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2015 Acclimation of Red Tide Dinoflagellate Karenia Brevis to Higher Temperatures Results in Abnormal Morphology and Changes in Growth Rates Daniel P. Owen

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ACCLIMATION OF RED TIDE DINOFLAGELLATE KARENIA BREVIS TO

HIGHER TEMPERATURES RESULTS IN ABNORMAL MORPHOLOGY

AND CHANGES IN GROWTH RATES

By

DANIEL PATRICK OWEN

A thesis submitted to the Department of Biological Sciences In partial fulfillment of the requirements for graduation with Honors in the Major for Marine Biology

Degree Awarded: Spring, 2015 Owen 2

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Introduction- Karenia brevis (formerly known as Gymnodinium breve and Ptychodiscus brevis) is a toxic, biflagellate marine dinoflagellate found in red tides and Harmful Algal Blooms (HABs) within the Gulf of Mexico, off the mid-East Florida coast, and up to the North Carolina coast. Toxic blooms occur almost annually in the Eastern Gulf of Mexico around the beginning of fall. In their vegetative cell phase, Karenia brevis has been reported to produce a natural neurotoxin known as brevetoxin. When large amounts of Karenia brevis cells aggregate together, as seen in full-blown red tide blooms, the brevetoxin content in the water column becomes high enough to affect other marine organisms within the bloom range. This results in the characteristic mass fish kills seen in red tides. When a Karenia brevis bloom reaches a coastal area, near-shore wave action disperses brevetoxins as an aerosol. Inhalation of aerosolized brevetoxins results in respiratory irritation in humans. Ingestion of brevetoxin-contaminated shellfish results in gastroenteritis and neurological symptoms characteristic of neurotoxic shellfish poisoning (Kirkpatrick et al. 2004). The annual economic impact of Florida Karenia brevis blooms results in an estimated $21-36 million (adjusted from rates reported in Kusek et. Al., 1999) of damages in years with heavy HAB activity. Vegetative Karenia brevis cells reproduce asexually through binary fission. Under certain conditions, hypothesized by Walker to be nitrogen limitation, variation in temperature, etc., heterothallic strains of Karenia brevis cells have been shown to transform into haploid gametes which then fuse to form a planozygote (Walker, 1982). Although there are contrasting reports, the identification of the next phase common in dinoflagellate lifecycles, the hypnozygote cyst (Figure 1), has not been confirmed in Karenia brevis, nor have possible Karenia brevis hypnocysts been found in natural sediment samples (Steidinger 2009, Persson et. Al., 2013). Owen 4

Figure 1. Diagram of the typical dinoflagellate life cycle. The formation of a pellicle cyst, a hypnocyst, has not yet been identified in the K. brevis life cycle. Image taken from tolweb.org.

Further, Karenia brevis has been observed to undergo drastic morphological changes induced through rapid changes in water temperature (Persson et. Al., 2013). Unfortunately, the methods of the most widely cited papers on the viable temperature range of Karenia brevis do not allow for proper cell acclimation time based on K. brevis average division rates (for example Magana and Villareal, 2006). Therefore, the work done on the viable temporal ranges of Karenia brevis can be interpreted as Karenia brevis reactions’ to temperature stress, instead of true acclimation to varying temperature. This may result in the typical high viable temperature limit of 30° C for K. brevis cited in more recent papers (Kusek et. Al., 1999; Magana and Villareal, 2006). This leaves many unanswered questions with respect to the effects of temperature change in the Gulf of Mexico, as a result of seasonal changes as well as climate change, on Florida Karenia brevis. Reported Karenia brevis blooms in the East Gulf of Mexico form annually during the months of August, September, and October. These three months show a change in average water temperature from 30ºC in August to 25ºC in October, with an average water temperature of 28ºC for the time period (NOAA 2014). The latest work from NOAA estimates an average increase in Gulf temperatures of ~2.5ºC by the year 2099 (Biasutti et Al., 2012). For Owen 5

