Copyright by Marco Agostoni

2010

The Thesis Committee for Marco Agostoni Certifies that this is the approved version of the following thesis:

Genetic analysis of nitrogen assimilation in the Texas brown tide Aureoumbra lagunensis

APPROVED BY SUPERVISING COMMITTEE:

Supervisor: Deana L. Erdner

Jerry J. Brand

Tracy A. Villareal

Genetic analysis of nitrogen assimilation in the Texas brown tide Aureoumbra lagunensis

by

Marco Agostoni, B.S.

Thesis Presented to the Faculty of the Graduate School of

The University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of

Master of Science in Marine Science

The University of Texas at Austin May 2010 Dedication

I dedicate this thesis to my parents. La mia scelta di studiare negli Stati Uniti ha sicuramente portato ad avere compromessi nella loro vita. Sebbene è difficile stare lontano da un figlio per così tanto tempo, non mi hanno mai fatto pesare la mia scelta. Acknowledgements

I am grateful to my advisor, Dr. Deana L. Erdner, for the opportunity she gave me to work on this very interesting microorganism and her valuable suggestions throughout my research. I am also thankful to the members of my committee, Dr. Jerry J. Brand and

Dr. Tracy A. Villareal, for their detailed comments that helped shape this thesis. I would especially like to thank Dr. Andrew Evans for his unwavering guidance, insight and encouragement. A special gesture of appreciation is extended to Patricia Garlough for her important role in analyzing the carbon and nitrogen composition of my samples. I thank Dr. Alfredo F. Ojanguren for sharing with me stories and ideas regarding statistic, basic science, and graphic design for my posters and presentations. I would like to thank my colleagues, Rodney Withall and Claudia Rocha for assisting me with numerous daily laboratory tasks. A final note of gratitude goes out to my parents, sister and friends, the unconditionally supportive people who motivated and pushed me through the difficulties of first time independent research.

May 7th 2010

v Abstract

Genetic analysis of nitrogen assimilation in the Texas brown tide Aureoumbra lagunensis

Marco Agostoni, MSMarineSci

The University of Texas at Austin, 2010

Supervisor: Deana L. Erdner

The initiation, persistence, and termination of harmful algal blooms (HABs) can all be influenced by nutrient availability. Recent studies have highlighted the role of both organic and inorganic nitrogen sources in HAB dynamics. The pelagophyte Aureoumbra lagunensis causes ecosystem disruptive algal blooms and is responsible for the longest recorded (1989-1997). Because of Aureoumbra‟s small size and its inability to use nitrate, it has been hypothesized that its ability to use ammonium and organic nitrogen, especially at low concentrations, contributed to the unusual persistence of this bloom. This project aimed to assess the response of Aureoumbra to inorganic and organic nitrogen sources by examining the expression of genes responsible for nitrogen assimilation, with an eventual intent of developing expression assays that are indicative of nitrogen source use and/or sufficiency in Aureoumbra. Large volume batch cultures of Aureoumbra were grown with either ammonium or as a nitrogen source.

-1 Physiological characteristics (C:N, cell , and Fv/Fm) were monitored throughout the growth period, and the expression of the AMT-1, AMT-2 and UREC genes was assayed at early-, mid- and late-exponential phases. The results show that

vi Aureoumbra can use both ammonium and urea, and that it is well adapted to low-nutrient environments. Only one gene, AMT-1, appeared to be transcriptionally regulated in response to changing nitrogen concentration, and only to ammonium. The results of this study contribute to our understanding of how algae in general cope with low nutrient availability and should ultimately help to define the dynamics of these HAB events.

vii Table of Contents

List of Tables ...... ix

List of Figures ...... x

CHAPTER 1 1

Genetic analysis of nitrogen assimilation in the Texas brown tide Aureoumbra lagunensis ...... 1 Introduction ...... 1 Materials & Methods ...... 6 Results and Discussion ...... 20 Conclusion ...... 33

Appendix ...... 35

Glossary ...... 37

Bibliography ...... 39

Vita …………………………………………………………………………….. 45

viii List of Tables

Table 1: Degenerate and specific primers for AMT, UREC, ACT, and EL1α 16

Table 2: Amplification characteristics of the gene-specific primer sets ...... 18

Table 3: Physiological measures...... 25

Table 4: Ratio of gene expression under different growth stages ...... 31

Table 5: Ratio UREC / AMT-1...... 32

ix List of Figures

Figure 1: Ammonium experiment...... 8

Figure 2: Urea experiment ...... 9

Figure 3: Alignment of the Aureoumbra AMT-1 and AMT-2 ...... 13

Figure 4: Alignment of the Aureoumbra UREC ...... 14

Figure 5: Alignment of the Aureoumbra ACT ...... 15

Figure 6: Physiological analysis...... 24

Figure 7: Expression of the AMT-1, AMT-2 and UREC genes ...... 28

x CHAPTER 1

Genetic analysis of nitrogen assimilation in the Texas brown tide Aureoumbra lagunensis

INTRODUCTION

The state of Texas experiences harmful algal blooms caused by several different organisms. The pelagophyte alga Aureoumbra lagunensis causes “brown tide” blooms in the coastal bend region of the state. Aureoumbra is an alga belonging to Phylum Heterokontophyta, Class , present in the Laguna Madre, Texas, but also noted in other parts of Texas, Florida and Mexico (Villareal et al., 2002). This harmful brown alga possesses other notable characteristics: it caused the largest HAB bloom ever recorded (9 years in the Laguna Madre; Buskey et al., 2001), and it has the highest reported critical N:P ratio (260 at a growth rate of 0.16 day-1; Liu et al., 2001). In addition, it can tolerate salinities from 10 through 90 psu (Liu and Buskey, 2000); can grow on organic phosphorus and glutamate (Muhlstein and Villareal, 2007); can produce large quantities of extracellular polymeric substance (EPS) under stress (Liu and Buskey, 2000); and cannot use nitrate (DeYoe and Suttle, 1994). Aureoumbra has a diameter of 3- 5µm, which could allow these small cells to be better competitors in conditions of low nutrients (Pedersen and Sand-Jensen, 1995). Furthermore, the size of these cells increases the chl a – specific absorption coefficient (Raven and Kubler, 2002), enabling them to absorb photons more efficiently. Aureoumbra blooms are an example of ecosystem disruptive algal blooms (EDABs; Sunda et al., 2006) - harmful algal blooms (HABs) that can modify or degrade ecosystems, altering the transfer of energy from the primary producers to higher trophic

