PATTERNS OF GROWTH AND CULTURING PROTOCOLS FOR SALPINGOECA ROSETTA TO BE USED IN INVESTIGATIONS OF THE ORIGIN OF ANIMAL MULTICELLULARITY A Thesis Presented to The Graduate Faculty of The University of Akron In Partial Fulfillment of the Requirements for the Degree Master of Science Ashley Wain May, 2011 PATTERNS OF GROWTH AND CULTURING PROTOCOLS FOR SALPINGOECA ROSETTA TO BE USED IN INVESTIGATIONS OF THE ORIGIN OF ANIMAL MULTICELLULARITY Ashley Wain Thesis Approved: Accepted: _______________________________ _______________________________ Advisor Dean of the College Dr. Francisco Moore Dr. Chand Midha _______________________________ _______________________________ Committee Member Dean of the Graduate School Dr. Richard Londraville Dr. George R. Newkome _______________________________ _______________________________ Committee Member Date Dr. Lisa Park _______________________________ Department Chair Dr. Monte Turner ii ABSTRACT 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 iii 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. iv TABLE OF CONTENTS Page LIST OF TABLES ............................................................................................................ vii LIST OF FIGURES ......................................................................................................... viii CHAPTER I. PROTOCOL DEVELOPMENT FOR THE CULTURING OF SALPINGOECA ROSETTA ...............................................................................................1 Introduction ..............................................................................................................1 Materials and Methods .............................................................................................7 Results ....................................................................................................................16 Discussion ..............................................................................................................22 II. TESTING THE VIABILITY OF PNA-MEDIATED CLAMPING TO DETECT RECOMBINATION USING A PROKARYOTIC TEST SYSTEM ..............................34 Introduction ............................................................................................................34 Materials and Methods ...........................................................................................37 Results ....................................................................................................................45 Discussion ..............................................................................................................51 LITERATURE CITED ......................................................................................................53 APPENDIX A ....................................................................................................................59 Introduction ............................................................................................................59 v Materials and Methods ...........................................................................................60 Results and Discussion ..........................................................................................62 APPENDIX B ....................................................................................................................68 Introduction ............................................................................................................68 Materials and Methods ...........................................................................................70 Results ....................................................................................................................73 Discussion ..............................................................................................................74 APPENDIX C ....................................................................................................................77 Introduction ............................................................................................................77 Materials and Methods ...........................................................................................77 Results and Discussion ..........................................................................................78 vi LIST OF TABLES Table Page 1 Doubling times for two distinct growth phases as well as for overall growth ..................................................................................20 2 SNP locations in 4 lines of E. coIi .........................................................................37 3 Expected outcomes and results of PNA-mediated PCR clamping ........................51 4 Results of nested ANOVA of counts from four boxes. .........................................80 5 Results of nested ANOVA of counts from eight boxes .........................................81 vii LIST OF FIGURES Figure Page 1 Trends in doubling time across media ...................................................................17 2 Comparison of cell density between media ...........................................................17 3 A growth curve for Salpingoeca rosetta ................................................................19 4 Growth trends: Still v. shaken cultures .................................................................22 5 Effect of freezing on cell density ...........................................................................23 6 The Allee Effect .....................................................................................................31 7 Comparison of DNA and PNA structure ...............................................................35 8 Primers and PNAs binding to template DNA ........................................................38 9 Elongation arrest ....................................................................................................39 10 HPLC sample for PNA 303 ...................................................................................45 11 MALDI Mass Spectrophotometry .........................................................................46 12 Amplification of PAB B using PABB 1B/2B primer set .......................................47 13 PNA-mediated clamping of FBGM 1 and 10 (Trial 1) ......................................... 48 14 Clamping of FBGM 1 with PNA 303 (Trial 2)..................................................... 49 15 Optimization of PNA 303 annealing temperature (Trial 3) .................................. 49 16 Optimization of PNA 303 concentration (Trial 4) ................................................ 50 viii 17 Initial optimization of PNA 612 annealing temperature (Trial 5) .........................51 18 Choanoflagellate cell incubated in rhodamine phalloidin for 30 minutes .............63 19 Choanoflagellate cell incubated in rhodamine phalloidin for 45 minutes .............63 20 Choanoflagellate cell incubated in rhodamine phalloidin for 60 minutes .............63 21 Choanoflagellate cell fixed with 6% acetone.........................................................64 22 Possible choanoflagellate colony fixed with 6% acetone and 1% formaldehyde .........................................................................64 23 Choanoflagellate cell fixed with 4% glutaraldehyde .............................................64