The Role of Temperature in the Distribution Of

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THE ROLE OF TEMPERATURE IN THE DISTRIBUTION OF

MIXOTROPHIC PROTISTS OF THE GENUS DINOBRYON

A Dissertation Submitted to the Temple University Graduate Board

In Partial Fulfillment Of the Requirements for the Degree of Doctor of Philosophy

By Adam Wesley Heinze May, 2009

Table of Contents

Page

ABSTRACT ...... IV LIST OF FIGURES ...... VI LIST OF TABLES ...... VII ACKNOWLEGMENTS ...... IX

Chapter

1. INTRODUCTION ...... 1

Basic morphology of the genus Dinobryon ...... 1

Mixotrophy the historical perspective ...... 3

Light requirements of mixotrophs ...... 4

Mixotrophy in food webs ...... 7

Relevance ...... 9

Temperature effects ...... 11

2. MATERERIALS AND METHODS ...... 14

Field collection and sample analysis ...... 14

Microscopic enumeration ...... 15

Chlorophyll protocol ...... 15

Nutrient concentrations ...... 16

Field experiments: Temperature and UV-B affects on phytoplankton...... 17

Laboratory Experiments ...... 18

General set-up temperature gradient incubator construction ...... 18

Culturing...... 22 II

Data analysis ...... 22

3. RESULTS ...... 28

Lake Lacawac field study ...... 28

Temperature increase experiment...... 39

Temperature gradient incubator ...... 47

4. DISSCUSSION ...... 56

5. CONCLUSION ...... 68

REFERENCES ...... 72

APPENDIX ...... 81

III

ABSTRACT

Mixotrophic organisms utilize both photosynthetic and phagotrophic nutritional modes and are increasingly recognized as important contributors to aquatic food webs. Dinobryon, a widespread freshwater genus of mixotrophs had dramatic seasonal and annual variation in Lake Lacawac, a mesotrophic kettle lake in northeastern Pennsylvania. Although light is required for population growth in Dinobryon, it does not appear to control vertical distribution patterns observed during a 3-year survey. One year, the maximum annual abundance was observed close to the surface, while the following year the maximum annual abundance occurred at 4 m depth where light levels were <2% of surface irradiance. The depth of maximum Dinobryon abundance during the second year overlapped with an oxygen maximum at 4 m, which strongly suggests effective photosynthesis at a relatively low light level. Analysis of the distribution data suggests that temperature may be a primary driver of

Dinobryon spp. distribution in the water column of this lake. Although annual variability in absolute abundance was high, peak Dinobryon biomass always occurred within a narrow range of temperature

(10°-16°C). This distributional study was validated by an early spring field experiment where temperature was raised with water heater to 16° C. The abundance of Dinobryon increased two-fold at the higher temperature. A novel laboratory instrument was developed and constructed that established a vertical temperature gradient with even lighting to test the ability of Dinobryon to congregate within that specific temperature range independent of light intensity. Dinobryon abundance in the column was maximum within the same range of water temperatures observed in the field study. Because lighting was even across the vertical water column, any effect of light attenuation in the lake distribution was eliminated and accumulation due to temperature effects was confirmed. With climate change

IV becoming more apparent, it is clear that understanding temperatures role in the distribution of the aquatic food web is timely and important.

LIST OF FIGURES

1.1 Photomicrograph of Dinobryon ...... 2 1.2 Dinobryon distribution in Lake Lacawac ...... 6 2.1 Temperature Gradient Incubator side view ...... 23 2.2 Temperature Gradient Incubator top view ...... 24 2.3 Photograph of Temperature Gradient Incubator ...... 25

2.4 Lake Lacawac aerial photograph ...... 26 3.1 Dinobryon distribution ...... 30 3.2 Temperature distribution ...... 31 3.3 BIC profile of Lake Lacawac on May 13, 2005 ...... 32 3.4 Metalimnetic Oxygen Maximum ...... 33 3.5 Temperature of maximum abundance vs. maximum abundance ...... 37 3.6 Scatter plot of temperature vs. abundance ...... 38 3.7 Temperature plot from April experiment ...... 42 3.8 April abundance of Dinobryon vs time ...... 43 3.9 April percentage of biovolume vs time ...... 44 3.10 Temperature intervals for column ...... 49 3.11 Temperature average for all columns ...... 50 3.12 Percentage of Dinobryon at any given depth ...... 52 A.1 Dissolved Oxygen distribution ...... 71 A.2 Asterionela distribution ...... 72 A.3 Strombidium distribution ...... 73 A.4 Synura distribution ...... 74 A.5 Uroglena colony distribution ...... 75 A.6 Biovolume of phytoplankter ...... 78

LIST OF TABLES

2.1 Sampling dates ...... 27 3.1 Secchi disk measurements ...... 34 3.2 Spearman’s rho of temperature vs Dinobryon for the field ...... 36 3.3 Two way chi square that shows Dinobryon more in middle of temperature at high abundance ...... 41 3.4 What biovolumes used ...... 45 3.5 Proportion of population and average cell count ...... 46 3.6 Temperature of the column per depth ...... 53 3.7 Spearman’s correlation of temperature ...... 54

VII

ACKNOWLEDGMENTS

First and foremost I would like to thank Tara Heinze for her love and support through this whole process. It is amazing to have a partner like her and I feel truly honored to be with her. Without her this process would have been impossible.

Dr. Robert Sanders who has been an excellent mentor. He has not only taught me to be a better scientist but also to be a better person by teaching me about myself and the world. He has always made time to improve my science and been more than gracious when life was difficult.

Dr. Edward Gruberg has been a constant encourager throughout this process. He allowed me to ask a wide variety of silly questions without making me feel silly which is refreshing. He loaned me a variety of electronics equipment and beat me handily at racquetball once a week.

Dr. Joel Sheffield has provided expert guidance to make my data more clear and analyzable. He has always been patient explaining in extensive detail how I could improve my ideas, and as a result I am a better scientist.

Dr. Wayne Coats for his helpful suggestions to make this document better.

Dr. Shohreh Amini for being available but not overbearing.

Amy Macaluso for giving me my ecology sea legs, and allowing me to ask questions I definitely should have known without outing me as the rookie I was.

Dr. Christa Speekmann for encouraging me to think about career development and suggesting that people can graduate with a Ph.D. and work doing something they enjoy.

VIII

Grier Sellers for stretching my science into phylogeny, which I hated until he arrived. My understanding is greater because of his gentle corrections and vast understanding of concepts I know little about.

Jonathan Swinden for propping me up at the end, and showing me that science can be exciting and worth doing. Also for giving me a chance to relieve some stress outside of lab.

Novneet Sahu for giving me a chance to understand that I do know things that are valuable and worth learning, and taking the time to learn them. Also, for stretching my conservative limits on experimentation.

Gloria Long for making me smile when I thought I had nothing to smile about, and making me aware that this work has real benefit to society.

Jon Oleshevski, and Willie Stichter for sampling help, in the winter, and under ice, when all my scientist friends ran for cover.

Paul Kohl for being a good friend and an excellent engineer, without his help the temperature column would have never been a reality.

Dr. Sandra Connley for being a good friend during long sampling trips and knowing when we needed to laugh and when we needed to buckle down and plow through.

Jason Porter for his advice and support during the early part of my degree process and having the confidence to make me a collaborator on a separate project.

Dr. Robert Moeller for teaching me the value of vigilance in science. That no is not a bad word, and for not succumbing quickly to peer pressure.

Dr. Janet Fischer, and Dr. Mark Olson for showing me that good family life and good science can exist simultaneously, it is not easy but if you prioritize both it can happen.

Dr. Pat Neale for being patient with a rookie, “know it all scientist”, and walking me VERY slowly through some complex ideas, my thinking has advanced greatly.

Dr. Wade Jeffrey for being a character who loves life and work. He illustrated to me that you don’t have to be subdued to have a successful science career.

Dr. J. Dean Pakulski for showing me that the way things have always been done is not the answer to many of life’s questions.

The biology department as an institution, it supported me financially and structurally and it took many members . I felt included in a community for the time I was here and that benefit has helped to shape my identity.

Marcus Sanderlin and Stefan Saunders to whom life has not always been fair but you have taught me what it means to persevere and emerge better even when you feel overwhelmed.

Sylvia and Rod Heinze for instilling in me the value of education and encouraging me to continue along this path.

Rachel Bowers who was always gracious enough to listen even when she knew more about the subject then I did.

Abigail Brigid Heinze for showing me that there is more to life then I used to think there was.

What used to be important is less important. It is hard to teach a young man perspective but you have.

Thanks.