1 levels. Sunda et al. (2006) hypothesized that the initiation and persistence of EDABs are influenced by nutrient availability, herbivore grazing, and nutrient regeneration. These three factors work synergistically and can alter the ecosystem dynamics. If the herbivore grazing rate on harmful algae is limited, grazer-mediated recycling of nutrients is reduced. Harmful algae that are well adapted to nutrient-stressed environments benefit from these positive feedbacks. Therefore, these factors contribute to EDAB formation and exacerbate the effects on the ecosystem. The Aureoumbra bloom of 1989-1997 is an oft-cited example of an EDAB. Herbivore grazing on Aureoumbra can be reduced due to the presence of the EPS layer; Liu and Buskey (2000) showed that EPS impeded digestion and clogged feeding apparatus. EPS can also capture charged molecules such as metals from the environment. This high concentration of metals can become toxic to grazers by bioaccumulation. The bound metals are also removed from the environment by EPS and consumed by this alga, reducing their availability for other species (Bhaskar and Bhosle, 2006). EPS production increased proportionally as salinity increased (Liu and Buskey, 2000), but also as a response to unbalanced C:N metabolism, as a sink for reducing power; when the amount of fixed carbon exceeds the nitrogen available in the medium, extracellular polysaccharides are synthesized (Otero and Vincenzini, 2004). The reduction in grazing resulted in reduced nutrient recycling in nutrient-poor the Laguna Madre system (Shormann 1992). During the severe freeze of December 1989, large numbers of and fish were killed, and the resulting decomposition could have been a large source of ammonium to the lagoon (Stockwell et al., 1993; DeYoe and Shuttle, 1994). Yet, it is unclear if this initial ammonium source could sustain the entire 9-year bloom, or if another nitrogen source had the principal nutrient role. However, one defining characteristic of EDAB algae is that they are very well adapted to nutrient-

2 stressed environments. Because smaller organisms have 1) a higher surface area to volume ratio and 2) a thinner boundary layer, Aureoumbra would seem to be well- adapted to low nutrient conditions just as a consequence of its size. anophagefferens is another brown tide alga present on the north-east coast of the U.S., but it seems to have expanded southward of the east coast of USA and to South Africa (Naidoo, 1999; Pitcher and Calder, 2000; Probyn et al., 2001). Molecular phylogenetic analysis by Bailey and Andersen (1999) placed both algae within the same class, the Pelagophyceae. Aureoumbra and Aureococcus share numerous other similarities. First, both are unicellular and second, they are small. Third, they have EPS layer external to the plasmalemma. Fourth, they share similar pigmentation with similar ratios (excepting 19'-butanoyloxyfucoxanthin:Chl a): chlorophylls a and c, β-carotene, fucoxanthin, diadinoxanthin, and 19'-butanoyloxyfucoxanthin. Finally, both have only a single with the structure that is specific for their class of Pelagophyceae (lamellae with three adpressed thylakoids and a girdle lamella) (DeYoe et al., 1997). Aureococcus blooms seem to be associated with high dissolved organic nitrogen (DON) to dissolved inorganic nitrogen (DIN) ratios (Berg et al., 2003; Pustizzi et al., 2004; Berg et al., 2008). Similar to the Aureococcus brown tide, the growth and development of Aureoumbra blooms may be influenced by the relative availability of inorganic and organic nutrient sources. Despite low inorganic nutrient inputs, the Laguna Madre ecosystem is highly productive. Dissolved organic carbon is released through seagrass exudation, while DON is released by leaching and other processes. Ziegler et al. (2004) analyzed samples from the Laguna Madre from 1996 and 1997 (during the long Aureoumbra bloom) and found that DON represented 96-98% of total dissolved nitrogen in the Laguna Madre. Muhlstein and Villareal (2007) studied the ability of Aureoumbra to grow in medium with organic nutrients such as urea and glutamic acid. Although the

3 experiment was conducted with bacterized culture, the results strongly suggest that Aureoumbra could effectively use organic nitrogen compounds. As mentioned, with its small size and its ability to use DON, Aureoumbra may be seen as a useful model for understanding present and future responses to low nutrient availability and adaptation to different sources of nitrogen. Schaefer & Alber (2007) described the relationship between temperature and nitrogen budget in coastal areas. For the authors, denitrification could act as the primary control on the nitrogen budget in coastal watersheds. They found a highly positive correlation between temperature and denitrification along 12 watersheds.In addition, LaRoche et al. (1997) suggested a correlation between groundwater discharge and subsequent inorganic nitrogen input to costal bays. Therefore a nitrogen loss may occur in coastal areas as a consequence of increasing temperatures and decreasing rainfall discharge. Increasing temperatures and decreasing rainfall discharge seems reasonable in accordance with Intergovernmental Panel on Climate Change (IPCC) (Houghton et al., 1995) model results that show a positive correlation between temperature and anthropogenic CO2 emission, and to a study conducted by Chen et al. (2001) that showed a negative correlation between increase of temperature and decrease of rainfall in Southern Texas. Furthermore, change in temperature does not just affect coastal regions, but also the open ocean. In these areas, changes in temperature would lead to ice melting in high latitude areas and increases in temperature water surface in low latitude regions. Consequently, stratification would be enhanced in both regions (Sarmiento et al., 1998). Falkowski et al. (1998) suggested that this scenario of events would increase the density contrast between the nutrient-depleted upper layer and the nutrient-rich lower layer, reducing nutrient fluxes to the ocean surface. A study conducted during the years 1998 to 2006 showed a high correlation between net and the stratification anomaly (Behrenfeld et al., 2006).