areas in which Karenia brevis blooms occur annually, this would mean an increase in average temperature from ~28ºC to ~30.5ºC during the months with heaviest bloom activity and possibly higher temperatures in the shallow waters along the coast with highest Karenia brevis accumulations (Brand et. Al., 2012). This paper addresses the effects of temperature on Karenia brevis. A clonal strain of Karenia brevis was acclimated to the currently estimated increase in Gulf temperatures over a period of time long enough to ensure proper acclimation of the experimental cultures. A long acclimation time was used to avoid temperature shock conditions for the culture and to more closely mimic natural temperature increases, such as those seen during seasonal transitions. Mimicking an actual climate change time-scale scenario would require longer times than are possible for the scope of this project, but the acclimation process allowed for at least one generation of cells to be generated under the increased temperature. Throughout the acclimation process, the morphology and growth rate of K. brevis was monitored. Results were compared to similarly treated, non-acclimated, control cells. Through these methods, this paper proposes that the longer acclimation time for K. brevis cultures to higher temperatures will allow for the cells to survive passed the reported 30° C temperature limit (Magana and Villareal, 2006). Further, since temperature shock treatments have been shown to produce abnormal cell morphologies (Persson et. Al. 2013), it is believed that the K. brevis cultures in this experiment acclimated to higher temperatures over relatively longer time intervals may produce abnormal cell morphologies due to temperature stress. Finally, based on earlier reports of the 30° C maximum viable temperature (Magana and Villareal, 2006), K. brevis cultures acclimated to 30 ° C will have significantly lower growth rates than cultures growing at 25° C. Together these analyses hope to provide a foundation for how long term increase in ocean temperature may affect the growth rates, physiology, and morphology in Karenia brevis.

Materials and Methods – Karenia brevis A clonal strain of Karenia brevis CCFWC-126 provided by the Florida Fish and Wildlife Conservation Commission based in St. Petersburg, Florida, was used in these studies. Karenia brevis CCFWC-126 was collected and isolated from a red tide bloom five nautical miles west of Owen 6

Stumps Pass, Florida (26.8871 N, 82.4406 W) in July 2006. Semi-continuous batches of CCFWC-126 were maintained for several months prior to the initiation of the acclimation phase of the experiment in sterile pyrex flasks in an open air light bank with light intensity ~80 µmol photons m-2 s-1, at ~25º C.

Culture Conditions For the acclimation phase of the experiment, all cultures were maintained in a 12:12 light/dark cycle, with experimental and control cultures kept in separate growth chambers (Thermo Precision Incubator 818) of 40Watt daylight fluorescent light bulbs, with a light intensity of 120 µmol photons m-2 s-1 (measured in water). All K. brevis cultures were initially grown in GP/2 media (Loeblich and Smith, 1968), with added Selenium in the form of H2SeO3, until all cultures experienced a culture crash (when the experimental cultures were being acclimated to 29° C) due to a media shortage. All cultures were then diluted with L1-S1 media (Guillard 1983) and allowed to recover for two weeks before the experiment continued. Control and experimental cultures were grown in autoclaved 500mL glass flasks in a semi-continuous batch approach and diluted with fresh temperature acclimated media. Control cultures were maintained at 25ºC throughout the acclimation process in duplicates, while experimental cultures were acclimated in triplicates with identical light settings. The experimental cultures underwent an increase of 1ºC every 5-7 days, from the initial 25ºC to the final temperature of 31ºC. There was an observed ~1° C temperature fluctuation in conjunction with the light cycle within the culturing chambers throughout the experiment (illustrated in Figure 2). While acclimating the experimental cultures to 30ºC, control and experimental cultures were diluted by 1:20 dilution (10mL of culture into 190mL of fresh L1-Si media) on the same dates throughout the acclimation phase. Dilutions during the acclimation phase were not performed at uniform intervals, or in a manner that would have ensured equal cell density in each culture with each dilution. All handling of cultures during dilution was performed in a sterile hood using sterile technique. L1-Si media for the culture dilutions was prepared as needed by adding the L1-Si nutrient recipe to distilled water, mixed with Instant Oceantm sea salt to maintain media at a salinity of 35 ppm, and filtered into sterile 1 L glass bottles. The salinity of fresh media was measured with a JBJ C-Scope Refractometer. Owen 7

Figure 2. Light and temperaturere chromatography. Blue line indicates the light intensity (µmmol photons m-2 s-1) and yellow line temperature (Centigrade)e) over a 6 hour period measured in the incubator. The arrowow indicates when the beginning of the daily dark cycle (6pm).. TThe data acquisition stopped due to a temperature sensor failurefai .

Morphological Observation Cell samples were observrved for changes in morphology one day before andan one day after temperature increases. A ~1mL aaliquot of cells from each experimental and contntrol culture was observed under an Omax light mmicroscope under 10x and 25x magnifications. Notes were taken during each observation and phototomicrographs and videos were taken with the ScopeImage9.0S program when cells with unusualal morphology or characteristics were observed. Photomicrographs of cells with nnormal cell morphology were also taken for commparative reference. These observations wewere made during exponential growth or lag phaseases depending on when the cultures were diluted in relation to the dates the cultures were observeed.d

Biomass Measurements duringng the Acclimation Phase During the acclimation phphase, the relative fluorescence of all current experimentalpe and control cultures was measured to estimate the cell density of the cultures over time.tim Relative Owen 8

Fluorescence Units (RFU) was measured by a Trilogy Fluorometer (model 7200-000, Turner Designs). Before measuring the RFU, the culture flasks where gently hand-swirled for 20 seconds within a sterile hood and two samples of 2mL were measured twice.

