Double-labelling methods used to diagnose apoptotic and necrotic cell degradations in copepod nauplii

Two double-labelling methods, Tunel+propidium iodide and Annexin V-FITC+propidium iodide, have been tested to diagnose cell degradation processes in N1-N2 nauplius stages produced by spawning females of the copepod Calanus helgolandicus fed either non-toxic (PRO: control) or toxic (TR1) diets under laboratory conditions. Observation of labelled samples with a confocal laser-scanning microscope revealed that the maternal-food effect, following absorption of the toxic diet, induced cell apoptosis and necrosis in 80-100% of the offspring, which sooner or later died. Similar cell damages were also observed in 18-50% of the nauplii produced by females incubated in filtered seawater. Such cell degradation processes and high mortality were not observed in nauplii produced by females fed the non-toxic diet. Our protocols allowed detection of cell degradations before death of unhealthy larvae, preferentially with a confocal microscope or a conventional visible-fluorescent-light microscope.