4 Although the effects of global warming on biota are problematic to study, ecosystem composition and physiological status would likely shift during conditions of high stratification (Litchman et al., 2006). Falkowski & Oliver (2007) predicted a reduction of numbers of large cells in the open ocean in response to a decrease in nutrient availability. Small primary producers that are able to fix atmospheric nitrogen, or use different sources of nitrogen, e.g. organic nitrogen, could play a bigger role in nutrient recycling

(Berg et al., 2003). Although the Laguna Madre is not expected to experience increased stratification with an average depth of 0.8 meter, studies of Aureoumbra will contribute to our understanding of how algae in general cope with low nutrient availability. Aureoumbra‟s ability to assimilate different nitrogen sources, especially at low concentrations, may be the key to its success in the environment. To better understand how Aureoumbra responds to its nutrient environment, this project aimed to develop a set of molecular markers for nitrogen source use in Aureoumbra. After cloning the genes involved in nitrogen assimilation, ammonium transporter (AMT) and urease C subunit (UREC), the expression of these genes was analyzed under laboratory conditions using quantitative real-time PCR (qPCR) with specific primers developed from the sequences. The primary aim of this project was to understand how expression of N assimilation genes varies between cells grown on different N sources and different N conditions, and if there was evidence of transcriptional control. These results then enabled me to assess whether gene expression assays could be used to determine the nitrogen source(s) and/or sufficiency in Aureoumbra.

5 MATERIALS & METHODS

Culture conditions

Aureoumbra lagunensis strain 1509, obtained from the Provasoli-Guillard Culture Collection, was used for all experiments. This strain was xenic; although it was listed as being axenic, PCR analysis showed the presence of bacteria in the cultures. Cultures were maintained in autoclaved 0.22µm- filtered Gulf of Mexico seawater (salinity 32 psu) enriched with f/2 nutrients (Guillard, 1975) without nitrate or silicate. Nitrogen was added to the medium as ammonium (50 µM) or as urea (25 µM; 50 µM in nitrogen). Cultures were grown in 9.5L glass bottles (Pyrex solution bottle, 1595-2X) with an initial medium volume of 8L. At the beginning of the experiment the same volume of Aureoumbra was added to each carboy to give a specific density, corresponding to a starting cell density of approximately 10,000 cells ml-1. Bottles were bubbled with 0.2µm- filtered air to maintain mixing. Cultures were maintained at 22°C on a 12:12 light:dark cycle, at an irradiance of approx. 96 µmol photons m–2 s–1. Cells were counted using a hemacytometer. The first experiment was conducted using culture growth in batch under N-

+ sufficient conditions on NH4 while the second experiment used urea as nitrogen source. For each nitrogen source, 3 replicates were used. Every day, a sample was withdrawn aseptically, and cell density and nitrogen concentration were measured. However, spectrophotometric analysis of N concentration was not performed until the day before the second extraction. The first sample for RNA analysis was collected on the 5th - 7th day after inoculation, at a cell density around 100,000 cells ml-1 (early log phase – T1). A concentration such as 100,000 cells ml-1 allowed for sufficient biomass to extract enough RNA and yet was low enough to still have sufficient N in the medium. On the day that the cultures were predicted to deplete the nitrogen (based on N disappearance in the 6 cultures, presumably two or three days after the first extraction), a second sample (in middle log phase – T2) was collected for RNA extraction. Finally, a third set of samples were collected at the end of the log phase (T3) (Figure 1,2). For RNA extraction, algae were collected by centrifugation at 1300 x g for 6‟ at 22°C, 3 hours after the beginning of the light cycle, and frozen at -80°C.

RNA extraction and cDNA synthesis

Total RNA extraction was performed using the UltraClean Plant RNA isolation kit (Mo Bio, Inc). Isolation of mRNA with oligo (dT) magnetic beads was conducted using the mRNA Enrichment kit (Mag-Bind). mRNA was quantified with an ND-1000 Spectrophotometer (NanoDrop). cDNA synthesis was performed using an oligo-dTVN primer (Integrated DNA Technologies, Inc.) and SuperScript III reverse transcriptase (Invitrogen catalogue 18080-093) following manufacturer‟s protocol, with incubation for

45‟ at 55°C.

7

45 1.6

1 -

T1 T2 1.2

+ 30 4

0.8 A1 Millions cells ml cells Millions µM NH µM 15 A2 T3 0.4 A3 A1 0 0 A2 3 5 7 9 11 A3 Days after inoculation

+ Figure 1: Ammonium experiment: cell densities and medium NH4 concentrations in the 3 replicate batch cultures A1, A2 and A3.

8

30 2

1 -

1.6

20 1.2 U1

0.8 ml cells Millions µM Urea µM 10 U2 T1 T3 U3 0.4 T2 U1 0 0 U2 3 5 7 9 11 U3 Days after inoculation

Figure 2: Urea experiment: cell densities and medium urea concentrations in the 3 replicate batch cultures U1, U2 and U3

9 Ammonium and urea assays

+ The assay for spectrophotometric determination of NH4 concentrations was adapted from Strickland & Parsons (1968) and Bolleter et al. (1961). Sample and standard assays were conducted in a 96-well flat-bottomed plate (Molecular Devices).

+ All samples and standards were run in triplicate, and NH4 standards of 5, 10, 20, 30, 40, 50, 60, 80, and 100µM were included on each plate. Each well contained 220µl of sample or standard, followed by the sequential addition 20µl of phenol, 20µl of 20mM sodium nitroprusside, and 40µl of oxidizing solution. Oxidizing solution was prepared by mixing 1 part bleach and 4 parts of alkaline solution (10g sodium citrate and 0.5g NaOH in 50ml ultrapure water). Color development was performed for 1h in the dark, at room temperature without mixing. The absorbance of the sample at 640nm was determined by spectrophotometer (SpectraMax 190, Molecular Devices Corp.). A linear regression was calculated from the A640 and concentration of the standards, and sample concentrations were calculated from the equation of the regression line (R2>0.99). The assay for enzymatic determination of urea concentrations was adapted from McCarthy (1970) and Parsons et al. (1984). All reagents were made according to the protocol with the exception of the enzyme solution. Crude urease (0.25g; Worthington Biochemical Corporation) was suspended in 15ml of 1% w/v EDTA solution and concentrated by centrifugation using Centriprep centrifugal filter devices (10,000 NMWL, Millipore), at 2060 x g for 20‟ for 3 times, at 20°C. Finally, concentrated urease was suspended in 45ml of 1% w/v EDTA solution, and mixed with 50ml of analytic reagent quality glycerol and 5ml of Cleland‟s reagent DTT (USB Corporation) according to the protocol. For each assay, 100µl of urease solution was mixed to 900µl of sample,