Chapter One

INTRODUCTION

Basic morphology of the genus Dinobryon

The genus Dinobryon is widely distributed in fresh waters and can also occur in brackish waters. Individual Dinobryon cells are surrounded by a vase shaped lorica that is likely composed of cellulose and protein. After cell division, one of the daughter cells moves to a new lorica that the daughter cell constructs using a spinning motion (Herth

1979). They usually form colonies of loricae attached base to lip. Typically, Dinobryon are

24-45 µm long and up to 15 µm in diameter at their widest point and have two unequal flagella used for locomotion (Ehrenberg 1834). The photomicrograph of the culture strain used in these laboratory experiments shows many of these components (Figure 1.1), although this particular strain of Dinobryon did not tend to be colonial. In addition to its wide distribution, the genus Dinobryon can numerically dominate phytoplankton assemblages at some depth, typically in early spring (Siver and Chock 1986; Sanders, Porter et al. 1989). Several species of Dinobryon have also been identified as mixotrophic, a nutritional strategy that combines photosynthesis and phagotrophic feeding on bacteria or other protists.

Lorica Flagellum

Figure 1.1 Photomicrograph of Dinobryon sp. (a non-colonial strain)

Mixotrophy the historical perspective

In the past, the idea was held that single-celled eukaryotes were either photosynthetic, photoautotrophic, or they obtained nutrients and carbon through phagocytosis, heterotrophic. Pfeffer (1897) appeared to be the first to recognize that a single organism can be both photosynthetic and phagocytic. This initial work has provided the foundation to studying mixotrophic organisms with the ability to photosynthesize and phagocytize. More recently, compelling electron micrographs of chloroplasts and digestive vacuoles in a single cell reveled structures for both photosynthesis and phagocyctosis

(Doddema and Veer 1983).

It is still unclear why an organism would maintain two distinct nutritional strategies.

Obtaining carbon from both phagotrophy and photoautotrophy may provide an energetic advantage (Fenchel 1982; Raven 1997). In freshwater systems, dissolved phosphorus often is the limiting resource to phytoplankton growth, so access to phosphorus may be a reason to consume bacteria that are relatively rich in phosphorus (Nygaard and Tobiesen 1993;

Horne and Goldman 1994; Rothhaupt 1997; Hitchman and Jones 2000). One study showed an increase in alkaline phosphotase suggesting phagotrophy may be a mechanism to gain phosphorus (Sawatzky, Wurtsbaugh et al. 2006). Observing multiple lakes, mixotrophic

Dinobryon spp. were most abundant during times of low ammonia concentration,

3 suggesting a potential advantage of mixotrophy to gather nitrogen as well(Taylor, Hern et al. 1979). A requirement for certain phospholipids was linked to phagocytosis in one mixotrophic genus (Kimura and Ishida 1989), and obtaining trace metals from bacteria also was indicated as a reason for some mixotrophs to phagocytize (Barbeau, Kujawinski et al.

2001). As with any feeding organism, the feeding behavior of mixotrophs may fulfill a wide variety of nutritional needs, that are not mutually exclusive.

Light requirements of mixotrophs

There is a wide continuum of nutritional strategies employed by mixotrophs, from those dependence mostly on photosynthesis to others dependence mostly on heterotrophy

(Jones 1994). Ochromonas sp. is a known mixotroph that can grow at high rates in total darkness. In fact, changes in light and nutrients seems to have little effect on its grazing rates (Sanders, Caron et al. 2001). Ochromonas grown in the dark with no bacteria dies quickly (Andersson, Falk et al. 1989). These data clearly indicate that Ochromonas is more on the heterotrophic end of the spectrum. The closely related Poteriochromonas malhamensis also is primarily phagotrophic and in the presence of abundant bacteria, photosynthesis contributes at most 7% of its carbon budget (Sanders, Porter et al. 1990).

On the other hand, some mixotrophs including Dinobryon are more phototrophic, with estimates that up to 100% of carbon can be derived from photosynthesis, but as much as

50% of carbon can come from phagocytosis (Bird and Kalff 1987; Bird and Kalff 1989; Raven

1997). Interestingly, Dinobryon is very difficult to culture without bacteria, axenically, and has been proposed as an obligate mixotroph. (Caron, Sanders et al. 1993) Because of their nutritional niche, determination of the light or food levels that impact grazing and photosynthesis has been a focus of study on a variety of mixotrophs, Poterioochromonas malhamensis, Dinobryon cylindricum Ochromonas sp., (Sanders and Porter 1988; Caron,

Sanders et al. 1993; Keller, Shapiro et al. 1994; Rothhaupt 1996; Boxhorn, Holen et al. 1998;

Holen 1999; Urabe, Gurung et al. 2000). Photosynthesis in the mixotrophic dinoflagellate

Fragilidium subglobosum was reduced by up to 49% when food was present showing that drastic changes in mixotrophic nutritional strategies are possible (Skovgaard, Hansen et al.

2000). Fragilidium subglobosum could also survive as an obligate phototroph, but had significantly higher growth rates when ingestable prey were present (Hansen, Skovgaard et al. 2000).

Previous work showed that Dinobryon spp. can be found at depths where attenuation has reduced light to very low levels (Figure 1.2 [(Siver and Chock 1986) used with permission).

However, light levels were not measured directly by Siver and Chock (1986) in their seasonal investigation of phytoplankton distribution with depth. Furthermore, in the laboratory it was shown that Dinobryon sp. was an obligate phototroph (Caron, Sanders et al. 1993). In an attempt to address the conflicting published information, I gathered field data for the current study to illuminate the questions concerning the relating to factors driving distribution of Dinobryon in nature. Three years of data collected seasonally

Used with permission of P. Siver

Figure 1.2 Dinobryon cylindricum distribution at Lake Lacawac at monthly intervals from

1980-81.

6 indicate that Dinobryon sp. is associated with a narrow range of temperature rather than a specific level of light. Using this field data to guide laboratory experiments, a series of questions related to temperatures effects on Dinobryon spp. were examined.

Mixotrophy in food webs

The role mixotrophs play in the food web is still not well understood, but is potentially quite important. In a traditional food chain model, bacteria are either primary producers, or absorb nutrients that, depending on concentrations, are not readily available to larger organisms. Heterotrophic protists eat the bacteria, zooplankton consume protists, fish eat the zooplankton, and larger fish eat the small fish. Mixotrophs are not only primary producers, but also can take up carbon, major nutrients, and micronutrients through phagotrophy. This can lead to significant differences in the food web dynamics.

One study showed the addition of mixotrophs reduced the number of picoplankton present, and heterotrophic flagellates were unaffected (Ptacnik, Sommer et al. 2004). This suggests that nutrients utilized by picoplankton, bacteria, and coccoid cyanobacteria, which are significant competitors for micronutrients, were more effectively moved to higher trophic levels in the presence of mixotrophs in systems that are nutrient limited.

A mesocosm, water contained in situ, experiment showed that the presence of mixotrophs was the cause of deep algal accumulations, and a deep chlorophyll maximum

(DCM), in an acidic lake (Titel, Bissinger et al. 2003). Bacterivory by mixotrophs can be so

7 pronounced that one study suggested during times of very low nutrient and bacterial abundance that significant heterotrophic flagellate grazing was excluded for months, and only mixotrophic grazing occurred (Hitchman and Jones 2000).

There are often times of the year where Dinobryon are more abundant and active than other protists (Siver and Chock 1986; Berninger, Caron et al. 1992). Dinobryon spp. alone have been shown to graze bacteria more than crustaceans, rotifers, and ciliates combined and also consumed bacteria at a rate at or higher than pure heterotrophs (Bird and Kalff 1986). Dinobryon made up a major portion of mixotrophs in blooms where they contributed up to 79% of community bacterivory (Sanders, Porter et al. 1989). This intense grazing can lead to shuttling of limiting micronutrients throughout the food web where they may have remained primarily in the lower trophic levels in the absence of high rates of bacterivory (Barbeau, Kujawinski et al. 2001). Mixotrophy efficiently moved dissolved organic phosphorous (DOP), carbon and dissolved organic carbon (DOC), through the food web (Jones 2000), and laboratory studies showed significant changes in carbon transfer to higher trophic levels with the addition or exclusion of a single mixotrophic species (Ptacnik,

Sommer et al. 2004). Dinobryon are ingested by smaller Daphnia and can be a major food in large-bodied cladoceran zooplankton (Tappa 1965; Sanders and Porter 1990). Dinobryon are an important component in the larger food web linking primary production and bacterial carbon to higher trophic levels. However, some Dinobryon spp. have also been shown to be difficult to eat by certain predators (Balseiro, Modenutti et al. 2001).

The determination of mixotrophy of an organism will not necessarily explain its role in the food web. The extent that the organism is either photosynthesizing or phagocytizing and what factors control that balance will alter its impact (Stickney, Hood et al. 2000). If one reduces the light or food below a certain level, the organism is no longer able to grow, or even survive. Dinobryon cylindricum has been shown to require both light and food to grow to maximum abundances. The percentage of carbon Dinobryon gains from photosynthesis is greater than 75% where phagocytosis only accounts for less then 25%

(Caron, Sanders et al. 1993). Other mixotrophs, like Ochromonas, can grow to nearly the same abundances in the dark as they can in the light as long as there are adequate bacteria concentrations (Sanders, Caron et al. 2001). Poterioochromonas has been shown to change its rate of ingestion due to light, the more light it has the less it ingests (Holen

1999). This strongly suggests that the balance of energy obtained from photosynthesis or phagocytosis is dependent on the availability of either light or ingestable prey, or both.