Biomass and Growth Measurements at 30ºC Once the experimental cultures were acclimated to 30ºC, the experimental and control cultures were diluted to under the cell density for exponential growth to occur (<1000cells/mL) and the RFU and cell number per mL were measured in each experimental and control culture every day over the course of 1 week (8 days for relative fluorescence and 7 days for cell density). Cell density counts and RFU’s were measured on the same date and at the same time in order to produce a growth curve for Karenia brevis in control and 30ºC acclimated cultures. Cell density was taken using a Beckman Coulter Z2 Coulter Particle Count and Size Analyzer. The number of particles counted in the size range between 17-35uM were all considered to Karenia brevis cells. Before taking cell counts, each culture was gently hand-shaken for 20 seconds and 14mL of the culture was poured into a single cuvette used for measuring the cell count. 4mL from this cuvette were then split between the two cuvettes used for the fluorescence reader. RFU was measured while the remaining 10mL sample was measured for cell density by the Coulter counter. A blank was measured between readings for each sample in order to ensure there were no remaining particles from the earlier sample and the culture counter was flushed with a chloride solution before and after the measurements took place.

Results Morphological Observations An abnormal, “rounded,” cell morphology (Figure 3) was first observed in cultures acclimated to 28º C. This rounded cell morphology became the dominant cell shape (>90% of all cells observed in each sample) in all of the experimental cultures through the duration of the rest of the acclimation phase of the experiment (from 28º C to 31º C). The rounded cell size still fell within the 15-35µm cell size range of normal K. brevis, however, the exact cell sizes of the abnormal cells were not measured. In both the cultures at 25° C and the experimental cultures acclimated to 30° C, the most common cell size was between 24µm-26µm. Cells with the rounded morphology demonstrated interesting characteristics compared to the cells with the Owen 9

normal K. brevis cell morphology (figure 4). Severely rounded cells had lost the cell wall furrows found on normal K. brevis cells. Both rounded cells with flagella and without flagella were observed. The rounded cells with flagella did not swim in in the typical K. brevis fashion. Instead of swimming in generally straight lines while the cell itself rotates, the rounded cells were observed to swim only in small circles without the characteristic cell rotations. Video examples of normal cell and rounded cell swimming patterns can be found in the video links in the appendix. This swimming pattern may be due to the more spherical shape of the rounded cell, and the possible loss of functionality of the rounded cell transverse flagella. Figure 5 illustrates that the shapes of the rounded cells were not uniform, with the symmetry of the normal morphology being lost in the more rounded abnormal cells. The rounded cell morphology was consistently observed in all cultures acclimated to 28° C and above. The rounded cell morphology dominant in the experimental cultures was never observed in the cultures kept at 25ºC.

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Figure 3. Example of novel “rounded” cell morphology first observed during acclimation phase at 28ºC. Cell is no longer in the typical disk shape. Swimming patterns of this cell type were irregular. They were only observed to swim in small circles without the typical spinning associated with the normal cell type. Picture taken at 25x on 1-20-2015 from experimental culture C at 30ºC.

Figure 4. Example of normal Karenia brevis cell morphology. Cell exhibits typical swim patterns with spiral rotation during swimming. Picture taken at 25x on 1-20- 2015 from control culture A at 25ºC.

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Figure 5. The novel “rounded” cell morphphology exhibited individual cell variations from the normall cellc morphology. All pictures were screen captures from videoeo taken at 10x magnification. A). Two examples of rounded cellc morphology from experimental culture A. The red arrow popoints to a flagella attached to the cell. B). Example of roundeded cell from experimental culture B. Notice the presenence of a flagella coming from the left side of the cell. C). Examample of a rounded cell more similar to the normal K. brevis ccell morphology. No obvious flagella was observed on this cell.ce

Growth Curves and Specific GGrowth based on Cell Density and RFU Figure 6 shows the averarage increase in cell density and Figure 7 showss thet average increase in RFU in each culturee oover an eight day growth period for cultures growrowing at 25° C and 30° C. All cultures of K. brevevis began exponential growth at ~1000cells/mL.L. After six days of exponential growth, all of thee ccultures growing at 30° C showed a decrease in measured cell Owen 12

density and RFU. This may indicicate the time period limit in which K. brevis acclclimated to 30° C can sustain exponential growth.. TThere was no relevance to the decrease in cell densityde and RFU to total culture density, as all off tthe cultures experiences the possible stationaryy phasep at the same time at different cell densities. No possible stationary growth phase was observeded after exponential growth in the controlrol cultures growing at 25°C during the 8 day growowth period.

