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.

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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

24 Effects of oxygen level on cell density over time ...... 74

25 Count averages for four-box samples ...... 79

26 Count averages for eight-box samples ...... 80

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CHAPTER I

PROTOCOL DEVELOPMENT FOR THE CULTURING OF SALPINGOECA

ROSETTA

Introduction

The transition from a world dominated by single-celled organisms to one that includes metazoans is a major evolutionary step upon which much of the diversity that has existed since is contingent. The identification of external factors that led to the evolution of a multicellular lifestyle is important to explain this process. Long Term

Experimental Evolution (LTEE) studies of the closest relatives of multicellular animals hold the potential to revolutionize our understanding of multicellular evolution. In order to gain that understanding, basic LTEE protocols need to be developed for these organisms.

The case for choanoflagellates as a study system

It is essential to understand how major changes in form and function can occur as the result of evolutionary processes as traits that were beneficial in the past are co-opted for present conditions. The important changes that become fixed, large and small, are not random and instantaneous, but can occur over thousands or millions of years as species

1 adapt to their environments. Investigation of the transition from unicellularity to multicellularity using LTEE will not only elucidate a driving force for macroevolutionary change, but will also provide information regarding the role of pre-adaptation and levels of selection. An interest in the transition to metazoan life dates back almost 150 years when James-Clark (1868) compared the feeding cells, choanocytes, of multicellular sponges with single-celled choanoflagellates, noticing that they shared a similar morphology.

This research has seemingly been rediscovered and has spurred increasingly advanced studies. Recently, several genome and protein-based studies have searched for the most likely ancestor to the metazoans (Snell et al. 2001, King 2004, Ruiz-Trillo et al.

2006, Steenkamp et al. 2006, King et al. 2008). While phylogenies based on ribosomal

DNA were not able to reliably elucidate the connection between choanoflagellates and metazoans, Snell et al. (2001) were able to demonstrate a robust relationship using Hsp70 protein sequences. That study spurned a number of other studies including a phylogenetic study of several genes, including Hsp70, based on data collected from multiple protist and metazoan samples (Steenkamp et al. 2006). They found gene sequences similar to those of animals as well as a 12 amino acid insertion in EF-1α, previously found only in metazoans. Overall, they demonstrated that choanoflagellates are part of a larger group, Opisthokonta, which also includes both animals and fungi.

King (2004) reviewed the available phylogenetic literature and choanoflagellate genomics to further elucidate the relationship between unicellular and multicellular organisms. She was able to demonstrate that while choanoflagellates have

2 characteristically protistan mtDNA with introns and intergenic DNA, they also have genes for distinctly animal proteins. Later, the genome of a particular species of choanoflagellate, Monosiga brevicollis, was sequenced (King et al. 2008). This allowed for the comparison of protein domains among M. brevicollis and several metazoans.

Although the physical linkages between the compared domains were often different, the presence of several shared domains coding for cell interaction proteins is indicative of a moderate degree of relatedness and encourages further research. UNICORN (Unicellular

Opisthokont Research Initiative), proposes to sequence the genomes of several animals and fungi along with the genomes of their closest unicellular relatives (Ruiz-Trillo et al.

2006). It is thought that opisthokonts may have a genetic predisposition for multicellularity and that comparative analyses of the sequenced genomes will further elucidate the possible evolution of metazoa from a protistan ancestor.

A functional link has also been demonstrated between choanoflagellates and metazoa with protein-based studies. Using protein annotation programs and studies of protein expression, King demonstrated that genes for cadherins, G protein-coupled receptors, tyrosine kinases, and C-type lectins in addition to protein-protein interaction domains are present and expressed in at least three choanoflagellate species (King et al.

2003, King 2004, King et al. 2008). Functional characterization of these proteins was also carried out in those studies. Tyrosine kinases, cadherins, and c-type lectins are among those proteins whose corresponding protozoan functions have most often been predicted.

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Tyrosine kinases, which are involved in intercellular signaling in multicellular organisms, have been shown to be involved in the detection of environmental conditions and to have important impacts on growth rates. Cadherin studies indicate that they are integral for colony formation and are also, along with c-type lectins, utilized for prey recognition and capture in choanoflagellates (King et al. 2003). Although both choanoflagellates and metazoans possess some or all of the domains for these proteins, as stated above, the purposes are different. It is thought that shuffling of these shared domains may have been responsible for the creation of new domain combinations and therefore the evolution of new protein functions in metazoans (Rokas 2008). According to Segawa et al. (2006), protein regulation also differs between the two groups and the evolution of a stable mechanism of regulation for tyrosine kinases may have corresponded with the transition to multicellularity.

Geology, habitats, and the origin of novelty

Late Proterozoic environmental change may have been a cue for the evolution of multicellularity from unicellular protists. Due to the dominance of unicellular life before this time, niche space was likely to be extremely limited and competition for resources high. However, if a clade had been pre-adapted for life at a different temperature, with more dissolved oxygen, etc., these environmental changes would obviously have opened a niche within which this pre-adapted clade could diversify. In this case, pre-adaptation refers to the possession of genes and gene combinations that facilitate adaptation within a novel environment. In addition, regulatory domain shuffling along with natural selection could have co-opted the function of existing proteins to fit the new environment.

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Many biotic and abiotic environmental changes, which may have been crucial to the origin of animal multicellularity, occurred during the late Proterozoic. These changes include increased oxygenation of the oceans, decreased global temperatures and possibly larger available prey and the presence of predators (Berkner and Marshall 1965, Stanley

1973, Knoll 1996, Hyde et al. 2000, Runnegar 2000, Hoffman and Schrag 2002).

Processes including increased volcanism and autolysis of water have been used to explain the initial increase in oxygen availability during the Proterozoic (Berkner and

Marshall 1965). Later, increased land mass and protection from ultraviolet radiation likely created new niches for photosynthesizing organisms, further increasing free oxygen within the oceans (Berkner and Marshall 1965). In addition, rapid burial of organic material as indicated by a peak in the ratio of 13C/12C would have made oxygen available for respiration (Knoll 1996).

Along with a sudden increase in oxygen, global temperatures dropped as a result of the “Snowball Earth” where the Earth was covered completely, or nearly so as shown by Hyde et al. (2000), with ice. The dissolution of the supercontinent Rodinia created new oceans and likely resulted in a carbon sink, decreasing CO2 globally and leading to drastically decreased temperatures (Hyde et al. 2000, Runnegar 2000, Hoffman and

Schrag 2002). This decrease in temperatures likely facilitated the oxygenation of the oceans as oxygen more readily dissolves in cold water than in warm water.

In addition to these strongly supported factors, biotic changes such as increased or newly evolved predation as well as increased prey size may also have played a role in the evolution of metazoa. As reviewed by Stanley (1973) the addition of a trophic layer in

5 the food chain during the late Precambrian likely lead to the diversification at the next lower level. In this case, it is possible that the evolution of predation on the common ancestor of protists and metazoans may have increased diversity at that level allowing for the evolution of multicellularity. On that note, predation by choanoflagellate ancestor likely led to diversification and the evolution of larger size in its prey which would have been supported by the recent increase in dissolved oxygen.

Future studies are required to test the extent to which choanoflagellates adaptively converge on metazoan trait states when exposed to environments conducive to change over long evolutionary periods. To test for such environmental influences I propose the following study for future research. Choanoflagellates will be reared for hundreds of generations in environmental conditions that may have driven the evolution of multicellularity to determine if physical and molecular changes, corresponding to evolution toward multicellularity, occur. These environments will differ in oxygen availability, temperature, available prey size, presence of predators, presence of signaling molecules, and in other environmental characteristics.

In order to carry out experiments of this character and magnitude, however, precise measurements regarding growth rates and average densities must be used to develop protocols appropriate for the long term propagation of choanoflagellates. Due to the lack of available published information regarding the general life history of choanoflagellates, this study was designed to collect such information. The experiments presented here examined differences in culture medium, the dynamics of growth, and the effects of agitation and freezing on cultured populations. While the main question

6 pertains to the effects of environment on evolution, the answers to these questions will allow for the development of precise and efficient protocols to answer the main question at hand.

Materials and Methods

Species used

All experimental populations were initiated either directly from a frozen culture of Salpingoeca rosetta provided by the King Lab (Berkeley, CA) or from a continuously cultured population initiated from that culture. The frozen sample was thawed at room temperature and the contents were added to a glass Petri dish containing 20 mL of sterile new barley media (artificial sea water infused with barley, described below). The culture was allowed to grow for 7 days in an incubator at 22.79±1.68˚C.

S. rosetta is often used as a model organism and was chosen for this line of experimentation due to its dual life stages; individuals are either solitary or colonial.

Since the ancestor to metazoa is expected to have consisted of a group of undifferentiated cells, this species serves as a suitable model organism for future experiments investigating the evolutionary transition to multicellularity. All cultures of S. rosetta were co-cultured with an undefined Gram negative strain of bacteria present in the original frozen sample. Cultures were maintained in Petri dishes and incubated at

24.8±1.3˚C.

Determination of cell density

Samples of cultures were taken regularly during each experiment for the determination of cell density. For each count, a 10 μL sample of culture was added to an 7

Improved Neubauer Levy Ultra Plane hemocytometer (Clay-Adams, Parsippany, NJ) below a glass coverslip. An Axiovert 100 inverted compound microscope (Zeiss,

Germany) at 500x with a 1.6x magnifier was used to view the cells. Choanoflagellates within a particular number of 0.0025 mm2 boxes within the grid were counted. Cells located on the right and bottom lines were included in the counts, while those on the left and top lines were excluded in order to standardize counts and prevent overestimation of cell density. The number of cells within each box as well as whether they were solitary or colonial was recorded for each count. The hemocytometer was thoroughly cleaned with ethanol between counts. All initial counts (immediately after inoculation) were later calculated by dividing the count taken before the previous transfer (end-cycle count) by the dilution factor, 11. See Appendix C for data regarding the reliability of cell enumeration using this technique.

The following equation was used to convert cell counts into concentrations of cells/mL:

.

At the end of each transfer cycle, when the density of cells was greater than 1x106 cells/mL, a sample of the culture was transferred.

Transfers

Samples of cultures were transferred every four days for all experiments in this study. After each end-cycle count, a 2 mL sample of each culture was transferred to a new Petri dish containing 20 mL of sterile media (1:11 transfers).

8

Doubling times

Doubling times were determined from log2 transformed counts from each transfer cycle plotted against the day of the experiment. Regressions over points corresponding to the first two days of growth, when growth rate was shown to be near its peak, were used to calculate the doubling time of cultures. The initial transfer cycle was not included in the determination of overall doubling time in the media comparison experiment because an initial cell density could not be calculated. The first two transfer cycles were not included in the determination of doubling time for both shaken and still cultures due to issues that may be attributable to the initial thawing of the culture and inconsistent cell counting.

Growth rates across media

Two novel media were created for this experiment to be tested against the traditionally used cereal grass medium. Four media: DI water, seawater broth, cereal grass medium, and new barley medium, were compared with each other. The media were compared based on average cell densities and doubling times of cultures growth within each medium. The ultimate goal of the experiment was to find an easily produced, versatile, and inexpensive medium appropriate for the long term culturing of the choanoflagellate species, Salpingoeca rosetta.

With increased popularity of choanoflagellates as a model organism for studies of metazoan origins comes a need for inexpensive and efficient culturing techniques and products. Although cereal grass medium has been used for some time, the product has recently become less available and has undergone formula changes that are thought to

9 have affected its quality as a culturing medium for choanoflagellates (King Lab, personal communication). It is therefore imperative that other types of media are developed and explored as possible replacements for cereal grass medium.

The medium chosen as a replacement for cereal grass medium must be at least as supportive of choanoflagellate growth and should be more economical. Cereal grass media is quite expensive to use for long term maintenance of many choanoflagellate cultures and cell culture costs could be reduced by as much as 95% if a suitable replacement is found. This estimate was based on the current (March 2011) cost of organic barley grass (Starwest Botanicals, Rancho Cordova, CA) and Ward‟s cereal grass medium (ScholAR Chemistry, West Henrietta, NY). As a culture medium it is important that it simulate the marine environment to which Salpingoeca rosetta is native.