Relevance

Because of mixotrophs’ ability to survive in harsh environments, some species can prove to be problem organisms for humans using aquatic resources. For instance, every year a bloom of Dinobryon divergens occurs underneath the ice just outside of Calgary causing water treatment problems (Watson, Satchwill et al. 2001). This leads to difficulty providing acceptable water for people as the bloom of D. divergens’ has a significant odor

9 associated with it (Satchwill, Watson et al. 2007). D. divergens mixotrophy likely contributes to its ability to survive under conditions below the ice where other photosynthetic organisms can’t maintain a niche.

There is a common and poorly understood phenomenon in many aquatic systems called the deep chlorophyll maximum (DCM). This is a depth below the surface where water has the highest chlorophyll concentration, generally due to an increased abundance of photosynthetic organisms (Wetzel 2001). This increase in photosynthetic organisms often creates a meta-limnetic oxygen maximum due to the increased photosynthetic oxygen production at that point in the water column. Even when Dinobryon spp. where deeper in the water column than typically associated with photosynthesis, a metatlimnetic oxygen maximum was created (Bird and Kalff 1989). This is particularly interesting because one study showed that a decrease in phosphorus increases the likelihood of a DCM

(Barbiero and Tuchman 2004). One study has even gone so far as to suggest that the DCM is formed by organisms moving just above the oxycline where they may be competing for nutrients mixed upwards from deeper nutrient-rich waters (Clegg, Maberly et al. 2003).

Titel et al. (2003) found the mixotrophs were up to fifty-fold more abundant than heterotrophs in one lake. The authors suggest that the combination of adequate light, low dissolved nutrient concentration and available food make an ideal habitat for mixotrophs.

However it is clear that not all DCM’s are formed by mixotrophs (Arenovski, Lim et al. 1995).

Other mixtrophs sometimes make up a large component of harmful algal blooms primarily in marine systems, and understanding their physiology may help humans manage the huge economic cost caused by red tides (Kimura and Ishida 1986; Kimura, Ishida et al.

1986).

Temperature effects

Temperature has far reaching impacts on aquatic organisms. Growth rates and photosynthetic rates are temperature dependent, as are other enzyme based processes ranging from the production of hydrogen to DNA repair (Eppley 1972; Goldman and

Carpenter 1974; Caron, Goldman et al. 1990; Mitchell 1997; Brush, Brawley et al. 2002;

Sanders, Macaluso et al. 2005; Zhang, Evans et al. 2007). Cold temperatures can decrease aquatic photosynthesis, or any other enzymatic process. A decrease of temperature by 10°

C reduced photosynthesis by half in two separate studies (Raven and Geider 1988; Davison

1991). However, photoinhibition can occur even at very low light levels with warmer water

(25° C), indicating that warmer temperatures can also inhibit photosynthesis (Raven, Kubler et al. 2000).

In addition, temperature modifies feeding rates, reproductive rates, and competition, which has wide implications for community structure. Reduced bacterial production at lower temperatures may increase the availability of nutrient resources to other organisms (Pomeroy and Deibel 1986). The differential ability of diatom species to

11 utilize silica effectively at different temperatures modified the outcome of competition

(Tilman, Mattson et al. 1981), and minor temperature change switched the dominance of different ecotypes of the cyanobacterium Prochlorococcus (Johnson, Zinser et al. 2006).

Temperature limited grazing rates of protozoa in the Southern Ocean (Becquevort, Menon et al. 2000), and algal blooms may be controlled by temperature around Antarctica due to effects on photosynthesis, heterotrophy, and bacterial production at specific temperatures

(Rose and Caron 2007). Interspecific differences of temperature effects on population growth, which could alter their relative distribution, were found in four species of co- occurring zooplankton (Ramos-Jiliberto and Aranguiz-Acuna 2007). Not only does temperature determine the rate at which organisms can grow, it affects the realized niche of many organisms in a variety of ways.

The behavioral component of organismal response to small temperature changes in aquatic systems has been difficult to study due to the limited availablity of tools for researchers. Past studies investigating temperature effects on plankton were limited to three basic approaches: experiments in natural systems; laboratory experiments where each temperature exposure occurred in a discrete water bath; or the use of plankton towers at the Max-Planck Institute for Limnology at Plön (Lampert and Loose 1992).

Experiments with temperature in natural systems often have confounding variables such as wind mixing, and they require enormous inputs of energy to establish changes from the natural setting that are difficult to tightly control (Baulch, Nord et al. 2003; Nouguier,

Mostajir et al. 2007). Furthermore, experiments in natural systems cannot separate the effects of changes in temperature from changes in light intensity and quality in stratified aquatic systems. This difficulty is illustrated by the observation that lowering temperature or increasing light both result in similar levels of net photosynthesis in the green alga

Chlorella (Maxwell, Falk et al. 1994). A different set of limitations apply to laboratory experiments that use separate incubators at discrete temperatures. Comparing incubators at different temperatures is valuable experimentally for certain growth and kinetics questions. The problem with this technique is that motile organisms cannot aggregate at a preferred temperature. There is evidence that Dinobryon can swim 1 m in a lake (Gilbert and Hampton 2001). This limits the investigation of motile organisms that may migrate to a depth of “ideal” temperature in a natural system. To address these limitations, an inexpensive, small-scale experimental apparatus was developed that allowed establishment of a gradual, measurable temperature gradient with other natural variables kept constant.

The experimental utility of this incubator was demonstrated by examining the vertical distribution of the mixotrophic alga Dinobryon sp. relative to temperature.

Chapter 2

MATERIALS AND METHODS

Field Collection and Sample Analyses

Samples were collected from Lake Lacawac (13 meters maximum depth) at 1 m intervals from surface to 11 m using a Van Dorn bottle. Sampling dates, spanning a three year period, are listed in Table 2.1. For most of this period, all collections were made at a single station in the deepest part of the lake. After 3/23/07, three additional stations along a 4 point transect (labeled A, B, C and D in Figure 2.4) were included. Station A was closest to the dock on the south side of the lake and was approximately 200 meters from station C which was marked with an anchored buoy. Station B was midway between stations A and

C, and station D was approximately 100 meters beyond station C. Stations A, B and C were sampled at depths of 1,3,6, and 9 meters, while station C was sampled every meter from the surface to 11m deep as on earlier dates. Water collected was emptied into a sample integrator and mixed. For plankton enumeration 125 ml subsamples from each depth were fixed with 3% Lugol’s iodine. Sampling dates following 7/17/2006, 125 mL samples were taken for chlorophyll determination, and 1 liter of water from each depth was collected for nutrient analysis. Water taken for chlorophyll analysis was kept out of light and at in situ temperature until processed immediately after returning to shore: Nutrient samples were collected in 1 liter high density polyethylene bottles filled to the brim, covered with

14 parafilm, and immediately frozen upon returning to the dock. Samples remained frozen until analyzed. Secchi depth was measured at each station, and temperature and oxygen concentrations at depth were determined using a YSI model 58 dissolved oxygen meter.

A flat-bottom aluminum rowboat was used to reach sampling sites, except when ice thickness could support equipment and investigators. When appropriate a hand powered 6 inch ice auger was used to make an opening large enough for the Van Dorn bottle.

Microscopic Enumeration

Lugol’s fixed samples were concentrated approximately 10 fold by settling 100 ml in a graduated cylinder overnight. Approximately 90 ml from the top of the cylinder was removed using a J-shaped aspirator, which left the settled material undisturbed on the cylinder bottom. The exact volume of the remaining sample was measured. Samples were examined in a Sedgwick-Rafter cell at 250 x magnification on a Zeiss Photo-Scope III.

Chlorophyll protocol

Shortly after returning to shore from sampling, 100 ml of whole lake water from each depth were filtered through 25 mm Whatman GF/F filters and the filters were then frozen until chlorophyll was extracted. Chlorophyll was extracted from the filters in 10 ml of 90% acetone-methanol mixture, which was vortexed for 1 minute and frozen at –80° C overnight. The mixture of the acetone methanol mixture were 750 mls acetone, 167 mls of

15 methanol, 83 mls of double distilled water, and 4 drops of concentrated ammonium hydroxide. Samples were then placed in a 60° C water bath for 2 minutes, vortexed for 2 minutes and returned to –80 ° C freezer overnight. Samples were thawed and centrifuged at 2500 rpm for 10 minutes. Fluorescence was measured using a Turner Designs

Fluorometer (TD-700). The TD-700 was calibrated using a solid chlorophyll standard (pn

7000-994) and the fluorometer runs an algorithm to determine the concentration of chlorophyll directly.