Figure 6. The average increasee oof cell density for K. brevis cultures growing at 25°C and 30°C. Averages and standard deviation were calculated fromm two K. brevis cultures at 25° C, and three cultures at 30° C.. ExperimentalE cultures were acclimated to 30° C by incrcreasing the ambient temperature of the culturing chamber by 1° C every 5-7+ days- allowing for the cultures to undergogo several divisions at each new temperature.

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Figure 7. The average increase of cecell density for K. brevis cultures growing at 25°C and 30°C. Averageges and standard deviation were calculated from two K. breviss ccultures at 25° C, and three cultures at 30° C.

Several steps were taken to determermine if there was a difference between the speciecific growth rates of K. brevis cultures growing att 225° C and cultures acclimated to 30° C. For eachch experimental and control culture, the growth rarate was determined for each day during the eightht day growth period. These growth rates were calculated using the equation:

B1 = celll ddensity or RFU on day 1

B2 = celll ddensity or RFU on day 2 t = time ininterval (1 day) The specific growth rates for eachach day were then averaged together for the controtrol cultures at 25° C and the experimental culturess aat 30° C. A standard deviation for all growth rateates was taken the same way. Figure 8 shows the avaverage specific growth rates of cultures growingng at 25° C and 30° C based on the cell density anand RFU measurements. Based on cell density, K.K brevis cultures at 25° C had a specific growth ratrate of 0.269 ± 0.161 [d-1], while cultures at 30° C had a specific growth rate of 0.322 ± 0.252 [d-1]. Based on the averages, cultures at 30° C appeaeared to have a Owen 14

slightly higher growth rate than cultures at 25° C. However when factoring in the standard deviation, this difference was not significant. Further, based on RFU/L, K. brevis cultures at 25° C had a specific growth rate of 0.345 ± 0.128 [d-1], while cultures at 30° C had a specific growth rate of 0.252 ± 0.308 [d-1]. In contrast to specific growth based on cell density, the specific growth based on RFU shows a decrease in average specific growth. With standard deviation taken into account, this difference is also not significant. In order to more closely examine the effect of temperature increase on the growth rates of K. brevis cultures in exponential growth phase, the specific growth of each culture per day over the eight day growth period was re-analyzed. The growth rate for days in which the growth rate became negative over the course of the eight days was replaced with an aggregate of the growth rates for the day before and the day after the outlier growth rate by the equation:

ln ln/

B1 = cell density or RFU on day before outlier

B2 = cell density or RFU on day after outlier t = time interval (2 days) The average specific growth and standard deviation for the cultures grown at 25° C and 30° C were then calculated again with the aggregated growth rates replacing the negative growth rates (see Figure 9). Many factors, such as unequal mixing of culture samples and the sensitivity of the instruments, could have resulted in growth measurements that did not fit the trend of exponential growth. The point of fitting growth rates with aggregate growth rates was to get a better picture of the differences of specific growth while minimizing the effect of errors in measurement. The new average specific growth based on cell density for cultures grown at 25° C was 0.294 ± 0.011 [d-1]. Cultures growing at 30° C had an average specific growth of 0.374 ± 0.027 [d-1]. These average growth rates were higher than the average growth rates shown in Figure 8a. As well, a significant increase in specific growth was shown in cultures growing at 30° C. The new average specific growth based on RFU for cultures growing at 25° C was 0.362 ± 0.037 [d-1]. Cultures growing at 30° C had a growth rate of 0.293 ± 0.032 [d-1]. Similar to the original calculated average specific growth based on RFU, these growth rates showed a decrease in average specific growth for cultures at 30° C. The effect of acclimating K. brevis to 30° C produces different trends in growth rate depending on how the measures of growth were taken. Owen 15

