ABSTRACT
IMPACT OF SIMULATED POLAR NIGHT ON ANTARCTIC MIXOTROPHIC AND STRICT PHOTOAUTOTROPHIC PHYTOPLANKTON
by Zev E. Cariani
Phytoplankton in polar regions experience long periods of continuous darkness annually during the polar night. Due to difficulties in performing field work during this period, it is largely unknown how phytoplankton endure this extreme transition from 24-hour daylight in the fall to several months of total darkness in the austral winter. The primary goal of this study was to compare physiological and photosynthetic responses of several Antarctic phytoplankton of variable trophic abilities (pure photosynthetic vs. mixotrophic) to simulated polar night conditions, including the transition seasons before and after winter. Two distinct responses were observed to extended darkness: (1) strict photoautotrophs (Chlamydomonas sp. ICE-MDV and Chlamydomonas sp. UWO241) exhibited functional downregulation their photosynthetic processes in the winter, followed by a lag phase of several days during mimicked spring, and (2) mixotrophs (Isochrysis sp. MDV and Geminigera cryophila) maintained functional photosynthetic apparatus, increased heterotrophy through the winter, and exhibited immediate growth upon return to light incubation. These differing responses to mimicked polar night conditions could represent two different strategies for surviving the long period of darkness in the phytoplankton’s natural environment.
IMPACT OF SIMULATED POLAR NIGHT ON ANTARCTIC MIXOTROPHIC AND STRICT PHOTOAUTOTROPHIC PHYTOPLANKTON
A Thesis
Submitted to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Masters of Science Department of Microbiology
by
Zev Emilio Cariani Miami University Oxford, Ohio 2018
Advisor: Dr. Rachael Morgan-Kiss Reader: Dr. D.J. Ferguson Committee member: Dr. Annette Bollmann Committee member: Dr. Mitchell Balish
©2018 Zev E. Cariani This thesis titled
IMPACT OF SIMULATED POLAR NIGHT ON ANTARCTIC MIXOTROPHIC AND STRICT PHOTOAUTOTROPHIC PHYTOPLANKTON
by
Zev Emilio Cariani
has been approved for publication by
Department of Microbiology
______
Advisor: Dr. Rachael Morgan-Kiss
______
Committee member: Dr. Mitchell Balish
______
Committee member: Dr. Annette Bollmann
Table of contents
P 1-18 Chapter 1 – Introduction to phytoplankton and the polar night
1. Phytoplankton metabolic and photosynthetic diversity
2. Survival of phytoplankton during the polar night
3. Phytoplankton in the McMurdo Dry Valleys
4. Phytoplankton isolates studied
5. Thesis objectives
Chapter 2 – Responses of Antarctic mixotrophic and strict photoautotrophic P 19-50 phytoplankton to mimicked polar night
1. Introduction and chapter objectives
2. Methods
3. List of experiments
4. Results
5. Discussion
6. Conclusions and significance
Chapter 3 – Physiological studies in Isochrysis sp. MDV and Geminigera cryophila P 51-61
1. Introduction
2. Methods
3. Results
4. Discussion
5. Conclusions and significance
References P 62-68
Appendix P 69-74
iii List of tables
Chapter 1
Table 1.1. Three major categories of phytoplankton trophic abilities. P 15 Table 1.2. Diversity of chlorophyte, haptophyte, and cryptophyte photosynthetic P 16 apparatus components.
Chapter 2
Table 2.1. Relative values for important photosynthetic and growth parameters P 43 by the end of the dark incubation.
Chapter 3
Table 3.1. Growth and photochemistry of Isochrysis sp. MDV during C source P 60 experiments in flasks under low light.
Table 3.2. Growth and photochemistry of G. cryophila during C source P 61 experiments in flasks under low light.
iv List of figures
Chapter 1
Figure 1.1. An example induction curve from the green algae, Chlamydomonas P 17 reinhardtii
Figure 1.2. Typical, under-ice daily PAR values recorded over the course of a P 18 year at 10m depth in Lake Bonney.
Chapter 2
Figure 2.1. Major cellular and photosynthetic processes in strict photoautotrophic P 39 and mixotrophic phytoplankton during the summer and winter.
Figure 2.2. Day number key for the mimicked seasonal photoperiod stimulus. P 40
Figure 2.3. The physiological indicators: growth (A, B), chlorophyll per cell (B), P 41 and FV/FM (maximum quantum yield) (C, D) of the strict photoautotrophic and mixotrophic cultures during mimicked polar night experiments of 28 days continuous darkness.
Figure 2.4. The photosynthetic functioning parameters: YPSII (effective quantum P 42 yield) (A, B) and NPQ (non-photochemical quenching) (C, D) of the strict photoautotrophic and mixotrophic cultures during mimicked polar night experiments of 28 days of continuous darkness.
Figure 2.5. Example induction curves recorded for UWO (A), ICE (C), Isochrysis P 44 (B), and G. cryophila (D) during mimicked polar night experiments.
Figure 2.6. Relative photosynthetic protein abundance of rubisco large subunit P 45 (rbcL) and PSII reaction center subunit (psbA) in cultures of chlorophytes (A and C) and mixotrophs (B and D) during mimicked polar night experiments.
Figure 2.7. Representative western blots used for quantification from UWO (A) P 46 and G. cryophila (B) during mimicked polar night experiments.
v Figure 2.8. Neutral storage lipid and chlorophyll autofluorescence in cultures of P 47
ICE (A and C) and the two mixotrophic species (B and D) during mimicked polar night experiments.
Figure 2.9. Algal cell diameter during mimicked polar night experiments. P 48
Figure 2.10. Cell viability (A), culture density and FV/FM (B), and 16S rRNA P 49 sequencing analysis (C) of ICE cultures during a mimicked polar night experiment with 20 days of continuous darkness.
Figure 2.11. β-gam activity of the two mixotrophic species under light and dark P 50 incubation time points during a mimicked polar night experiment.
Chapter 3
Figure 3.1. Relative amounts of neutral storage lipids (A), chlorophyll P 58 autofluorescence (B), culture density (C), and FV/FM (D) results from a light intensity experiment with ICE, Isochrysis, and G. cryophila.
Figure 3.2. β-gam activity of Isochrysis cultures incubated under conditions of P 59 replete, low N, and low P.
vi Acknowledgements
Thank you Miami University Microbiology Department for providing a funded Masters of Science degree program at a time when it seems many other universities are turning away from these degrees in favor of Ph.D. This aspect was a major draw for me entering your program and I hope that you continue to offer and develop this path for students in the future. During my time here, I thoroughly enjoyed taking such varied classes as medical mycology and limnology taught by professors who did a great job engaging all of their students.
Thank you Annette for providing mentorship during the classes I TA’d for you. I appreciated the organized manner in which you ran the lab sections and taught Microbial Physiology; it always felt as though you welcomed whatever questions we had.
Thank you DJ for reading this thesis so quickly and allowing me to defend before I left Oxford. I really appreciate it and enjoyed reading through the lighthearted comments.
Thank you Rachael for starting your research program on Algae living in inland Antarctic lakes. You never turned down ideas for experiments are were always willing to try new techniques. I learned so much doing research in your lab and enjoyed learning the nuances of all of the different instruments. Wish you the best of luck in your research moving forwards. Microbial ecology, phytoplankton, and photosynthesis are all amazing fields of new research and I am grateful for the opportunity to experience them in your lab.
Thank you Isha, Shasten, Greg, and Isaiah for being great lab mates. Isha, I am so grateful for your organization in keeping the lab up and running. It would have been hard to get anything done without you! Shasten you were so much fun to work with preparing for the Antarctic field season and dealing with all of the singular issues the Phyto-pam presented. Thank you for always keeping a lighthearted and positive outlook.
Thank you everyone else in the Microbiology Department who went to all of the preruns, classes, and seminars the past few years. I am going to miss all of you and hope that everyone is able to forge ahead through all of the changes going on in the building. Cheers and best of luck!
1 Chapter 1 - Introduction to phytoplankton and the polar night
Thesis objective
The primary goal of this study was to compare physiological responses of several Lake Bonney phytoplankton of variable trophic abilities to simulated Antarctic winter conditions (i.e. polar night). Lake Bonney is a well-studied, perennially ice-covered lake located in the McMurdo Dry Valleys (MDV) of Antarctica. It contains an entirely microbial food web driven by the primary production of eukaryotic phytoplankton. An important, environmental stressor affecting these phytoplankton is the polar night period of 4-5 months of continuous darkness. MDV lake phytoplankton are known to have diverse trophic abilities, which could influence their photosynthetic and physiological strategies for enduring the polar night stressor. Studying algae wintertime survival strategies also has significance for better understanding primary producers in polar regions more broadly.
