Analysis of nucleic acids in Daphnia: development of methods and ontogenetic variations in RNA-DNA content

New sensitive fluorochromes, PicoGreen and RiboGreen (MolecularProbes, Inc.), were used to detect ng ml^-1 concentrations ofnucleic acids (RNA and DNA) in single Daphnia individuals ofall developmental stages. Nucleic acids were assayed by fluorescencewith a microplate reader using (i) RiboGreen (non-specific dye)and PicoGreen (DNA-specific dye) together and (ii) RiboGreenwith and without RNase. The two methods yield similar valuesfor DNA and RNA. The second method has the advantage of determiningboth RNA and DNA within a single sample aliquot, allowing maximumuse of available sample. In parthenogenically reproducing Daphniapulex, RNA and DNA concentrations were related to changes inmetabolic activity associated with moulting cycle and ontogeneticdevelopment. In juveniles, elevated DNA content was observedin the early postmoult, followed by an increase in RNA duringintermoult and premoult. RNA concentration peaked at the lateststages of embryonic development, followed by a gradual declineduring juvenile development, with the lowest values in adultfemales (eggs removed). Similarly, DNA concentrations were highestduring the early phase of postnatal development, decreasingwhen body size increased. Our results suggest that ontogeneticvariations have implications for the use of nucleic acids asa measure for growth in zooplankton and may provide useful insightsinto mechanisms of growth on the cellular level.