Based on cell density, K. brevis has an increased growth rate at 30° C. In contrast, based RFU measurements, K. brevis has a decreased growth rate at 30° C. As the experimental cultures were being acclimated to 29° C, a crash occurred in all cultures due to a shortage in GP/2 media. All of the cultures were then diluted into L1-Si media and allowed to recover for 2 weeks before the acclimation phase of the experiment began again. New dilutions were made from the current cultures using L1-Si media for the remainder of the experiment. Three days of RFU measurements were taken before the crash from the experimental and control cultures in exponential growth phase while the experimental cultures were being acclimated to 27° C (before the switch to L1-Si media). This allowed for a calculation of average specific growth for K. brevis growing in GP/2 media. Figure 10 shows the average specific growth rate for K. brevis cultures growing in GP/2 media compared to the exponential-fitted, average specific growth based on RFU for the eight day growth period of K. brevis grown in L1-Si media. At 25º C, cultures growing in GP/2 media had a specific growth of 0.498 ± 0.011 [d-1]. This growth rate is significantly higher than the specific growth of 25º C cultures growing in L1-Si media. The K. brevis cultures in GP-media had an average specific growth 0.136 [d-1] higher than the cells in L1-Si media. The cultures growing in GP-media, acclimated to 27º C, were found to have an average specific growth of 0.570 ± 0.109 [d-1]. This was the highest average specific growth calculated over the course of the experiment.

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Fig 8. The average specific growowth of K. brevis cultures grown at 25° C and 30° C. Averageses and standard deviations were determined from the growrowth rate calculated for each day. (A) the average specific grorowth based on cell density measurements. The increase in ggrowth rates of cultures growing at 30° C, although this differference is not significant based on standard deviations.s. (B) The average specific growth based on RFU measuremenents. In contrast to graph (A), graph (B) shows an apparent ddecrease in average growth rates of cultures growing at 30° C,C although this decrease is also not significant based on sstandard deviation. Owen 17

Figure 9. Graphs of the averagege specific growth fitted for exponential growth of K. brevis culturescu grown at 25° C and 30° C. Averages and standard deviviations were calculated from the growth rate of each day. AnyAn day that showed a specific growth that did not fit an exponenential trend was replaced with an aggregate specific growth fromf the day before and the day after the outlier growth rate.te. This was done in order to reduce errors in calculating the effecte of temperature on K. brevis growth rates during exponenential growth. (A) The average specific growth based on cell densityde measurements. A statistically significant increase in growowth rates was observed in cultures growing at 30° C. (B) The average specific growth based on RFU measurements. Inn contrast to (A), (B) shows a significant decrease in averagee growthg rates of cultures growing at 30° C. Owen 18

Fig 10. The differences in averagrage specific growth rates in K. brevis cultures grown in GP/22 mediam and L1-Si media. Cultures grown at 25° C in GP/22 mmedia had a significantly higher average growth rate than culturescu grown in L1- Si media at 25 ° C. Cultures growing in GGP/2 media at 27° C had the highest average specific growthh measured during the experiment, although it is not significantltly different from the cultures growing in GP/2 media at 25° C.

Calculated Chlorophyll a per ccell The amount of Chlorophyhyll a (Chl a) per cell was calculated from the RFFU measurements taken over 7 days in conjunctionn with the cell counts in both the two control cultltures at 25ºC and the three experimental cultures at 30ºC. RFU measurements for each day were divideddi by the calibration factor of 283363 RFUU per 1 mg Chl a/L in order to obtain mg Chl a/L for each day. The measurement mg Chl a/L for each day was divided by the corresponding celell density (converted to cells/L) in order to obtain the daily mg Chl a/cell. The daily measusurements were then multiplied by 1x109 in orderer to obtain the daily pg Chl a/cell. All daily pg ChlC a/cell for cultures at 25ºC were averaged totogether, and standard deviation taken, resultingg ini an average of 18.7 ± 3.13 pg Chl a/cell. As shohown in Figure 11, the cultures at 30ºC were founund to have an average of 17.7 ±3.50 pg Chl a/c/cell. The chlorophyll content of the K. brevis cellells at 25ºC and 30ºC were not found to be statistiistically significantly different from each other. BasedBa on this data, acclimation of K. brevis to 30ºCC ddoes not affect the amount of chlorophyll α presresent in each cell. Owen 19

Figure 11. The calculated chlororophyll a per cell in cultures growing at 25ºC and acclimatedd to 30ºC. The RFU measurements of 7 days during growth by the calibration factor of the fluorescence machine resultinging in mgChl a/L were divided by the corresponding measured ccell count/L with mg converted to pg in order to get pgChl a/cell/ for each day. Measurements for each day were averageged together for the cultures growing at 25ºC and 30ºC and theth standard deviation was taken for these averages.