1. Phytoplankton metabolic and photosynthetic diversity
1.1 Trophic diversity of eukaryotic phytoplankton Early studies of plankton often placed them into either strictly autotrophic or heterotrophic functional types much like those used to describe plants and animals. Since the 1980s and 90s, the true characteristics of plankton as existing along a continuum between the two nutritional modes has been increasingly appreciated (e.g. Sanders 1991, Stoecker 1998). Heterotrophs with no ability to utilize light energy rest at one end of the continuum and purely photoautotrophs with no ability for organic C uptake lie at the other (Flynn et al. 2013). Unlike plants, virtually all phytoplankton are osmotrophic to some extent, which means that they can utilize dissolved organic compounds (DOC) (Flynn et al. 2013). A classic example of a photo-osmotroph is the green algae, Chlamydomonas reinhardtii, which is primarily phototrophic but can also metabolize a range of small carbon compounds (Singh et al. 2014). The most common representatives of osmotrophic phytoplankton are cyanobacteria and diatoms (Flynn et al. 2013). As this type of nutrition is common, researchers often do not consider osmotrophic protists to be true “mixotrophs”. Mixotrophs acquire additional carbon and nutrient
1 through phagocytosis and digestion of smaller prey cells (Table 1.1) (Stoecker et al. 2016, Flynn et al. 2013, Mitra et al. 2014). Mixotrophic algae are diverse in their modes of living and can be categorized into constitutive versus non-constitutive (Mitra et al. 2016). Constitutive mixotrophs acquire chloroplasts from parental cells during cell division. An example of this group are most Antarctic cryptophytes, which are bacterivorous as well as phototrophic through chloroplasts well integrated into their life cycles (Bell and Laybourn-Parry 2003). Non-constitutive mixotrophs do not inherit parental chloroplasts. An example of non-constitutive mixotrophs are oligotrich ciliates who feed on algae, but often retain chloroplasts from the ingested algae for phototrophy (Stoecker et al. 2016). These chloroplasts are continually degraded and acquired through phagocytosis rather than from parental cells (Stoecker et al. 2016). Among constitutive mixotrophs, there are generally two ways of balancing phototrophy and heterotrophy (Stoecker et al. 1998). The first mechanism is obligate phototrophy, where photosynthesis is essential for growth and heterotrophy is largely supplemental. An example of this type can be found in the chrysophyte, Dinobryon cylindricum, which requires light for growth and ceases bacterivory in darkness (Caron et al. 1992). The second mechanism is obligate heterotrophy, where phagotrophy is obligate but phototrophy is facultative. An example of this type is the chrysophyte, Poterioochromonas malhamensis, which was found to be reliant on phagotrophy at all times and phototrophy was induced only during conditions of low bacteria concentration (Sanders et al. 1990). 1.2 Functioning of the photosynthetic apparatus
Photosynthetic bacteria, algae and plants acquire light energy through the use of diverse membrane-bound pigment-protein complexes termed the photosynthetic apparatus. In eukaryotic phototrophs (plants and algae), the photosynthetic apparatus is associated with thylakoid membranes inside the chloroplast. Photosynthesis begins with absorption of light energy by light harvesting antenna, which transfer energy to one of two reactions centers (photosystem I, PSI; photosystem II, PSII). These antennae consist of pigments to perform light absorption and light harvesting proteins to assist in correctly orienting the pigments. During linear electron flow, photosynthesis begins when light energy is captured by PSII antenna and transferred to the reaction center of PSII where it is used to hydrolyze of water and perform charge separation. Electrons are then transferred to cytochrome b6f, excited again at PSI, and
2 eventually used to reduce NADP+ to NADPH. ATP is also produced through ATP synthase, which is powered by the concurrent generation of a proton gradient between thylakoid lumen and stroma. Algae have several methods of regulating photosynthesis and avoiding the damaging effects of light energy capture, such as generating reactive oxygen species. The first major mechanism is called the xanthophyll cycle and is induced by pH gradients produced during high light intensities. Excess excitation energy is dissipated in a regulated manner as heat through antennae complex rearrangements and the conversion of certain pigments to forms that are able to perform photoprotection (Ruban 2016). A second mechanism, state transition quenching, helps to balance excitation energy in the photosynthetic electron transport chain and consists of changing light harvesting complex (LHC) associations between PSII and PSI (Finazzi 2005). Finally, sustained downregulation of photochemical energy generation can result from degradation of functioning PSII reaction centers, which are normally a large source of reactive oxygen species (Malnoë 2018). 1.3 Diversity of the photosynthetic apparatus
Light harvesting systems are the most variable aspect of photosynthesis due to the importance of being adapted to different light environments (Nyogi and Truong 2013). Different classes of algae contain characteristic pigment profiles of the three major classes of pigments: chlorophylls (all algae contain Chl a), carotenoids, and phycobillins. The major types of proteins present in antennae are light harvesting type proteins (plants and most algae) and phycobiliproteins (cyanobacteria, cryptophytes, and red algae). The three types of algae studied in this thesis have differing components in their photosynthetic apparatus (Table 1.2). Chlorophytes are the best characterized due to their relatedness to land plants and use the major accessory pigment chlorophyll b for light harvesting. Chlorophyll b is primarily bound to PSII antenna proteins. Ratios of Chl a/b are reliable estimators of relative antenna size with low vs. high Chl a/b indicating a large vs. small antenna, respectively (Kitajima and Hogan 2003). Chlorophytes have several different types of light harvesting proteins and have flexible processes of reducing photochemical output through the xanthophyll cycle with the pigment violaxanthin. The light harvesting complex stress-related (LHCSR) protein has been shown to be necessary for xanthophyll cycle processes (Nyogi and Truong 2013).
3 In contrast with chlorophytes and plants which acquired their chloroplast via primary endosymbiosis of a cyanobacteria, haptophytes and cryptophytes likely acquired their chloroplasts from engulfing red algae during the process of secondary endosymbiosis (Burki et al. 2012). Haptophytes have the major accessory pigments chlorophyll c1, chlorophyll c2, and fucoxanthin, the last of which gives them their brown color. Haptophyte LHC proteins are altered compared to chlorophyte LHCs as they associate with these different pigments (Neilson and Durnford 2010). Haptophytes are thought to have a xanthophyll cycle using the pigment diatoxanthin and an LHCSR homolog (Nyogi and Truong 2013).
Cryptophytes have the major accessory pigment chlorophyll c2 and unique types of phycoerythrin and phycocyanin, which give them a pink coloration (Scholes et al. 2006). They use unique LHCs and phycobiliproteins in their antennae. Surprisingly, rather than having the phycobiliproteins arranged in phycobilisomes as is the case with red algae and cyanobacteria, cryptophytes have their phycobiliproteins densely packaged in the thylakoid lumen (Scholes et al. 2006). Cryptophytes have been found to have rapid and reversible processes similar to the xanthophyll cycle, but the mechanisms by which this occurs are still being investigated as it appears to be a novel type. None of the usual xanthophyll pigments are present, but this process does appear to be pH dependent and occur in the LHCs rather than in the phycobiliproteins (Prasil et al. 2012). 1.4 Studying photosynthetic processes: chlorophyll fluorescence analysis
A popular method for monitoring photosynthetic performance in algae is chlorophyll fluorescence analysis. Both photochemistry and energy dissipation mechanisms are monitored via fluorescence quenching. The possible pathways for energy transfer after pigment excitation have been categorized into photochemical quenching (qP), non-photochemical quenching (qN), and fluorescence. Photochemical quenching refers to the use of light energy for electron transport and chemical energy production. Non-photochemical quenching consists of other processes which compete with fluorescence, such as xanthophyll cycles (often indicated by the parameter NPQ) and state transitions.
At room temperature, fluorescence emitted by photosynthetic systems varies in a predicable way when stimulated by certain light intensities. There are several methods for fluorescence monitoring, one of most commonly used is pulse amplitude modulated (PAM) chlorophyll fluorescence. Photosynthetic organisms are probed for their fluorescence yield by a
4 weak measuring light. This measuring light flashes at a particular frequency which the detector is also tuned into. In this way, the fluorescence yield resulting from the measuring light stimulation is the only thing recorded and noise from fluorescence stimulated by other sources are cancelled out (Schreiber 2004). Consequently, the propensity of the photosynthetic apparatus for fluorescence is able to be determined during the course of changing light regimes. The majority of this fluorescence comes from PSII and thus this analysis can be considered a probe for PSII activity alone (Kalaji et al. 2017). Induction curves are commonly used to measure photosynthetic parameters and are started with dark adapting the sample. Dark adaption is done to oxidize all of the PSII reaction centers such that they are primed for the initial measurements of maximum photosynthetic efficiency, FV/FM (Baker 2008). FV/FM is first measured by probing the system after dark adaption with the measuring light to determine FO and then with a saturating pulse to determine FM (Fig.
1.1). FV is the difference between FM and FO. FO is often low in the case of healthy organisms that transfer light energy efficiently from their antenna to the associated oxidized reaction centers. FM represents the maximum possible fluorescence yield because it is measured during saturating pulse, which reduces all of the reaction centers and increases fluorescence by inhibiting photochemical quenching. FV/FM thus gives insights into the proportions of PSII reaction centers that can perform photochemistry, which is a useful stress indicator because PSII has been shown to be highly sensitive to changing environmental conditions (Murchie and Lawson 2013). NPQ and ΦPSII are other important parameters for measuring photochemical processes and are derived from the fluorescence measurements FO’ and FM’. FO’ is the baseline fluorescence under actinic light illumination, which is a light designed to drive photosynthetic electron transfer at an intensity selected by the user. FM’ is the fluorescence recorded from a saturating pulse under actinic illumination. The difference between FM’ and FM is used to calculate the parameter NPQ, which gives insights into the strength of xanthophyll cycle type processes occurring (Ruban 2016). Another parameter calculated from these fluorescence values is ΦPSII, which is the effective quantum yield of PSII. ΦPSII is an indicator of how efficient phototrophs are at using a given intensity actinic light for photochemistry.