+ and the sample was incubated at 50°C for 10‟ to reduce the urea to ammonium. NH4

10 concentration was then determined using the spectrophotometric assay described above. All samples and standards were run in triplicate, and urea standards of 0, 2.5, 5, 10, 15, 20, and 25µM were included on each plate. This method was preferred over a direct spectrophotometric assay for urea, as the results were immediate and the same reagents were used as for the ammonium assay (Revilla et al., 2005). However, to verify the

+ efficiency of this method, a measurement of 50µM of NH4 was compared with enzymatic determination of 25µM of urea. The enzymatic method was 93% efficient at converting urea to ammonium.

Gene cloning

For the identification of the AMT gene, degenerate primers were designed using known gene sequences from a variety of eukaryotic unicellular organisms. Conserved regions were identified by eye, and degenerate primers were selected to include no more than 4 degeneracies. For AMT, sequences of Cylindrotheca fusiformis, Thalassiosira weissflogi, Chlamydomonas reinhardtii, and Trichodesmium erythraeum (http://www.ncbi.nlm.nih.gov/ database) were used. For UREC, published degenerate primer sequences were used (Baker et al., 2009). Any resulting PCR amplicons were cloned into the pCR2.1-TOPO vector (Invitrogen) following the manufacturer‟s protocol. Individual clones were tested for the presence of the gene fragments using clone PCR, and positive clones were sequenced at the University of Texas ICMB Core Facility. Sequences were edited by eye using Sequencher (GeneCodes Corp.). The resulting sequences were verified by 1) determining the similarity to other genes through tblastx searches against the NCBI database; and 2) assessing their affiliation with eukaryotic genes by phylogenetic analysis based on multiple sequences both from eukaryotic and prokaryotic organisms (Geneious, Biomatters Ltd.; Figure 3,4,5). If the partial sequences corresponded to the gene of interest and were determined to be of eukaryotic origin, 11 specific primers were designed (Table 1). Degenerate primers for AMT identified and amplified two different ammonium transporters belonging to Aureoumbra. Specific primers were designed to target the two gene fragments. Although the two ammonium transporter gene fragments, designated AMT-1 and AMT-2, were similar in sequence, we ensured that the primers of the AMT-1 were specific for their own sequence and vice versa for the primers of AMT-2. In addition, a partial sequence of the actin gene (ACT) and elongation factor 1 alpha gene (EF1α) that are commonly used as housekeeping or reference genes (Le Bail et al., 2008), were cloned, sequenced, and verified. However, EL1α was cloned from cDNA, thus a phylogenetic analysis to assess its affiliation with eukaryotic genes has not been run.

12

AMT-1

AMT-2

Figure 3: Alignment of the Aureoumbra AMT-1 and AMT-2 with multiple sequences from eukaryotic (blue) and prokaryotes (red). The tree was constructed using PhyML (Guindon and Gastuel, 2003) with an input tree generated by BIONJ with the MtREV model of amino acid substitution. PhyML bootstrap trees were constructed from 100 resampled datasets without gamma-corrected distances, and likelihood log of -7204.42665.

13

Figure 4: Alignment of the Aureoumbra UREC with multiple sequences from eukaryotic microalgae (blue), fungi (green), and prokaryotes (red). The tree was constructed using PhyML (Guindon and Gastuel, 2003) with an input tree generated by BIONJ with the MtREV model of amino acid substitution. PhyML bootstrap trees were constructed from 100 resampled datasets without gamma-corrected distances, and likelihood log of -3309.41939

14 .

Figure 5: Alignment of the Aureoumbra ACT with multiple sequences from eukaryotic microalgae (blue), fungi (green), and prokaryotes (red). The tree was constructed using PhyML (Guindon and Gastuel, 2003) with an input tree generated by BIONJ with the MtREV model of amino acid substitution. PhyML bootstrap trees were constructed from 100 resampled datasets without gamma-corrected distances, and likelihood log of -7612.45242.

15 Table 1: Degenerate and specific primers used for amplification of AMT, UREC, ACT, and EL1α genes from Aureoumbra.

Gene Degenerate Primer Specific Primer

AMT-1 For: CATGCAMGCTGGMTTYGCSATGC For: AGCTATTGCTGAGCGTGCTACCTT

Rev: GGGTTGAAKCCRTACCAKCCGAACC Rev: TTTCCATCAGCGGACCATCCCATA

AMT-2 For: CATGCAMGCTGGMTTYGCSATGC For: GCCAAGACATATGGTCAAGCGCAA

Rev: GGGTTGAAKCCRTACCAKCCGAACC Rev: ACATCTTTCAGCTACGGCACCAGA

UREC For: GCAATACCATGCGCAATCGCAATCGCNGCNG For: AGCTATGGGTCGTGTTGGTGAAGT

Rev: GTCATCGGTCTCAAACTTCAYGARGA Rev: AATCGCAGCTGGGTTAATCGTCAC

ACT For: TGGGAYGAYATGGAGAAGAT For: AGGTTATGCTCTTCCTCACGCCAT

Rev: GTASARRTCCCTRCGRATGTC Rev: AGTGGTACCGCCAGAAAGGACAAT

EL1α For: GGYATCGAYAAGCGTACCAT For: TCTGTTGGTGGATTTGAGGCTGGA

Rev: GTGGTGCATYTCGACGGACTT Rev: TCGAGCTTCACCATACTTGACCGA

16 Real Time PCR assay

In order to confidently compare qPCR results between different genes, all primer sets needed to have similar amplification efficiency. To assay the efficiency of the specific primers, qPCR was performed using each primer set and a dilution series of Aureoumbra cDNA and plasmid containing the cloned gene fragments (1:1; 1:10; 1:100;