Results Summary An abnormal, rounded, cecell morphology was produced in K. brevis culturtures acclimated to 28° C and persisted in cultures acacclimated through 28° C to 31° C. As well, specicific growth rates of cultures growing at 25° C andd acclimated to 30° C differed depending on whetether the average growth rates were derived from cculture cell density or RFU measurements. K. brevisbr cultures grown in GP/2 media had significificantly higher average growth rates based on RFUFU measurements than cultures growing in L1-Si mmedia. Cultures growing at 25° C and 30° C did notn have significantly different chlorophylyll a content per cell. Figure 12 provides a summmary of the growth rates and chlorophyll a cocontent of Karenia brevis presented in this paper. Owen 20

Acclimated Specific growth Specific growth Specific growth pg Chl a /cell temperature based on cell based on RFU based on RFU (in L1-Si of K. brevis density [d^-1] measurements measurements media) cultures (in L1-Si Media) [d^-1] (in L1-Si [d^-1] (in GP/2 Media) media) 25°C 0.294 ± 0.011 0.362 ± 0.037 0.498 ± 0.011 18.7 ± 3.13

27°C N/A N/A 0.570 ± 0.109 N/A

30°C 0.374 ± 0.027 0.293 ± 0.032 N/A 17.7 ±3.50

Figure 12. Summary of average specific growth rates and chlorophyll a per cell content of K. brevis cultures acclimated to different temperatures maintained in either GP/2 or L1-Si media.

Discussion Abnormal cell morphology overview The rounded cell morphology was consistently observed in all cultures acclimated to 28° C and above. This may mean that the abnormal cells were still undergoing normal asexual division throughout the acclimation process and produced subsequent generations of abnormal cells. The abnormal rounded cells observed in cultures acclimated to 28º C and above did not resemble the reported possible temporary cyst form of K. brevis (Bouchard and Purdie 2011) or the possible sexual morphological cells (Persson, et. Al., 2013) occasionally observed in cases of positive and negative temperature shocks. The rounded cells observed in this experiment were unusual in several respects to the abnormal temperature shock cell shapes. First, no structure resembling a pellicle cell wall was observed on the outside of the rounded cells. Also, a majority of the rounded cells maintained their flagella and mobility. Finally, the rounded cells remained viable and present in every generation of cells after the initial observation of the novel cell morphology. Further exploration into whether the formation of the rounded cell morphology consistently occurs in asexually reproducing K. brevis cultures acclimated to 28º C and above should be done. If the rounded cell morphology is consistently produced in higher temperature acclimated cultures, the observed morphological variations of K. brevis may account for some of the possible unidentified dinoflagellates species reported to occur within natural K. brevis red- tide blooms (B. Richardson, personal communication, May 7, 2014).

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Discrepancies in average specific growth It is interesting that the average specific growth based on RFU for cultures growing at 30ºC showed a decrease in growth rates compared to cultures grown at 25º C, while the average specific growth based on cell density showed a significant increase in growth rates for the same cultures. As well, specific growth in general was higher based on RFU than cell density. It is extremely difficult to determine what caused the discrepancy between the final average growth rates for the K. brevis cultures. There are several possible sources for the observed discrepancies observed in the growth rate data. Although, the calculated pg Chl α/cell was about twice the amount reported for similar strains of K. brevis (Corcoran, et. Al., 2014), there was no significant difference found between the RFU/cell for each culture (as well as pg Chl α/cell measurements) growing at 25º C and 30º C in L1-Si media. That implies that there was not a lower amount of activated pigments per cell measured by the Fluorometer in cultures predominantly composed of the abnormal rounded cells versus the normal morphology cells. On average, cells growing at 25º C and 30º C did not have significantly different amounts of pigments accounting for the measured RFU per cell, and cannot account for difference in significant growth based on RFU and cell density. Further, cultures with a measured sample density of ~≥2000cells/mL would commonly result in a high masking error in the Coulter counter readings. The high masking error may have been avoided by diluting samples from each culture before being measured, but the high masking error was also occasionally produced by samples well under the 2000cells/mL density. In cases of high masking, it can be inferred that the measured cell count is an underestimate of the actual cell density of the sample. This error would result in an increase in specific growth based on cell density, but would not account for the differences in the trend growth rate based on cell density and RFU between the experimental and control cultures. It is probable that there were simply not enough replicates of measurements of RFU and cell density for 25º C and 30º C cultures. Only duplicates of cultures growing at 25° C and triplicates of cultures eventually acclimated to 30° C were maintained. There was only a single, eight day trial performed in order to measure growth data. In order to determine if there is a consistent, significant difference between specific growth based on RFU and specific growth based on cell density for the same cultures, it is recommended that many more replicates of the measurements be taken if this experiment is repeated. With additional trials, it may be possible to Owen 22

apply other statistical analyses to determine if the observed discrepancies between growth rate trends for K. brevis at 25° C and 30° C were a result of errors during cell growth measurements or because of differences in cell physiology.