5 2. Survival of phytoplankton during the polar night
2.1 Overview of phytoplankton dark survival strategies
Algae in aquatic environments are regularly exposed to long periods of darkness through vertical mixing out of the photic zone or during the Arctic and Antarctic polar nights. They survive these long periods of darkness through spore/cyst production, adaptive nutrient acquisition, and physiological/photosynthetic changes (McMinn and Martin 2013). Spores (single cells) or cysts (group of cells) are structures resistant to a variety of environmental stressors. They have been known to persist through the polar night in sea ice or sink down into the sediment and germinate upon the return of light (Backhaus et al. 2003, Fryxell 1989, Keafer et al. 1992). Forming these structures is a particularly common strategy among polar marine dinoflagellates and diatoms, but in the McMurdo Dry Valley lakes where those taxa are rarer, a chlorophyte (Chlamydomonas species) was still observed to form high numbers of spores during the middle of the polar night (Howes et al. 2000). Spores/cysts have also been shown to have reduced photosynthetic abilities (Anderson et al. 1987) and increased amounts of energy storage products (Doucette and Fryxell 1983). Alternative metabolic processes in algae can allow for sustained respiration in the absence of light. Osmotrophic algae can have flexible metabolisms such that they can survive extended darkness by the uptake of dissolved organic carbon (DOC), such as Chlamydomonas reinhardtii metabolizing acetate (Johnson and Alric 2013). In polar areas, Antarctic diatoms have been shown to increase uptake of various DOC compounds in response to low light (Rivkin and Putt 1987). Mixotrophic algae have been observed to increase bacterivory in response to low nutrient or light levels, indicating the benefits of having dual trophic modes for mitigating stressful conditions like long periods of darkness (Nygaard and Tobiesen 1993, Floder et al. 2006). Algae often undergo other physiological changes to survive long periods of darkness, which include reductions in metabolism and breakdown of energy storage products. Using the broad-spectrum metabolic measurement, fluorescein diacetate (FDA) hydrolysis on a number of algal species, Jochem (1999) found two differing responses to darkness: one group reduced metabolic activity to less than 10% of initial values and the other group remained unchanged. In contrast, several mixotrophic species were found to increase their metabolic activity during darkness when cultured with heat killed bacteria (Jochem 1999). Increases in energy storage molecules (e.g. lipids and starch) have been well documented for marine algae at the onset of
6 winter (Fryxell 1989, Zhang et al. 1998). High amounts of starch have also been observed in several MDV lake protists during the transition into and middle of the polar night (Howes et al. 2000, Laybourn-Parry 2002). One freshwater cyptophyte species was found to survive extended darkness through slow respiration of stored carbohydrate storage compounds (Morgan and Kalff 1975). Another Arctic diatom species was measured to respire stored neutral lipid compounds (triacylglycerol or TAG) during a dark period of 8 weeks (Schaub et al. 2017). Photosynthetic processes are usually downregulated during periods of metabolic dormancy, which is possibly part of a strategy to avoid oxidative stress from light intensities experienced during this time. An example of this are dormant, overwintering evergreen trees where PSII reaction centers are degraded, while the light harvesting antenna increase their capacity for energy dissipation as heat (Gilmore and Ball 2000, Öquist and Hüner 2003). PSII is often degraded during photosynthetic downregulation because oxidative stress comes primarily from PSII reaction centers (Shigeoka and Foyer 2011). 2.2 In-situ studies of marine phytoplankton during the polar night Algae living in polar oceans have been found to persist through the polar night in-situ, albeit at low densities. Chlorophyll concentrations measured in surface waters during the arctic polar night are extremely low (e.g. <0.06 μg/L; Berge et al. 2015). Identification of phytoplankton during polar winters has shown similar species composition compared to other seasons, suggesting minimal change in species distribution seasonally (Niemi et al. 2011, Rokkan and Seuthe 2011). A study around the arctic island of Svalbard at the height of the polar night detected two important bloom-forming algae, Micromonas (chlorophyte) and Phaeocystis (haptophyte), distributed over widespread areas and depths (Vader et al. 2014). Evidence for the persistence of similar species of polar algae throughout the year beckons the question of how they are surviving the period of continuous darkness. In general, larger algae (e.g. diatoms and dinoflagellates) show a greater propensity for producing dormant, resting stages than pico or nanoflagellates (Vader et al. 2014). However, one study in Greenland incubated a number of freshly sampled algae in darkness and concluded that cyst/spore formation was not as important as the breakdown of storage metabolites and nutritional versatility (Zhang et al. 1998). Mixotrophy has been found to be particularly important in Arctic regions with contributions to bacterivory sometimes estimated to rival or exceed those of traditional heterotrophic consumers (Sanders and Gast 2011). In the oceans off Antarctica,
7 mixotrophy has also been identified as an important adaptation for phototrophs surviving the extreme environment with major stressors such as the polar night (Moorthi et al. 2009). Understanding wintertime survival strategies is crucial to studying primary production in polar oceans. Populations which persist through the polar night are important for seeding spring and summer blooms (Palmisano and Sullivan 1983, Niemi et al. 2011). Blooms in polar regions are often of such massive scale as to rival those anywhere else in the world; therefore, polar phytoplankton contribute significantly to global C cycling (Westberry et al. 2008). There is a need for more detailed studies to understand the physiology of basic phytoplankton survival strategies. Also, a better understanding of how algae behave seasonally could help researchers assess possible changes in the future under predicted conditions of earlier ice off dates and higher light intensity in the water column (McMinn et al. 2010). Since there is limited understanding of how polar primary producers survive the winter darkness, predicting impacts of future changes remains difficult. 2.3 Lab controlled studies of photosynthetic responses to dark incubation
Several laboratory studies have taken the approach of incubating polar algal isolates in extended darkness to observe their wintertime survival strategies. This approach avoids difficulties of doing fieldwork in polar regions during complete darkness and also allows for additional physiological experiments which are not feasible in natural communities. Thomas and Peters (1996), investigated the response of five diatom species from the Southern Ocean to long term darkness and found that all of the species remained in a vegetative stage rather than forming resting spores. Additionally, the diatoms retained a low capacity for photosynthetic carbon assimilation throughout the darkness, and chlorophyll remained steady after initially decreasing upon dark incubation (Thomas and Peters 1996). Another study on marine Antarctic diatoms found that they had maximum darkness survival times of just 30-60 days (Reeves et al. 2011). Extended dark incubation also had large effects on their photosynthetic parameters, such as reducing maximum quantum yield (FV/FM) from optimal levels to near 0 (Reeves et al. 2011). The chlorophyte, Koliella antarctica, is one of the best studied psychrophilic algae for winter adaptations. It survives long-term dark incubation and resumed growth after a dark incubation of 60 days (Ferroni et al. 2007). This chlorophyte also exhibited substantial chloroplast reorganization during the period of darkness with reductions in functional PSII, but relative stability of LHCs (Ferroni et al. 2007). Similarly, when a yellow-green snow filamentous alga was
8 incubated in the dark for two months it degraded PSII cores, but maintain associated LHCs (Baldisserotto et al. 2005). It is possible that the strategy of structurally downregulating PSII while maintaining the antenna in a functionally downregulated state provides protection from oxidative stress while maintaining major photosynthetic apparatus components. Interestingly this overwintering response was similar to that of evergreen trees, which suggests a broad phylogenetic distribution of this particular strategy (Öquist and Hüner 2003). The Antarctic red macroalgae Palmaria decipiens incubated in 6 months of darkness experienced minimal photosynthetic change for three months, after which FV/FM reduced dramatically (Lüder et al. 2002). Substantial pigment reductions in chlorophyll and phycobilins were observed after 4 months of darkness (Lüder et al. 2002). Commonalities observed between all of these photosynthetic responses include reductions in photosynthetic processes. These studies did focus exclusively on strict photoautotrophs while the photosynthetic response of mixotrophic algae could be comparatively unique. Most ex-situ studies did not mimic natural conditions of the polar night transition (i.e. the fall season) prior to dark incubation, which is an important factor to consider as doing so has been shown to aid several algae in surviving dark incubation. Palmisano and Sullivan (1982, 1983) showed that preconditioning marine algae by mimicking natural conditions during the transition from fall to winter prior to dark incubation enhanced survival. Incidentally, a major survival strategy of the diatoms in this study was to increase storage and use of their cellular carbon reserves, and upregulate heterotrophic processes (Palmisano and Sullivan 1982).
3. Phytoplankton in the McMurdo Dry Valleys
3.1 McMurdo Dry Valley ice covered lakes While the continent of Antarctica is almost completely covered by a thick sheet of ice, there are several ice-free regions where sunlight is able to reach liquid water on the continent’s surface and provide energy for simple food webs therein. The largest ice-free regions are the McMurdo Dry Valleys (MDV) of Southern Victoria Land. The MDV harbor numerous ice-covered lakes, which contain microbial ecosystems. Lake Bonney represents one of the most intensively studied lakes in the area, owing to more than two decades of activities as part of the MDV Long Term Ecological Research (LTER) program.
