Meeting Abstract
Variations in DNA ploidy have been observed in Lumbriculus, a freshwater oligochaete, as well as in other annelids. Interpretation and application of experimental results using these animals may be impacted as ploidy levels affect protein expression, reproductive behavior and response to stressors. Ploidy is typically determined by chromosome spreads, a time-consuming and inefficient method. We adapted flow cytometry protocols used on vertebrates and plants to determine ploidy levels in Lumbriculus. Worms were from an Environmental Protection Agency lab, Aquatic Foods, and natural habitats. To isolate nuclei, Lumbriculus homogenates were filtered to remove cell debris and centrifuged through density gradients. Nuclei were recovered, treated with RNAse, and stained with propidium iodide. Flow cytometry of the labeled nuclei showed Lumbriculus from natural habitats in Minnesota and Iowa were diploid. Populations from natural habitats in California were highly polyploid as were the EPA and Aquatic Foods worms. Flow cytometry results were verified using chromosome spreads, confirming that flow cytometry provided a rapid, reliable way to determine Lumbriculus ploidy levels. We anticipate that this method could readily be applied to analysis of DNA content in other annelids. To further compare the populations, proteins in worm homogenates were subjected to isoelectric focusing gel electrophoresis. Distinct protein profiles were seen; one was shared in common by the diploid worms, the other was characteristic of polyploid populations. Diploid worms could also be distinguished from polyploid worms based on differences in hemoglobin linker proteins, modes of reproduction, and metabolic rates. The results further support classifying the diploid and polyploid forms of Lumbriculus as different species.