Nutrient Concentrations

A spectrophotometric method was used to determine nitrate (Morris and Riley

1963). Briefly, 100 ml of lake water was placed in a 125 ml Erlenmeyer flask. Ammonium chloride (2ml) was added and the sample was then run through a cadmium column that quantitatively reduced nitrate to nitrite. The first half of the sample was discarded and the remaining 50 ml was combined with 1 ml of sulfanilamide for between 2 and 8 minutes, followed by 1 ml of naphthylethylenediamine. The sample was then allowed to sit for 10 minutes and measured in a 1-cm cuvette at 543 nm on a Shimadzu UV-160 spectrophotometer. The nitrate per liter was calculated using the formula:

µg-at N/l = corrected extinction x (F – 0.95 C) where C is the concentration of nitrite in the sample in microgram-atoms per liter and F determined by

F= 20/Es

where Es is the extinction of the standard. This procedure gave nitrate plus nitrite. To determine nitrite only, the procedure was repeated without the cadmium column, and the amount of nitrite was calculated using:

µg-at N/l = corrected extinction x F

Where F = 2.00/Es, and Es is the extinction of the standard. Total nitrate was calculated from subtraction of the nitrite determination from the nitrate plus nitrite determination.

Phosphate was also determined using a colorometric method (Murphy and Riley

1962), but was below the detection limit for the method using a one centimeter cuvette.

Field experiments: temperature and UV-B effects on phytoplankton

General design

The purpose of this experiment was to study the effects of changing temperature and UV on the phytoplankton community of a clear oligotrophic lake during spring, as light intensity, day length, and temperatures were all increasing. An integrated water sample

(surface to 3 M) was collected from Lake Giles, an oligotrophic lake in northeastern

Pennsylvania at 7:00 am on April 10th 2006. The water was filtered through a 60 µm Nitex mesh screen to exclude larger zooplankton. The water was then distributed into one gallon

UV transparent Bitran bags and stored in coolers that contained water from 3 m depth for

17 transportation to outdoor incubators. The temperature of the outdoor incubators was regulated by thermostatically controlled circulating water baths, a flow through system utilizing water from Lake Lacawac. One incubator was maintained at 5.5° C, the temperature of lake Giles at a depth of 3m on the day of collection, and the other incubator was gradually raised to 15° C. Temperature was recorded every hour over the 10 day experimental period. The water was incubated under full sun, with a film of water covering the Bitran bags to allow the maintenance of temperature and insure maximum potential UV exposure. For each temperature treatment, one set of replicates was shielded from UV radiation using OP3 Arylite, while another set was exposed to UV under OP4 Arylite.

Triplicate samples were taken at 8:00 am on days 0, 1, 4, 7, and 10. Samples from the warmer treatment were not taken on day 1 because the planned gradual rise to the target temperature was not complete.

To determine treatment effects on phytoplankton populations, cells were fixed and enumerated using light microscopy as previously described. Four transects of the

Sedgwick-Rafter cell were viewed and enumerated, which constituted 20% of the entire sample.

Laboratory experiments

General set-up and temperature gradient incubator construction

An extruded Plexiglass tube (US plastics cat. num 44550, 65-inch X 6-inch outer diameter, 0.125-inch thick wall) was attached to a 12-inch square Plexiglass base using weld-on #16 cement according to manufacturer’s instructions, and then a bead of 100% silicone caulk was added along the seam after the cement had hardened. The base was reinforced by attaching it to a 1-inch thick plywood stand with leveling feet. The Plexiglass base of the column was attached to the wooden stand using 0.25-inch bolts with washers.

Sampling ports consisted of 6.5 mm Tygon tubing placed through holes drilled 15 cm apart on the Plexiglass column (Figure 1) and attached using the weld-on cement. After curing the weld-on cement a silicone bead was applied around the seal. Tubing extended with 20 cm outside of the column for each sampling port and extended 7.6 cm into the column to extract samples from the center of the column when full of water (Figure 2). A drain was installed 2 cm from the bottom using the same method. The column held approximately 30 liters of liquid in this configuration.

Thermistors were used to accurately measure temperature at the point of sampling.

The thermistors (Sensor Scientific WM502C) where prepared by first insulating wires with a nonconductive compound so that they did not short circuit when touched. To allow the thermistors to be positioned in the center of the column at the point of water sampling, long leads were soldered on using a heat sink to avoid damage to the thermistors. A thermistor was inserted directly adjacent to each sampling tube and affixed to the tube using weld-on #16 cement. Prior to installation, the thermistors where tested in 4 water

19 temperatures to ensure that their resistance corresponded accurately to the curve generated from the Sinehart-Hart equation:

1/T = a + b ln(R) + c (ln(R))3, where T is temperature in degrees Kelvin, R is the resistance of the thermistor in ohms, and a, b and c are thermistor specific constants. For the WM502C thermistors, a =

0.0012873851, b = 0.00023575235, and c = 0.00000009497806. A digital volt ohm meter

(Tektronix TM5003 with DM502A) was used to measure the resistance of the thermistors during the experiment, but any ohm meter capable of measuring in the kilo ohm range would be appropriate with this set of thermistors. The resistances were measured within 5 minutes of sampling the plankton.

A single heated water bath with a submersible pump circulated warm water into the column through a continuous series of Tygon tubing loops (Figure 1). The water temperature at depth thus depended on the number of tubing loops present: the surface water had the most loops of circulating warm water, and the coolest bottom water had no tubing running through it. The water bath was a 34-liter insulated, covered container heated by a 150 W submersible heater (Jager 3606 Heater). The Tygon tubing ran through small holes drilled directly into the top of the water bath and into the uncovered Plexiglass column. The water was circulated through the Tygon tubing using a single submersible pump (Maxi-Jet MJ1000 (1000 l/h)). Tubing (1.9-cm o.d., 1.3-cm i.d) was attached directly

20 to the output jet of the submersible pump and connected to two additional tubes of the same dimensions using a Y-fitting (US Plastics). Reducers and y-fittings were used to establish 4 loops (0.25-inch o.d., 0.125-inch i.d.) of heated water. The length of each loop was adjusted so that water velocity in each heating line was the same. The length of the first loop (25 cm) reached the first port. The next loop extended 40 cm into the water column, covering the next two sampling ports, and the final two loop lines descended 80 cm and 100 cm into the column. The heating loops were weighted using 4 cm sections of

PVC pipe (1.5-inch i.d.). The water bath was thermostatically controlled and set to 23˚ C for these experiments. The apparatus was assembled in a walk-in environmental room at a temperature of 10˚C.

To light the column, six 24-inch 20 W fluorescent fixtures, with cool white bulbs were staggered vertically along each side of the temperature gradient incubator. The lights projected 30 cm above and below the column (the base was elevated off the ground).

Overlapping the fixtures was necessary to create an evenly lit water column, since the light is brightest in the center of the fluorescent bulb. A continuous even lighting regime with depth was selected to reduce the variables in these experiments, but it would be simple to vary the lighting to replicate more natural lighting regimes by lighting exclusively from the top of the column and using a timer for lighting.

The temperature gradient incubator was run for three separate experiments used as replicates.

Culturing

Dinobryon sp. were obtained from University of Toronto Culture collection (strain

#392), the same strain as strain # 2267 in University of Texas at Austin culture collection.

Cells were maintained under a 12:12 light:dark cycle at 20° C in DY IV media (Sanders, Caron et al. 2001). The light during the culturing process ranged from 80 µEm-2s-2, to 30 µEm-2s-2.

New stock cultures were started once a month, and the organisms used in experiments were from cultures in exponential growth.

Data analysis

To analyze the data, several software packages were used. Contour plots were generated using Matlab. SPSS 15.0 was used to determine the correlation values. Minitab was used to graphically represent much of the data during the initial stages of the analysis.

Excel 2007 was used to organize data, determine the initial descriptive statistics, and do much of the graphing represented in this paper. Because the values were not normally distributed, Spearman’s rho was calculated.

Figure 2.1 Side view of temperature gradient incubator. Values in centimeters

15 cm

Figure 2.2 Top view of temperature gradient incubator

Figure 2.3 Photograph of temperature gradient incubator

50 m

Figure 2.4 Aerial photograph of Lake Lacawac

Table 2.1 Sampling Dates

2005 4/9, 5/13, 6/13, 7/11, 8/2, 10/1, 11/3, 12/22

2006 1/23, 2/22, 3/23, 4/20, 5/13, 6/22, 7/17

2007 3/23, 4/13, 5/5

Chapter 3

RESULTS

Lake Lacawac field study

Populations of Dinobryon spp. had early summer peaks and were below the detection limits for much of the year over the course of three years in Lake Lacawac (Figure

3.1). In 2005, the maximum Dinobryon abundance was found in June at a depth of 4 meters. In 2006, Dinobryon reached similar abundances, but peaked closer to the surface, at one meter, in April (Figure 3.1). The greatest abundance of Dinobryon spp. observed during this study was 4604 cells ml-1 at 1 meter.