9 Lake Bonney is permanently covered in approximately 4 m of ice and contains steep chemical gradients with hypersaline bottom waters (Spigel and Priscu 1996). Due to these characteristics, the lake is among the most consistently stratified bodies of water on the planet with stable layers of properties such as dissolved nutrient concentration and water temperature (Priscu et al. 1999). Temperatures in Lake Bonney range from -10C to 60C and nutrient concentrations are oligotrophic with nutrients increasing at and below the permanent chemocline (15-20 m depth) (Priscu et al. 1999). Besides nutrients, light often limits primary production as well with irradiances rarely above 10 µmol photons m-2 s-1 due to attenuation from the ice cover. Primary production in Lake Bonney is performed by photosynthetic nanoflagellates (e.g. chlorophytes, haptophytes, and chryptophytes) which provide DOC for the microbial loop (Li et al. 2016). Tertiary predators are heterotrophic nanoflagellates and ciliates, which are the highest trophic level supported in this shortened food web (Li et al. 2016). From a microbiological perspective, the extreme environmental conditions of the MDV lakes are worth studying because they provide unique examples of cellular adaptation under the environmental limits of life. Bacteria exhibit low-temperature adapted enzymes, antifreeze proteins, and high amounts of polyunsaturated fatty acids (Laybourn-Parry 2002). The green algae, Chlamydomonas sp. UWO241 is currently being studied as a model for low temperature/hypersaline adapted photosynthesis (Dolhi et al. 2013). MDV lakes are also sentinels for climate change with trends recorded in the multi-decade datasets of the LTER program. Drastically changing conditions, such as lake level rise in Lake Bonney, are visible reminders that even recently discovered pristine environments at the bottom of our planet cannot escape anthropogenic impacts (Spigel and Priscu 1996). 3.2 McMurdo Dry Valley phytoplankton during the polar night
Photoautotrophs in MDV lakes experience extreme seasonal shifts in photosynthetically active radiation (PAR) with the polar night period sometimes exceeding 5 months of continuous darkness (Morgan-Kiss et al. 2016). In Lake Bonney’s water column, the transition into the polar night involves PAR at shallow depths declining from ~20 to 0 µmol photons m-2 s-1 in the span of just one month. Photoperiods change from continuous sunlight to declining light-dark cycles and continuous darkness during this time (Fig. 1.2) (Bielewicz et al. 2011). These seasonal changes in PAR, especially when combined with other extremes such as hypersalinity, oligotrophy and low
10 temperature stress, are some of the most stressful conditions phototrophs must cope with (Morgan-Kiss et al. 2016). During the transition between fall and winter, phytoplankton communities in Lake Bonney undergo shifts in relative abundance and alterations in photosynthetic processes. Phytoplankton photosynthetic rates in-situ decreased rapidly in the fall as PAR declined (Kong et al. 2012). However, community chlorophyll a levels increased during this period, suggesting that populations of photoautotrophs became more shade adapted prior to the start of continuous darkness (Kong et al. 2012, Morgan-Kiss et al. 2016). In a study of protist populations during the polar night transition, Bielewicz et al. (2011) found that some protists predicted to be mixotrophic increased in abundance relative to non-mixotrophs (e.g. stramenopiles increased relative to chlorophytes). Mixotrophy is an important characteristic for surviving the polar night in MDV lakes. Mixotrophic algae are the dominant primary producers in many MDV lakes, such as cryptophytes in Lakes Hoare and Fryxell (Laybourn-Parry et al. 2005). Grazing studies have shown that mixotrophs contribute significantly to total rates of bacterivory, often exceeding traditional bacterivore like heterotrophic nanoflagellates (Thurman et al. 2012). Mixotrophic taxa increase in population size at the end of the polar night before the return of light, which is thought to allow them to enter the short summer season with active populations (Howes et al. 2000, Laybourn-Parry 2002, Thurman et al. 2012). Although the importance of mixotrophy for surviving the polar night in the extreme environments of MDV lakes has been documented, gaps remain in our understanding of the physiology and photochemistry of this important group of algae during long periods of darkness.
4. Phytoplankton isolates studied for this project
4.1 Rationale for studying MDV lake isolates Isolates from MDV lakes are well-suited for studying their responses to the polar night ex-situ. As Palmisano and Sullivan showed (1982, 1983), algae survival during dark incubation is enhanced by first preconditioning the cells (i.e. mimicking natural changes experienced in late fall). Mimicking fall conditions of marine environments ex-situ involves changing multiple variables like temperature, salinity, and nutrients. In contrast, all of those factors are remarkably stable annually in Lake Bonney: variations in PAR is one of the few changing environmental
11 parameters (Priscu et al. 1999). Photoperiods in MDV lakes are also among the most extreme on earth, which likely results in more observable, drastic survival strategies in the phototrophs living there than by members living at lower latitudes. To our knowledge there have not been many detailed photosynthetic studies on mixotrophic algae incubated in long term darkness. This is an important topic as mixotrophic algae are abundant in polar habitats and play important roles in the global carbon cycle. In addition, physiological and photosynthetic responses of mixotrophs are likely to be unique compared with the better studied, osmotrophic algae. Thus, there are open questions regarding wintertime survival adaptations in this important group of algae. For example, to what extent do mixotrophic algae rely on heterotrophic processes? Do mixotrophs downregulate their photosynthetic apparatus in the winter or do they maintain it in a functional state? The latter possibility could give them an advantage in being able to immediately use light upon its return at the end of the polar night. Indeed, mixotrophic species have often been observed increasing in numbers and dominating phytoplankton assemblages during polar spring (Gast et al. 2018). 4.2 MDV lake phytoplankton in this project The present study investigated the response of two species of mixotrophic algae to mimicked polar night photoperiods. The first species, Isochrysis sp. MDV, is an abundant phytoplankton in Lake Bonney, which dominates the chemocline (Kong et al. 2012). It has been shown to possess an acidic food vacuole as well as two chloroplasts, but the extent of its mixotrophic abilities are still largely unknown (Li et al. 2016). The second species which will be the focus of this study is a marine cryptophyte, Geminigera cryophila, which is closely related to a cryptophyte species in Lake Bonney (Li et al. 2016). G. cryophila has been shown to ingest bacteria at rates similar to those in MDV lakes and increase ingestion in response to dark incubation (McKie-Krisberg et al. 2015). G. cryophila is also important prey for larger heterotrophic protists (Gast et al. 2018). The chlorophyte isolates studied are strict photoautotrophs and include the psychrophilic Chlamydomonas sp. UWO241 (UWO) and Chlamydomonas sp. ICE-MDV (ICE). There are open questions concerning the basic wintertime survival strategies of both chlorophytes, such as whether or not they form spores or breakdown energy storage compounds. ICE was recently isolated and is the most abundant chlorophyte in Lake Bonney, particularly at shallow depths (Teufel et al. 2016). UWO was isolated more than 20 years ago from the hypersaline, deep photic
12 zone of Lake Bonney. UWO represents one of the best studied psychrophilic, pure photosynthetic algal strains, which includes several studies considering winter survival and adaptation (Morgan-kiss et al. 2006). 4.3 Impact of polar winter on the Lake Bonney alga, Chlamydomonas sp. UWO241 The response of UWO to loss of light stimulation has been investigated through experiments of varied light qualities, which could have implications for its survival during the polar night (Morgan-Kiss et al. 2005). When cultures of UWO were incubated in red light alone, cells unexpectedly ceased growth, increased respiration rates, and underwent numerous photosynthetic changes (Morgan-Kiss et al. 2005). These changes included functionally downregulated PSII and a shift in LHCII 77K fluorescence to shorter wavelengths, suggesting LHCII uncoupling from PSII. Morgan-Kiss et al. (2005) postulated that this unusual response to red light may be due to an inability to sense red light. The natural light in Lake Bonney is blue- green in spectrum with no red wavelengths (440-580 nm; Lizotte and Priscu 1992). Therefore, responses of UWO in lab experiments to red light may be similar to physiological changes in response to polar night. With the return of light, the LHCII antennae could be re-associated with PSII to rapidly upregulate photosynthetic processes. In the low temperature and oligotrophic environment of Lake Bonney, retaining, rather than degrading, the photosynthetic apparatus through the polar night may be crucial to the survival of strict photoautotrophs (Morgan-Kiss et al. 2006). However, this functional model was never tested under true mimicked winter conditions. The photosynthetic response of UWO to the polar night was also observed in-situ by transplanting isolate cultures back into their natural environment for several weeks during the fall-winter transition (Morgan-Kiss et al 2016). As winter approached, UWO became more shade acclimated and reduced RubisCO protein (RbcL) abundance, but was found to retain substantial levels of PSII reaction center (PsbA) protein. This indicates downregulation of C-fixation potential, while the photosynthetic apparatus was maintained (Morgan-Kiss et al. 2016). These results are in agreement with the previously introduced model for UWO’s wintertime photosynthetic response as well as natural population photosynthetic rates and chlorophyll concentrations. Finally, seasonal trends in expression of major photosynthetic genes (rbcL, psbA) closely matched that of the natural populations in the lake, which also validated the effectiveness
13 of representing phytoplankton populations in Lake Bonney with pure cultures (Morgan-Kiss et al. 2016). 5. Thesis objectives The primary goal of this study was to compare physiological responses of several Lake Bonney phytoplankton of variable trophic abilities to simulated Antarctic winter conditions (i.e. polar night). Lake Bonney is a well-studied, perennially ice-covered lake located in the McMurdo Dry Valleys (MDV) of Antarctica. It contains an entirely microbial food web driven by the primary production of eukaryotic phytoplankton. An important environmental stressor affecting these phytoplankton is the polar night period of 4-6 months of continuous darkness each year. I hypothesized that strategies for surviving this darkness would range from metabolic and photosynthetic downregulation to increased heterotrophic activity. Chapter 2 details the experiments conducted for this investigation, which were divided into two aims based on algal trophic modes. The first aim focused on how strict photoautotrophs adjust their photosynthetic and physiological processes in response to the polar winter. The second aim investigated the strategies utilized by mixotrophic phytoplankton. Chapter 3 details the experiments conducted with a secondary goal of characterizing both of the mixotrophic species, which are both representative of dominant phytoplankton groups in MDV lakes.