1:1000; 1:10000). Standard curves of all the primer sets showed efficiency above 93% (Table 2). In addition, melting curve analysis after qPCR showed that only one PCR amplicon was present for each reaction. Moreover, to verify the accuracy of the data two identical replicate analyses of each experimental sample were performed. The cycle threshold (Ct) values of these replicates resulted to have a standard deviation <0.3. To alleviate the need for inclusion of housekeeping gene, gene expression levels were normalized to the same amount of starting cDNA (Libus and Štorchová, 2006). Quantification of single-stranded cDNA was performed using RiboGreen RNA quantification assay (Invitrogen) which allowed for precise determinations of ssDNA concentrations. To analyze the relative gene expression, qPCR was performed with an Eppendorf Realplex Mastercycler instrument (Eppendorf) in the presence of SYBR-green master mix dye (Invitrogen) to bind the cDNA. The statistically significant effects of nitrogen conditions on gene expression were determined using analysis of variance

(ANOVA). Statistical analyses (n=3 per each N condition) were performed utilizing 95% confidence intervals (p < 0.05). All statistical analyses were computed using SPSS 16.0 software (SPSS Inc. IL).

17 Table 2: Amplification characteristics of the gene-specific primer sets, when used with a standard dilution series of Aureoumbra cDNA or cloned genes.

Gene Slope Efficiency R2

AMT-1 -3.39 97% >0.99

AMT-2 -3.40 96% >0.99

UREC -3.49 93% >0.99

ACT -3.49 94% >0.99

EL1α -3.51 92% >0.99

18 Physiological analysis

Analyses of carbon to nitrogen ratio per cell, Fv/Fm ratio, and total chlorophyll were conducted at the same time (±0.5h) every day from the early-log phase to the end- log phase. The aim was to better understand the physiological responses of Aureoumbra to its environment and verify that Aureoumbra was in N limitation. The C:N ratio allowed me to estimate the balance between these two elements. Fv/Fm was used as indicator of photosystem II efficiency. Decreases in total chlorophyll are empirically correlated with nitrogen stress. For the C:N ratio, at least 12,000,000 cells were filtered onto an ashed GF/F filter (Whatman) and dried over night at 60°C. The samples (n=1 for each bottle) were analyzed using a Carlo-Erba NC 2500 element analyzer (Carlo Erba Strumetazione,

Milan, Italy). Total chlorophyll and Fv/Fm analyses were performed with a Turner Designs Model 10-AU digital fluorometer. A total of 5ml of culture (n=3 for each bottle) were filtered through a GF/F filter (Whatman) to analyze the total chlorophyll. The filters were transferred to 10ml of 100% methanol and extracted for at least 24h at 4°C in the dark. The photochemical efficiency (Fv/Fm) of 3ml of Aureoumbra culture (n=3 for each bottle) was measured using a modification of Vincent et al. (1984). After dark adaptation for 30‟, Fo was read at 15s. The samples were then exposed for at least 30s to a final concentration of 20µM of DCMU (3‟,4‟-dichlorophenyl-1,1-dimethylurea, Sigma-

Aldrich, St Louis, MO, USA) dissolved in 100% ethanol, to reduce QA, the first electron acceptor of photosystem II. In the presence of DCMU the fluorescence represents the maximal fluorescence (Fm). Fm was read in the fluorometer after 15s. Fv was calculated as

Fm-Fo.

19 The statistically significant effects of nitrogen conditions on physiological responses were determined using analysis of variance (ANOVA). Statistical analyses

(n=3 per each bottle for total chlorophyll and Fv/Fm; n=1 per each bottle for C:N ratio) were performed utilizing 95% confidence intervals (p<0.05). All statistical analyses were computed using SPSS 16.0 software (SPSS Inc. IL).

RESULTS AND DISCUSSION

This work is the first attempt to study gene expression in the brown tide Aureoumbra lagunensis. The primary aim of this project was to understand how the expression of genes associated with nitrogen assimilation varies between cells grown on different N sources and under different N conditions. The first step was to isolate fragments of our target genes via PCR using degenerate primers. In total, five genes were isolated and identified: UREC, ACT, EF1α and two fragments of similar sequence that both encode ammonium transporters: AMT-1 and AMT-2. AMT and UREC are involved in the assimilation of ammonium and urea, respectively, whereas ACT and EF1α were selected as potential housekeeping genes (Le Bail et al., 2008) for the qPCR assays. The expression of AMT and UREC genes was determined under N-sufficient and N-deficient conditions, with inorganic and organic nitrogen sources. The results provide information on the transcriptional regulation of N assimilation in Aureoumbra and show how Aureoumbra growth and physiology respond to different nitrogen sources.

Aureoumbra growth and physiology

From the 3rd day after inoculation (around 25,000 cells ml-1), Aureoumbra started growing exponentially and stopped after 11-12 days. It behaved somewhat unexpectedly, as it continued to grow for 2-3 divisions after the medium nitrogen was depleted, both in

20 the NH4+-grown and urea-grown cultures. It has been shown that different microalgae are able to store nitrogen and other nutrients. Quantity and quality of nitrogen stored may control cell division rate, thus uncoupling the relation between cell division and environmental limiting nutrient concentration (Cunningham and Maas, 1978; Sciandra 1991). Aureoumbra could be seen as a “storage specialist” since it was able to store nitrogen in intracellular pools enough to exponentially grow for 2-3 divisions. These storage specialists may have an ecological advantage compared with other species when nutrient inputs are episodic; indeed these species can accumulate nutrients and survive between nutrient pulses (Sommer 1985, Grover 1991). Gene expression and the physiological responses were analyzed during 3 stages of growth. The first samples were collected 2-3 days before medium nitrogen was depleted, in early log phase (T1), thus these samples were considered to be nitrogen- sufficient, as external nitrogen concentration was sufficient to support several more cell divisions. The second sample was collected one day after medium nitrogen was depleted, in middle log phase (T2); yet the cells continued to divide and grow exponentially after this point. The continued growth indicated that the cells had sufficient nitrogen internally to continue dividing. The last sample was collected during the end of the exponential phase (T3), where cell growth had slowed substantially or ceased.