This is important not only for the maintenance of osmotic balance, but also in the recreation of evolutionary conditions for LTEEs. In order to elucidate factors leading to the evolution of multicellularity, environmental conditions must be accurately replicated in the experimental system. The medium should also be capable of sustaining bacteria, the main food source of choanoflagellates. Bacteria must be maintained at densities that will not overwhelm choanoflagellate cultures.

Lysogeny Broth is readily available and relatively inexpensive as compared to cereal grass medium. The medium, prepared traditionally, supports bacterial growth very well over short time periods. However, bacteria tend to grow so quickly in the medium that they become overwhelmed by their own growth and cultures begin to decline due to lack of oxygen and accumulation of metabolites (Losen et al. 2004). For this study, the

10

LB prepared contained more salt than is normal and was diluted to 5% of its original strength in order to make it ideal for choanoflagellate growth. Traditionally prepared LB would be too nutrient-rich and would facilitate rapid growth of bacteria, thus overwhelming the choanoflagellates. Dilution served to keep bacteria at a tolerable density.

Media tested

Seawater Broth

Seawater broth was prepared by dissolving 33.42 g Reef Crystals (Aquarium

Systems, Mentor, OH), 0.5 g tryptone (BD, Franklin Lakes, NJ), and 0.25 g of yeast extract (EMD, Darmstadt, Germany) in 1 L of deionized (DI) water. The solution was autoclaved for at least 45 minutes at 121˚C and following sterilization, 0.25 g Na2HPO4 was added. The traditional protocol (Miller 1972) for the preparation of lysogeny broth

(LB) was adapted for the preparation of the medium. In order to prevent rapid de- oxygenation uncontrolled microbial growth, the concentration of the traditional medium was decreased drastically (1:20) while the salinity was increased to that of seawater, so as to support the growth of the marine species.

Cereal Grass Medium (CGM)

The cereal grass medium used consists of artificial sea water infused with cereal grass. The protocol described by King et al. (2009) was used with modifications. The unaltered protocol is as follows: 10g of Ward‟s cereal grass was added to 2 L of boiling natural sea water and allowed to steep for 3-5 hours. The medium is then filtered through a double layer of Whatman filters to remove particulate matter and then filter sterilized

11 through 0.22 micron Millipore Stericups. For this experiment the following protocol was followed in order to decrease cost and to increase the effectiveness of the medium. 33 g

Reef Crystals were dissolved in 500 mL of DI water while 2.5 g Cereal Grass Media were added to a separate flask containing 500 mL of DI water. Both flasks were autoclaved for 20 minutes. 50 mL of DI water was then added to each flask to compensate for evaporation and the water infused with cereal grass was filtered twice through qualitative filter paper (VWR, West Chester, PA). The filtered cereal grass solution was then combined with the artificial sea water and 0.25 g Na2HPO4 was added.

The combined solution was autoclaved for at least 45 minutes at 121˚C.

New Barley medium (NBM)

New Barley medium, artificial sea water infused with organic barley grass powder, was prepared for use in this and other experiments following the same protocol as described for cereal grass medium. Organic barley powder (2.5 g) in 500 mL DI water along with 33 g Reef Crystals in 500 mL DI water (in a separate flask) were autoclaved for 20 min at 121˚C. Following sterilization, 50 mL of DI water was then added to each flask to compensate for evaporation, the barley solution was filtered twice through qualitative filter paper, and then combined with the artificial sea water. Na2HPO4 (0.25 g) was then added and the solution was autoclaved for at least 45 minutes at 121˚C. Both the cereal grass medium and the new barley medium were checked throughout the duration of the experiment for contamination as autoclave sterilization can cause both media to form precipitates which can be mistaken for contamination.

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Choanoflagellate Counts

The demographic traits of S. rosetta in four media: deionized water, seawater broth, cereal grass medium (CGM), new barley medium (NBM), were compared. From a continually cultured population of S. rosetta, 2 mL samples were taken to inoculate 12

Petri dishes (n=3), each filled with 20 mL of 1 of the 4 media. Mid- and end-cycle counts were performed to determine choanoflagellate density during each transfer cycle. Cells were counted from 8 haphazardly chosen boxes, corresponding to an area of 0.02 mm2.

The experiment was carried out over four transfer cycles. The density of choanoflagellates maintained in each medium were compared using analysis of variance

(ANOVA) and growth rates of cultures grown in CGM and those grown in NBM were compared with a t-test.

Growth Curve of Salpingoeca rosetta

The densities of replicate cultures of Salpingoeca rosetta were measured over 246 hours in order to determine an overall growth curve for the species when cultured in new barley medium. Points were taken every 6 hours for the first 96 hours of the experiment and every 24 hours until hour 246.

Five replicate cultures were initiated from samples of a constantly cultured population of Salpingoeca rosetta. Following inoculation, counts of samples from each replicate were performed every 6 hours to determine choanoflagellate density at regular time intervals in order to get a precise growth curve. Cells located within 8 haphazardly chosen 0.0025 mm2 boxes were counted. Counts at 6 hour intervals continued for a total of 96 hours. After the first 96 hours, cultures were counted every 24 hours starting at

13 hour 126 with a final count at 246 hours post-inoculation (hpi). Values were combined and graphed to create a growth curve for the species under defined conditions. Densities

(cells/nL) were log2 transformed in order to allow for examination of different growth phases.

Values of r and K for S. rosetta were calculated from the growth curve. The phase of log-linear growth between 0 and 30 hpi was assumed to be uninhibited growth.

A regression provided the rate of change (dN/dt) for that phase. The growth rate, r, was calculated from the following formula:

N was estimated from the average number of cells during that phase. After r was calculated, it was used in the determination of K, the carrying capacity, using the following formula:

A regression of the stationary phase values provided the rate of change (dN/dt) and N was determined by the average number of cells during the phase of growth.

Test of Culturing and Freezing Protocols

Two culturing protocols, one in which cells were cultured in still Petri dishes and one in which cells were grown in Erlenmeyer flasks shaken at 100 rpm, were compared in order to improve overall culturing technique for the choanoflagellate species,

Salpingoeca rosetta. Also investigated was the effect of the periodic freezing of culture

14 samples. The average cell density of still cultures undergoing periodic freezing every two transfer cycles was compared to that of still cultures undergoing continuous transfers.

Two mL samples of the initial culture were transferred to 7 identical, glass Petri dishes and 3 50 mL Erlenmeyer flasks, each containing 20mL of sterile new barley media. Of the 7 cultures grown in dishes, 3 were designated to undergo continuous transfers and counts every 4 days (cultures 6 and 7; initially every 3 days) with 1 of the 3 counted every day (culture 1). Prior to each transfer (and every day for culture 1) the number of choanoflagellates within a 0.01 mm2 area, 4 haphazardly chosen 0.0025 mm2 boxes, on the hemocytometer was counted. Each culture was transferred 14 times.

Cultures S1, S2, and S3 were cultured in flasks at room temperature and shaken at

100 rpm for 23 hours per day. End-cycle counts were compared to those of still cultures, cultures 1, 6, and 7, with a t-test.

Cultures 2-5 were periodically frozen during the experiment. Each culture was frozen and thawed 3 times. Prior to the first freeze, the cultures had been transferred twice. After the third transfer cycle, 900 μL samples of each culture were added to centrifuge tubes along with 100 μL DMSO instead of being transferred to fresh medium.

The tubes were then frozen overnight at -80˚C and then moved to liquid nitrogen for long-term storage as per King et al. (2009). Cultures were kept in liquid nitrogen 1-4 days and then thawed at room temperature. The thawed contents were added to glass

Petri dishes containing 20 mL of sterile new barley media and allowed to grow in the incubator at 24˚C for 1 week before they were transferred. Each culture was transferred a total of 8 times and two transfers occurred between each freezing. Average counts of

15 frozen cultures were compared to those of continuous cultures, cultures 1 (end-cycle counts), 6, and 7, with a t-test.

Results

Growth rates across media

The doubling times of cultures were not significantly different between media as determined by an analysis of variance (ANOVA) (p=0.575). The removal of data corresponding to cultures grown in DI water did not make the difference significant

(p=0.378). ANOVAs performed for each media type found no significant change in doubling time after sequential bonferroni correction (Rice 1989) (Figure 1).

Although doubling times were not shown to differ, results from an ANOVA indicate that the average choanoflagellate density measured from replicates of cultures is significantly different between seawater broth, CGM, and NBM (p=2.1207E-4). T-tests directly comparing the cell density of CGM to seawater broth and NBM indicated that differences were significant (p=7.23E-9 and p=0.005, respectively, df=22). While cell density was shown to be significantly higher in CGM than in seawater broth, cultures grown in NBM had the highest density of choanoflagellates overall (Figure 2).

Growth Curve of Salpingoeca rosetta

An analysis of growth over a period of 246 h was included in this study. Overall, choanoflagellate density increased quickly initially and then more gradually until approximately 96 hours post-inoculation (hpi). After 96 hours the density of the majority of the replicates decreased abruptly before leveling out (Figure 3a). Growth within the culture appears to have occurred in two distinct phases. Log2 transformation of the 16 counts results in linearization of the curve between 0 and 30 and 36 and 96 hours post- inoculation (Figure 3b). Using the information provided in Figure 3b, doubling times were calculated for each phase of growth as well as for overall growth (Table 1).

30 25 20 Water 15

1:20 LB (hours)

10 CGM Doubling Time Doubling 5 NBM 0 0 1 2 3 4

Transfer Cycle

Figure 1. Trends in doubling time across media. The doubling times of cultures grown in all media types remained stable throughout the

experiment. Error bars represent 95% confidence intervals.

25

20

15 CGM NBM 10 Seawater Broth

5 Average Cell Cell Average Count(cells/nL)

0

Figure 2. Comparison of cell density between media. Cultures grown in NBM produced the densest cultures, overall. Error bars represent 95% confidence intervals. 17

Replicates 1-3 followed this overall trend. However, replicates 4 and 5 grew more slowly and increased in density after 96 hours as the density of other replicates dropped off. Replicates 4 and 5 did eventually achieve a maximum density followed by a decline as the other replicates had.

On average, the replicate cultures observed in the study grew in density for the first 96 hours they were in culture and then declined more slowly until an equilibrium of cell division and death was reached. During the 96 hour growth phase, growth appears to occur in two distinct phases. For the first 30 hours cultures of S. rosetta appear to undergo rapid, log-linear growth. Throughout this phase, cultures are capable of doubling their density in only 13.4 hours.

At 36 hours post-inoculation (hpi), the growth changes from a log-linear pattern to one that is more obviously exponential in nature. The growth rate in this period declines to the point at which populations are capable of doubling in 34.6 hours, much longer than during the initial phase of growth. However, cultures did grow substantially during this period, reaching a density of 36.9 cells/nL at 96 hpi, more than 300 percent of the 30 hour population density of 11.5 cells/nL. The presence of two growth phases is supported by the study investigating culturing methods. In that study, the majority of the increase in density over time occurred during the first 24 hours after which growth slowed.

Growth occurred on average from 0 and 90 hpi. The total duration of the growth period was determined by the intersection of the trend lines fit to points from 36-96 hpi

18 and 126-246 hpi. The average overall doubling time between 0 and 90 hpi was determined to be 32.3 hours.

40 35 30 25 20 15 10 5 0

Density Density (cells/nl) 0 50 100 150 200 250 Hours Post-Inoculation a.

6 5 4 3 2

(cells/nl) 1 Average Cell Density

2 2 0

log 0 50 100 150 200 250 300 Hours Post-Inoculation

1st Growth Phase 2nd Growth Phase Stationary Phase b.

Figure 3. A growth curve for Salpingoeca rosetta. (a) Average cell density of Salpingoeca rosetta grown over 246 hours. On average, the cultures grew

in size for the first 96 hours, then decreased in size until death rates matched the rate at which new cells were created. (b) Log transformation results in 2 linearization of the curve from hours 0 to 30 (blue diamond) and also 36 to 96 (green triangles) hpi. Purple Xs represent death/stationary phase.

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The condition of the choanoflagellates in culture mirrored the growth trends.

While cultures 1-3 grew in size quickly, the choanoflagellates were large with many occurring in colonies or actively swimming. However, after cultures reached their peak densities, cultures consisted of more non-motile, substantially smaller individuals. The same was true for cultures 4 and 5. Initially, individuals were small and sedentary, but then appeared larger and more active as cultures began to grow in size after the first 96 hours of the experiment.