14 Table 1.1. Three major categories of phytoplankton trophic abilities.
Trophic Mode Description Major constituents
Photo-osmotrophy Primarily phototrophic with Diatoms, Coccolithophores, (i.e. strict ranging capacities of nutrient Cyanobacteria, most chlorophytes, photoautotrophy) uptake by osmosis (non- and many other phytoflagellates phagocytic)
Constitutive Phagotrophy coupled with Mixotrophic phytoflagellates (e.g. Mixotrophy phototrophy. Well integrated haptophytes, cryptophytes, some (Mitra et al. 2016) and inherited chloroplasts chlorophytes) and most dinoflagellates
Non-constitutive Acquired phototrophy by Mixotrophic rhizaria (e.g. mixotrophy heterotrophic plankton foraminifera, radiolaria), (Mitra et al. 2016) through ingestion and mixotrophic ciliates, and some retention of chloroplasts dinoflagellates
15 Table 1.2. Diversity of chlorophyte, haptophyte, and cryptophyte photosynthetic apparatus components.
Algal group Major accessory PSII antenna NPQ mechanism pigments
Chlorophytes Chl b Chlorophyll a/b, major and minor Xanthophyll cycle LHC proteins with violaxanthin
Haptophytes Chl c1, Chl c2, Chlorophyll a/c LHC proteins (also Xanthophyll cycle fucoxanthin called fucoxanthin-chlorophyll LHC) with diatoxanthin
Cryptophytes Chl c2, Unique chl a/c LHC and phycobilin pH dependent, but phycobilins proteins. Phycobilins form a matrix uncharacterized structure in thylakoid lumen rather than phycobilisome.
16
Figure 1.1. An example induction curve of the green algae, Chlamydomonas reinhardtii shows the determination of the 4 basic fluorescence measurements: FO, FM, FO’, and FM’. ML refers to measuring light, SP to saturating pulse, and AL to actinic light.
17
Figure 1.2. Typical, under-ice daily photosynthetically active radiation (PAR) values recorded over the course of a year at 10 m depth in Lake Bonney. The polar night period of continuous darkness lasts for 5-6 months at this depth from April to September (MDV-LTER dataset).
18 Chapter 2 – Responses of Antarctic mixotrophic and strict photoautotrophic phytoplankton to mimicked polar night
1. Introduction and chapter objectives
Phytoplankton in marine environments experience long periods of darkness both when mixed out of the photic zone and during the winter period of continuous darkness in polar regions (i.e. the polar night). Those species which are best able to survive the polar night provide the basis for spring blooms (Palmisano and Sullivan 1983, Niemi et al. 2011). These blooms contribute significantly to global carbon cycling (Westerberry et al. 2008). Since sampling for algae during polar winters remains challenging, researchers have often turned to incubating isolates species under long periods of darkness in laboratory experiments. These studies have shown that photo-osmotrophic (i.e. strict photoautotrophic or not capable of phagotrophy) phytoplankton species typically endure this stressor by downregulating metabolic and photosynthetic processes without undergoing drastic morphological changes to form resting stages (Peter and Thomas 1996, Ferroni et al. 2007, McMinn and Martin 2013).
Phytoplankton isolates from McMurdo Dry Valley (MDV) lakes are ideal candidates for performing mimicked polar night incubation studies. MDV lakes are ecological treasures due to their unique characteristics, but have been vastly better characterized in the summer season than winter due to issues in accessibility (Morgan-Kiss et al. 2016). In contrast with most aquatic environments, MDV lakes are permanently ice-covered and stratified. This provides stable abiotic conditions during the fall to winter transition (apart from changing light intensities), which makes simulating seasonal transitions easier. Mixotrophic phytoplankton (i.e. combine photosynthesis with phagotrophy) are also often the dominant primary producers in MDV lakes (Laybourn-Parry et al. 2005). Mixotrophs could have unique photosynthetic and metabolic responses to the polar night, but representative isolates have been understudied in this regard even though they are known to be important throughout polar regions (Moorthi et al. 2009, Gast et al. 2018).
The goal of this chapter was to investigate the response of phytoplankton isolates from the MDV Lake Bonney to simulated polar night conditions. This goal was divided into two aims
19 based on algae trophic modes, which are likely key to understanding their wintertime survival strategies. The first aim was focused on how strict photoautotrophs adjust their photosynthetic and physiological processes in response to the polar night. The second aim investigated the strategies utilized by mixotrophic phytoplankton to survive the polar night.
The objective of aim one was to study the response of Lake Bonney strict photoautotrophs, Chlamydomonas sp ICE-MDV and Chlamydomonas. sp. UWO241 to mimicked polar night conditions. Other experiments on strict photoautotrophs incubated in extended darkness have shown that phytoplankton often downregulate their photosynthetic and metabolic processes (Reeves et al. 2011, Jochem 1999, Ferroni et al. 2007). The use of energy storage products to fuel respiration during extended darkness has also been observed (Fryxell 1989, Zhang et al. 1998). A sampling study at MDV Lake Fryxell during the middle of the polar night found that a Chlamydomonas species produced high numbers of spores, which implies a distinct possibility that the two Lake Bonney chlorophytes in this study could produce resting stages as well (Howes et al. 2000). Finally, UWO represents one of the best studied psychrophilic, pure photosynthetic algal strains, which includes several studies considering UWO’s photosynthetic wintertime survival strategies (Morgan-kiss et al. 2006).
The mesophilic, Chlamydomonas reinhardtii was also studied in aim one as a control chlorophyte to compare with ICE and UWO. C. reinhardtii was selected because it is a model organism, taxonomically similar to the Lake Bonney chlorophytes, and un-adapted to the polar night stressor. It was thus helpful in differentiating responses to the polar night from generic responses chlorophytes have to extended darkness.
The goal of Aim 2 was to determine the photosynthetic and heterotrophic response of Antarctic mixotrophs, Isochrysis sp. MDV and G. cryophila, to mimicked polar night conditions. Both of these organisms have the ability to ingest a range of carbon compounds and in the case of G. cryophila has also been shown to phagocytize bacteria (McKie-Krisberg et al. 2015). These alternative methods of nutrient and energy acquisition could allow this group of phytoplankton to remain metabolically active during extended darkness in their natural environment. The utilization of different trophic strategies between the mixotrophs and the strict photoautotrophs is anticipated to be reflected in differential physiological responses to summer versus winter conditions (Fig. 2.1).
20 I hypothesize that the Antarctic strict photoautotrophs (i.e. ICE and UWO) and the mixotrophs (i.e. Isochrysis sp. MDV and G. cryophila) will exhibit distinct physiological responses in response to mimicked polar night conditions. Furthermore, based on previous studies, I predict that ICE and UWO will exhibit: (1) functional downregulation of their photosynthetic apparatus, (2) maintenance of major photosynthetic components, and (3) degradation of energy storage products to better regain activity following extended darkness. In contrast, I predict that the Antarctic mixotrophic algae will respond to mimicked polar night by: (1) exhibiting relatively minimal changes in their photosynthetic apparatus, and (2) increasing heterotrophic activity as an alternative energy source to compensate for the loss of light.
2. Methods
2.1 Growth conditions and experimental stimulus mimicking the polar night
Cultures of Chlamydomonas sp. UWO241 (CCMP 1619), Chlamydomonas sp. ICE-MDV, and Chlamydomonas reinhardtii (UTEX 89) were grown in Bold’s Basal Medium. Cultures of Isochrysis sp. MDV were grown in F/2 Medium with 30% seawater and Geminigera cryophila (CCMP 2564) was grown in L1 Medium with 100% seawater. Both of the two mixotroph species were non- axenic while the chlorophytes were all axenic. All Antarctic species were grown and incubated at 4-60C while C. reinhardtii was grown and incubated at 200C.
For mimicked polar night experiments, cultures were grown first in sterile 200 ml tubes with glass bubblers under low blue light intensity (10-20 μmol photons m-2 s-1). This light intensity was selected to mimic the natural light quality and irradiances of Lake Bonney’s water column. To acclimate algae to this light intensity they were grown and subcultured at least once in low blue light prior to start the mimicked winter experiments. Prior to extended dark incubation, all cultures were put through a mimicked fall photoperiod stimulus to precondition them to continuous darkness. This preconditioning stimulus consisted of 2-day cycles of 24 hours, 18 hours, 12 hours, and 6 hours of light each day before continuous darkness (Fig. 2.2). After cultures reached the stage of continuous darkness they were transferred to sterile flasks, wrapped several times in foil, and incubated on slow shakers at their respective growth temperatures. Periods of continuous darkness ranged from 16-28 days depending on the experiment. After the dark incubation was over, cultures were transferred back to 200 ml tubes, mixed 1:1 with their growth
21 media, and placed under continuous low blue light intensity. This was done to provide the best possible conditions for the cultures to recover.
All cultures were in log phase at the start of the mimicked winter incubations to ensure that stationary physiological processes did not interfere with the measurements. Additionally, all measurements were done after the cultures had been incubated in at least one hour of darkness. This was done to ensure that normal processes occurring after short periods of darkness did not interfere with the objective of measuring long term changes occurring after extended darkness. Cultures were sampled under dim green light to avoid light stimulation during the extended dark incubation. Culture density was monitored using a spectrophotometer at 750 nm to avoid chlorophyll absorbance. Finally, unless otherwise noted, all measurements and cultures were done in biological replicates.