In both batch cultures, chlorophyll cell-1 decreased over time (Figure 6A,B),

+ whereas the cellular C:N ratio increased (Figure 6C,D). In NH4 -grown cells, the C:N ratio at T1 was 5.9 (SD ±0.3) and increased to 16.7 (SD ±2.2) by the end of the log phase (T3). Under growth on urea, the C:N at T1 was 7.7 (SD ±0.1) and increased to 23.9 (SD ±0.3) by the end of log phase (Table 3). An increase of C:N may be due to production of a large quantity of EPS, which is a common response to unbalanced C:N metabolism; under N limitation algae may produce extracellular polysaccharides as sink for reducing

21 power (Otero and Vincenzini, 2004). However, this explanation was not consistent with the observed changes in C and N per cell. Overall, the increase in C:N ratio was due to a strong decrease of total N cell-1 and a slight decrease of total C cell-1 (data not shown).

+ -1 -1 Total chlorophyll of NH4 -grown cells decreased from 0.32 pg cell (SD ±0.05 pg cell ) at T1 to 0.12 pg cell-1 (SD ±0.02 pg cell-1) at T3. In the urea cultures, total chlorophyll decreased from 0.2 pg cell-1 (SD ±0.01 pg cell-1) at T1 to 0.14 pg cell-1 (SD ±0.02 pg cell-

1) at T3 (Table 3). Based on the decreases in chlorophyll cell-1 and increases in C:N, Aureoumbra became N stressed over time in the batch cultures. These changes were already evident at the mid-log time point, when medium nitrogen had become depleted. Despite the physiological indications of N stress, the cells continued to grow through several more divisions, indicating a divergence between growth and nitrogen uptake. This incongruence complicates the determination of the physiological state of the Aureoumbra cells at T2 and T3. In my opinion, since the growth rate remained exponential even after the medium N was depleted (external N deficiency), I concluded that Aureoumbra became internally N deficient only in the end of the log phase.

Although the Fv/Fm ratio is generally a very sensitive indicator of nitrogen limitation in microalgae (Parkhill et al., 2001), there were no changes in the efficiency of

PSII during the experiments, indicating that Aureoumbra was somehow able to acclimate and maintain photosynthetic function under nitrogen stress. Whereas Cullen et al. (1992) and MacIntyre et al. (1997) showed that Thalassiosira pseudonana and Alexandrium tamarense respectively maintained steady Fv/Fm values under N-limitation, Cruz et al.

(2003) suggested that when chlorophyll decreases in response to N limitation but Fm/Fm remains stable, organisms are able to use different mechanisms for N repartition within

22 + the cell. In both the urea- and NH4 –grown cells, Fv/Fm was close to 0.8 (Table 3) - an indication of good physiological status (Figure 6E,F).

23 0.4 0.3 T3 0.3 T3 0.2

0.2

T1 0.1 0.1 T1

T2 T2

Total Chlorophyll pg/cell Chlorophyll Total Total Chlorophyll pg/cell Chlorophyll Total 0.0 0.0 7 8 9 10 11 12 5 6 7 8 9 10 11 A B

20 40

15 30

10 20

C:N ratio C:N C:N ratio C:N 5 10

0 0 7 8 9 10 11 12 5 6 7 8 9 10 11 C D

1 1

0.8 0.8

0.6 0.6

0.4 0.4

Fv/Fm ratio Fv/Fm Fv/Fm ratio Fv/Fm 0.2 0.2

0 0 7 8 9 10 11 12 5 6 7 8 9 10 11 E Days after inoculation F Days after inoculation

+ Figure 6: Physiological analysis of cells grown with NH4 (A, C, E) and urea (B, D, -1 F). (A, B) C:N ratio (C, D) total chlorophyll cell (E, F) Fv/Fm ratio. The arrows show when the RNA samples were collected during the experiment. Vertical bars represent standard deviation.

24 + Table 3: Physiological measures of urea- and NH4 -grown cells, at the three time points used for gene expression analysis. Total Chlorophyll and Fv/Fm ratio were replicated 3 times for each bottle, for a total of 9 measurements of each condition. C:N ratio was measured once for each bottle, for a total of 3 replicates of each condition. Standard deviations are shown in parentheses.

N source Sample Chlorophyll ng/cell C:N Fv/Fm

T1 0.32 (0.05) 5.9 (0.3) 0.77 (0.08)

T2 0.22 (0.03) 9.7 (1.2) 0.73 (0.10) + NH4 T3 0.12 (0.02) 16.7 (2.2) 0.75 (0.08)

T1 0.20 (0.01) 7.7 (0.1) 0.78 (0.06)

UREA T2 0.16 (0.02) 15.9 (0.4) 0.77 (0.01)

T3 0.14 (0.02) 23.9 (0.3) 0.77 (0.02)

25 Genetic analysis

Determining changes in the expression of genes involved in N assimilation as well as their relative abundance might provide a means to assess N use and sufficiency in Aureoumbra. Five genes were initially isolated for this analysis: UREC, ACT, EF1α, AMT-1 and AMT-2. AMT and UREC are involved in nitrogen assimilation, and ACT and

EF1α were selected as potential housekeeping genes (Le Bail et al., 2008) for the qPCR assays. To determine if ACT and EL1α genes might be used as reference genes, qPCR was performed using the same quantity of cDNA from T1, T2, and T3. Unfortunately, the expression of the two candidate reference genes repeatedly varied (approx. 10-fold) under different conditions, such that they could not be considered as suitable housekeeping genes. Thereafter, expression of UREC, AMT-1 and AMT-2 was normalized to the starting amount of cDNA for each reaction (Libus and Štorchová, 2006). AMT-1 was the only one of the three genes regulated by changing nitrogen

+ availability, and only by ammonium (Figure 7). When the medium NH4 was depleted, the expression of AMT-1 increased, and then expression decreased as the cells progressed to late-exponential/stationary phase. However, even in late exponential phase (T3) the relative expression level was higher than in early exponential (T1). In contrast,

+ expression levels of all 3 genes in urea-grown cells, and AMT-2 and UREC in NH4 - grown cells, showed a drastic decrease in expression over time. In urea-grown cells,

+ expression of all the genes was drastically reduced throughout time, whereas in NH4 - grown cells, the decrease in expression occurred only at T3. In early exponential phase

+ (T1), the expression of all three genes was similar in both the NH4 - and urea-grown cells.