Differences in average growth rates due to media type Cultures grown in GP/2 media had significantly higher specific growth based on RFU than the cultures grown in L1-Si media. This may be a result of how long the Karenia brevis cultures were grown in GP/2 media compared to L1-Si media. The Karenia brevis parent cultures used to generate the experimental and control cultures for this experiment were maintained in GP/2 media for several months prior to the initiation of this project. It wasn’t until the experimental cultures were acclimated to 29° C, that all of the cultures were switched to and began growing in the L1-Si media. The quick switch to L1-Si may have given the cultures a media shock treatment. All of the cultures may not have been given a long enough time to acclimate to the difference in media composition, and this may have resulted in sub-optimal growth rates for the control and experimental cultures when the experimental cultures were finally acclimated to 30° C. As well, a relatively small data set was used to find average specific growth of cells grown in GP/2 media compared to cells grown in L1-Si media. A majority of papers report using L1-S1, or slightly altered L1-Si media, for growth of K. brevis cultures. If a consistent, significant change in growth rates for cultures growing in GP/2 media and L1-Si media were found, further studies should examine the component that causes the difference in growth rates. This would tell us under what nutrient condition K. brevis grows best in, and may help us understand what environmental conditions may result in the production and sustainability of natural Red Tide blooms.

The upper temperature range of Karenia brevis The 30ºC upper acclimated temperature cap on K. brevis cell viability, reported in recent papers previous to this work (Kusek et. Al., 1999), was not observed over the course of this experiment. K. brevis cultures acclimated to 30º C grew and were maintained for several weeks. Cultures acclimated to temperatures >30º C did not result in previously reported rapid cell death (Magana and Villareal, 2006). However, cultures growing at 25° C, appear to have a higher carrying capacity and can undergo exponential growth for a longer amount of time than cultures Owen 23

acclimated to 30° C. At the end of this experiment cultures acclimated to 31º C, and ~32º C from temperature fluctuations in the culture chamber, were maintained for 15 days without any culture mass cell mortality events. This experiment result conflicts with the conclusion of a K. brevis high temperature viability limit of 30º C (Magana and Villareal, 2006), and instead support earlier reports of the temperature limit being closer to 34º C (Hitchcock 1976). The observation of a higher temperature limit for K. brevis viability may be due to the longer acclimation time cultures underwent over the course of this experiment. Whereas most papers reporting the temperature range of K. brevis rely on what may be considered a temperature shock method of acclimation, experimental cultures in this project were allowed to undergo several rounds of division at 1º C increments. It would be extremely interesting to see the maximum high temperature limit of K. brevis based on the longer term acclimation method used in this experiment. The resultant limit may allow researchers to understand the viability and morphology of K. brevis over the course of seasonal changes in surface ocean temperature.

A learning experience Several errors, highlighted throughout the paper, in the methods and techniques used to conduct this experiment may have resulted in and/or confounded the results of this work. The techniques needed to perform this experiment properly were being developed as the project progressed. Several oversights were made in the initial phases of the experiment that could have been avoided, and were actively compensated for towards the end of the experiment. The number of days the experimental cultures were acclimated to a new temperature should have been consistent throughout the experiment. The switch from GP/2 media to L1-Si media over the course of the experiment was not intentional and should have been avoided. Cultures should have grown in a consistent type of media throughout the experiment to possibly maintain optimal growth rates for all of the cultures. Culture crashes, in which K. brevis cultures experienced mass cell mortality events, occurred occasionally throughout the experiment due to insufficient media preparation or improper culture maintenance. Only 1 trial of the 8 day growth measurements for cultures acclimated to 25° C and 30° C was able to be taken, because the cultures growing at 30° C for replicates crashed due to improper culture chamber conditions. Further, all of the cultures that were measured for the 8 day growth trial should have been diluted to equal cell densities at Owen 24

the beginning of the measurement period. This would have greatly reduced the variation in measurements of cell density and RFU for each day between each culture. Finally, because the control cultures at 25° C were run in duplicate, the standard deviation for all of the control measurements averages is only based on 2 samples. The resulting standard deviations for the control culture readings are not statistically valid. All of these errors, can be and should be accounted for in future work with Karenia brevis that may be similar to this overall project.

Conclusion Despite the problems evident in this project, several new insights into Karenia brevis can be taken from this work. An abnormal, but functional, rounded cell morphology was produced by acclimating a clonal strain of K. brevis to water temperatures observed during peak red tide bloom activity. If this rounded morphology can be produced consistently, it may offer new perspectives into the typical forms of K. brevis during the dinoflagellate life cycle. Further, the longer-term acclimation strategy used in this project may have shown that K. brevis has a much larger viable temperature range than previously considered. To expand on this work, the physiology, such as oxygen consumption, and growth rates of K. brevis acclimated through the techniques used in this experimental should be more closely examined. In conjunction with the rounded cell morphology, the higher temperature range, and the future physiological observations, researchers will be able to understand what may be happening to populations of K. brevis throughout seasonal temperature variations. Finally, all of these techniques can be used to more thoroughly characterize other kinds of Harmful Algal Bloom forming organisms, and may help with prevention and remediation of HABs in the future.