2.2 Chlorophyll fluorescence analysis
Steady state chlorophyll fluorescence measurements were performed using a Dual-PAM 100 chlorophyll fluorometer (Heinz Walz, Germany) with a 10 mm quartz glass cuvette and micro magnetic stirrer. These measurements were performed at 80C in the case of the Antarctic strains and 200C in the case of C. reinhardtii. For these measurements, samples were first dark-adapted by exposing them to far red light for 2 minutes. Induction curves consisted of 10 or more saturating pulses with a red actinic light set to 27 μmol photons m-2 s-1 in the case of Antarctic strains and 38 μmol photons m-2 s-1 in the case of C. reinhardtii. Dual-PAM software (v1.9) was used to record data and calculate photochemical parameters. Maximal PSII quantum yield, FV/FM
= (FM – FO)/FM. Non-photochemical fluorescence quenching, NPQ = (FM -FM’)/FM’. Effective PSII quantum yield, Φ(PSII) = (FM’ - F)/FM’. 2.3 Stain procedures and flow cytometry analysis Flow cytometry was performed on a BD Acurri flow cytometer. Samples were passed through a 40 μm filter, various stains were applied, and measurements were taken for a minimum of 30,000 events. Analysis of the events consisted of first gating for singlet cells by graphing FSC- H vs FSC-A. Second, the average fluorescence intensity for the algae populations was measured for the stained and unstained samples on the appropriate channels. Finally, the unstained fluorescence was subtracted from the stained fluorescence to determine average net fluorescence from the stain, which corresponds to average, relative fluorescence intensity per cell.
22 Propidium iodide (535/617) was used to assess cell viability. 500 μl of culture was incubated with 4.5 μM of propidium iodide in black tubes on ice for 15 min prior to analysis. Increases in fluorescence on channel FL2 (488/585) indicated non-viable cells and results were expressed as the percentage of viable cells vs the total amount of cells analyzed. BODIPYTM (493/503; 4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene) was used to determine relative levels of neutral lipids. In black tubes, 500 μl of culture was incubated with 50 μl of 100% DMSO and 1.5 μl of 2 mM BODIPYTM stock solution on ice for 10- 20 min before analysis. Fluorescence was monitored on channel FL1 (488/533). SYBR® Green I was used to count bacteria following the methods of Prest et al. (2014) by adding 10 μl of working stock (1:100 dilution in DMSO) to 500 μl of sample in black tubes, inverting 10 times, and incubating on ice for 10 or more minutes before measuring on FL1. Chlorophyll autofluorescence (~430/670) was monitored on unstained samples on channel FL3 (488/>670). 2.4 Protein extraction and western blot analysis
Samples for protein extraction (5 to 30 ml) were collected from cultures at various time points. Samples were centrifuged for 8 min at 3,500 rpm, 40C and pellets were stored at -200C until further processing. Pellets were resuspended in 0.75 ml of 1x LDS extraction buffer containing 100 mM Tris (pH 7.8), 10% glycerol, 2 mM EDTA, 2% LDS, and 1 μM aminocaproic acid. Samples were homogenized using a bead beater for 3 cycles of 30 s beating with ice incubations between cycles. The lysate was spun down briefly to remove large debris and transferred to a fresh tube. Protein concentration was determined using a DC compatible Bradford assay (Bio-Rad) with BSA in LDS buffer as concentration standards. Samples were stored at -200C until SDS-PAGE was performed. For protein electrophoresis, samples were normalized to the same protein concentration, 20 mM DTT was added, and they were incubated for 7 min at 700C and spun down briefly before loading onto the gel. Four μg total protein was loaded per lane on 12% SDS polyacrylamide resolving gels (Bio-Rad TGX™ FastCast™), and electrophoresis was done at a constant 150-250 volts. Gels were transferred to nitrocellulose paper using a transfer apparatus on ice set to a constant, 100 volts. Blots were blocked with 5% non-fat milk powder and probed with RbcL or PsbA (Agrisera) 10 and rabbit 20 antibodies at varying concentrations. ECL (Thermo-Fischer) and detection on X-Ray film was performed following antibody incubation.
23 Quantification of relative protein levels was done on scanned X-Ray film images using ImageJ software. Images were converted into grayscale and then net protein densitometry was assessed by subtracting the mean gray value of the background below each lane from the protein in the lane. These net values were inverted and presented as a ratio of the later time points to the first time point to assess changes during the experiment. 2.5 β-N-Acetyl-glucosaminidase heterotrophic enzyme assay
β-N-Acetyl-glucosaminidase (β-gam) assays were performed using a kit (Sigma-Aldrich CS0780) to monitor a representative enzyme of heterotrophic activity. β-gam is an enzyme found across many taxa that breaks down complex organic compounds such as chitin and peptidoglycan in bacteria cell walls (Strojsova and Dyhrman 2008, Sherr and Sherr 1999). Culture extracts were prepared by sampling (10-20 ml) at various times during the mimicked winter incubations. Samples were centrifuged at 3,500 rpm for 8 min (40C). Pellets were re-suspended in 0.75 ml acidic extraction buffer (0.1 M Acetate, 0.2% lauryl ether [Brig 35], pH 4.6) and homogenized using a bead beater with 3 cycles of 30 seconds beating and incubations on ice in between. Protein concentration was determined on cell lysates using Bradford assay with bovine serum albumin in extraction buffer used as protein standards. Cell lysates were normalized for protein concentration. 70 μl of normalized cell lysate was incubated with 630 μl of NAG substrate for 2 hours during the enzyme assay. Readings were taken immediately and then again at 2 hours by adding 667 μl of stop solution to 333 μl of sample and measuring absorbance at 405 nm. The initial readings were subtracted from the 2-hour time point to determine change in absorbance due to enzyme activity. A standard curve of known product (4- Nitrophenyl-N-acetyl-β-D-glucosaminide or NAG) concentrations in stop buffer was used to convert the sample absorbance values to product concentrations, which were later normalized to total protein concentration. 2.6 DNA library preparation and 16S rRNA amplicon sequencing Samples from Chlamydomonas sp. ICE-MDV were collected in 10 ml amounts at various time points during a 20-day darkness experiment and centrifuged at 3,000 rpm, 40C for 10 minutes to pellet the cells. DNA extraction was performed on the pellets using a FastDNA spin kit for soil (MP Biomedicals, OH) following manufacturer’s protocol. DNA concentrations from the extraction were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, DE). PCR amplification was done in triplicate for each sample on the hypervariable regions V4 of
24 the 16S rRNA gene using barcoded and linked primer sets encoding F515/R806. Library preparation and sequencing reactions were done following the CBFG-tailored version of the Earth Microbiome Project 16S amplicon sequencing protocol an Illumina MiSeq Platform (300 cycles, forward and reverse) located in the Center for Bioinformatics and Functional genomics (CFBG, Miami University). After the sequencing reaction was complete, filtering and demultiplexing was performed using QIIME (v 1.8.0). Sequencing analysis was conducted in CLC Genomics Workbench using the Microbial Genomics Module. The sequencing analysis closely followed the Qiagen OTU clustering steps tutorial and the OTU clustering was performed using a 97% similarity threshold compared to the standard 16S_97_otus_GG.clc database included in the module. 2.7 Chlorophyll extraction (in acetone)
2ml samples were centrifuged for 3 min at 13,000 rpm to pellet cells. Pellets were resuspended in 1 mL 90% (v/v) acetone on ice and homogenized using a bead beater for 3 cycles of 30 s of beating with ice incubations between cycles. The extracts were then centrifuged for 2 min at 13,000 rpm to pellet cell debris and the supernatant was transferred to a cuvette for chlorophyll determination at 647 nm, 664 nm, and 630 nm absorbance following the methods of Jeffery and Humphrey (1975).