The growth rate, r, of S. rosetta calculated from the growth curve was determined to be 0.04252 h-1. The carrying capacity of each 22 mL culture was determined to be

4.53x108 cells, or 20.6 cells/nL.

Table 1. Doubling times for two distinct growth phases as well as for overall growth.

Growth Interval Regression Coefficient Doubling Time (hours)

0-30 hpi 0.0747± 0.036 13.387

36-96 hpi 0.0289± 3.3E-3 34.602

overall: 0-90 hpi 0.031± 6.8E-3 32.258

Test of Culturing and Freezing Protocols

Test of culturing protocol

All cultures were maintained for the duration of the study with little, if any, loss of cell density. Growth in all cultures maintained in a continuous fashion appears to be stable with cell counts increasing from inoculation to the end of the transfer cycle.

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Growth was rapid for the first 24 hpi and occurred more slowly until samples were transferred to fresh medium. This was true of all transfer cycles of continuous cultures quantified daily.

The end-cycle density in both shaken and still treatments did not change across cycles. A comparison of the average S. rosetta cell density per nL indicates that the two culturing methods differ in their overall ability to maintain choanoflagellate density. Still cultures (avg.=16.897) were shown to support significantly more cells than those that are shaken (avg.=14.872) at 100 rpm (T-test p=0.0065, df=76).

A comparison of doubling times indicates that, while cell density differs between treatment groups, agitation has no significant effect on the doubling time of choanoflagellate cultures (T-test p=0.381, df=24). Shaken cultures maintained an average doubling time of 14.79 hours, while still cultures took, on average, 14.32 hours to double in size. Growth rates for both still and shaken cultures display a trend of nearly zero change over time (Figure 4).

The two culturing protocols also resulted in at least one difference in the constituents of the cultures maintained under them. Colonies were frequently observed in still cultures, while colonies in shaken cultures were extremely rare and when present, tended to be smaller than those found in still cultures, primarily consisting of only a few individuals. No colonies were observed in shaken cultures until day 48 of the experiment.

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25

20

15

10 Still

5 Shaken Doubling Time Doubling (hours) 0 0 2 4 6 8 10 12 14 Transfer Cycle

Figure 4. Growth trends: Still v. shaken cultures. Doubling times of still and shaken cultures changed very little over the course of the experiment.

Test of freezing protocol

Data corresponding to the effects of freezing on cultures of S. rosetta were also included in the analyses. Counts of periodically frozen cultures were compared to those of continuously transferred, still cultures. Overall, the periodic freezing of choanoflagellates was shown to have no significant effect on their average cell density when cultures are given a one week recovery period after thawing (T-test p=0.434, df=91; Figure 5).

Discussion

Growth rates across media

A comparison of available media was done in order to find a culture medium for

Salpingoeca rosetta and other choanoflagellate species that is both inexpensive and effective at supporting dense cultures. Overall, cultures grown in the seawater broth 22 medium maintained higher cell density than did control cultures grown in deionized water, but much lower than cultures grown in either cereal grass medium or new barley medium. Seawater broth is therefore is not a suitable replacement for CGM. Throughout the experiment, high bacterial loads were observed during most counts in seawater even though it was dilute. A more dilute concentration of the medium may have been more conducive to choanoflagellate growth.

25

20

15 frozen continuous 10

5 Average Cell Cell Average Density (cells/nL)

0

Figure 5. Effect of freezing on cell density. Average cell densities of cultures that experienced periodic freezing and those that were cultured continuously. Error bars represent 95% confidence intervals.

Barley grass is another practical nutrient source for the production of media.

Organic barley grass powder can be purchased in large quantities at low cost and it is similar enough to cereal grass that a comparable protocol can be followed to prepare growth medium. While NBM is suitable for bacterial growth, it is less nutrient-rich than

LB so that predation by choanoflagellates is capable of controlling bacterial load.

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NBM is capable of maintaining cultures of S. rosetta over long periods of time assuming that cultures are regularly transferred to fresh media. Over the four transfer cycles in this study, NBM out-performed all other media, including cereal grass media, in maintenance of choanoflagellate density. This finding could have an impact on studies of choanoflagellates. With cheaper supplies available, choanoflagellate research may become an option for labs limited financially or by unfamiliarity with the differences between potential media.

Published protocols recommend cereal grass medium for the maintenance of marine choanoflagellate species like Salpingoeca rosetta (King et al. 2009a). In this and other studies, stable cultures of choanoflagellates have been maintained using this medium. However, as stated above, the formula of the cereal grass component that is available commercially has been altered and the medium itself is expensive, 22 times more expensive than possible alternatives such as barley grass. The medium used in this study was purchased prior to the formula change and thus its performance is comparable to that of the other media.

Cultures grown in cereal grass media grew exceptionally well when compared to those grown in seawater broth and in deionized water. The medium is suitable for the maintenance of bacteria at appropriate levels and the media doesn‟t tend to become turbid allowing clear photographs to be taken while cultures remain in Petri dishes.

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Growth Curve of Salpingoeca rosetta

The difference in growth rates between the initial and subsequent growth phases is likely due to competition between the choanoflagellates that arises as the cultures exponentially increase in density. Seemingly unlimited growth which occurred during the log-linear period may be due to the lack of competition for resources during this time, including their food source, bacteria, which likely grow rapidly when introduced to a fresh nutrient source. This phase of growth was used to calculate the uninhibited growth rate, r, for S. rosetta.

At 30 hpi, cultures had reached about 50% of their carrying capacity. Although the growth continued to increase exponentially, it occurred over a much longer time period than did the initial growth phase. A decrease in growth is likely due to competition for limited resources such as oxygen and space. It is unlikely that choanoflagellates are competing for food in this system. Bacteria grow extremely well in

NBM, possibly to the point that they can overwhelm choanoflagellate cultures by overconsumption of available oxygen. Competition likely increased over time as cultures were shown to have overshot their carrying capacity (K) of 20.6 cells/nL during exponential growth.

The data from this study provide necessary information for future research.

Growth rates and measures of average density over time inform protocols for the flow rate when using chemostats and the timing of transfers, important factors LTEEs. The optimization of flow rate of media into and out of a chemostat is necessary for the maintenance of stable population sizes and is essential for a successful long term

25 experiment. Cultures appear to have undergone a phase of unlimited growth for at least the first 30 hpi. Because the constant supply of nutrients in chemostat would simulate unlimited resources, the μmax, maximum growth rate, is used to determine the proper flow rate within chemostat environments. The growth rate obtained during the period of seemingly unlimited growth would be used in the determination for a chemostat housing

S. rosetta under the conditions described. Assuming that the growth during that phase was truly unlimited, the calculated flow rate would allow for the maintenance of high density, yet stable cultures over long periods of time. This technique, applied to other choanoflagellate species could be extremely beneficial as well.

Also important are the pattern of growth up to 96 hpi and the determination of the overall growth rate. This informs protocols for transfer regimes in LTEEs not involving the use of a chemostat, regimes such as the ones described previously. Similar to a chemostat flow rate, the volume and frequency of transfers determine the stability of evolving cultures. Alterations from optimization can lead to populations that become too small and die out due to stochastic changes or populations transferred too late may no longer be in a growth phase and may decline even in the presence of abundant nutrients.

Under the conditions described, it appears that cultures can be transferred to fresh media at least every 48 hours, if not more frequently. At this point, cultures are doubling most rapidly and, once transferred, will continue to grow immediately as there is no evidence of a lag period. With a doubling time of approximately 14 hours, S. rosetta could be transferred every 48 hours with a 1:8 transfer ratio in order to maintain density over time.

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Healthy cells are also an essential component to the maintenance of successful long-term cultures. It was observed in this study that density of cultures declined once the cultures had reached their maximum density at 96 hpi. Not only would fewer cells be transferred in samples after this time, but the cells transferred in death or stationary phase may not be as healthy. Cultures inoculated with cells from these phases would be expected to be less productive than those inoculated from healthy, growing cultures.

Altogether, observation of choanoflagellate growth over 246 hours has provided much needed information for future studies requiring successful long term cultures. However, as some replicates grew more slowly than others in this study, care should be taken to monitor the growth of cultures throughout LTEEs.

Test of Culturing and Freezing Protocols

According to published protocol for the maintenance of Monosiga brevicollis, another marine choanoflagellate, cultures should be transferred at a ratio of 1:15 every 2-

3 days (King et al. 2009a). However, under the conditions required for long term evolution, transferring cultures every 2 or even 3 days has not been conducive to the maintenance of Salpingoeca rosetta cultures over long periods of time. Experience has shown that cultures quickly decline in density when diluted 1:15 at that rate. Data previously described regarding the growth pattern of S. rosetta show that, under particular conditions, cultures continue to grow and do not reach maximal density until

96 hours post inoculation. Transferring samples every four days in this study allowed cultures to become dense enough so that a sufficient number of cells were transferred during each inoculation. The timing also ensured that cells were still in the growth phase,

27 thus they continued to grow upon transfer to fresh resources. In addition to the timing of transfers, the optimization of dilution during transfers is necessary. The dilution factor,

1:11 was large enough that cells were not in immediate competition with each other upon transfer, but low enough that culture densities remained stable and did not dissipate over time.

Although the particular regime described was successful, the data presented from previous experiments supports more frequent transfers, with the creation of less dilute cultures upon transfer. Frequent transfers, during or near the log-linear phase of growth would allow for more generations to pass per day, thus improving an LTEE, as cells transferred during this phase are growing rapidly and will continue to do so upon transfer.

Choanoflagellates within shaken cultures are included in the cultures that maintained both cell density and growth rate for the duration of the study. Although agitation is not addressed in published protocols, bacterial growth has been shown to increase when cultures are shaken (Losen et al. 2004). As discussed previously, evidence has shown that choanoflagellate growth may be limited by oxygen availability due to bacterial growth. In this study, a set of 3 replicate cultures were shaken at 100 rpm so as to aerate the culture without damaging the fragile choanoflagellate cells (King et al.

2009b). If aeration had been a limiting factor under the conditions described, shaken cultures should have had improved densities and growth rates over still, non-agitated cultures. This was not observed in this study, however; average density was significantly lower than those of still cultures. Turbulence at the level described may have put additional stress upon the choanoflagellates in the cultures, thus decreasing growth and

28 cancelling the oxygen advantage. In addition, the turbulence appears to also limit colony formation. While colonies were frequently observed within still cultures, observational data show that colonies were extremely rare in shaken colonies and colonies, which tended to be small compared to those in still cultures, were observed only late in the experiment. In all, shaking does not appear to be essential to the cultivation of choanoflagellates and may be detrimental, especially for long term experiments and those where colony formation is encouraged.

The effect of freezing on cell density was also investigated. It has been shown that frozen cultures did not differ significantly in cell density under the conditions described. The periodic freezing of cultures did not significantly affect choanoflagellate density over time as compared to replicate cultures which were transferred continuously throughout the experiment. This has important implications for long term studies. If cells are frozen improperly and damage to cells does occur, growth rates and densities would be affected.

Periodic freezing of samples is essential to any long term evolution experiment.

Traditionally, samples of cultures at several different generations are frozen so that they can later be analyzed for phenotypic and/or genetic changes that occurred over the duration of the experiment in comparison to an ancestor population. Freezing is also necessary for culture recovery. For example, if a culture becomes contaminated, it typically becomes unusable for the study. If frozen samples are available, one can resume the experiment from the point of the last freezing. It is essential, however, that

29 frozen samples are equivalent to a culture that has been cultured continuously in order to incorporate freezing as a part of the normal passage regime.

Cultures from which samples were frozen were given a one week recovery period after being removed from the liquid nitrogen and thawed. This may have given healthy cells time to grow to normal densities and also time for damaged cells to degrade. Also, although the one week time period was a successful addition to the protocol, the recovery time should be optimized. Long term evolution experiments take place over hundreds of generations and while cells are recovering from being frozen, they are not in their experimental environments. Thus, optimization of recovery may allow one to include more generations in a LTEE.

Allee Effect

A great emphasis has been put on the necessity of maintaining population size in choanoflagellate cultures. If population size is not maintained and cultures decline in size over time, they are likely to become extinct even when the individuals that constitute the cultures are healthy. This can be explained by the Allee Effect. Usually used to describe sexual populations, the Allee Effect refers to the increased likelihood that a population‟s size will tend toward zero when a minimum population size is not met (Allee 1927, reviewed in Stephens et al. 1999). In the case of sexual populations, this can be due to the inability of individuals to find mates. As they have not yet been shown to reproduce sexually, this is likely not the case for populations of choanoflagellates (Carr et al. 2010).