A contour plot of temperature in Lake Lacawac shows typical seasonal warming of the surface waters in spring, as day length and solar intensity increase, and then subsequent cooling of surface water during the fall and winter (Figure 3.2). Oxygen concentrations varied seasonally, as expected for a mesotrophic system like Lake Lacawac.

Water was well mixed and high in oxygen from top to bottom in winter, with the bottom waters gradually becoming anoxic by summer. During spring when Dinobryon spp. were abundant, there were metalimnetic oxygen maxima, as exemplified by data from June 13th

2005, which also illustrates the vertical distribution of Dinobryon spp. on that date (Figure

3.3). The oxygen maximum of 13 ppm dissolved oxygen at 4 meters corresponds to the depth of Dinobryon spp. maximal abundance. Relative to other phytoplankton present on this date, Dinobryon spp. was the most closely associated with the oxygen maximum layer.

However, this was the only incidence in the study where Dinobryon and the oxygen maximum were found to coincide.

Light intensity at depth was determined from a Biosphericals Instruments profiling radiometer (BIC) and plotted as a percentage of surface irradiance (Figure 3.4). The 1% light level of photosynthetically active radiation (PAR) was at 4.3 m at midday on May 13, 2005.

Penetration of ultraviolet radiation (UV-B, 280 – 320 nm, UV-A 320-400 nm) was represented by BIC recordings in several narrow bandwidths (305, 320 and 380 nm). Light in the 305-380 nm range was reduced to 1% of surface irradiance at a depth of less than 1 meter in Lake Lacawac. This was of particular importance to this study because other evidence suggests that Dinobryon are not very resistant to ultraviolet radiation (UVR). BIC instrumentation was not generally available for this study, however Secchi disk measurements were taken at every sampling date (Figure 3.5). When multiplied by a factor of 2.7, Secchi depth gives a reasonable estimate of the depth of 1% surface PAR (Poole and

Atkins 1929). In general, sampling dates in 2005 and 2006 had similar Secchi depth measurements. Samples from 2007 had a deeper Secchi depth, indicating greater water clarity in the early spring that year, but as noted previously Dinobryon spp. were not found in high abundances in early spring during either 2005 or 2006.

Figure 3.1 Dinobryon spp. seasonal and vertical profile from Lake Lacawac. Black dots indicate points when and where samples were taken.

Figure 3.2 Seasonal and vertical profile of temperature in Lake Lacawac. As in Figure 1, hash marks on top and bottom represent dates that sampling occurred.

Lake Lacawac, May 13, 2005 Irradiance as % of Subsurface 1 10 100 0

Depth (m) Depth 3 305nm 320 nm 4 380 nm PAR 5

Figure 3.3 Biospherical Instruments profiling radiometer data from Lake Lacawac May 13th 2005. X axis is log graph of % irradiance compared to sensor just covered in water from the lake. Y is depth into Lake Lacawac in Meters

Figure 3.4 Dissolved Oxygen and Dinobryon Abundance vs. Depth on June 13th 2005 from Lake Lacawac. Shows a metalimnetic oxygen maximum from June 13th 2005. ‘s are the Dinobryon spp. abundances and X’s show dissolved oxygen.

Date Secchi Disk Depth 4/9/2005 2.8 5/13/2005 2.7 6/13/2005 2.6 8/2/2005 2 10/1/2005 3 11/3/2005 2.5 2/22/2006 2.2 3/23/2006 2 4/20/2006 2.6 5/13/2006 2.5 6/22/2006 2.3 7/17/2006 2.3 3/23/2007 3 4/13/2007 3.3 5/5/2007 4.3 5/23/2007 4.5 6/22/2007 4.5 720/2007 4.3 9/7/2007 5.3 Table 3.1 Secchi disk depth measurements over the course of sampling

There was a highly significant negative correlation between Dinobryon abundance and temperature when samples without Dinobryon are excluded (Spearman’s rho, p=0.009,

Table 3.1). Because Dinobryon spp. were not evenly distributed in the water column (e.g.,

Figure 3.3), data were examined by plotting the maximum abundance for each sampling date versus the temperature of the water at the corresponding depth for each date that

Dinobryon spp. were present (Figure 3.6). Although Dinobryon were present at temperatures ranging from 2.9 to 22.8°C, abundances exceeded 1,000/ml only when temperature at depth was between 10.5 and 18.2°C.

Temperature Spearman’s rho Dinobryon Correlation Coefficient -.195 Sig. (2-tailed) .009 N 180

Table 3.2 Spearman’s rho bivariate correlation of Temperature and Dinobryon from the entire experiment. N is only 180 due to not counting values where Dinobryon abundance is zero.

5000

4500

4000

3500

3000

2500

2000 Dinobyron(cells/ml) 1500

1000

500

0 0 5 10 15 20 25 30 Temperature (°C)

Figure 3.5 Temperature vs Dinobryon abundance for all abundances greater than 1 cell/ml.

120

100

40 % ofMaximum %abundance 20

0 0 5 10 15 20 25 30

Temperature (°C)

Sanders Hitchman Flint Bird Kamjunke Lacawac

Figure 3.6 All sampling data from every depth and every sampling date from Lake Lacawac.

Each point represents one depth on one sampling date. Zero values are included in this graph.

To examine whether there was a subset of temperatures for which Dinobryon abundance tended to be at higher levels, a two-way chi-square test of Dinobryon cells ml-1 to Temperature was performed for all the samples in the Lake Lacawac study. Samples were taken within a temperature range of 0 to 27.5° C, and so temperature values were divided into three equal subsets, 0-9.1, 9.2-18.3, and 18.4-27.5° C. The range of values for abundances had a clear division at 358 cells ml-1 which was rarely exceeded. Consequently, the ranges for Dinobryon abundances included three groups: 0, 1-358, and 359-4750 cells ml-1. These values were chosen because 0 is a critical important placeholder, and 358 was the average of all no zero time points. Table 3.2 shows the actual incidences and expected incidences calculated from the data for occurrences of the three abundance groups within each temperature range. Abundances less than 358 cells ml-1 (including 0) were never significantly different from predicted. However, incidences of high abundance occurred significantly more frequently than expected in the temperature range of 9.2 – 18.3° C

(Pearson chi-square, p<0.0001). The Pearson’s correlation is used to evaluate if the chi- square values are correlated. If the Pearson’s correlation is significant then the values in the chi-square have some non-random distribution, as is obvious in the 9.2-18.3° C range.

Field experiment: temperature increase and UV protection

To investigate the effect of warming spring temperature along with increasing irradiance on Dinobryon populations, lake water temperature was increased gradually in

39 the treatment from 5.5 to 16° C over a 3 day period (Figure 3.8). The control incubation and the experimental incubation, once its target was reached, remained at stable temperatures for the duration of the experiment (Figure 3.8). Light quality also was altered using UVR- blocking plastic shielding on some treatments, and Dinobryon abundances and relative population biovolume were affected both by changes in light quality and temperature

(Figures 3.9 and 3.10). It is clear that irrespective of temperature, the Dinobryon population was negatively affected by UVR. When not sheltered from surface levels of UVR, populations remained static or declined, while those protected from UVR reached higher abundances (Figure 3.9). Over the 10 day experimental period, Dinobryon populations grew most in the warmer temperature, if protected from UVR. (Figure 3.9).

The increases in Dinobryon population size were at the expense of other phytoplankton groups. Dinobryon abundance relative to other phytoplankton increased

(Table 3.3), but the increase in terms of relative biovolume in the community was even greater. Biovolume, calculated from microscopic measurements represents a measurement of biomass. Though Dinobryon is only of moderate size relative to many of the other phytoplankton species present (Table 3.4), its population biovolume as a portion of community biovolume increased, indicating that it became the dominant phytoplankter in terms of community biomass both when sheltered from UVR and also with an increase in temperature (Figure 3.10). This change due to heating is simply a trend as two way ANOVA shows no significance.

Temperature 0-9.2°C 9.2-18.3°C 18.3-27.5°C Dinobryon 0 Count 52 15 6 (cells/ml) Expected Count 50.5 14.5 8.0 >0-358 Count 103 19 18 Expected count 96.9 27.8 15.4 359-4700 Count 9 13 2 Expected Count 16.6 4.8 2.6

Value df Asymp. Sig. (2-sided) Pearson Chi-Square 22.722 4 .000

Table 3.3 Two way chi-square for Temperature and Dinobryon spp. abundances from all sampling dates at Lake Lacawac.

20 18 16 14 12 10

8 Temperature (C) Temperature 6 4 2 0 0 50 100 150 200 250 300 Time (hours)

Figure 3.8 Temperature of experimental containers measured hourly over 10 days for temperature and UV experiment. Water from the water bath was measured not the sampled water.

