Flow cytometry analysis of DNA ploidy levels and protein profiles distinguish between populations of Lumbriculus (Annelida: Clitellata)

• Published in: Invertebrate biology 2016-12, Vol.135 (4), p.385-399
• Main Authors: Tweeten, Kay A., Morris, Samantha J.
• Format: Article
• Language: English
• Subjects: Annelida; Clitellata; hemoglobin; heterochromatin; isoelectrofocusing; Lumbriculus; Lumbriculus clades
• ISSN: 1077-8306; 1744-7410
• DOI: 10.1111/ivb.12150

Variations in DNA ploidy have been observed in Lumbriculus, a freshwater annelid, as well as in other clitellates. Interpretation and application of experimental results using these animals may be impacted as ploidy levels affect the 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 the ploidy levels in different populations of Lumbriculus, including a laboratory strain (Environmental Protection Agency), a commercial strain (Aquatic Foods), and worms collected from natural habitats. To isolate nuclei, worms were homogenized, filtered to remove cell debris, and centrifuged through OptiprepTM density gradients. Nuclei were recovered, treated with RNAse, and stained with propidium iodide. Flow cytometry of the labeled nuclei showed that Lumbriculus from natural habitats in Minnesota and Iowa were diploid, with an estimated genome size of 2.7 pg. Populations from natural habitats in California and Oregon were highly polyploid, as were the laboratory and commercial strains. Chromosome spreads verified the high ploidy levels indicated by flow cytometry results, but also suggested that flow cytometry may be underestimating the DNA content levels. Staining of nuclei with diamidino-2-phenylindole indicated that this may be due to high levels of heterochromatin in nuclei from polyploid forms of Lumbriculus. To further compare the populations, proteins in worm homogenates were subjected to isoelectrofocusing 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. The results further support taxonomic classification of the diploid and polyploid forms of Lumbriculus as distinct species.