3. List of mimicked polar night experiments
The first mimicked polar night experiment was done with ICE cultures incubated in continuous darkness for 20 days and never returned into the light. During earlier iterations of these mimicked polar night experiments, bacteria were observed microscopically in ICE cultures after extended darkness, which was likely due to contamination during repeated sampling. The goal of this experiment was to observe the changes in bacteria growth in ICE cultures more closely using time series 16S rRNA amplicon sequencing alongside cell viability assays and culture density measurements in ICE. It was thought that those portions of non-viable ICE cultures were likely leaking organic C to allow for increased bacteria growth during the mimicked polar night. Time series fluorescent stains were measured on the flow cytometer and included BODIPYTM, propidium iodide, chlorophyll fluorescence, and cell counts. Time series DNA extractions were performed for 16s rRNA amplicon sequencing on associated bacteria with the cultures, which were thought to have originated through processes occurring during repeated sampling. Finally,
25 chlorophyll fluorescence parameters were measured in time series as well to monitor culture health and photosynthetic processes. The second mimicked polar night experiment was done with Isochrysis sp. MDV and G. cryophila cultures incubated in continuous darkness for 16 days before being returned to the light. Time series fluorescent stains were measured on the flow cytometer and included BODIPYTM, sybr green, and chlorophyll fluorescence. Chlorophyll fluorescence parameters were measured in time series monitor culture health and photosynthetic processes. Samples were taken for protein extraction and western blotting of major photosynthetic proteins at four time points during the experiment. The third mimicked polar night experiment was done with ICE, UWO, and C. reinhardtii cultures incubated in continuous darkness for 28 days before being returned to the light. Chlorophyll fluorescence parameters were measured to monitor photosynthetic processes. Samples were taken in time series for protein extraction and western blotting of major photosynthetic proteins. Spectrophotometry was used to monitor culture densities during the entire experiment. The fourth mimicked polar night experiment was done with Isochrysis sp. MDV and G. cryophila cultures incubated in continuous darkness for 28 days before being returned to the light. Chlorophyll fluorescence parameters were measured to monitor photosynthetic processes. Spectrophotometry was used to monitor culture density and the Phyto-PAM II was used to measure chlorophyll concentrations during the entire experiment. The chlorophyll concentrations were measured using individual references for each strain made for the Phyto- PAM II based upon concentrations determined by chlorophyll extraction in acetone. The final mimicked polar night experiment was done with Isochrysis sp. MDV and G. cryophila cultures grown in flasks and incubated in continuous darkness for 11 days before the final measurements were taken. Initially, the acidic food vacuole stain, LysoTracker® Green DND-26, was used to measure possible changes in food vacuole size or acidity. This stain was highly variable however and did not change significantly across a variety of conditions tested in the two mixotrophic organisms (data not shown). Next, ingestion rates of 0.5 µm fluorescent beads was attempted to be measured in both mixotrophic species, but ingestion rates measured in this particular case were found to be very close to 0 under all conditions tested (data not shown). Finally, the activity of the heterotrophic enzyme N-Acetyl-β-d-glucosaminidase (β-gam)
26 was selected as the best representative of heterotrophic activity during mimicked polar night experiments. β-gam is an enzyme found across many taxa and breaks chemical bonds of amino sugars, which are present in a variety of organic compounds such as chitin and glycoproteins. Isochrysis sp. MDV cultures for this experiment were grown in both autotrophic and mixotrophic media (5% cereal grass or 10% rice extract added) while G. cryophila was grown in autotrophic media alone. The goal of using the two C supplements was to provide cues for stimulating possible heterotrophic responses than may not have been present without complex organic C substrates in the media. G. cryophila was not cultured with either cereal grass or rice extract as no increases in growth were observed with G. cryophila cultures under those conditions. The measurements taken were primarily two time points of β-gam enzyme assays, which require a large sample volume. Optical density and induction curves were also taken to monitor culture health and density
4. Results 4.1 Changes in culture optical density during mimicked polar night incubation
Culture density was monitored using optical density measurements in chlorophytes cultured under a preconditioning photoperiod stimulus (i.e. mimicked fall), mimicked polar night darkness period (i.e. mimicked winter), and a post darkness recovery period (i.e. mimicked summer). During mimicked fall (Fig. 2.3A, days 0-10), all three chlorophyte species grew rapidly.
OD750 levels ceased to increase upon the onset of continuous darkness and then declined throughout that period (days 10-38), falling to 55-65% of their initial values by the end (Fig. 2.3A, Table 2.1). During the mimicked summer period when cultures were returned to continuous low blue light, they were mixed 1:1 with growth media, which accounts for the initial decline in OD750 (Fig. 2.3A, days 38-50). However, all three strains were able to resume growth several days after the start of this recovery period. UWO and C. reinhardtii were observed to resume growth on the 4th day of recovery and ICE resumed growth on the 6th day (Table 2.1).
Culture density was also monitored in the mixotrophic species Isochrysis sp. MDV and G. cryophila during similar mimicked Antarctic seasonal transitions as described above. Both mixotrophs reached culture density values typical of late log phase before they entered continuous darkness (Fig. 2.3B). Similar to the chlorophytes, the optical density values dropped steadily during this period with G. cryophila declining to 61.4% and Isochrysis sp. MDV to 45.1% of their day 10 OD750 values by day 38 (Table 2.1). Upon return to light incubation, both Isochrysis
27 sp. MDV and G. cryophila began to grow immediately, which was in contrast to the several day lag phases observed in the chlorophytes. It should be noted that one of the three replicates of G. cryophila failed to recover and was removed from the calculations at this time.
4.2 Photosynthetic measurements during mimicked polar night incubation
FV/FM, maximum quantum yield, is a common stress measurement as well as photosynthetic parameter. During the mimicked fall season, FV/FM values in all three chlorophyte species were consistently high (e.g. >0.6). In response to the onset of polar winter, FV/FM steadily declined (Fig. 2.3C) to 33-45% of their initial values (Table 2.1). ICE and UWO exhibited higher
FV/FM values than C. reinhardtii on average throughout the dark incubation, but ICE cultures had the lowest average FV/FM on day 38 at 0.147. During the recovery phase, FV/FM increased immediately in all three strains.
High FV/FM values for both mixotroph species were measured during the mimicked fall season; although maximum FV/FM values were ~20% lower compared to the chlorophytes (Fig.
2.3D). In contrast with the chlorophytes, FV/FM values in both mixotrophic species were remarkably stable during the entire experiment with values falling to just 80% of their original values (e.g. >0.4) at the end of mimicked winter (Fig. 2.3D, Table 2.1). FV/FM of both mixotrophs transiently decreased by 33% upon return to light incubation. This is likely due to a change in culture housing from flasks to tubes, which the mixotrophs are more sensitive to than the chlorophytes.
Φ PSII is a measure of photosynthetic efficiency under steady state conditions. Φ PSII values in the chlorophyte species were consistently high (e.g. >0.5) during mimicked fall. However, once cultures were shifted to the dark Φ PSII declined to below detection limits in all strains after ~2 weeks incubation (Fig. 2.4A). Upon return to the light during the mimicked summer, ΦPSII recovered slower than FV/FM in all strains; although, it eventually did return to fall levels (Fig. 2.4B). Values were lower in the mixotroph species than the chlorophytes during the preconditioning period, but never fell below 0.3 during the mimicked polar night (Fig. 2.4B).
Similar to the trends in FV/FM, ΦPSII values of the mixotrophs were also more stable during dark incubation compared to those of the strict photoautotrophs (Fig. 2.4B).
NPQ is a fluorescence parameter which is a measure of energy loss due to non- photochemical heat dissipation. NPQ values in all three strict photoautotrophic species were
28 consistently low during the mimicked fall period but increased once cultures were transferred to extended darkness conditions (Fig. 2.4C). During this period, UWO consistently showed the highest NPQ (max NPQ of 1.40 on day 27), ICE the second highest (max NPQ of 0.852 on day 18), and C. reinhardtii the lowest (max NPQ of 0.62 on day 38). These differences in NPQ between the three species could also be seen by viewing the saturation pulse analysis during extended darkness (Fig. 2.5A and C). The decline in FM’ upon actinic illumination relative to FM was highest in UWO compared to ICE or C. reinhardtii. NPQ measurements declined during mimicked summer to near 0 in all three species.
In contrast with the strict photosynthetic stains, both mixotroph strains exhibited NPQ levels at or near zero, regardless of the mimicked season (Fig. 2.4D). Interestingly, both mixotrophs often exhibited FM’ levels which were higher than FM, (Fig. 2.5B and D). These data suggest that despite several attempts to full dark adapt the mixotroph samples (including a far red light incubation, data not shown), the electron transport pool was not always fully oxidized. This may indicate the presence of relatively high dark sources of electron donors in the mixotrophs, making full dark adaptation difficult to achieve.
Cellular chlorophyll concentrations from both mixotrophic species were measured during the mimicked polar night experiment. Chlorophyll levels remained stable in both species throughout transitions between mimicked seasons (Fig. 2.3B). Note that G. cryophila exhibited higher cellular chlorophyll relative to Isochrysis sp. MDV, which is likely due to having a larger average cell size. Cellular chlorophyll was not monitored for the chlorophyte species because the Phyto-PAM II machine was not available at that time.
4.3 Photosynthetic protein abundance during mimicked polar night
Western blots for PSII reaction center D1 protein (PsbA) and RubisCO large subunit (RbcL) were conducted on whole cell protein extracts during the 28-day and 16-day dark incubation for the chlorophytes and mixotrophs, respectively. The chlorophytes ICE and UWO exhibited significant reductions in RbcL during the mimicked winter period on day 22 and day 38, (Fig. 2.6A). RbcL levels in both species increased during mimicked summer to match levels during the fall incubation period (day 43; Fig. 2.6A). Both mixotroph species also exhibited reductions in RbcL during dark incubation, which was significant on both day 11 and day 22 for G. cryophila and day 22 for Isochrysis sp. MDV (Fig. 2.6B). PsbA remained at levels similar to those
29 during mimicked fall in both the Lake Bonney chlorophyte species as well as the mixotrophs throughout the experiment (Fig. 2.6C and D). In C. reinhardtii however, PsbA declined after day 22. Representative western blots used for these quantifications are shown in Figure 2.7.
4.4 Neutral lipids, cellular chlorophyll, and average cell size during mimicked polar night
Neutral storage lipids and chlorophyll in cultures of ICE and both mixotroph species were monitored during mimicked polar night conditions (Fig. 2.8). The mimicked polar night experiments were 16 days of continuous darkness before return to light incubation in the case of the mixotrophs and 20 days of darkness with no return to light incubation in the case of ICE. Note that these experiments were preliminary and we were unable to repeat them due to failure of the flow cytometer instrument.
During log phase growth in low blue light, ICE cells were measured to have ~4 fold less neutral lipid concentration and ~2-fold higher chlorophyll concentration than both mixotroph species. Cultures of ICE increased levels of neutral lipids during the mimicked fall. Upon dark incubation, lipid levels in ICE cultures declined rapidly from 7x105 to 3.1x105 relative fluorescence units (RFU) and stayed low throughout dark incubation (Fig. 2.8A). In contrast, Isochrysis sp. MDV exhibited a transient decline in neutral lipids from 2x105 to 1.5x105 RFU from days 5 to 11 but remained relatively stable throughout the dark incubation and returned to previous high values upon return to the light (Fig. 2.8B). Neutral lipid levels in G. cryophila remained consistent throughout the experiments (Fig. 2.8B).
