Name: measurement of survival time in brachionus rotifers: synchronization of maternal conditions
Uploaded: 2016-07-22T13:00:00
Description: Watch this Scientific Journal Video about Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions at JoVE.com

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>
	<li>Wallace, R. L., Snell, T. W., Ricci, C., Nogrady, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Rotifera Vol.1: Biology, ecology and systematics. , (2006).</li><li>Segers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Annotated checklist of the rotifers (Phylum Rotifera), with notes on nomenclature, taxonomy and distribution. , (2007).</li><li>Mark Welch, D. B., Meselson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+the+evolution+of+bdelloid+rotifers+without+sexual+reproduction+or+genetic+exchange.">Evidence for the evolution of bdelloid rotifers without sexual reproduction or genetic exchange.</a> Science. 288 (5469), 1211-1215 (2000).</li><li>Suga, K., Mark Welch, D., Tanaka, Y., Sakakura, Y., 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=Analysis+of+expressed+sequence+tags+of+the+cyclically+parthenogenetic+rotifer+Brachionus+plicatilis.">Analysis of expressed sequence tags of the cyclically parthenogenetic rotifer Brachionus plicatilis.</a> PLoS ONE. 2, e671 (2007).</li><li>Denekamp, N. Y., 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=Discovering+genes+associated+with+dormancy+in+the+monogonont+rotifer+Brachionus+plicatilis.">Discovering genes associated with dormancy in the monogonont rotifer Brachionus plicatilis.</a> BMC Genomics. 10, 108 (2009).</li><li>Lee, J. -. S., 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=Sequence+analysis+of+genomic+DNA+(680+Mb)+by+GS-FLX-Titanium+sequencer+in+the+monogonont+rotifer,+Brachionus+ibericus.">Sequence analysis of genomic DNA (680 Mb) by GS-FLX-Titanium sequencer in the monogonont rotifer, Brachionus ibericus.</a> Hydrobiologia. 662 (1), 65-75 (2010).</li><li>Flot, J. -. F., 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=Genomic+evidence+for+ameiotic+evolution+in+the+bdelloid+rotifer+Adineta+vaga.">Genomic evidence for ameiotic evolution in the bdelloid rotifer Adineta vaga.</a> Nature. 500 (7463), 453-457 (2013).</li><li>Dunn, C. W., 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=Broad+phylogenomic+sampling+improves+resolution+of+the+animal+tree+of+life.">Broad phylogenomic sampling improves resolution of the animal tree of life.</a> Nature. 452 (7188), 745-749 (2008).</li><li>Yoshinaga, T., Kaneko, G., Kinoshita, S., 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=The+molecular+mechanisms+of+life+history+alterations+in+a+rotifer:+a+novel+approach+in+population+dynamics.">The molecular mechanisms of life history alterations in a rotifer: a novel approach in population dynamics.</a> Comp. Biochem. Physiol. B Biochem. Mol. Biol. 136 (4), 715-722 (2003).</li><li>Dahms, H. -. U., Hagiwara, A., Lee, J. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecotoxicology,+ecophysiology,+and+mechanistic+studies+with+rotifers.">Ecotoxicology, ecophysiology, and mechanistic studies with rotifers.</a> Aquat. Toxicol. 101 (1), 1-12 (2011).</li><li>Snell, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rotifers+as+models+for+the+biology+of+aging.">Rotifers as models for the biology of aging.</a> Int. Rev. Hydrobiol. 99 (1-2), 84-95 (2014).</li><li>Snell, T. W., Johnston, R. K., Gribble, K. E., 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=Rotifers+as+experimental+tools+for+investigating+aging.">Rotifers as experimental tools for investigating aging.</a> Invertebr. Reprod. Dev. 59, 5-10 (2015).</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=Molecular+characterization+of+Mn-superoxide+dismutase+and+gene+expression+studies+in+dietary+restricted+Brachionus+plicatilis+rotifers.">Molecular characterization of Mn-superoxide dismutase and gene expression studies in dietary restricted Brachionus plicatilis rotifers.</a> Hydrobiologia. 546, 117-123 (2005).</li><li>Yoshinaga, T., 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=Insulin-like+growth+factor+signaling+pathway+involved+in+regulating+longevity+of+rotifers.">Insulin-like growth factor signaling pathway involved in regulating longevity of rotifers.</a> Hydrobiologia. 546, 347-352 (2005).</li><li>Ozaki, Y., Kaneko, G., Yanagawa, Y., 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=Calorie+restriction+in+the+rotifer+Brachionus+plicatilis+enhances+hypoxia+tolerance+in+association+with+the+increased+mRNA+levels+of+glycolytic+enzymes.">Calorie restriction in the rotifer Brachionus plicatilis enhances hypoxia tolerance in association with the increased mRNA levels of glycolytic enzymes.</a> Hydrobiologia. 649 (1), 267-277 (2010).</li><li>Kailasam, M., 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=Effects+of+calorie+restriction+on+the+expression+of+manganese+superoxide+dismutase+and+catalase+under+oxidative+stress+conditions+in+the+rotifer+Brachionus+plicatilis.">Effects of calorie restriction on the expression of manganese superoxide dismutase and catalase under oxidative stress conditions in the rotifer Brachionus plicatilis.</a> Fish. 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. 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 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 ...

Watch this Scientific Journal Video about Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions at JoVE.com

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

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

Measurement of Survival Time in 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 ...

Research

JoVE Journal

Biology

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is ...

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

Measurement of Lifespan in Drosophila melanogaster

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 ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

The cell cycle is important for growth, genome replication, and development in all cells.  In bacteria, studies of the cell cycle have focused largely on ...

Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...