A single-step staining method to evaluate egg viability in zooplankton

A simplified method for viability analysis of zooplankton eggs by staining of nonviable eggs with a fluorescent nucleic acid stain TO-PRO-1 iodide is proposed here as a further development of fluorescence-based egg viability assays. This is one-step analysis with no intermediate steps for chorion removal. The method was calibrated using predetermined mixtures of viable and nonviable eggs (rotifers and copepods), and validated using hatching experiments (copepods) and egg development assay (cladocerans) as reference measurements. In these tests, eggs of several zooplankton species, Brachionus plicatilis (Rotatoria), Daphnia magna (Cladocera), Nitocra spinipes (Harpacticoida), Acartia tonsa (Calanoida), were used. Moreover, staining efficiency was not affected by storage of samples for up to 1 month in -80 degrees C, making the assay suitable for egg viability assessment in field and laboratory studies. To illustrate usefulness of the method, it was applied to evaluate how absence of re-mating affects production of viable eggs in females of A. bifilosa (Calanoida). In females separated from males, proportion of sterile eggs increased in 3 d after the separation and no viable eggs were produced after 5 d. The effects of mating frequency on egg viability are important to understand when designing egg production experiments and interpreting field data on egg viability in populations with skewed sex ratios.