800

700

600

500

400

300 Dinobryon(cells/ml) 200

100

0 0 2 4 Time (days) 6 8 10 12 5° without UV 16° without UV 5° with UV 16° with UV

Figure 3.8 Abundance of Dinobryon measured over time. Diamonds are 5° C without UV, Squares are 16° C without UV, triangles 5° C with UV, and Xs are 5° C with UV. Error bars are Standard Error n = 3

5 % ofDinobryon %biovolume in community 0 0 2 4 6 8 10 12 Temperature (C°)

5° with UV 5° without UV 16° with UV 16° without UV Figure 3.9 Percentage of Dinobryon biovolume as measured from entire community. Squares are 5° C with UV, asterisk are 16° C without UV, triangles 5° C without UV, and Xs are 16° C with UV. Error bars are Standard Error n = 3

Biovolume um3 Chrysophyta Flag 6-10 um 69 11-20 um 262 20-25 um 1578 Dinobyron 359 D. Lorica Synura 262 Cryptophyta 6-15 um 217 16-40 um 1367 Chlorophyta Sphaerocystis 87 Ankistrodesmus 42 Schroederia 272 6-10 um 69 8-20 um spiny 166 8-20 um round 262 Elakatotrix 2 cell -25 um 196 Scenedesmus 118

Kirchneriella 23 Oocystis (parva sp.) 572 Selenastrum minutum 131 Dinoflagellates Gymnodinium sp. 3611 P. limbatum 27847 Cyanobacteria Merisimopedia 180 Bacillariophyta pennate 6-8 um 48 20 um 360 50 um 540 synedra 692 Asterionella 613

Table 3.4 Biovolume values used to calculate biovolmes for figure 3.10. (Olenina, Hajdu et al. 2006)

Average Std Deviation of Proportion of Dinobryon Count the Dinobryon population Count Dinobryon constitutes T0 240 139 1.53 Day 10, 5.5°, - UV 288 85.2 5.73 Day 10, 5.5°, +UV 6.69 11.6 0.863 Day 10 16.0°,- UV 549 186 9.97 Day 10 16.0°, + UV 9.82 10.0 0.593

Table 3.5 Count values for field experiment done in triplicate. Proportion of population done by cell counts.

Temperature gradient incubator

The temperature gradient incubator held a consistent temperature over at least seven days (Figure 3.11). Measurements were taken at 24 hour intervals at each thermistor on the incubator during the week long experiment (Table 3.5). The average temperature at each thermistor depth after 48 hours of heating the column for three different experiments indicated that the incubator also was reliable from experiment to experiment (Figure 3.12).

The vertical distribution of Dinobryon in the temperature gradient incubator indicated a response to changing temperature (Figure 3.13). At the beginning of the experiment before the heater was turned on, the temperature and the % of Dinobryon cylindricum at any given depth were approximately the same (Figure 3.13A). After the heating tubes had been on for 48 hours and there was temperature stratification in the column, a larger percentage of the D. cylindricum population was found at the port 90 cm from the top where the temperature was 19.0° C (Figure 3.13B). The relative abundance of

D. cylindricum in 19.7° C water at the 75 cm port was also elevated from initial (Figure

3.13B). After the heater was turned off for one week, both the temperature and the D. cylindricum population became more evenly distributed again (Figure 3.13C).

The results of the experiment reported in Figure 3.13B suggest that D. cylindricum tend to favor an intermediate temperature of approximately 15. When data recorded at 24 hours for three experiments were correlated, Spearman’s rho analysis showed there was a

47 strong negative correlation for temperature and Dinobryon when examined over the narrow experimental range(p<0.02, Table 3.6). Furthermore, averaging the changes in population size from initial conditions at a given depth to temperature stratified conditions at the same depth at 24 hours, there is evidence of movement away from water that reaches temperatures above 19°C (Figure 3.14).

Temperature (C) 13 15 17 19 21 23 0

Depth (cm) Depth 80

100

120

Figure 3.10 Temperature gradient in the TGI recorded at approximately 24h intervals over 7 days.

Temperature (C) 13 15 17 19 21 23 0

Depth (cm) Depth 60

100

120

Figure 3.11 Average temperature of three TGI experiments, after 48 hours. Error bars are one standard deviation.

A Dinobryon (cells/ml) 0 500 1000 1500 2000 2500 3000 3500 0 22.3˚ 23.3˚ 20 23.36 40 22.4˚ 60 22.5˚

Depth(cm) 22.3˚ 80 22.6˚ 100 22.5˚ 120 22.4˚

B Change in Dinobryon at given depth (cells/ml) -2000 -1500 -1000 -500 0 500 1000 1500 0 21.1˚

22.8˚ 20 22.0˚ 40 21.4˚

60 20.7˚

Depth(cm) 19.7˚ 80 19.0˚ 100 16.0˚

120 14.4˚

C Dinobryon (Cells/ml) 0 500 1000 1500 2000 0 14.6˚ 15.2˚ 20 14.6˚ 40 14.5˚ 60 14.5˚

Depth(cm) 14.5˚ 80 14.6˚ 100 14.5˚ 120 14.0˚

Figure 3.13 Distribution of Dinobryon sp. as a percentage of total abundance in the column (A) Initial distribution, (B) distribution after 24 hours, (C) distribution after the heater has been turned off for 7 days. Error Bars are Standard Error n of 3.

Table 1 Temperture variation over 7 days.

Depth (cm) Average Standard Temperature (°C) Deviation of Temperature (°C) Over 7 days over 7 days

0 22.1 0.101

15 22.4 0.385

30 21.9 0.214

45 21.2 0.269

60 20.5 0.202

75 19.6 0.073

90 18.9 0.101

105 15.9 0.065

120 14.5 0.280

Table 3.6 Temperature values of one TGI experiment with standard deviation over the 7 days of the experiment. Manufactures tolerance of the thermistor is +/- 0.2°C.

Temperature Spearman’s Rho Dinobryon Correlation Coefficient -.449 Sig (2-tailed) .019 N 27

Table 3.7 Correlation from all three TGI experiments at the 24 hour time point.

Abundanceafter 48 hours 6

2 Dinobryon

0 14 15 16 17 18 19 20 21 22 23 -2

-4

Percent ChangePercentin -6

-8 Temperature (°C)

Figure3.13 g Average of three TGI experiments percentage change after 48 hours of heating column. Vertical standard deviation is percent change over time and horizontal error bars is the standard deviation of the temperature variation.

Chapter 4

DISCUSSION

A variety of factors influence the spatial and temporal occurrence of phytoplankton in nature. Algae are adapted to fresh or salt water, but generally not to both. Dissolved nutrient concentrations can limit growth of phytoplankton species differentially, and the ratio of several potentially limiting nutrients also can alter competitive outcomes (Tilman

1977). Susceptibility to predators affects absolute and relative abundance of phytoplankton

(Roberts and Laybourn-Parry 1999; Sanders, Berninger et al. 2000; Cadotte, Jantz et al.

2007; Meyer and Kassen 2007; Randa 2007). However, changing light regimes will clearly be a major factor affecting seasonal and depth distribution of photosynthetic organisms, and temperature also has fundamental effects on growth of single-celled organisms. This work tracks the seasonal distribution of a genus of mixotrophic algae, Dinobryon, in a mesotrophic lake in North Eastern Pennsylvania and examines how solar radiation and temperature may affect this distribution.

The two main seasonal peaks of Dinobryon abundance observed during this three- year study occurred during spring, but with maximum abundances at different depths

(Figure 3.1). A previous study in Lake Lacawac also identified a seasonal spring peak of

Dinobryon, but at a somewhat different time and depth (Siver and Chock 1986). In all three cases, peak abundances were at an intermediate depth in the lake. As a mixotroph,

Dinobryon is not dependent on photosynthesis for its sole source of carbon, but laboratory evidence suggests that it is an obligate phototroph that cannot survive long periods in the dark (Caron, Sanders et al. 1993). Fieldwork in Antarctica showed Dinobryon sp. present only in the summer, which Mcknight et al. (2000) attributed to the extended darkness of

Antarctic winter, he suggested they die in the dark. Likewise, Dinobryon below our level of detection during the reduced day length and light intensities of winter in Lake Lacawac.

Additionally, Dinobryon were rarely found below 4 m in this 13 m deep lake at any time of the year, likely due to the attenuation of photosynthetically active radiation (PAR) in the surface waters (Figure 3.4).

During periods of Dinobryon blooms, the photic zone depth (1% surface PAR irradiance) from BIC measurements was around 4.5 meters in Lake Lacawac. Cells below this depth would not have enough light for photosynthesis to balance respiration. Secchi disk measurements, while not exact indicators of the photic zone, have been shown to correlate well with water clarity and thus light penetration. Secchi depth measurements were consistent during bloom periods. When light penetration estimated using Secchi depth measurements was calculated in Lake Lacawac and compared to data from a submersible profiling radiometer (BIC) on the same day, the extinction coefficients for the

Secchi disk and BIC were 4.59 and 4.38 m-1, respectively. Both approaches suggested that light levels below 5 m approached complete darkness and would exclude active populations an obligate phototroph like Dinobryon.