However, the effect may still apply to their cultures. As stated before, cultures that do not maintain a particular population size will tend toward a population size of zero, but

30 those that maintain densities above the critical value will tend toward a stable population size (Figure 6). This fate assigned to small asexual populations is likely due to the larger impacts of stochastic events on smaller populations than on larger ones (Stephens et al.

1999).

Figure 6. The Allee effect. Populations smaller than the critical density will decline in number toward extinction, while those above the critical abundance will tend toward an equilibrium abundance, U. (from Stephens et al. 1999)

The lack of facilitation between conspecifics can also increase the likelihood of population extinction at low population densities (Courchamp et al. 1999). Facilitation refers to the potentially unconscious interactions between conspecifics that increase survival. The degree to which conspecific interactions benefit the population by increasing population size determines the impact that the lack of facilitation has on extinction probability. If, for example, colony formation in choanoflagellates increases their survival, it is likely that even if the population size is at or slightly above the critical value, the lack of facilitation will lead to population extinction.

Future Research

The three studies described have important implications for future research. With the data obtained as well as data to be obtained from additional choanoflagellate species using the methods described, LTEEs will be devised. In the near future, several LTEEs

31 will be initiated with cultures of Salpingoeca rosetta. The growth rates as well as the information obtained regarding the effects of freezing will be essential for the development of protocols for long term evolution. Precise and accurate growth rates are essential to the calculation of the number of generations cultures are capable of reaching between transfers. Stable growth rates ensure that changes can be accurately correlated with time.

Additionally, the growth curve of other species, such as Monosiga brevicollis, must be determined. M. brevicollis is a non-colonial species of marine choanoflagellate and is an important component of future studies investigating the effects of environment on colony formation and components of multicellularity along with the colonial S. rosetta. M. brevicollis has proven in past studies to be somewhat difficult to transfer using the protocol described in this study because they adhere to the substrate and must be scraped before they are transferred. Therefore, other techniques must be used to eliminate issues such as cell death and contamination. One option is to use chemostats to maintain evolving cultures of this species. Chemostats maintain a constant environment, can allow for maximal growth rates, and their use does not require that substrate-attached cells be scraped off prior to transfer. Maximal growth rates, decreased cell death, and decreased risk of contamination, increase the number of generations that can be included in an LTEE and also protects cells from damage due to scraping. The flow rate, based on

μmax, can easily be determined with observation of growth over time.

The studies described have also provided information needed to control some costs associated with future studies. Knowing that inexpensive medium can be used and

32 has shown to be more effective than the traditionally used, allows for more replicates to be used in future studies. Inefficient use of time can also be expensive. Knowledge of not only S. rosetta’s patterns of growth, but also the appearance and behavior of healthy cultures will help to eliminate possible complications. Familiarity with the subject will allow for the alleviation of potential issues before they become detrimental to future long-term projects.

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CHAPTER II

TESTING THE VIABILITY OF PNA-MEDIATED CLAMPING TO DETECT

RECOMBINATION USING A PROKARYOTIC TEST SYSTEM

Introduction

Knowledge of an organism‟s mode of genetic transmission is crucial for the understanding of their evolutionary history and changes that occur as they are evolved in a laboratory setting. Choanoflagellates, a protist clade sister to metazoans, are not at present known to reproduce sexually, although they have been shown to possess a majority of the genes associated with meiosis (Carr et al. 2010). This indicates that choanoflagellates have the potential for sexual reproduction and detection of recombination using molecular techniques would provide further evidence as to their mode of reproduction. Peptide nucleic acid (PNA)-mediated PCR clamping may be capable of detecting recombination. In order to test their ability to detect sequence changes in DNA, preliminary studies have been initiated investigating the effectiveness of PNAs to discriminate sequence polymorphism in four strains of E. coli.

Since its development in 1993 (Orum et al.), PNA-mediated PCR clamping has been used in many studies to selectively amplify rare polymorphisms, such as those

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maintained by mutation-selection balance. This technique has been shown to have diverse applications, especially in the non-invasive diagnosis of diseases such as cancers and β-thalassemia and the characterization of microbial diversity (von Wintzingerode et al. 2000; Luo et al. 2006; Galbiati et al. 2008). The mechanism by which PCR clamping works depends on characteristics of the PNAs used; the thermal stability of PNA-DNA complexes as well as the selectivity with which PNAs bind to complimentary DNA affect amplification during PCR cycling (Orum et al. 1993).

PNAs, although very similar to DNA primers, have a polyamide backbone rather than a sugar-phosphate backbone (Nielsen et al. 1991; see figure 7). Whereas the phosphate groups intervening between sugars in DNA result in an overall negative charge, the amide groups are neutral. The neutral charge on the backbones of PNAs eliminates the repulsion, however small, that occurs between complimentary DNA strands, therefore allowing PNAs to outcompete DNA primers for binding locations on

Figure 7. Comparison of DNA and PNA structure. The polyamide backbone of PNAs contribute to their overall neutrality and increased thermal stability of PNA-DNA complexes. (from Neilsen et al. 1991)

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template DNA. Because PNAs are not recognized by DNA polymerase, they cannot act as primers and thus prevent amplification during PCR (Orum et al. 1993).

The selective binding of PNAs to template DNA is what makes them useful for the inhibition of PCR reactions. PNAs bind only to their exact compliments; differences, as small as single base pair mutations, disrupt binding between PNAs and their DNA template (Orum et al. 1993). Thus, PNAs prevent the amplification of completely complimentary DNA, while allowing amplification of mutated or recombined templates.

This blockage of DNA replication within PCR reactions is commonly referred to as PCR clamping.

Using a prokaryotic test system, the utility of PNA-clamping to differentiate between strains with sequence polymorphisms will be tested for future use in a choanoflagellate system. Clonal populations of choanoflagellates, differing in the presence of SNPs, can be readily established and will be identified based on the pattern of clamping of gene amplification. Assuming that PNAs can be used successfully and consistently to identify certain strains based on the locations of their polymorphisms, the technology can be used to identify choanoflagellate clones that have undergone recombination based on changes in patterns of amplification.

Bioinformatics-based software, such as RDP2, can be used to find sites where recombination events are likely to occur (Martin et al. 2005). PNAs, complimentary to what is designated as the wild type sequence of gene segments surrounding likely recombination sites, can be used to clamp PCR amplification of those genes in which the

36 wild type sequence is present while those containing SNPs will be amplified.

Comparison of amplification patterns obtained from samples of mixed populations with those of the component clones can be used to identify rare recombinants. Sequence comparisons can confirm the presence of recombinant DNA.

Materials and Methods

In this study, PNA-mediated PCR clamping was used to inhibit the amplification of wild type sequences of the PAB B gene, while allowing the amplification of sequences with single base pair polymorphisms in several strains of E. coli with the goal of obtaining a specific amplification pattern by which each strain can be differentiated.

FBGM 1 is designated as the wild type (WT) strain with no single nucleotide polymorphisms (SNPs) in the PAB B gene, FBGM 10 contains both SNPs (GT at position 303 and TA at position 612), FBGM 19 contains the SNP at 303, and FBGM

20 contains the SNP at 612 (Figure 8 and Table 2). Mutations were located within sequences produced previously in our lab and aligned using BioEdit (Hall, 1999).

Table 2. SNP locations in 4 lines of E. coli. --- represents the WT and X represents the presence of a single base pair mutation at the specified location.

303 612

FBGM 1 (WT) ------

FBGM 10 X X

FBGM 19 X ---

FBGM 20 --- X

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Elongation arrest, rather than traditional competitive clamping, was used to accomplish selective amplification. This method was chosen due to the availability of primers which could be used to amplify amplicons encompassing two single base pair mutations found in the PAB B gene of various strains of E. coli. Elongation arrest involves the premature termination of amplification when DNA polymerase reaches a bound PNA, whereas traditional clamping involves competition between primers and

PNAs for binding locations as described above. As shown in Figure 9, the early termination of elongation of either the sense or non-sense strand results in an overall decrease in amplification. Due to the premature termination of elongation, the primer is not able to bind in future cycles and thus amplification is decreased drastically as only the original template strands are available for primer attachment and elongation.

Figure 8. Primers and PNA binding to template DNA. PNA-DNA complexes are much more stable than DNA-DNA complexes, so PNAs will always bind to WT sequences to which they are complimentary (top). However, PNAs are highly specific and the annealing of PNAs is prevented by even a single base pair mutation (bottom).

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Figure 9. Elongation arrest. (a) PNA 1 binds in the absence of a mutation at 303. (b) The presence of the bound PNA arrests elongation of the new strand initiated by the reverse primer. (c) Due to the arrested elongation, the new template strand does not contain the binding sequence for the forward primer and amplification is prevented. Original template strands are black, while elongated copies are blue.

PNA synthesis

Two PNAs were synthesized in the lab of Dr. Leeper at the University of Akron using Fmoc chemistry with the following sequences (Casale et al. 2004):

PNA 303: CAACGCGACCGAT-Lys PNA 612: GACAAATTTACTG-Lys

In order to create a substrate for PNA synthesis, 30 mg of Fmoc-Lys-CLEAR amide resin was soaked in DCM overnight (1 h for PNA 612). After the resin had swelled, 5 equivalents of each monomer were dissolved in NMP to produce 0.2M monomer solutions which were then separated into 0.285 mL aliquots for use in synthesis. A 0.5M

HATU solution in DMF, 0.5M base (0.2M DIEA, 0.3M lutidine) in NMP, and capping reagent (5%Ac2O/6% DIPEA) in DMF were prepared for use in pre-activation and capping reactions. PNAs were synthesized from the C-terminus to the N-terminus and

39 the column was placed on a rotator for all incubations. The steps for synthesis were as follows:

1. Initial deprotection: the resin was washed with DMF x3 and then incubated in deFmoc reagent (20% piperidine in DMF) for 5 minutes to remove the protective Fmoc group from the lysine to allow coupling to monomer. Excess reagent was then removed with vacuum filtration and the resin was incubated in deFmoc reagent for 15 min. The resin and column were washed with DMF x3, DCM x3, and DMFx3.

2. Pre-activation: the first monomer (PNA 303: T, PNA 612: G) was prepared for coupling to the lysine residue with addition of 110 μL of HATU solution and 285 μL of 0.5M base with a subsequent 2.5 min incubation.

3. Coupling: the activated monomer was added to the column along with the de- protected resin and incubated for 7.5 min to form a bond between the monomer and the lysine residue.

4. Capping: excess fluid was removed and the column was filled with capping reagent and incubated for 3 min to cap growing PNAs to which monomer was not successfully added during coupling. After removal of the capping reagent, the resin and column was washed with DMF x3, DCM x3, and DMF x3.

5. Deprotection: the column was again filled with deFmoc reagent and incubated for 2 min. During the incubation, the second monomer was pre-activated. The deFmoc reagent was then drained and the resin and column were again washed with DMF x3,

DCM x3, and DMF x3.

40

The process was repeated until all 13 monomers had been added to each resin.

When synthesis was not continuous, as with PNA 612, the cycle was always stopped after capping and the column was drained and stored in the refrigerator. Before synthesis was resumed at the deFmoc step the resin was brought up to room temperature and washed with DMF x3, DCMx3, and DMF x3. Synthesis of PNA 612 was delayed after the addition of the 6th and the 11th monomers. After the final deFmoc step, the resin and column were again washed with DMF x3, DCM x3, and DMF x3 and the resin was dried and stored in the refrigerator.

The PNA, along with the lysine residue, was then cleaved from the resin. After preparation of reagent B (0.1 g phenol, 100 μL H20, 40 μL TIS, and 1.76 mL TFA) it was added to the column containing the resin-bound PNA and incubated on the rotator for 1 h.

The solution containing the cleaved PNA was then drained into a 15 mL centrifuge tube and cold 30% acetic acid was added to bring the total volume up to 8 mL. The solution was vortexed and 5 mL of cold ether was added. After the solution was shaken and vortexed, it was centrifuged to separate the two layers for 5 min at 5000 RPM. The ether layer was then removed and another 5 mL of cold ether was added. The extraction was carried out a total of five times. Subsequent to extraction, the solution was frozen in a bath of dry ice and propanol and then dried in a lyophilizer overnight.

