Staging egg development of marine copepods with DAPI and PicoGreen®

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.

Myofibril Changes in the Copepod Pseudodiaptomus marinus Exposed to Haline and Thermal Stresses

“…Such a study could be done using Confocal Laser Scanning Microscopy (CLSM) considering their sensitivity to excitation by visible wavelength proven in the literature and in our study. However, most imaging studies of copepods with a confocal microscope were focussing on the morphology of adults [16] or on the identification of different stages of egg development [17] using lipophilic probes and fluorescent nucleic acid stains as markers of nuclei. Double labeling methods (Annexin or Tunel with propidium iodide) have allowed the detection of cell degradation before the death of nauplii produced by females fed on toxic diets [18].…”

Visualisation of the copepod female reproductive system using confocal laser scanning microscopy and two-photon microscopy

“…CLSM has been applied in a number of copepod studies to examine cuticle morphology using auto-fluorescence (Buttino et al, 2003;Michels, 2007;Zirbel et al, 2007). More generally CLSM has been used to examine nerve terminals of muscles in crayfish (Harrington and Atwood, 1995) and various neurons within the nervous system in lobsters (Oginsky et al, 2010).…”

“…More generally CLSM has been used to examine nerve terminals of muscles in crayfish (Harrington and Atwood, 1995) and various neurons within the nervous system in lobsters (Oginsky et al, 2010). Coupling with molecular probes such as DiOC 6 has proved successful in developmental studies including observations of embryonic development (Buttino et al, 2003;Zirbel et al, 2007). 4 6 diamidino-2-phenylindole (DAPI) has been previously used for labelling copepod eggs and nauplii (Zirbel et al, 2007).…”

“…Coupling with molecular probes such as DiOC 6 has proved successful in developmental studies including observations of embryonic development (Buttino et al, 2003;Zirbel et al, 2007). 4 6 diamidino-2-phenylindole (DAPI) has been previously used for labelling copepod eggs and nauplii (Zirbel et al, 2007). These powerful imaging techniques have enabled whole-mount observations of developmental processes within the copepod body without time-consuming manual dissection.…”

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

“…Moreover, as hardness of the chitinous wall varies between zooplankton species and embryonic developmental stages (Cesar 1989;Fryer 1996), the chitinase treatment might cause a differential damage when embryos at different developmental stages and/or of different species are analyzed together. Recently, Zirbel et al (2007) devised a method to stain nuclei in formalin-preserved copepod eggs with semi-permeant dyes DAPI and PicoGreen without destroying the chorion. The method is based on creating an osmotic imbalance between egg matrix and the external staining medium by using de-ionized water as a staining vehicle, which stresses the egg chorion enough to allow fluorescent stain molecules into the egg and nuclei.…”