well as a characteristic wheel-like ciliary apparatus called the corona, which is used for locomotion and feeding. Most rotifers belong to classes Monogononta or Bdelloidea, which contain about 1,600 and 500 species, respectively 2. Monogonont rotifers generally have both sexual and asexual reproductive phases (cyclical parthenogenesis), while bdelloid rotifers reproduce by obligatory parthenogenesis 3. It is thus possible to obtain genetically identical rotifer individuals, which ensures high reproducibility in experiments. In addition, they have several other advantages as model organisms, such as a short lifespan, ease of culture, availability of genomic and transcriptomic sequence data 4-7, and a unique phylogenetic position distant from arthropods and nematodes 8. Rotifers are therefore promising invertebrate models in ecological, toxicological, and aging studies 9-12.
The survival time under exposure to environmental stress or chemicals is a frequently measured parameter in these research fields 13-19. However, caution is needed when measuring the survival time of rotifers because it is susceptible to environmental conditions of their mothers. Namely, in the monogonont rotifer Brachionus manjavacas, female offspring from aged mothers have a shorter lifespan than those from young mothers; however, maternal calorie restriction (CR) partially offsets the deleterious effects of advanced maternal age 20. In B. plicatilis, maternal CR provides offspring longevity, long survival time under starvation, and high oxidative stress resistance associated with enhanced expression of antioxidant enzymes 21,22. The maternal age effect has also been observed in bdelloid rotifers 23. Therefore, the conditions of experimental rotifers should be carefully synchronized over several generations before measurements of survival time.
Here we provide a protocol for measurement of survival time in Brachionus rotifers following synchronization of culture conditions over several generations. Intermittent fasting (IF), a variation of CR where rotifers are fed periodically, was applied to reveal the effect of synchronization due to the well-known effects of IF on longevity 22,24.

<table border="0" cellpadding="0" cellspacing="0" width="688">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Sodium chloride</td>
			<td style="width:135px;">Wako</td>
			<td style="width:119px;">190-13921</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Magnesium chloride</td>
			<td style="width:135px;">Wako</td>
			<td style="width:119px;">136-03995</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Magnesium sulfate</td>
			<td style="width:135px;">Wako</td>
			<td>131-00427</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Potassium chloride</td>
			<td style="width:135px;">Wako</td>
			<td style="width:119px;">168-22111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Calcium chloride</td>
			<td style="width:135px;">Wako</td>
			<td style="width:119px;">035-00455</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Sodium bicarbonate</td>
			<td style="width:135px;">Wako</td>
			<td style="width:119px;">199-05985</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Sodium bromide</td>
			<td style="width:135px;">Wako</td>
			<td style="width:119px;">190-01515</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Membrane filter (0.45 &#181;m pore size)</td>
			<td style="width:135px;">Millipore</td>
			<td style="width:119px;">HAWP04700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Culture plate, 6-well, non-treated</td>
			<td style="width:135px;">Thomas Scientific</td>
			<td style="width:119px;">6902D01</td>
			<td>Flat bottom</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Culture plate, 48-well, non-treated</td>
			<td style="width:135px;">Thomas Scientific</td>
			<td style="width:119px;">6902D07</td>
			<td>Flat bottom</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:268px;">Tetraselmis, Living</td>
			<td style="width:135px;">Carolina Biological Supply Company</td>
			<td style="width:119px;">152610</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">PRISM 6</td>
			<td style="width:135px;">GraphPad Software</td>
			<td style="width:119px;">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.

Figure 1 shows representative survival curves of poorly synchronized populations (out of two replicates). In this experiment, rotifers were either fed everyday [ad libitum (AL) group] or every other day (IF group). Median survival was 13 and 18 days in the AL and IF groups, respectively. Although it is well known that IF extends the lifespan of the rotifer, this experiment failed to detect a statistically significant difference between lifespans of the AL ...

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

Rotifers are microscopic zooplankton used as models in ecotoxicological and aging studies. Here we provide a protocol for powerful and reproducible measurement of survival time in Brachionus rotifers. Synchronization of culture conditions over several generations is of particular importance because maternal condition affects life history of offspring.

Measurement of Survival Time in 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 ...

The overall goal of this procedure, is to accurately measure the survival time of Brachionus Rotifers. By offsetting possible deleterious effects of maternal generations. This method can help answer key questions in aging research.By providing a reliable way to measure the survival time of rotifers. The main advantage of this technique is that it is reproduceable, and have high statistical power. To begin, to 4.5 liters of distilled or deionized dilution water, add 454 millimolar sodium chloride, 26 millimolar magnesium chloride, 27 millimolar magnesium sulfate, ten millimolar potassium chloride, and ten millimolar calcium chloride.Prepare a 0.48 molar stock solution of sodium bicarbonate. And add 25 milliliters of it to the salt solution just prepared. Prepare a 0.4 molar stock solution of sodium bromide.And add ten milliliters of it, to the salt solution. Then, with distilled water, bring up the salt solution to five liters. And, using a 0.45 micrometer filter membrane, filter the solution.To culture laboratory raised, or wild captured rotifers, place them in a sterile 100 milliliter beaker, of artificial sea water. And culture them at 25 degrees Celsius. According to the methods of Snell, culture dietary micro algae species, in artificial sea water.Insuring that the same lot of micro algae are used in all experimental groups. Maintain a stock population of rotifers in a batch culture by feeding them every two days. And changing the medium every week.Many neonates and adults bearing two to three eggs, can be observed when rotifers are in optimal conditions. To synchronize rotifers by pre-culture, select a single rotifer from the stock population. And culture it in artificial sea water in a 100 milliliter beaker as just described for about two weeks.After the incubation, collect egg bearing rotifers from the sub-population. Culture them as a single cohort by placing approximately 50 rotifers per milliliter of sea water in the wells of a six well plate, and feed them ad libitum. When the eggs have hatched, transfer neonates to newly prepared culture medium.Repeat the culturing and transferring of neonates over two to three generations. To measure survival time, place neonates hatched within a three hour period of time, in the wells of 24 or 48 well plates containing one milliliter of artificial sea water. At 12 or 24 hour intervals, transfer the rotifers to newly prepared culture medium.Record the number of offspring, and whether each individual is dead or alive. Recording the rotifer as dead when the cilia movement of the corona has completely stopped. When experimental rotifers are actively reproducing, remove neonates since they grow rapidly and are sometimes difficult to distinguish from experimental rotifers.Carry out data analysis according to the text protocol. This figure shows survival curves from two replicates of poorly synchronized rotifer populations. Fed either ad libitum, shown as A-L, or every other day shown as Intermittent Fasting, I-F.Median survival was 13 and 18 days respectively, in the two groups. Although it is well known that intermittent fasting extends the life span of the rotifer, the differences were not statically significant. In this experiment, rotifer conditions were optimal and well synchronized.And the animals tended to survive until the later phase of the experiment. Median survival was 13 days for AL rotifers, and 20 days for the IF groups. Which was statically significant.After watching this video, you should have a good understanding of how to synchronize rotifer conditions. We hope that this protocol enhances of additive rotifers, of the model organism and aging research.

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8.3K visualizações.  The University of Tokyo. The overall goal of this procedure, is to accurately measure the survival time of Brachionus Rotifers. By offsetting possible deleterious effects of maternal generations. This method can help answer key questions in aging research.By providing a reliable way to measure the survival time of rotifers. The main advantage of this technique is that it is reproduceable, and have high statistical power. To begin, to 4.5 liters of distilled or deionized dilution water, add 454 millimolar sodiu...