Subsurface abundance peaks, like those observed in Lake Lacawac, have been reported previously for members of the genus Dinobryon (Siver 1986), It is also possible that the low abundances close to the surface are a response to ultraviolet radiation (UVR).

When lake water was incubated directly at the surface in the spring experiment that manipulated UVR and temperature, a decrease in absolute and relative abundance of

Dinobryon was noted in treatments exposed to natural levels of UVR (Figures 3.9 and 3.10).

This suggested that UVR placed Dinobryon at a competitive disadvantage tog other phytoplankton. In Lake Lacawac, this effect of UVR on Dinobryon would probably be important only in early spring at the surface, because UVR penetration was quite low (>1% of surface at 1 m) at a time when the photic zone extended to > 4 m (Figure 3.4). Dinobryon was observed near the surface in April 2006, but these cells may have been mixed or swam below the level of high UVR during parts of the day, or been more resistant to UVR then the cells tested in this experiment. Based on the response seen in the presence of UVR,

Dinobryon sp. would not be expected to be abundant in the shallow water column in clearer lakes where UVR penetrates deeper than in Lake Lacawac. This is supported by previous work showing that UVR altered other protists in plankton communities and microbial food webs (Rae and Vincent 1998; Doyle, Saros et al. 2005; Sanders, Macaluso et al. 2005). The occurrence of a deeper population as a UVR avoidance mechanism has been demonstrated for mixotrophic ciliates and crustacean zooplankton (Modenutti, Balseiro et al. 2005).

Due to the primarily photosynthetic nature of Dinobryon, penetration of solar radiation into Lake Lacawac and its attenuation set limits on where and when the species occurred in the lake. But, light intensity did not appear to be the single critical component driving vertical distribution in the water column. Surface irradiance was similar at the time of the two abundance peaks in the current study, and the depth of maximum Dinobryon abundance varied considerably despite generally consistent light penetration and summation of surface irradiance of 32,690 w/m3 on May 13, 2005 and 29,407 w/m3 on April

20, 2006 (Figure 3.5)(Hargreaves 2006). Temperature was suggested as an important regulatory component of Dinobryon occurrence (Bird and Kalff 1988). Seasonal changes in protist abundances were well documented in many lakes, and temperature was implicated in partially causing these changes (Siver and Chock 1986; Bennett, Sanders et al. 1990; Kifle and Purdie 1993; Bernard and Rassoulzadegan 1994; Ducklow, Carlson et al. 2001), and the data from Lake Lacawac also suggest a role for temperature. The temperature distribution in Lake Lacawac is typical of a dimictic lake, a lake that goes through two stratification events a year, with stratification interspersed with a well-mixed water column in spring and autumn. Although the general seasonal temperature isoclines are similar from year to year

(Figure 3.2), warming and thermal stratification occur at slightly different times. Dinobryon abundance peaked in spring/early summer. Sampling over the same time period as the previous year caught slightly different Dinobryon peaks on the similarly shaped but time- shifted temperature curve. Although Spearman’s correlation coefficient has only moderate

59 strength (Table 3.1), the values are highly significant and negative, suggesting that lower temperature yields growth of Dinobryon within a naturally occurring temperature range.

This does not fit a strictly kinetic model of higher temperature yielding higher growth, but niches are dictated by more than kinetics as this study demonstrates. There were many sampling dates on which Dinobryon abundance was zero – due not just to cold temperature, but to ice cover, low light, or competition from other plankton – and all data points were used to generate the negative Spearman’s correlation. Furthermore, a comparison of Dinobryon distribution and temperature (Figures 3.1 and 3.2) suggests that

Dinobryon was also absent during periods of higher temperature, so a strict interpretation of the negative correlation is misleading. Finally, additional experimentation discussed shortly clearly illustrated that very cold water was not ideal for a thriving population of

Dinobryon, and potentially showed a temperature range where they were best suited to survive.

Seasonal studies that included Dinobryon showed that abundances can vary widely

(Hilliard 1968; Lehman 1976; Siver and Chock 1986; Lehman 1988; Hitchman and Jones

2000; Watson, Satchwill et al. 2001; Todonleke, Jugnia et al. 2002; Titel, Bissinger et al.

2003; Kamjunke, Henrichs et al. 2007). Temperature was recorded for several of these studies, and those data suggest that temperature may play an important role in distribution of Dinobryon. When plotted together with my data on a graph of percent maximum

Dinobryon abundance versus temperature, a correspondence of larger Dinobryon

60 populations and an intermediate temperature range was apparent (Figure 3.7). Low

Dinobryon abundances were observed at temperatures above 20° C and below 8°C, and maximum abundances occurred between 10° C and 14° C in all studies (Figure 3.7). In each study various species of Dinobryon were looked at. In the Sanders study it was D. cylindricum, in Kamjunke D. divergens, in Flint D. sertularia and the others are not mentioned. Although the sample size is too small to draw any conclusions the difference in species could contribute to the changes in temperature ranges. In Lake Lacawac, abundances over 1000 cells ml-1 were observed only at temperatures between 18.2° and

11.2° C (Figure 3.6). The highly significant values (p< 0.001) in a Pearson two-way chi- square evaluation further illustrated central clustering (Table 3.2) and indicated that

Dinobryon abundance was actually associated with a particular temperature range. The chi- square analysis predicted that for the temperature range between 9.2° C and 18.3° C only

5.2 samples would fall into the 358 to 4700 cells ml-1 category, but there were 14 observed occurrences. These data were offset by the reduced number of field samples with highly abundant cells in the colder water, where the analysis predicted the most samples with high abundance.

In the field experiment where temperature was increased in one set of treatments, the community response showed that Dinobryon was a better competitor when the water was 16° C as opposed to 5° C (Figures 3.8 and 3.9). This supported the combined field data showing consistent clustering of high Dinobryon abundance at temperatures between 15° C

61 and 20° C (Figure 3.7). Although this is only suggestive of a discrete preferred temperature range of Dinobryon sp. in the field, it does point toward the idea that colder water is not the ideal environment for Dinobryon to compete (Figure 3.9 and 3.10). When the water was warmed to 16° C, twice as many Dinobryon sp. were present in a mixed community, and the proportion of biomass (as biovolume) that Dinobryon contributed to the phytoplankton community also nearly doubled. This is important because Dinobryon could gain and maintain a competitive advantage in the early spring as water temperatures warmed with increased solar radiation. It is also clear that UVR has a strong negative impact on the growth of Dinobryon. Two way ANOVA shows a strong negative correlation of UVR and the growth rate of Dinobryon suggesting UVR may play an important role in keeping Dinobryon out of the surface waters (p>.001).

Some models have suggested that higher temperature equals higher growth rate

(Muller and Geller 1993), but the data presented here suggest a maximum environmentally relevant temperature causes a decrease in growth rate of Dinobryon. This appears to be true not only in the field, where community structure and competition may have an effect, but also in the laboratory in a monoculture environment (Figure 3.13 B). The effects of temperature on planktonic organisms has been an area of active research, and the recognition of upper and lower temperatures causing reduced growth in protists is not new

(Finlay 1977). However, the potential changes in thermal structure of aquatic systems due to global climate change is likely to increase interest in this area. Even small temperature

62 changes have been associated with differential responses in aquatic systems, but it has been difficult to separate the role of temperature versus other environmental factors that also change with depth in a stratified water column.

Earlier laboratory approaches have been limited to incubations at several temperatures in separate experimental containers, which eliminated potential behavioral swimming responses known to be important to plankton. The unique facility at Plön,

Germany (Lampert and Loose 1992), in which some of the control of a laboratory experiment can be combined with the scale of a small field experiment, is economically and logistically impractical for most ecologists. Another approach that investigated temperature effects on protists in the field was to grow the organisms up in mass and release them to the natural system (Jeong, Kim et al. 2003). This has several obvious problems for studying temperature. First, there is little control of temperature, and just as in other manipulated field experiments, the many other variables involved make it difficult to isolate temperature effects. Also, there is the potential of introducing an unwanted species to a watershed.

I approached this problem by constructing a temperature gradient incubator (TGI) that satisfied the need for a low cost, moderately sized instrument to manipulate water column temperature independently from a light gradient. The TGI consistently held a measurable temperature range (Figures 3.11 and 3.12) and was shown to effectively create

63 a temperature gradient to which a motile planktonic algae responded (Figure 3.13). A highly significant negative correlation with temperature and Dinobryon abundance was seen in the

TGI (Table 3.7), again indicating that colder water is an environment that Dinobryon prefer.