26 The ability of Aureoumbra to grow with urea and the presence of UREC

+ transcripts strongly suggest that Aureoumbra is able to hydrolyze urea to NH4 (even though the cultures contained bacteria - DAPI analysis not shown). Yet, greater reductions in gene expression, higher C:N and lower chl cell-1 in urea-grown cells suggests, not surprisingly, that Aureoumbra may favor ammonium as an N source.

Indeed, Muhlstein and Villareal (2007) calculated the maximum growth rate µmax under

+ different N sources in Aureoumbra (type strain TBA-2) and showed that NH4 supported higher growth rates than urea. In contrast, I did not find a difference in growth rate between the two nitrogen sources (data not shown).

Furthermore, the standard error of the Ct values was much higher in the urea-

+ grown cells than in cells grown on NH4 . At T3 this result could be explained by the different growth rate of bottle U3 in the last two days compared with the other two replicates. Indeed, gene expression followed similar Ct values in bottles U1 and U2, but not in bottle U3. However, it does not explain the difference at T2, where the growth rate of the three bottles was similar, but the Ct values were different among all of the three replicates. Possible explanations for this variability include stress or the presence of bacteria. If bacteria were competing with Aureoumbra for the urea, and if bacteria types or densities varied between the culture replicates, a different gene expression response might occur among the three replicates. Also, physiological characteristics (based on the lower chlorophyll cell-1 and higher C:N) of urea-grown cells suggest that these cells was

+ more stressed than in NH4 -grown cells, and that could lead to a greater variation in gene expression.

27 14.00 AMT-1 17.32

20.64

Ct NH4 23.96 Urea

27.28

T1 T2 T3

14.00 AMT-2 17.32

20.64

Ct NH4 23.96 Urea

27.28

T1 T2 T3

14.00 UREC 17.32

20.64

Ct NH4 23.96 Urea

27.28

T1 T2 T3

+ Figure 7: Expression of the AMT-1, AMT-2 and UREC genes in cells grown on NH4 (black bars) or urea (gray bars). Transcript levels are expressed as Ct (cycle threshold), which is inversely proportional to initial amount of cDNA target. Vertical bars represent the standard error. Major units in the Ct axis correspond to 10-fold change in relative gene expression.

28 Gene expression assay

A secondary goal of the project was to determine whether gene expression assays could be used to determine nitrogen source and sufficiency in Aureoumbra. Relative gene expression levels could provide a tool to assess the physiological status of the cells with

+ + respect to NH4 and urea assimilation. In NH4 -grown cells, AMT-1 was the only gene

+ increased by changes in NH4 availability. Because of its contrasting pattern of expression, AMT-1 is the best candidate as a gene indicator of N status, especially when compared to AMT-2 or UREC expression. Table 4 shows the different Ct ratio between

+ the three genes in NH4 -grown cells during T1, T2, and T3. It is not possible to establish a useful ratio in urea-grown cells, both because of the high standard error in relative gene expression, and because the genes showed the same expression pattern during the three physiological conditions. From these results, the UREC/AMT-1 ratio shows the largest changes, and is thus the most promising indicator. To assess the utility of this ratio for verifying N sufficiency or deficiency under

+ NH4 growth of Aureoumbra, the first step was to verify significant differences among the three growth conditions for each replicate bottle. The standard deviation among the three bottles was 0.4 or less (Table 5), and the ratio of UREC/AMT-1 was significantly different between all three time points (p<0.001) but not between bottles within a single time point. However, whereas this ratio seems useful for evaluating the condition of Aureoumbra in lab experiments when grown with ammonium, it has definite limitations. At this point, I only have data for three discrete points in the growth curve, so it is impossible from my data to know how the expression of these genes may vary at other growth stages, e.g. very early log phase. In addition, I do not know how the ratio changes (if it changes) when Aureoumbra grows under steady-state nitrogen limitation, as

29 opposed to the N starvation that occurs in batch culture. Moreover, these three ratios do not change consistently with time or the severity of N stress. From T1 to T2 there is an increase from 1.07 to 1.31, yet at T3 it decreases to 1.20 (Table 4). It will therefore be hard to determine if any value between 1.20 and 1.31 corresponds to the progression from N sufficiency to external N deficiency (T1-T2) or from external N deficiency to N starvation (T2-T3). Hence, I cannot determine the N status of the cells from one measurement. To resolve these issues I would need to perform additional culture experiments to verify very early log phase and steady-state limitation gene expression. Also perhaps I would need to perform some “blind” analyses with field samples, along with nutrient measurements.

30 Table 4: Ratio of gene expression under different growth stages in cells grown on + NH4 .

AMT-2 / AMT-1 UREC / AMT-1 UREC / AMT-2 N condition (Ct/Ct) (Ct/Ct) (Ct/Ct)

+ T1 (NH4 ) 1.07-1.12 1.06-1.09 0.97-1.00

+ T2 ( NH4 ) 1.24-1.32 1.28-1.35 0.98-1.09

+ T3 ( NH4 ) 1.19-1.27 1.20-1.25 0.99-1.01

31 + Table 5: Ratio UREC / AMT-1 in each experimental replicate under different NH4 conditions.

