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

Abstract

New sensitive fluorochromes, PicoGreen and RiboGreen (Molecular Probes, Inc.), were used to detect ng ml−1 concentrations of nucleic acids (RNA and DNA) in single Daphnia individuals of all developmental stages. Nucleic acids were assayed by fluorescence with a microplate reader using (i) RiboGreen (non-specific dye) and PicoGreen (DNA-specific dye) together and (ii) RiboGreen with and without RNase. The two methods yield similar values for DNA and RNA. The second method has the advantage of determining both RNA and DNA within a single sample aliquot, allowing maximum use of available sample. In parthenogenically reproducing Daphnia pulex, RNA and DNA concentrations were related to changes in metabolic activity associated with moulting cycle and ontogenetic development. In juveniles, elevated DNA content was observed in the early postmoult, followed by an increase in RNA during intermoult and premoult. RNA concentration peaked at the latest stages of embryonic development, followed by a gradual decline during juvenile development, with the lowest values in adult females (eggs removed). Similarly, DNA concentrations were highest during the early phase of postnatal development, decreasing when body size increased. Our results suggest that ontogenetic variations have implications for the use of nucleic acids as a measure for growth in zooplankton and may provide useful insights into mechanisms of growth on the cellular level.