After the product had been dried, it was dissolved in sterile water, filtered using a syringe and a 0.2 μm filter. The concentration of PNA 303 was found using absorbance, determined using a Genesys 6 UV/Vis spectrophotometer (Thermo-scientific, Waltham,

MA) and the extinction coefficient while that of PNA 612 was found using a NanoDrop

41 spectrophotometer. High-performance liquid chromatography (HPLC) as well as matrix- assisted laser desorption/ionization (MALDI) mass spec was performed on PNA 303 to check for purity and composition, respectively.

The dry PNAs as well as those dissolved in water were stored at -20˚C.

Bacterial cultures

E. coli strains FBGM 1, FBGM 10, FBGM 19, and FBGM 20 were obtained from the lab of Dr. Francisco Moore. Working lines were prepared from his frozen stocks.

After inoculating 15 mL of sterile LB broth with each line, the lines were allowed to grow overnight in an incubator-shaker at 37˚C and 120 rpm. Samples of each culture were then frozen in 80% glycerol (85% culture/15% glycerol). All working lines were stored in liquid nitrogen.

PCR (to check primers)

Forward primer PABB 1B (5‟-CTGCACAGCGGYGAYTGCTATCAGG-3‟) and reverse primer PABB 2B (5‟-CTTTATCAAAAGTTTCCTGATATTCCG-3‟) were initially tested for their ability to amplify a 721 bp amplicon within strains FBGM 1 and

FBGM 10. DNA extracted following the protocol included with the InstaGene Matrix

DNA extraction kit (BIO-RAD, Hercules, CA) was used as the template. Isolated DNA was quantified using a NanoDrop spectrophotometer (Thermo Scientific) and then diluted to 100 ng/μL. PCR reactions were run in 25μL containing 12.5 μL Ready PCR Mix 2x

(Amresco, Solon, OH), 0.5 μL forward/reverse primers, 1 μL of template DNA, and 10.5

μL of water. Cycles were run as follows: 94˚C 2 min, 40 cycles of 94˚C 30s, 63˚C 30s, and 72˚C 2 min, followed by 72˚C 5min and 4˚C 5 min.

42

The optimum annealing temperature for PABB 1B and PABB 2B was later found after a gradient of annealing temperatures (43-65˚C) was used in an additional PCR run following the same protocol, but in 10μL reaction volumes.

Subsequent to amplification, PCR products were separated out on a 1% agarose gel containing ethidium bromide by electrophoresis. The gel was viewed using a G:Box gel imaging system (Syngene, Frederick, MD) and checked for the presence of bands corresponding to an amplicon of 721 bp. A photograph of the gel was taken using

GeneSnap (Syngene, Frederick, MD).

PNA-mediated PCR clamping

Bacterial cells were harvested from each of the four strains of E. coli as 1.5 mL samples of each culture were centrifuged at 5,000 x g for 10 minutes. After the supernatant was discarded, DNA was isolated using an InstaGene Matrix DNA extraction kit (BIO-RAD). Extracted DNA was quantified using a NanoDrop spectrophotometer.

Trial 1: Test of both PNAs on FBGM 1 and FBGM 10

Trial 1 was performed with both FBGM 1 and FBGM 10. PCR reactions were run in 25μL volumes and both PNAs were heated to 56˚C prior to their addition to eliminate aggregates. 12.5 μL Ready PCR Mix 2x (Amresco), 0.5 μL forward/reverse primers, 0.5 μL of each PNA included, and 1 μL of template DNA, with the remaining volume taken up by water. Cycles were run as follows: 94˚C 2 min, 40 cycles of 94˚C

30s, 68˚C 30s, 56˚C 30s, 72˚C 2min, followed by 72˚C 5 min and 4˚C 5 min.

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Trial 2: Test of PNA 303 on FBGM 1

Trial 2 followed the same protocol as trial 1, except that only DNA from FBGM 1 was included in the PCR reactions. Trials 3 and 4 utilized only DNA from the FBGM 1 strain along with PNA 303 in order to optimize both the annealing temperature and the concentration of the PNA in the reaction mixture.

Trial 3: Optimization of PNA 303 annealing temperature

In trial 3 the PCR cycle was run as follows: 94˚C 2 min, 40 cycles of 94˚C 30s,

70-80˚C for 30s, 56˚C 30s, 72˚C 1 min, followed by 72˚C 5 min and 4˚C 5 min. The

PNA annealing temperature was set on a gradient with individual reactions run on the same cycles with different PNA annealing temperatures. PNA 303 was used at a concentration of 100 μM and heated to 65˚C prior to use.

Trial 4: Optimization of PNA 303 concentration

In trial 4, the concentration of PNA 303 was varied between 125 and 250 μM and added to reaction mixtures at a volume of 1 μL/25μL reaction. The PCR cycle was run as follows: 94˚C 2 min, 40 cycles of 94˚C 30s, 77.3 ˚C for 30s, 56˚C 30s, 72˚C 1 min, followed by 72˚C 5 min and 4˚C 5 min.

Trial 5: Initial optimization of PNA 612 annealing temperature

Trial 5 was run with DNA from FBGM 1 and PNA 612 to find an appropriate annealing temperature for the PNA. A 567 μM concentration of PNA 612 was added at a volume of 1 μL (per 25 μL reaction) to reaction mixtures. The PCR cycle was run as follows: 94˚C 2 min, 40 cycles of 94˚C 30s, 70-94˚C, 56˚C 30s, 72˚C 1 min, followed by

72˚C 5 min and 4˚C 5 min. Again, the PNA annealing temperature was run on a gradient

44 in order to optimize the temperature for the step. All products were separated out on a

1% agarose gel with ethidium bromide added and photographed as described previously.

Results

Assessement of Purity and Structure

High-performance liquid chromatography (HPLC) was run on a sample of PNA

303 dissolved in sterile water to separate and quantify the components of the mixture

(Figure 10). PNA 303 was eluted at 21.12. The sample was shown to be 91.39% pure and to be the most abundant substance in the solution. In addition, matrix-assisted laser desorption/ionization (MALDI) mass spec was performed to assess the composition of

PNA 303 (Figure 11). The strongest peak, at 3682.766 m/z, corresponds with the total mass of PNA 303 including the lysine residue. Additional peaks at 3431.926, 3157.079,

2882.192, etc. represent incomplete versions of PNA 303.

Figure 10. HPLC sample for PNA 303. The sample was determined to be 91.39% pure. % B refers to the concentration of acetonitrile with 0.1%

formic acid. mAU: milli-absorbance unit

45

The absorbance of PNA 303 was determined to be 0.813 at 260 nm. With an extinction coefficient of 124,700 L/(mol x cm), the concentration of PNA 303 in solution used for PNA-mediated PCR clamping was determined to be approximately 0.65 mM.

PNA 612 was also shown to have peak absorbance at 260 nm. The average concentration of PNA 612 was determined to be 689.5 ng/μL or approximately 0.2 mM.

Figure 11. MALDI mass spectrophotometry. MALDI mass spec was performed on PNA-1 to ensure correct sequence. The peak representing the full mass of PNA 303 is labeled.

PCR

As shown in Figure 12, 721 base pair amplicon was amplified in both FBGM 1 and FBGM 10 using the PABB 1B/2B primer set. The optimal annealing temperature was determined to be 56˚C with later experiments.

PNA-mediated PCR clamping

Trial 1

Products of clamped PCR reactions are shown in Figure 13. Figure 13a represents PCR reactions run with FBGM 1. Bands in lanes 3 and 4 represent

46 amplification that occurred in the presence of PNA 303 while those in lanes 5 and six represent amplification that occurred when PNA 612 was included in reaction mixtures.

Lane 7 represents a control reaction run without either PNA. While amplification of the

721 base pair amplicon representing the PAB B gene did occur in all reactions, bands representing those run with PNA 303 are less intense than those run in the absence of

PNAs. Bands from reactions run with PNA 612 are equally intense as those from reactions run without PNAs.

Figure 12. Amplification of PAB B using PABB 1B/2B primer set. The amplicon is 721 bp in length.

Figure 13b represents reactions run with FBGM 10. Bands in lanes 3 and 4 represent amplification from reactions including PNA 303 while those in lanes 5 and 6 represent reactions including PNA 612. The band in lane 6 represents amplification within a reaction mixture containing neither PNA. Amplification of the desired 721 base pair amplicon occurred in all reaction mixtures run out in lanes 3-7.

47

a. b.

Figure 13. PNA-mediated PCR clamping of FBGM 1 and 10 (Trial 1). (a) FBGM 10: Bands of intensity equal to controls resulted from inclusion of either PNA. (b) FBGM 1: Bands of reactions including PNA 303 are noticeably lighter than those of reactions including either PNA 612 or neither PNA.

Trial 2

Products from reactions run with FBGM 1 are presented in Figure 14. Lanes 3 and 4 are representative of reactions run in the absence of PNA and Lane 5 represents the no template control. Lanes 6 and 7 contain the products from reactions run with PNA

303 while bands in lanes 8 and 9 represent products of reactions which included PNA

612. No amplification occurred in the no template control. The intensity of the bands in lanes 6 and 7 are noticeably less intense than those representing reactions run in the absence of either PNA or those that included PNA 612.

Trial 3

Products from optimization reactions run with FBGM 1 and PNA 303 are shown in Figure 15. Lane 3 represents the unclamped PCR product and lane 4 contains the no template control for comparison. The optimal PNA 303 annealing temperature, 77.3˚C, 48 was determined from the average of the temperatures at which clamping of the PCR reaction occurred.

Figure 14. Clamping of FBGM 1 with PNA 303 (Trial 2). Results were consistent with previous runs. Lanes 3 and 4 contained non- clamped products, lane 5 contains the no template control, lanes 6 and 7 contain product from reactions run with PNA 303, and lanes 8 and 9 contain products from reactions with PNA 612.

Figure 15. Optimization of PNA 303 annealing temperature (Trial 3). Clamping occurred at 76˚C, 77.5˚C, and 78.5˚C (lanes 5-7). Lanes 5-9 contain products run with PNA 303 annealing temperatures of 76, 77.5, 78.5, 79.3, 79.8, and 80˚C, respectively.

49

Trial 4

PCR products from reactions run to optimize the concentration of PNA 303 are presented in Figure 16. Duplicate reactions are mirrored in the top and bottom rows of the gel. Clamping occurred in all wells containing PCR products from reactions including PNA 303. The greatest inhibition of DNA amplification occurred in lane 4 corresponding to the addition of a 150 μM concentration of PNA 303. Lane 5 represents the unclamped PCR product for comparison.

Figure 16. Optimization of PNA 303 concentration (Trial 4). Product clamped to the greatest degree in lane 4 (150 μM). Lane 3=125μM, lane 6=175μM, lane 7=200μM, lane 8=250μM.

Trial 5

Results of the initial annealing temperature optimization reaction for PNA 612 are presented in Figure 17. Lane 8 contains the unclamped PCR product for comparison.

Clamping occurred in lanes 12-14 corresponding to PNA annealing temperatures of 92.2,

93.5, and 94˚C. A summary of all 5 trials is presented in Table 3.

50

Figure 17. Initial optimization of PNA 612 annealing temperature (Trial 5). Clamping occurred in lanes 12-14 (92.2, 93.5, and 94˚C, respectively). Lanes 2-7 and 8-11 correspond to PNA annealing temperatures of 70-90.2˚C.

Table 3. Expected outcomes and results of PNA-mediated PCR clamping.

Strain Site 303 Site 612 PNA 303 PNA 612 Amplification Amplification Expected? Observed? FBGM 1 wildtype wildtype clamped clamped no no

FBGM 10 SNP SNP none none yes yes

FBGM 19 wildtype NT NT no NT SNP (not tested) FBGM 20 wildtype SNP NT NT no NT

Discussion

All data from HPLC, MALDI mass spec, and spectrophotometer readings indicate that the PNAs were abundant and that PNA 303 was synthesized correctly. Because

HPLC and MALDI mass spec have yet to be performed on PNA 612, it is possible that

51 any issues experienced during clamping could reflect inconsistencies between the desired and actual structure and composition of the PNA.

All trials were performed with primers designed for the amplification of a 721 bp amplicon including the PAB B gene. PABB 1B and PABB 2B performed equally well in the amplification of FBGM 1 as well as FBGM 10 and is expected to initiate amplification in the other strains as well as they are highly similar in sequence.