UREC / AMT-1 A1 A2 A3 SD (Ct/Ct)

T1 1.08 1.09 1.06 0.02

T2 1.35 1.30 1.28 0.04

T2 1.22 1.20 1.25 0.02

32

CONCLUSION

Predicting the influences of nutrient availability on phytoplankton communities is fundamental to understanding the present and future structure of marine food webs and how physical forcing and nutrient limitations affect them. Since the gene expression of

UREC provided information on organic N assimilation in Aureoumbra, it is relevant to consider the implications of enriching waters with urea. Urea is a fertilizer and feed additive whose use has increased 100-fold in the last 40 years (Glibert et al., 2008). As Aureoumbra can use urea to support its own N metabolism, discharge of this compound to coastal waters may lead to an increase in algal blooms and could provide an advantage to Aureoumbra in the field. Tracy Villareal (personal information, data not published) monitored the presence of urea in the Laguna Madre from February 1997 to August 1998, near the end of the long Aureoumbra bloom. Urea was consistently present in the Laguna

+ - - Madre together with NH4 and NO3 +NO2 . Urea is an organic compound containing two amine groups. Thus on a per molecule or per mole basis it provides twice as much N as

+ - - + NH4 or NO3 +NO2 . For example, in one sample the NH4 concentration was twice that of urea, such that the two compounds provided equimolar amounts of N. In July 1998, the

+ - - urea concentration was 4 times higher than NH4 and 7 times higher than NO3 +NO2 ; in

+ - - August 1998, urea was 132 times higher than NH4 and 22 times higher than NO3 +NO2 . Urea, therefore, may have been an important N source during the bloom.

Future directions

Similar experiments may be conducted with cultures using nitrite or amino acids as the N source. Muhlstein and Villareal (2007) demonstrated Aureoumbra’s ability to grow using these two different N sources. Fragments of the genes encoding Nitrite

33 Reductase (NiR) and Amino Acid Transporter (AAT) still remain to be cloned from Aureoumbra. While analyses of AMT and UREC provided important information on genes involved in inorganic vs. organic N assimilation, information on the presence and regulation of AAT and NiR would improve our understanding of the physiology of this alga. In particular, it would be very useful to determine if Aureoumbra possesses genes involved in amino acid uptake. It is now recognized that amino acids, and DON in general, can promote harmful algal blooms (Lewitus, 2007), but despite these advances, there is a lack of marine food web models that incorporate DON and phytoplankton. It would also be informative to determine if Aureoumbra has any uptake preference when

+ multiple N forms are present in the medium, e.g. both NH4 and urea. By measuring disappearance rate for each N form, I can understand the nitrogen preference in Aureoumbra. By analyzing gene expression and physiological responses similarly to the experiments above described, I can better understand how Aureoumbra copes in natural environment where different nitrogen sources are present.

34 Appendix

Culture axenization

Since our project was focused on inorganic and organic nitrogen uptake, the bacteria present in xenic cultures could compete with Aureoumbra for the substrate and interfere with the analysis of nitrogen use. Therefore, we tried to axenize our cultures following a variation of the protocol of Berg et al. (2002). Aureoumbra strain CCMP1509 was exposed to the following antibiotics sequentially during exponential growth: Penicillin G (1.0mg ml-1), Neomycin (0.25mg ml-1), Gentamicin (0.25mg ml-1), Kanamycin (0.5mg ml-1), Penicillin G (2.0mg ml-1) (all antibiotics purchased from VWR), and finally Ciprofloxacin (0.1 mg ml-1) (Eaton Veterinary Laboratories). The cultures were kept in antibiotic solution for no more than 15h. The day after exposure, 1 ml of the culture was added to 25ml antibiotic-free medium. The presence of bacteria was determined by conducting PCR using universal bacterial primers SR-FWD 5‟- AGAGTTTGATYMTGGC-3‟ (forward, positions 4–19 of Pasteurella haemolytica) and SR-REV 5‟-GYTACCTTGTTACGACTT-3‟ (reverse, positions 1505–1488 of Pasteurella haemolytica) (Davies et al., 1996). At the end of the multiple antibiotic treatments, bacteria were still present in the cultures.

The antibiotic treatments may not have worked since the EPS layer could act as refuge for heterotrophic bacteria, as shown by the fluorescent stain DAPI. A different approach was tried to eliminate EPS from Aureoumbra. Cells were collected by centrifugation (Eppendorf 5810R) at 800 x g for 4‟ and resuspended in a solution of 100 ml sterile 0.1M EDTA. The cells were then filtered onto a 3.0µm polycarbonate filter (Whatman). Penicillin G (0.5mg ml-1), Neomycin (0.2mg ml-1), Gentamicin (0.2mg ml-1), and Ciprofloxacin (0.1mg ml-1) were added. The day after treatment, 1ml of sample was 35 added to 25ml antibiotic-free medium. However the cells did not grow after EDTA treatment and filtration.

36 Glossary

ACT – Aureoumbra actin gene AMT-1 – Aureoumbra ammonium transporter gene 1 AMT-2 – Aureoumbra ammonium transporter gene 2

C:N – carbon to nitrogen ratio Chl – chlorophyll cDNA – complementary DNA

Ct – cycle threshold DAPI - 4',6-diamidino-2-phenylindole DCMU - 3‟,4‟-dichlorophenyl-1,1-dimethylurea

DIN – dissolved inorganic nitrogen DNA - Deoxyribonucleic acid DOC – dissolved organic carbon DON – dissolved organic nitrogen EDAB – ecosystem disruptive algal blooms EF1α – Aureoumbra elongation factor 1 alpha gene

EPS – extracellular polymeric substance

Fv/Fm – Photosystem II efficiency HAB – harmful algal blooms mRNA – messenger RNA N – nitrogen

+ NH4 - ammonium cation

- NO2 - nitrite ion 37 - NO3 - nitrate ion PCR – polymerase chain reaction PSII - photosystem II qPCR – quantitative real-time PCR RNA – Ribonucleic acid T1 – early log phase

T2 – middle log phase T3 – end log phase UREC – Aureoumbra urease C subunit gene

µmax – maximum growth rate

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44 Vita

Marco Agostoni was born in Milan, in the country of Italy on the 7th of February, 1977 to Claudio Agostoni and Rosanna Ginevoli. He attended the ITIS Feltrinelli for his high school education and. He started his undergraduate education at the University of Ancona, Italy in 2003 from where he graduated in November 2006 with a B.S. in Marine Biology. During his senior year at the University of Ancona, Marco worked on the development of FTIR method for the taxonomic characterization of algal samples and for biodiversity studies. In September 2007, Marco started a masters program in the University of Texas at Austin Marine Science Institute.

Permanent address: Via Ramponi 20, Canonica D‟adda (BG), 24040, Italy This thesis was typed by the author.

45