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.
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.
Rotifers are microscopic cosmopolitan zooplankton (<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.
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.
2. General Culture Conditions
3. Synchronization of Rotifers by Pre-culture
4. Measurements of Survival Time
5. Data Analysis
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 and IF groups. Empirically, insufficient synchronization results in early mortality and gradual declines in survival rate as observed in this experiment. Damage to rotifers caused by inappropriate treatment or low water quality for the sub-population tends to yield similar results.
When rotifer conditions are optimal and well synchronized, early mortality is hardly observed and accordingly rotifers tend to die in a synchronized way during the later phase of the experiment (Figure 2). Median survival was 13 and 20 days in the AL and IF groups, respectively. Although fewer animals were used than the experiment in Figure 1, the difference in lifespan between these groups was statistically significant. This is the representative results from more than five experiments that have been published previously 22.
Figure 1: Kaplan-Meier curves for poorly synchronized individuals subjected to intermittent fasting (IF). The AL group was fed ad libitum throughout the experiment, whereas the IF group was fed every other day. N = 11 and N = 12 for the AL and IF groups, respectively (N refers to number of individual used in the experiment). The experiment was performed at 25 °C. No significant difference in lifespan was detected when log-rank test was used (P = 0.1207). However, this data is difficult to interpret because log-rank test should not be used to compare two crossing survival curves although the test is known to be robust 36. No established methods are currently available for crossing survival curves with censored data. Please click here to view a larger version of this figure.
Figure 2: Kaplan-Meier curves for synchronized individuals subjected to IF. The rotifer cohort, obtained by pre-culture, was subjected to the same IF schedule (fed every other day). N = 6 and N = 8 for AL and IF groups, respectively. The experiment was performed at 25 °C. Log-rank test, P = 0.0057. Please click here to view a larger version of this figure.
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 power. Synchronization is also expected to increase reproducibility of survival time measurements – because mothers are cultured under optimal conditions, the current protocol offsets possible deleterious effects of maternal generations. If early mortality is still observed after careful synchronization, consider using newly prepared culture media, another lot of feeding algae, or a newly established experimental cohort (i.e., start from protocol 3.1).
A limitation of this protocol is that the well-synchronized rotifers are potentially over-sensitive. For example, upon screening of chemicals that extend lifespan, some chemicals screened by this protocol may fail to detect significant effects on lifespans of poorly synchronized rotifers (e.g., individuals from wild and batch-cultured populations). Thus, the results of such experiments should be interpreted with caution.
The effect of maternal age on offspring survival time has also been reported in other invertebrate models including the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans 39,40. Although it is more time-consuming in these long-lived models, the synchronization procedure over several generations would be useful for these animals to decrease experimental variations in measurement of survival time.
The authors have nothing to disclose.
We are grateful to George Jarvis, Martha Bock, and Bette Hecox-Lea, Marine Biological Laboratory, for their help in filming.
Sodium chloride | Wako | 190-13921 | |
Magnesium chloride | Wako | 136-03995 | |
Magnesium sulfate | Wako | 131-00427 | |
Potassium chloride | Wako | 168-22111 | |
Calcium chloride | Wako | 035-00455 | |
Sodium bicarbonate | Wako | 199-05985 | |
Sodium bromide | Wako | 190-01515 | |
Membrane filter (0.45 µm pore size) | Millipore | HAWP04700 | |
Culture plate, 6-well, non-treated | Thomas Scientific | 6902D01 | Flat bottom |
Culture plate, 48-well, non-treated | Thomas Scientific | 6902D07 | Flat bottom |
Tetraselmis, Living | Carolina Biological Supply Company | 152610 | |
PRISM 6 | GraphPad Software | Version 6.0d |