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

Summary

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.

Abstract

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.

Introduction

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 rotif....

Protocol

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.

1. 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 all other salts.
2. Prepare the 0.48 M NaHCO₃ stock solution (200x concentration). Add 25 ml of it to the above solution. The final co....

Results

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 an.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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