Clamping trials 1 and 2 brought consistent results. However, clamping was not entirely successful. Clamping runs with FBGM 10 have consistently resulted in amplification with reactions run with either PNA resulting in as much amplification as those run without a PNA (Figure 13a). This is appropriate as the strain contains both single base pair mutations, preventing either PNA from binding to the template strand.

PCR clamping reactions run with FBGM 1 have consistently shown PNA 303 to cause some inhibition of amplification. While there is considerable leakiness in its clamping, as has been shown in other experiments utilizing elongation arrest PNA- mediated PCR clamping, the PNA clearly decreases the quantity of amplified DNA (von

Wintzingerode et al. 2000). Optimization of PNA annealing temperature and concentration in trials 3 and 4 improved the effectiveness of PNA 303 and decreased the leakiness of reactions.

Although very little data has been collected on the clamping ability of PNA 612, it was shown to effectively clamp PCR reactions with FBGM 1 at annealing temperatures higher than 90˚C. Future work must be done to optimize this temperature and also the concentration at which the PNA is added to reaction mixtures.

52

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Chapter I

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Allee, W.C. 1927. Animal aggregations. The Quarterly Review of Biology 2: 367-398.

Berkner, L.V. and L.C. Marshall. 1965. On the origin and rise of oxygen concentration in the Earth‟s atmosphere. Journal of Atmospheric Sciences 22: 225-261.

Carr, M., B.S.C. Leadbeater, and S.L. Baldauf. 2010. Conserved meiotic genes point to sex in the choanoflagellates. Journal of Eukaryotic Microbiology 57: 56-62.

Courchamp, F., T. Clutton-Brock, and B. Grenfell. 1999. Inverse density dependence and the Allee effect. Trends in Ecology in Evolution 14: 405-410.

Hoffman, P.F. and D.P. Schrag. 2002. The snowball Earth hypothesis: Testing the limits of global change. Terra Nova 14: 129-155.

Hyde, W.T., T.J. Crowley, S.K. Baum, and W.R. Peltier. 2000. Neoproterozoic „snowball Earth‟ simulations with a coupled climate/ice-sheet model. Nature 405: 425-429.

James-Clark, H. 1868. On the spongiae cliatae as ifusoria flagellata. Memoirs Read Before the Boston Society of Natural Histoy 1: 305-340.

King, N., C.T. Hittinger, and S.B. Carroll. 2003. Evolution of key cell signaling and adhesion protein families predates animal origins. Science 301: 361-363.

King, N. 2004. The unicellular ancestry of animal development. Developmental Cell 7: 313-325.

53

King, N., M.J. Westbrook, S.L. Young, A. Kuo, M. Abedin, J. Chapman, S. Fairclough, U. Hellsten, Y. Isogai, I. Letunic, M. Marr, D. Pincus, N. Putnam, A. Rokas, K.J. Wright, R. Zuzow, W. Dirks, M. Good, D. Goodstein, D. Lemons, W. Li, J.B. Lyons, A. Morris, S. Nichols, D. J. Richter, A. Salamov, JGI Sequencing, P. Bork, W.A. Lim, G. Manning, W. T. Miller, W. McGinnis, H. Shapiro, R. Tjian, I.V. Grigoriev, and D. Rokhsar. 2008. The genome of the choanoflagellate Monosiga brevicollis and the origin of metazoans. Nature 451: 783-788.

King, N., S.L. Young, M. Abedin, M. Carr, B.S.C. Leadbeater. 2009a. Starting and maintaining Monosiga brevicollis cultures. in Emerging Model Organisms: A Laboratory Manual. CSHL Press, Cold Harbor, NY.

King, N., S.L. Young, M. Abedin, M. Carr, B.S.C. Leadbeater. 2009b. Visualizing the subcellular localization of actin, beta-tubulin, and DNA in Monosiga brevicollis. in Emerging Model Organisms: A Laboratory Manual. CSHL Press, Cold Harbor, NY.

Knoll, A.H. 1996. Breathing room for early animals. Nature 382: 111-112.

Losen, M., B. Frolich, M. Pohl, and J. Buchs. 2004. Effect of oxygen limitation and medium composition on Escherichi coli fermentation in shake-flask cultures. Biotechnology Progress 20: 1062-1068.

Miller, J.H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

Rice, W.R. 1989. Analyzing tables of statistical tests. Evolution 43: 223-225.

Rokas, A. 2008. The molecular origins of multicellular transitions. Current Opinion in Genetics and Development 18: 472-478.

Ruiz-Trillo, I., G. Burger, P.W.H. Holland, N. King, B.F. Lang, A.J. Roger, and M.W. Gray. 2006. The origins of multicellularity: A multi-taxon genome initiative. TRENDS in Genetics 23: 113-118.

Runnegar, B. 2000. Loophole for snowball Earth. Nature 405: 403-404.

Segawa, Y., H. Suga, N. Iwabe, C. Oneyama, T. Akagi, T. Miyata, and M. Okada. 2006. Functional development of Src tyrosine kinases during evolution from a unicellular ancestor to multicellular animals. Publications of the National Academy of Sciences 103: 12021-12026.

Snell, E.A., R.F. Furlong, P.W.H. Holland. 2001. Hsp70 sequences indicate that choanoflagellates are closely related to animals. Current Biology 11: 967-970. 54

Stanley, S.M. 1973. An ecological theory for the sudden origin of multicellular life in the late Precambrian. Proceedings of the National Academy of Sciences USA 70: 1486-1489.

Steenkamp, E.T., J. Wright, and S.L. Baldauf. 2006. The protistan origins of animals and fungi. Molecular Biology and Evolution 23: 93-106.

Stephens, P.A., W.J. Sutherland, R.P. Freckleton. 1999. What is the Allee effect? Oikos 87: 185-190.

Chapter II

Casale, R., I.S. Jensen, and M. Egholm. 2004. Synthesis of PNA oligomers by Fmoc chemistry. Pp. 61-76 in P.E. Nielsen, ed. Peptide Nucleic Acids: Protocols and Applications. Cromwell Press, Trowbridge, Wilts.

Galbiati, S., B. Foglieni, M. Travi, C. Curcio, G. Restagno, L. Sbaiz, M. Smid, F. Pasi, A. Ferrari, M. Ferrari, and L. Cremonesi. 2008. Peptide-nucleic acid-mediated enriched polymerase chain reaction as a key point for non-invasive prenatal diagnosis of β-thalassemia. Haematologica 93: 610-614.

Hall, T.A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95-98.

Luo, J.D. E.C. Chan, C.L. Shih, T.L. Chen, Y. Liang, T.L. Hwang, and C.C. Chiou. 2006. Detection of rare mutant K-ras DNA in a single-tube reaction using peptide nucleic acid as both PCR clamp and sensor probe. Nucleic Acids Research 34: e12.

Martin, D.P., C. Williamson, and D. Posada. 2005. RDP2: Recombination detection and analysis from sequence alignments. Bioinformatics 21: 260-262.

Nielsen, P.E., M. Egholm, R.H. Berg, and O. Buchardt. 1991. Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science 254: 1497-1500.

Orum, H., P.E. Nielsen, M. Egholm, R.H. Berg, O. Buchardt, and C. Stanley. 1993. Single base pair mutation analysis by PNA directed PCR clamping. Nucleic Acids Research 21: 5332-5336.

55 von Wintzingerode, F., O. Landt, A. Ehrlich, and U.B. Gobel. 2000. Peptide nucleic acid-mediated PCR clamping as a useful supplement in the determination of microbial diversity. Applied and Environmental Microbiology 66: 549-557.

Appendix A

Abedin, M. and N. King. 2008. The premetazoan ancestry of cadherins. Science 319: 946-948.

Fox, C.H., F.B. Johnson, J. Whiting, and P.P. Roller. 1985. Formaldehyde fixation. Journal of Histochemistry and Cytochemistry 33: 845-853.

King, N., C.T. Hittinger, and S.B. Carroll. 2003. Evolution of key cell signaling and adhesion protein families predates animal origins. Science 301: 361-363.

King, N. 2004. The unicellular ancestry of animal development. Developmental Cell 7: 313-325.

King, N., S.L. Young, M. Abedin, M. Carr, B.S.C. Leadbeater. 2009b. Visualizing the subcellular localization of actin, beta-tubulin, and DNA in Monosiga brevicollis. in Emerging Model Organisms: A Laboratory Manual. CSHL Press, Cold Harbor, NY.

Appendix B

Canfield, D.E., S.W. Poulton, and G.M. Narbonne. 2007. Late-Neoproterozoic deep- ocean oxygenation and the rise of animal life. Science 315: 92-95.

James-Clark, H. 1868. On the spongiae cliatae as ifusoria flagellata. Memoirs Read Before the Boston Society of Natural Histoy 1: 305-340.

King, N., C.T. Hittinger, and S.B. Carroll. 2003. Evolution of key cell signaling and adhesion protein families predates animal origins. Science 301: 361-363.

King, N. 2004. The unicellular ancestry of animal development. Developmental Cell 7: 313-325.

56

King, N., M.J. Westbrook, S.L. Young, A. Kuo, M. Abedin, J. Chapman, S. Fairclough, U. Hellsten, Y. Isogai, I. Letunic, M. Marr, D. Pincus, N. Putnam, A. Rokas, K.J. Wright, R. Zuzow, W. Dirks, M. Good, D. Goodstein, D. Lemons, W. Li, J.B. Lyons, A. Morris, S. Nichols, D. J. Richter, A. Salamov, JGI Sequencing, P. Bork, W.A. Lim, G. Manning, W. T. Miller, W. McGinnis, H. Shapiro, R. Tjian, I.V. Grigoriev, and D. Rokhsar. 2008. The genome of the choanoflagellate Monosiga brevicollis and the origin of metazoans. Nature 451: 783-788.

Marchant, H.J., J. van den Hoff, and H.R. Burton. 1987. Loricate choanoflagellates from Ellis Fjord, Antarctica including the description of Acanthocorbis tintinnabulum sp. nov. Proceedings of NIPR Symposium, Polar Biology 1: 10-22.

Michod, R.E. and D. Roze. Cooperation and conflict in the evolution of multicellularity. Heredity 86: 1-7.

Ruiz-Trillo, I., G. Burger, P.W.H. Holland, N. King, B.F. Lang, A.J. Roger, and M.W. Gray. 2006. The origins of multicellularity: A multi-taxon genome initiative. TRENDS in Genetics 23: 113-118.

Segawa, Y., H. Suga, N. Iwabe, C. Oneyama, T. Akagi, T. Miyata, and M. Okada. 2006. Functional development of Src tyrosine kinases during evolution from a unicellular ancestor to multicellular animals. Publications of the National Academy of Sciences 103: 12021-12026.

Snell, E.A., R.F. Furlong, P.W.H. Holland. 2001. Hsp70 sequences indicate that choanoflagellates are closely related to animals. Current Biology 11: 967-970.

Steenkamp, E.T., J. Wright, and S.L. Baldauf. 2006. The protistan origins of animals and fungi. Molecular Biology and Evolution 23: 93-106.

Appendix C

Berkson, J. 1939. The error of estimate of the blood cell count as made with the hemocytometer. The American Journal of Physiology-Legacy Content 128: 309- 323.

King, N., S.L. Young, M. Abedin, M. Carr, B.S.C. Leadbeater. 2009c. Isolation of Single Choanoflagellate Cells from Field Samples and Establishment of Clonal Cultures. in Emerging Model Organisms: A Laboratory Manual. CSHL Press, Cold Harbor, NY.

57

APPENDICES

58

APPENDIX A

THE OPTIMIZATION OF FLUORESCENT STAINING PROTOCOLS FOR

SALPINGOECA ROSETTA

Introduction

Microscopy has been of great importance for research on choanoflagellates. In particular, epifluorescence and confocal microscopy have been indispensible.

Immunofluorescence microscopy has allowed for the localization of the expression of particular proteins and can be used to observe changes in the expression levels over time and with experimental treatments. However, in order to use this tool effectively, staining protocols must be optimized to ensure not only that staining is specific, but that materials are used efficiently.

