Rapid assessment of copepod (Calanus helgolandicus) embryo viability using fluorescent probes

Vital fluorescent probes have routinely been used to distinguish viable from non-viable embryos in various veterinary and aquaculture studies. Here, we present new protocols to rapidly detect embryo viability in the copepod Calanus helgolandicus using three of these probes, fluorescein diacetate (FDA), SYTOX green and 7-aminoactinomycin D (7-AAD), and the confocal laser scanning microscope. The percentage of fluorescent-FDA embryos and non-fluorescent SYTOX green and 7-AAD embryos were compared with the percentage of hatched unstained embryos and with the percentage of embryos that had been stained, washed, and allowed to hatch. Results showed that all three dyes accurately predicted embryo viability and could be used to rapidly calculate C. helgolandicus egg-hatching success. We also tested the possible applications of SYTOX green in egg-production/egg-hatching assays in which the dinoflagellate Prorocentrum minimum or the diatom Skeletonema costatum are used to investigate for the possible negative impact of diatoms on embryo viability. Other possible applications for fluorescence methods in studies on the reproductive biology of zooplankton, and in particular of copepods, are discussed.