Long Term Experimental Evolution (LTEE) studies can be used to understand major evolutionary events such as the origin of multicellularity in animals. Such studies require a thorough understanding of the characteristics of the evolving organism as well as a reliable nutrient resource, an efficient transfer regime, and knowledge of the growth rate of the organism. The intention to carry out such experiments using several species of choanoflagellates, including Salpingoeca rosetta, has led to the examination of conditions necessary for their long term propagation.
A new medium was developed as an inexpensive and readily prepared growth resource. A comparison of cell densities and doubling times demonstrated that new barley medium (artificial sea water infused with barley) is a suitable long term growth medium. Cell counts performed at regular intervals determined the growth curve of S. rosetta at 24°C in new barley media. Two distinct growth phases were observed within the growth curve for which doubling times were determined. Finally, testing of a four day transfer protocol as well as a freezing and recovery protocol supported the previous finding and demonstrated effectiveness in the maintenance of choanoflagellate populations. The experiment demonstrated that maintenance does not require and is actually negatively affected by the use of shaken-flask cultures. In addition, a comparison of periodically frozen samples with those which were transferred regularly showed that freezing does not present a detriment to cell culture density.
Knowledge of transmission genetics is also crucial to the development of a more complete understanding of choanoflagellates and their role in metazoan evolution. Although not yet observed, genetic recombination in choanoflagellates is likely, based on the presence of conserved genes for meiosis. To test for recombination, I examined the effectiveness of a protocol using peptide nucleic acids (PNAs). PNA-mediated PCR clamping may allow for the detection of rare recombinant DNA within mixed populations. I present preliminary data regarding the effectiveness of PNAs designed against strain specific sequences in E.coli that will be used to design an experiment to detect sexual reproduction from the amplification of rare recombinant DNA using PNA-mediated PCR clamping.