The typical morphology of a choanoflagellate includes a round cell with a cone- shaped, actin-based collar and a single flagellum consisting of β-tubulin. These structures can be easily visualized. The DNA and collar can be visualized with simple stains, DAPI and rhodamine phalloidin, respectively. The flagellum as well as the cell body are visualized a primary antibody, α-β tubulin, along with a fluorescent secondary antibody. Protocols describing this process have been published for Monosiga brevicollis and they are available from past presentations for Salpingoeca rosetta (Abedin and King

59

2008, King et al. 2009b). These protocols may not be optimal for every situation, however, and have been modified for use in current experiments.

Materials and Methods

Published protocols were altered in order to optimize staining for the conditions present in our lab and for the cultures that are maintained. Two features of the protocols were targeted: fixation and the staining of the actin-based collar with rhodamine phalloidin. The study utilized samples of a culture of Salpingoeca rosetta that had been cultured continuously since its use in a previous experiment. The population was cultured in new barley medium (artificial sea water infused with barley grass) in an incubator at approximately 24˚C.

Glass coverslips (18 mm2; Corning, Lowell, MA) were prepared prior to staining with HistoGrip (Invitrogen, Carlsbad, CA), rather than the poly-L-lysine recommended, and allowed to dry overnight. Just prior to staining, the prepared coverslips were sprayed with 100% ethanol and allowed to dry. After drying, the coverslips were added to incubation chambers consisting of a glass Petri dishes containing moist filter paper covered with a piece of Parafilm large enough that the coverslips were separated by at least 2.5 cm. All incubations took place in these incubation chambers. Samples (700 μL) of S. rosetta culture was then added to the 6 coverslips and allowed to incubate for 30 minutes. After the incubation period the excess fluid was very carefully aspirated from the glass. Care was taken as to keep the cells adhered and to not destroy their delicate structures. Six percent acetone (125μL) was then added to each coverslip, allowed to incubate for 5 minutes and then gently aspirated from the same corner of the coverslip as 60 before. One percent formaldehyde was then added to each coverslip following the same protocol as described for the acetone. After the formaldehyde had been aspirated, 125μL of blocking solution (1xPBS, 1% BSA, 0.3% TritonX-100) was added to each coverslip and incubated for 30 minutes. After removal of the blocking solution, 125μL of DAPI was added to each slide, incubated for 5 minutes in the dark, and aspirated. Rhodamine phalloidin (100 μL) at a concentration of 6U/mL was added to each coverslip and allowed to incubate for either 30, 45, or 60 minutes in the dark. Two coverslips were incubated for each amount of time. Coverslips were then washed twice with 1xPBS and allowed to dry almost completely in the dark, then placed face down on glass slides with

10μL of ProlonGold (Invitrogen), and allowed to equilibrate for 30 minutes after which they were viewed on a Olympus BX60 light microscope (Olympus America, Center

Valley, PA) and photographed using an Olympus DP71 digital camera (Olympus

America). This was repeated for 2 more replicates at each rhodamine phalloidin incubation time.

The same protocol with modification was followed in order to observe the effects of alterations in the portion pertaining to cell fixation. Following the removal of the incubated culture from the coverslips, 125μL of 6% acetone followed by 125 μL of 1% formaldehyde, 125 μL 6% acetone, or 125 μL of 6% acetone followed by either 1% or

4% glutaraldehyde were added to 2 prepared coverslips each and incubated for 5 minutes.

Those followed by formaldehyde or glutaraldehyde were incubated an additional 5 minutes after the removal of the acetone. The remaining 2 coverslips were heat fixed after the liquid remaining on the coverslip had dried completely. The remainder of the

61 protocol was followed as described. Cells were incubated in rhodamine phalloidin for 30 minutes in the dark.

Results and discussion

Both the DNA and actin were stained successfully on several cells when stained with DAPI and incubated in rhodamine phalloidin for 30, 45, or 60 minutes. The photos below (Figures 18-20) are examples of successful staining. Although staining was successful for all incubation times, examples incubated for 30 minutes are equivalent to, if not more clearly stained, than those with longer incubation times. In the example shown in Figure 18, the DNA in the nucleus is clearly stained and while faint it is clear that the arrow is pointing at the attachment sight of the actin-based collar in both pictures.

The same is true for Figures 19 and 20, although the actin staining is less bright after a 60 minute incubation period.

The outcomes of alterations in the fixation protocol were more mixed. S. rosetta cells were successfully stained when fixed in 6% acetone or in 6% acetone followed by

1% formaldehyde (Figures 21 and 22). Cells were also stained with 6% acetone and 4% glutaraldehyde, but the staining appeared to be non-specific as the flagella and cell bodies of the choanoflagellates, as well as the surrounding bacteria were stained with the rhodamine phalloidin along with the actin collars (Figure 23). Stained choanoflagellate cells were not observed when cells were heat fixed or when 1% glutaraldehyde was used subsequent to 6% acetone.

62 a. b.

Figure 18. Choanoflagellate cell incubated in rhodamine phalloidin for 30 minutes. (a) DNA in nucleus is stained with DAPI. (b) Arrow is pointing to the collar stained with rhodamine phalloidin.

a. b.

Figure 19. Choanoflagellate cell incubated in rhodamine phalloidin for 45 minutes. (a) DNA stained with DAPI. (b) Arrow is pointing to collar stained with rhodamine phalloidin.

a. b.

Figure 20. Choanoflagellate cell incubated in rhodamine phalloidin for 60 minutes. (a) Nucleus of choanoflagellate stained with DAPI. (b) Arrow is pointing to faint, red collar stained with rhodamine phalloidin.

63 a. b.

Figure 21. Choanoflagellate cell fixed with 6% acetone. (a) DNA of choanoflagellate as well as that of surrounding bacteria stained with DAPI. (b) Choanoflagellate collar stained with rhodamine phalloidin. The picture appears to be of overlapping choanoflagellates.

a. b.

Figure 22. Possible choanoflagellate colony fixed with 6% acetone and 1% formaldehyde. (a) DNA of colony as well as that of attached bacteria stained with DAPI. (b) Collars of choanoflagellates stained with rhodamine phalloidin.

a. b.

Figure 23. Choanoflagellate fixed with 4% glutaraldehyde. (a) Nucleus of cell with DAPI-stained DNA. (b) Non-specific staining of cell body and collar with rhodamine phalloidin.

64 stained at all. Although this may have been due to cell damage, it is more likely due to inefficient staining methods.

In order to alleviate this issue, the incubation time of cells with rhodamine phalloidin was increased to 30, 45, and 60 minutes, rather than the recommended 15 minute incubation time. While all of the increased incubation times were successful, the

60 minute incubation actually produced cells that were more faintly stained. The 30 and

45 minute incubations were equally successful so in order to be time efficient, a 30 minute incubation was used in later experiments.

Cell fixation is another hindrance of successful fluorescent staining that was addressed in this study. While the published protocols recommend the use of 4% formaldehyde to fix choanoflagellates prior to staining, it has been my experience that a concentration of formaldehyde that high destroys the cells of protists, making them impossible to stain clearly. When the concentration of formaldehyde was changed to 1%, the cells remained in tact and were very clearly stained. In fact, when 6% acetone was used along with 1% formaldehyde for fixation, colonies remained in tact and were stained along with many individual cells. Also surprising was the successful staining of cells fixed in acetone alone. However, the lack of cross-linking that occurs due to the formaldehyde is likely essential to the preservation of the cell structure and for the precise staining and localization of specific structures and proteins (Fox et al. 1985).

While no stained cells fixed in 1% glutaraldehyde subsequent to 6% acetone were observed during this study, the addition of 4% glutaraldehyde after 6% acetone has been removed results in stained cells with very clearly defined structures. However, the

65 staining is non-specific. Whereas rhodamine phalloidin should only stain the actin-based collar, it appears to stain the cell bodies and flagella of cells as well as the bacteria on which they feed. This was not observed in any other experiment performed in this study.

It is likely that a concentration between 1 and 4% will be more appropriate for use.

Other than the 6% acetone/1% glutaraldehyde, the attempts made at heat fixing the cells were also unsuccessful. It is likely that allowing the coverslip to dry damaged the cells in itself. Also, even if heat fixation had been successful at adhering the cells to the coverslip, it is likely that proteins would have been denatured by the heat and therefore impossible to localize.

Overall, the fixation of cells in 6% acetone prior to 1% formaldehyde and then later the incubation in rhodamine phalloidin appear to be successful and appropriate alterations for the staining of Salpingoeca rosetta. However, fixation methods will continue to be tested as glutaraldehyde appears to be extremely promising with the detail that can be observed with its use.

Future studies investigating the location and degree of certain proteins in several choanoflagellate species, including S. rosetta have been planned. PRCDH1, a cadherin protein expressed in S. rosetta, is one of the proteins. Cadherins are cell adhesion proteins and as such are associated with multicellularity. However, as stated before, they are expressed in choanoflagellates, single-celled organisms (King et al. 2003; King

2004). Because of this, future experiments involving choanoflagellates in environments supposedly conducive to multicellularity may have an effect on the expression of such proteins. One way to analyze this change is with fluorescent techniques. The detail

66 provided by glutaraldehyde fixation is essential for such analyses, but non-specific staining such as occurred in this study would lead to inaccurate results. Thus, while the outlook is promising, the technique must continue to be improved.

67

APPENDIX B

THE DEVELOPMENT OF PROTOCOLS FOR A LONG TERM EVOLUTIONARY

EXPERIMENTS WITH CHOANOFLAGELLATES

Introduction

The origin of multicellularity was a major macroevolutionary step that altered subsequent evolutionary dynamics. Several studies have investigated the transition from free living unicellular organisms to multicellular organisms with collections of cooperating cells. In 1868 James-Clark compared the collar cells of multicellular sponges with single-celled choanoflagellates, noticing that they share a similar morphology

(James-Clark 1868). More recently, several genome and protein-based studies have searched for the most likely ancestor to the metazoans (Snell et al. 2001, King 2004,

Ruiz-Trillo et al. 2006, Steenkamp et al. 2006). These studies support choanoflagellates as a sister group of metazoa, indicating that multicellularity may have arisen in an ancestor shared by choanoflagellates and metazoans. This possibility will be explored with experiments intended to test the extent to which choanoflagellates adaptively converge on metazoan trait states when exposed to environments conducive to change over long evolutionary periods. Several choanoflagellate species will be evolved for hundreds of generations in environmental conditions that may be conducive to the evolution of multicellularity. Changes in the transcription and expression of genes 68 associated with multicellularity, yet possessed by choanoflagellates will be used to analyze changes that occur.

Eventually, several environmental parameters will be altered and included as evolutionary environments. These include prey size, the presence of predators, chemical signals and temperature. However, only oxygen level was addressed in this preliminary study. Oxygen level was investigated because low oxygen levels during the early

Proterozoic may have constrained body size (King 2004). Without circulatory systems, choanoflagellate ancestors would have relied on the diffusion of oxygen across membranes for respiration and thus may have remained simple and small to maintain sufficient oxygen exchange.

It is likely that these environmental changes will lead to upregulation or downregulation of several proteins depending on whether the condition would favor unicellularity or multicellularity. The proteins include cadherins, c-type lectins, and tyrosine kinases. Although they are not utilized in exactly the same way in choanoflagellates and all metazoans, it is likely that they functioned in a single role in their common ancestor and later co-opted, due to processes such as domain shuffling, for different uses in multicellular organisms (King 2003, King 2004, Ruiz-Trillo et al. 2006,

Segawa et al. 2006, King 2008). Environmental change may have enabled group selection for cooperation among cells to overcome the conflict involved with selection among individual cells once the necessary changes in protein function had occurred

(Michod and Roze 2001).

69

The goal for this study was to test the effectiveness of changes that had been made to the protocol as well as to find where adjustments must be made for future use of this system and the protocols that have developed for it.

Materials and Methods

A preliminary trial run of a long term evolution experiment (LTEE) was run over approximately 50 generations of Salpingoeca rosetta in order to test the functionality of the system in place. The system is set up for an LTEE testing the effects of oxygen level on the evolution of the choanoflagellate species. It is essential that the system be conducive to the long term maintenance of choanoflagellate cultures under various environmental conditions so that effects of treatment can be determined.