However, the colder water in these experiments was within the range where Dinobryon reached highest abundances in the field study. Within 48 hours, the most Dinobryon grew or migrated to a depth corresponding to temperatures between 14°C and 16° C in three experiments using the TGI (Figure 3.14). Although highly variable, abundances showed a positive trend at temperatures below 20° C, while in general the trend was for a decrease in abundance within 48 h at temperatures equal to or greater than 20° C (Figure 3.14).

The TGI is a valuable tool for understanding aspects of seasonal and depth distributions of planktonic organisms that occur as a result of changes in temperature because it allows them to respond behaviorally to a range of temperatures. For example, it enabled us to address questions about the seasonal and depth distribution of Dinobryon sp.

(which correlated with temperature in a Lake Lacawac) in a manner that was not available prior to the construction of this instrument. Simple modifications in the placement of light sources for the instrument will also allow investigators to examine interactions of light and temperature changes with depth. In addition, experiments with multiple species could elucidate how predation or competition may be affected by differential responses in a temperature/light gradient. These questions are pertinent to understanding how species

64 may respond to modifications in local aquatic environments driven by global climate change.

Individually, these experiments show interesting trends, but taken together they provide additional complementary evidence about the important role temperature can play as a driver of distribution of Dinobryon. This field distribution study suggested that a temperature range between 9.3° C and 18.2° C is the non-random range of maximum distribution of Dinobryon. The field temperature-shift experiment also illustrated that warming water from 5° C to 16° C led to an increase in the absolute and relative abundance and biomass of Dinobryon present in a mixed phytoplankton community. This strengthens the case that a simple change in temperature can create a change in the distribution and competitiveness of organisms over a relatively short period of time. Finally, the TGI shows that warming of surface waters in spring can increase the prevalence of Dinobryon in deeper cooler water. Although none of these experiments and observations alone separately define the temperature range at which Dinobryon will thrive, in concert they suggest a temperature range in which Dinobryon populations will grow. These data do not suggest that temperature is the only factor to control distribution of Dinobryon in the water column. Light, competition for nutrients, and predation will likely play critical roles in their distribution. The role of mixotrophy in their nutrition was not investigated, but also is likely to influence Dinobryon populations. The TGI column experiments removed some environmental factors, such as light, nutrient limitations and predation, to determine

65 whether temperature within the natural range in the lake was a feasible driver of population distribution.

The intermediate temperatures at which I found the highest Dinobryon abundances in Lake Lacawac are typical of the depth of the spring oxygen maximum (Sanders, et al. unpublished data). Although chlorophyll data was not collected on 13 June 2005, it is clear from the Lugol’s counts that Dinobryon sp. was the most abundant species larger than 4 µm in length. At the depth of maximum Dinobryon population size on that date, there was a metalimnetic oxygen maximum often associated with a deep chlorophyll maximum (DCM) when oxygen is released as a biproduct of photosynthesis by phytoplankton (Figure 3.3).

The maximum abundance of Dinobryon sp. on this date occurred under light conditions <5% of surface PAR, and since they were the most abundant phytoplankton >4

µm the data suggest that they play an important role in changing the chemocline of the lake

– at least for oxygen (Figure 3.4). Bird and Kalff (1989) also found Dinobryon associated with a metalimnetic oxygen maximum and hypothesized that Dinobryon could not sustain bacterial consumption at the rate recorded because over an extended period of time all bacterial biomass in the DCM would be consumed. This would further emphasize not only the role of mixotrophs in the metalimnetic oxygen maximum, but also have larger relevance in the water column as water is mixed through this layer and could be effectively grazed by mixotrophs. Although stable populations of mixotrophs and a varying population of

66 autotrophs have been shown, this work also highlights the potential importance of mixotrophs in the food web (figure 3.1) (Sanders 1995).

Mixotrophs have been shown often to be important in food web dynamics. Removal of mixotrophs from food web systems can cause dramatic shifts in the food web dynamics

(Porter, Sherr et al. 1985). But mixotrophs are an important grazer that can have important impacts on bacteria (Sanders, Porter et al. 1989). There are many other studies that show the importance of mixotrophs in the food web (Havens and J. 1985; Bird and Kalff 1986;

Jones 1994; Hitchman and Jones 2000; Jones 2000; Todonleke, Jugnia et al. 2002; Titel,

Bissinger et al. 2003; Jost, Lwarence et al. 2004; McManus, Zhang et al. 2004; Ptacnik,

Sommer et al. 2004). This study illustrates that not only is Dinobryon the dominate phytoplankter at specific points in Lake Lacawac, but also it may be sensitive to a specific temperature stress. As we look at the impacts of a changing climate, these organisms may serve as sentinels to help predict larger changes in the near future.

Chapter 5

CONCLUSIONS AND FUTURE STUDIES

Dinobryon is a mixotrophic protist that sometimes feeds rapidly on bacteria as well as carries out photosynthesis. Previous studies have examined the possible that changes in nutritional strategies of mixotrophy toward more phototrophy or phagotrophy are based on environmental factors (Sanders, Porter et al. 1990; Caron, Sanders et al. 1993; Leeper and Porter 1995; Rothhaupt 1996; Holen 1999; Hansen, Skovgaard et al. 2000; Sanders,

Berninger et al. 2000; Skovgaard, Hansen et al. 2000; Urabe, Gurung et al. 2000; Sanders,

Caron et al. 2001). This is particularly important when attempting to determine the grazing impacts by mixotrophs, if that grazing is variable. For instance, Dinobryon has been shown to eat more in the ocean at 40 m than it does at 5 m at the same sampling point on the same day (McKenzie, Deibel et al. 1995). Temperature has been shown to affect photosynthesis so temperatures effect on this “switch” in nutritional strategies may lead to a better understanding of temperature effects on food web interactions.

There is a possibility that Dinobryon may change its nutritional strategy based on the conditions that the organism is found in. There are some suggestions that morphological changes in Dinobryon may occur with changes in the nutritional strategies, whether it be changes in the caudal spine, or changes in the lorica (Ishida and Kimura 1986). In another alga, Epiphyxias, a stalk-like appendage forms to allow it to capture prey that is not present

68 during time when it is simply autotrophic (Andersen and Wetherbee 1992). Similar changes that may occur as a result of temperature have been poorly studied in Dinobryon.

There is also evidence to suggest that higher temperatures may lead to encystment in Dinobryon sp. (Sandgren 1981; Sandgren 1986). A trend in the TGI suggested that warmer water may lead to increased encystment of Dinobryon sp, although it was not statistically significant, and an alternative hypothesis that algal encystment may offer protection from bacteria (Mayali, Franks et al. 2007) was not explored in this work. Cysts have been found in large abundance in marine sediments, and it seems only logical that the cysts could be circulated in the water column during spring turnover (Lewis, Yentsch et al.

1979).

Future studies could use the TGI to look at multiple trophic levels. For instance, diel vertical migration of zooplankton to avoid fish predators at the surface (Williamson,

Sanders et al. 1996; Gilbert and Hampton 2001; Ptacnik, Sommer et al. 2004). Can the migration of organisms be changed by temperature as opposed to light (Gilbert and

Hampton 2001)? The alga studied in this work appears to stratify in the water column on the basis of temperature not light, so can one set up a scenario where a small increase in surface temperature pushes the alga below the range of upward migration that zooplankton typically take to find the algal food at night.

The final idea that I think could be explored as a result of this work is to study changes in algal protein expression as a result of temperature change. Initial experiments suggested a change in protein expression with a change in temperature. These experiments would first be conducted by changing the temperature of Dinobryon, extracting the algal proteins and running an agarose gel to visualize the proteins. At that point bands, could be removed and sequenced, or analyzed with mass-spectroscopy to determine how protein expression changes with changes in temperature.

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APPENDIX

A.1 Dissolved oxygen seasonal and vertical profile from Lake Lacawac.

A.2 Asterionela seasonal and vertical profile from Lake Lacawac.

A.3 Strombidium. seasonal and vertical profile from Lake Lacawac.

A.4 Synura seasonal and vertical profile from Lake Lacawac.

A.5 Uroglena colonies seasonal and vertical profile from Lake Lacawac.

16-40µ 10-20µ round Cryptophyta Chlorophyta

8-20µ spiny Chlorophyta

Flag 6-10 µ Chrysophyta Oocystis

6-15µ Cryptophyta

16-40µ Chrytophyta

C Flag 6-10 µ Chrysophyta

10-20µ round Chlorophyta Dinobryon

6-10 µ round Chlorophyta

Synedra

Ankistrodesmus

6-10 µ round Chlorophyta 10-20µ round Chlorophyta

10-20µ round Chlorophyta Dinobryon

6-10 µ round Chlorophyta Synura

A.6 Percent Biovolume of phytoplanter sampled in the field UV temperature experiment. A. is at the start of the experiment. B is 5˚ C water with UV present. C is 5˚ C water without UV present. D is 16˚ C water with UV present. E is 16˚ C water without UV present .