Staging egg development of marine copepods with DAPI and PicoGreen®

Marnie Jo Zirbel, Charles B. Miller,Harold P. Batchelder

Limnol. Oceanogr. Methods 5:106-110 (2007) | DOI: 10.4319/lom.2007.5.106

ABSTRACT: Calanoid copepod eggs have a robust, chitinous outer chorion, which makes field-collected, formaldehydefixed eggs difficult to penetrate with stains or molecular probes. Egg development studies in copepods have involved physical, chemical, and enzymatic treatments to remove the chorion. We present an efficient, onestep method for staining copepod eggs with the fluorescent nucleic acid stains DAPI and PicoGreen®. Nuclei in treated eggs are clearly visible for examination and counting with compound and confocal microscopy, so that eggs can be rapidly classified with respect to developmental stage. The method is effective for eggs of Calanus, Metridia, and Centropages. Both stains were effective after 24-h exposure for eggs that were fixed from several days to 8 years. Early stages are distinguished by complete counts of nuclei. A blastula phase and gastrulation are distinctive. Calanus pacificus and Calanus marshallae embryos spend more than half the development period in the later ‘gray ball’ and limb bud stages. Stage classification by this method is useful for studies of copepod egg mortality in the field.