Experimental Conditions

In order to elucidate the effects of oxygen level on the evolution of choanoflagellates two separate conditions were created. Cultures maintained in a hypoxic environment were kept in tightly sealed coplin jars while those maintained in a hyperoxic evolutionary environment received additional oxygen from an oxygen generator (Invacare Mobilaire V, Elyria, OH). The generator was connected pumped oxygen at a rate of <0.5 L/min through rubber tubing to a manifold with multiple output nozzles controlled independently of one another. Each utilized nozzle was connected to a

25 mm Easy Pressure syringe filter holder (PALL Life Sciences, Ann Arbor, MI) containing a 0.45 μm filter through rubber tubing. Filters were changed as needed during the duration of the experiment and holders were regularly autoclaved along with the filters. Each filter holder was connected to a 21 gauge 1 inch needle which had been 70 inserted into silicone tubing with a 1/8” diameter. The other end of the tubing had been inserted into each high oxygen evolution environment. All tubing was the same length for each jar. Each evolution environment was kept in a plexiglass holder to keep them upright and metal caps were placed over the caps of the jars to help prevent contamination. Cultures were incubated at approximately 24˚C for the duration of the study.

This preliminary experiment was run with five replicates and one control for each of the two environmental conditions. One control environment was maintained for each of the two conditions. They were treated just as the experimental cultures are during transfers. Contamination in either control jar would have indicated that contamination had occurred in the experimental cultures. However, this was never found to be the case.

Preparation of Evolutionary Environment

For the duration of this experiment, cultures of S. rosetta were kept in plastic coplin jars. These jars were prepared 1 day prior to the start of the study and then 1 day prior to each transfer. Jars were filled with 25 mL of sterile barley media and then autoclaved for 20 minutes at 121˚C and 15psi. Jars prepared for cultures grown in high oxygen environments were autoclaved along with silicone tubing and needle used to connect them to the oxygen source. After the volume lost to evaporation in the autoclave, the volume in each jar was approximately 24.25 mL, the desired volume for accurate transfers and generation counts. The caps of each jar were covered in aluminum foil prior to autoclaving in order to prevent contamination. The filters contained in

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25mm holders as well as the metal caps used to cover the coplin jars were autoclaved for

20 minutes separately on the vacuum setting so as to reduce moisture.

Transfers

Each jar was initially inoculated with a 0.75 mL sample from a culture of S. rosetta that had been previously used in another experiment and had since been regularly transferred to new media. The original culture had been co-cultured with an undefined strain of Gram negative bacteria. This strain was the food source for cultures in this experiment.

Once started, samples of each culture were transferred to new vials every four days. Just prior to each transfer cultures were thoroughly mixed by gently pipetting the cultures and then, every 10 generations, cells within four haphazardly chosen 0.0025mm2 boxes on the grid of an Improved Neubauer Levy Ultra Plane hemocytometer were counted. Cells located on the bottom and right gridlines were included in the counts while those on the top and left lines were not. See Appendix C for data regarding the reliability of cell enumeration using this technique. Samples (0.75 mL) were then transferred to fresh, sterile media. After transfers the jars were returned to the incubator and oxygen flow was returned to the high oxygen jars. The transfer ratio allowed for up to 5.05 generations between each transfer.

At one point the experiment had to be delayed. At that point cells were counted and samples were frozen as described below. When the experiment was resumed, the samples were thawed at room temperature and the contents were added to Petri dishes containing 20 mL of sterile new barley medium each. Cultures were allowed to grow for

72 one week at approximately 24˚C before evolution was resumed. At that point coplin jars containing approximately 24.25 mL of sterile new barley medium were inoculated with

0.75 mL samples of the cultures, cultures were returned to the incubator, and oxygen flow was restored. The recovery time allowed after thawing was not included in the generation count. Changes in the average densities of both high and low oxygen cultures were each tested with an ANOVA and the difference in the average density of high and low oxygen cultures was analyzed with a t-test.

Freezing

Samples from all cultures, excluding controls, were frozen every 25 generations during the experiment and also when the experiment had to be delayed. At these points cultures were still counted and transferred, except when the experiment was delayed.

After samples had been counted and transferred, 0.9mL samples of each culture were added to sterile centrifuge tubes. 100μL of DMSO was then added to each and the tubes were stored in a -80˚C freezer overnight. Samples were maintained long term in liquid nitrogen.

Results

The differences in evolutionary environment resulted in differences between the cultures evolved over 50 generations in hyperoxic and hypoxic conditions. Although all cultures survived throughout the duration of the experiment, the average density of all replicates for the duration of the experiment was significantly higher for cultures grown in a high oxygen environment than for those grown in hypoxia (T-test p=1.53E-7, df=38).

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Also, while samples from high oxygen cultures regularly contained colonies of choanoflagellates, colonies were not found in samples from low oxygen cultures.

While there were differences between cultures, the averages of replicates for each count did not differ significantly between counts for either the hyper- (p=0.151) or hypoxic (p=0.329) environments according to ANOVA. As shown in Figure 24, the density of hypoxic cultures varied very little throughout the experiment and the overall trend in density over time was slightly positive. The average density of hyperoxic cultures did vary over time, but the change over time is nearly zero.

18 16 14 12 10 8 High 6

4 Low Average Cell Cell Average Density

(choanoflagellates/nL) 2 0 0 1 2 3 4 5 Transfer Cycle

Figure 24. Effect of oxygen level on cell density over time. The average of replicates‟ measured choanoflagellate density throughout the experiment. Density is consistently higher in hyperoxic cultures than in hypoxic cultures.

Discussion

This preliminary study has offered some interesting results regarding choanoflagellate growth in different environments. Research on other choanoflagellate species has shown them to be more abundant in hypoxic waters with high bacterial loads

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(Marchant et al. 1987). However, the data obtained in this study indicate that choanoflagellate cultures grew more readily in a hyperoxic environment than in hypoxia.

Although it seems intuitive that organisms grow better in the presence of oxygen, oxygen can be a poison. Also, it is possible that different species of choanoflagellate thrive in very diverse conditions. The species described were loricates and possess a different life history than the species featured in this analysis. This could have a profound effect on their oxygen tolerance. Although the observations were not specifically analyzed for this project, colonies were observed frequently in hyperoxic environments, though not at all in hypoxic ones. Further study will elucidate whether or not this may be due to the increased levels of oxygen or if colony formation simply requires a minimum level of oxygen to occur. If it is true that an increase in oxygen induces the formation of

“multicellular” structures, it is possible that a global increase in oxygen may have allowed for the evolution of multicellular animals. However, it will be necessary to compare hyperoxic cultures not only to hypoxic ones, but also to normoxic cultures in the future.

Also, although the data indicate that hypoxic cultures have significantly lower counts than ones evolved in hyperoxia, the analyses of variance indicate that the averages observed are not significantly different between counts and thus the cultures appear to be stable over time. In addition, the trend in the averages over time for hypoxic cultures is slightly positive, so it is not likely that they are in danger of dying out due to their environmental conditions although it would be preferable to have similar densities in both environments. In order to achieve this, it may be necessary to vent the low oxygen jars to

75 allow gasses to escape and even to periodically add oxygen to the environments so that cultures do not become anoxic which, in the past, has been shown to be the case in this system.

Ideally, oxygen levels would match those found before and after the increase in oxygen during the late Proterozoic (Canfield et al. 2007). In order to achieve this, oxygen flow would have to be more carefully controlled as the system in place saturates the medium with oxygen rather than keeping it at a specific concentration. It is important to keep in mind however, that even if the oxygen levels do not mirror exactly what occurred in the past, a change in the genetics or phenotype of evolved choanoflagellates is still indicative of their response to their environment which is ultimately what would have led to the evolution of multicellularity in animals.

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APPENDIX C

RELIABILITY OF CELLS COUNTS FROM HAPHAZARDLY CHOSEN BOXES ON

HEMOCYTOMETER GRID

Introduction

The hemocytometer is a convenient tool for the quantification of cells in liquid culture. Originally used to enumerate blood cells, hemocytometers have since been used to count a diversity of cells in culture, including choanoflagellates (Berkson 1939, King et al. 2009c). Used throughout the studies described in Chapter I and Appendix B, the hemocytometer was instrumental to the collection of data. The counting technique utilized in studies assessing media, determining growth dynamics, and testing transfer and freezing regimes was itself assessed for consistency. As cell counts are the basis for many of the results presented in this thesis, it is essential that methods used to obtain them produce reproducible results. Demonstration of reliability will solidify this protocol as acceptable for the enumeration of cells in culture.

Materials and Methods

Three replicate cultures were initiated from a regularly transferred culture of

Salpingoeca rosetta. The parent culture was chosen from a set of three continuous

77 cultures based on its relatively high density of choanoflagellate cells. After 3 days of incubation at 24˚C, cells from samples of each replicate were quantified.

Cells from twelve samples of each replicate culture were counted. The densities of 6 samples were determined based on counts from 4 haphazardly chosen 0.0025 mm2 boxes and the remaining densities were determined based on counts from 8 haphazardly chosen boxes of the same size since both protocols were used in the experiments described in Chapter I and Appendix B. For each count, a 10 μL sample of culture was added to an Improved Neubauer Levy Ultra Plane hemocytometer (Clay-Adams) below a glass coverslip. An Axiovert 100 inverted compound microscope (Zeiss) at 500x with a

1.6x magnifier was used to view the cells. Cells located on the right and bottom lines were included in the counts, while those on the left and top lines were excluded in order to standardize counts and prevent overestimation of cell density. The hemocytometer was thoroughly cleaned with ethanol between counts.

Nested ANOVA was used to demonstrate the consistency of the technique.

Results and Discussion

Nested ANOVA was used to analyze the results of both trials involving counts from 4 0.0025 mm2 boxes and those involving counts from 8 0.0025 mm2 boxes.

Comparisons were made at three levels within a hierarchy: between replicate populations, between samples nested within replicates, and between box counts within samples. The lower level nested value was used as the denominator for all F-ratio tests.

Count averages at the levels of population and sample are shown in Figures 25 and 26. In trials for which cells in 4 boxes were counted for each sample samples nested within 78 populations and boxes nested within samples were non-significant sources of variance.

The replicate population from which samples were taken was a significant source of variation. More than twice the variance can be attributed to counts from individual boxes than from the other levels of the hierarchy (Table 4).

30

25

20

15

10 Average Cell Cell Average Count 5

0 Population 1 Population 2 Population 3 a.

14 12 10 8 6

4 Average Cell Cell Average Count 2 0 Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 b.

Figure 25. Count averages for four-box samples. (a) Average counts of replicate populations. (b) Average counts of samples taken from Population 1. Error bars represent the 95% confidence interval calculated based on error from the next lower nested level, sample and box, respectively.

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60

50

40

30

20

Average Cell Cell Average Counts 10

0 Population 1 Population 2 Population 3 a.

10 9 8 7 6 5 4 3 Average Cell Cell Average Count 2 1 0 Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 b.

Figure 26. Count averages for eight-box samples. (a) Average counts of replicate populations. (b) Average counts of samples taken from Population 1. Error bars represent the 95% confidence interval calculated based on error from the next lower nested level, sample and box, respectively.

Table 4. Results of nested ANOVA of counts from four boxes.

Source DF Type III SS Mean F Ratio Prob. > F Square Population 2 14.1111111 7.0555556 7.51 0.01 Sample(Population) 10 9.3888889 0.9388889 0.05 1.00 Box(Sample) 18 344.4166667 19.1342593 1.11 0.3839 Error 36 621.8333333 17.2731481

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Replicate populations and samples nested within replicates were not found to be significant sources of variance. Boxes nested within samples were shown to be a marginal source of variance. More than four times as much variance can be attributed to counts from individual boxes than to any other level tested (Figure 5).

Table 5. Results of nested ANOVA of counts from eight boxes.

Source DF Type III SS Mean F Ratio Prob. > F Square Population 2 9.4305556 4.7152778 2.60 0.1234 Sample(Population) 10 18.1527778 1.8152778 0.09 0.9998 Box(Sample) 42 820.5833333 19.5376984 1.56 0.0420 Error 84 1050.416667 12.504860

These results demonstrate that samples taken from replicate populations are not significant sources of variance when cell counts are performed following the described protocol indicating that the cell counts performed in the experiments described in Chapter

I and Appendix B are valid. Although population was shown to be a significant source of variation in the four-box counts, this could be due to divergence that took place during the three days between population initiation and the counts. Counts from individual boxes were a marginally significant source of variance when eight-box samples are used, this is expected due to random error and is not an issue as multiple boxes are counted for each sample. Statistical power, however, can likely be increased by increasing the number of boxes from which cells are counted.

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