Acknowledgements The author of this paper would like to thank Dr. Bill Richardson, Dr. Karen Steidinger, and Dr. Alina Corcoran of the Florida Fish and Wildlife Conservation Commission, as well as Dr. Laura Keller, Dr. Sven Kranz, and Dr. Trisha Terebelski of Florida State University for their support on this project.

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Works Cited - Biasutti, M., A.H. Sobel, S.J. Camargo, T.T. Creyts. 2012. Projected Changes in the Physical Climate of the Gulf Coast and Caribbean. Climatic Change 112 (3-4): 819-845.

Bouchard, J.N., D.A. Purdie. 2011. Temporal variation of caspase 3-like protein activity in cultures of the harmful dinoflagellates Karenia brevis and Karenia mikimotoi. Journ. Plankton Research 33 (6): 961-972.

Brand, L.E., L. Campbell, E. Bresnan. 2012. Karenia: The biology and ecology of a toxic genus. Harmful Algae 14: 156-178.

Corcoran, A.A., B. Richardson, L.J. Flewelling. 2014. Effects of nutrient-limiting supply ratios on toxin content of Karenia brevis grown in continuous batch. Harmful Algae 39: 334-341.

Figueroa, R.I., E. Garces. Dinoflagellate Life Cycles (picture). Tree of Life Project. Web. 24 April 2015. < http://tolweb.org/notes/?note_id=5512>

Guillard, R.R.L. 1983. Culture of phytoplankton for feeding marine invertebrates. In: Culture of marine invertebrates. Selected readings. (Ed. by C.J. Berg Jr). pp. 108-132. Hutchinson Ross Publ. C.; Stroudsberg, Pensylvania.

Hitchcock, W.S., 1976. Effects of temperature and surfactants on the proliferation of the Florida red tide organism Gymnodinium breve, MS Thesis, University of South Florida, Tampa, FL, pp 1-84.

Loeblich, A. R. and Smith, V. E. 1968 Lipids, 3: 5-13.

Kirkpatrick, B., L.E. Fleming, D. Squicciarini, L. Backer, R. Clark, W. Abraham, J. Benson, Y.S. Cheng, D. Johnson, R. Pierce, J. Zaias, G.D. Bossart, D.G. Baden. 2004. Literature Review of Florida Red Tide: Implications for Human Health Effects. Harmful Algae 3(2): 99-115.

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Kusek, K.M., Vargo, G., Steidinger, K., 1999. Gymnodinium breve in the field, in the lab, and in the newspaper – a scientific and journalistic analysis of Florida red tides. Contr. Mar. Sci. 34, 1- 229.

LaRoche, J., B. Rost, A. Engel. 2010. Bioassays, batch culture and chemostat experimentation, Approaches and tools to manipulate the carbonate chemistry., Guide for Best Practices in Ocean Acidification Research and Data Reporting. In: Riebesell U., Fabry V. J., Hansson L., Gattuso J.- P. (Eds.), 81-94.

Magana, H.A., T.A. Villareal. 2006. The effect of environmental factors on the growth rate of Karenia brevis (Davis) G. Hansen and Moestrup. Harmful Algae 5: 192-198.

NOAA-ESRL Physical Sciences Division. Water Temperature Table of the Eastern Gulf of Mexico. National Oceanographic Data Center. [updated 2014 Oct 8; accessed 2014 Oct 8]. https://www.nodc.noaa.gov/dsdt/cwtg/egof.html.

Persson, A., B.C. Smith, S. Morton, A. Shuler, G.H. Wikfors. 2013. Sexual life stages and temperature dependent morphological changes allow cryptic occurrence of the Florida red tide dinoflagellate Karenia brevis. Harmful Algae 30: 1-9.

Steidinger, K.A. 1983. A re-evaluation of toxic dinoflagellate biology and ecology. Progress in Phycological Research 2: 147-188.

Steidinger, K. A. 2009. Historical perspective on Karenia brevis red tide research in the Gulf of Mexico. Harmful Algae 8: 549-561.

Walker, L.M. 1982. Evidence for a Sexual Cycle in the Florida Red Tide Dinoflagellate, Ptychodiscus brevis (=Gymnodinium breve). Trans. Am. Microsc. Soc., 101(3): 287-293.