During the mimicked fall period when light levels were decreasing, ICE increased chlorophyll from 4.5x106 to 5.6x106 RFU. Following transfer to continuous darkness chlorophyll levels steadily declined in ICE cultures from 5.6x106 RFU on day 12 to 2.1x106 RFU by day 30 (Fig. 2.8C). In contrast, both mixotrophic species were observed to have consistent chlorophyll levels throughout the entire experiment (Fig. 2.8D). This result is consistent with the chlorophyll concentration measurements using the Phyto-PAM II (Fig. 2.3B).
Forward scatter was used to approximate changes in cell size during the course of the same experiment. ICE, G. cryophila, and Isochrysis sp. MDV all underwent decreases in average cell size from measurements taken during log phase growth compared to 16 days of continuous dark incubation (Fig. 2.9). Of these three species, ICE cells exhibited the greatest decline in cell size from 9.5 to 5.9 µm diameter on average. It is worth noting that these cell sizes are
30 approximations from a calibration ladder measured with spherical beads. G. cryophila in particular is far from being spherical in shape so cell sizes in reality could be much different than those calculated using forward scatter.
4.5 Cell viability, culture density, and associated bacteria in ICE cultures during mimicked polar night
Occasionally, bacteria were observed to be associated with the chlorophyte cultures (in particular, the ICE strain) during extended dark incubation, despite no evidence of bacteria in the chlorophyte cultures when grown in continuous light. Therefore, we monitored changes in the bacterial community in ICE cultures during 20 days of continuous darkness. Algal cell viability assays and culture density measurements were also monitored (Fig. 2.10A). ICE cells were observed to have high viabilities (e.g. >95%) during mimicked fall conditions and until day 24 of the dark incubation, whereupon cell viability declined transiently to nearly 30% viable (Fig. 2.10A, arrow). This decline was followed by a recovery in cell viability at day 30. During this experiment both culture densities and FV/FM values were observed to decrease during dark incubation (Fig. 2.10B). FV/FM decreased initially from 0.675 to 0.40 upon the transition into darkness and then drastically fell to near 0 by day 24 and 30. Cell counts were done on the flow cytometer for this experiment and declined from 2050 cells µl-1 on day 12 to 1700 cells µl-1 on day 30 (Fig. 2.10B).
To monitor associated bacteria, two biological replicates of ICE cultures were selected for 16S metagenomic sequencing (Fig. 2.10C). Initially, a high percentage of chloroplast 16S reads related to a Chlamydomonas sp. ICE-L were observed, which was highest at day 12 (94%), indicating that the vast majority of the culture was dominated by ICE. Gammaproteobacteria species began increasing by day 15 and constituted 55% of the reads by d21. Flavobacteria were observed to increase later in the dark incubation, reaching 43% of reads by the final measurement on day 30. It is worth noting that the three bacteria classes observed in ICE cultures were virtually all one species each representing the class, which constitutes a very low diversity of bacteria. The majority of Gammaproteobacteria reads were from an uncultured Pseudomonas sp., Flavobacteriia reads from Chryseobacterium sp. OV259, and Alphaproteobacteria reads from Brevundimonas bullata.
31 4.6 Heterotrophic activity, assessed through the representative enzyme β-gam, in mixotroph species during mimicked polar night
Mixotrophic species can rely on alternative energy sources through phagotrophic ingestion of bacterial prey or particulate carbon, followed by particle digestion in a digestive organelle, the food vacuole. Heterotrophic activity in both mixotroph species during mimicked polar night conditions was assessed using a β-gam assay (Fig. 2.11). Mimicked polar night experiments were performed in autotrophic Isochrysis sp. MDV cultures, and those supplemented with complex organic carbon in the form of either 5% dehydrated cereal grass or 10% rice extract. Due to limited reagents for this assay and high culture volumes required, two time points were used to assess changes in enzyme activity during mimicked polar night: normal log phase growth in the light compared with 11 days of dark incubation.
β-gam activity in both Isochrysis sp. MDV cultures supplemented with complex C sources were always higher than autotrophic cultures (Fig. 2.11). Additionally, both cereal grass and rice extract cultures increased significantly upon 11 days of dark incubation from values near 3 to above 6 nM product µg protein-1 hr-1. Values of autotrophic Isochrysis sp. MDV cultures also increased from 1.70 to 2.38 nM product µg protein-1 hr-1 upon dark incubation. G. cryophila in contrast displayed constitutively low enzyme activity compared with Isochrysis sp. MDV and did not significantly change activity upon dark incubation.
4.7 Microscopic observations during mimicked polar night experiments
General results from microscopic observations were that cells went through the dark incubation with few morphological changes and little evidence of spore/cyst formation. Caveats to this were that in an early experiment, all three biological replicates of ICE formed very high numbers of small spores that could be seen by day 16, both microscopically and by measuring them on the flow cytometer. This phenomenon was not observed at any point subsequently however for ICE or any other species. Bacteria contaminants did not appear to be significant during any of the 28-day extended darkness experiments, which were done with a bigger emphasis on aseptic technique than the initial experiments. In general, the mixotrophic species appeared to move more during the darkness than the chlorophytes. However, there were several time points when the chlorophytes did appear to be swimming quickly during extended darkness, such as ICE cells by day 33.
32 5. Discussion
Studies in this chapter compared the response of strict photoautotrophic and mixotrophic cultures of Antarctic phytoplankton to mimicked polar night. I hypothesized that: (i) strict photoautotrophs would downregulate their photosynthetic and metabolic processes during the period of extended darkness, and (ii) the mixotrophs respond to the polar winter by maintaining a functional photosynthetic apparatus and increasing heterotrophic activity.
5.1 Photosynthetic processes during the mimicked polar night
All of the strict photoautotrophs (i.e. chlorophytes) responded to the polar night by downregulating photosynthesis; however, the mechanism varied between the mesophile C. reinhardtii vs. the Antarctic phytoplankton, C. spp. UWO241 and ICE-MDV. Maximum photochemical efficiency (FV/FM) and quantum yield of PSII (PSII) values declined rapidly after 2-3 weeks of continuous dark incubation. These results suggest that in response to winter conditions, all three of the strict phototrophs exhibited functional downregulation of photosynthesis. In C. reinhardtii, a loss in function of photosynthesis was accompanied by reduced levels of the PSII core protein, PsbA, suggesting structural downregulation of PSII (Fig. 2.6C; Murchie and Lawson 2013). Acclimation in evergreen trees also involves both structural and functional downregulation of PSII to avoid oxidative damage during the winter when temperatures fall below freezing but light is available (Gilmore and Ball 2000, Öquist and Hüner 2003).
Both Antarctic chlorophytes maintained steady levels of PsbA protein throughout the mimicked polar night experiment (Fig. 2.6C). This is consistent with earlier studies which proposed that UWO functionally downregulated its photosynthetic apparatus during the polar winter, but generally maintains many of the major proteins (Morgan-Kiss et al. 2005, 2006, 2016). Maintenance of PSII reaction centers in the Antarctic strains suggests that they may rely on photoprotection of PSII, such as antennae quenching. In support of this suggestion, winter NPQ values were significantly higher in both psychrophiles relative to C. reinhardtii (Fig. 2.4C). NPQ is associated with activation of the xanthophyll cycle dissipation of excess excitation energy as heat (van de Poll et al. 2011). In addition, Morgan-Kiss et al. (2005, 2006) suggested that UWO may rely on uncoupling of the light harvesting antennae from PSII cores. Both of the Antarctic photoautotrophs UWO and ICE could possess the ability to keep and protect their photosynthetic
33 machinery during the polar winter. This would be an adaptive advantage for these organisms, as they would be prepared to quickly switch to a functional state when light becomes available in the spring (Morgan-kiss et al. 2006).
Alterations in the organization and function of the photosynthetic apparatus has also been observed in other species of polar algae during dark incubation. Antarctic macroalgae and diatoms have been seen to reduce FV/FM in response to mimicked winter (Reeves et al. 2011, Lüder
2002). The chlorophyte Koliella antarctica exhibited substantial chloroplast reorganization, accompanied by reductions in functional PSII, but relative stability of LHCs (Baldiserotto et al. 2005, Ferroni et al. 2007). A yellow-green, snow filamentous algae was also observed to degrade PSII but maintain associated light harvesting complexes in response to dark incubation (Baldisserotto et al. 2005). These researchers also hypothesized that it would be beneficial for algae to retain LHCs through the polar night to begin light capture as soon as possible afterwards (Ferroni et al. 2007).
The mixotrophic species Isochrysis sp. MDV and G. cryophila exhibited differential responses at the level of the photosynthetic activity under all incubations. First, PSII and FV/FM were significantly lower in the mixotrophs vs. chlorophyte species in the fall, suggesting that the mixotrophs possess a lower photosynthetic capacity. However, once the mixotrophs were shifted to darkness, both strains maintained stable levels of photosynthetic activity (PSII and FV/FM) throughout the mimicked winter (Fig. 2.3B and 2.4B). NPQ levels were also at or near zero, suggesting that capacity for energy dissipation in both strains was very low (Fig. 2.4D). In addition, both mixotroph species maintained similar levels of the PSII core protein and PsbA and cellular chlorophyll throughout the entire experiment (Fig. 2.3B and 2.6B). These results indicated that the mixotrophs do not significantly change their photosynthetic processes during the period of extended dark incubation. A major advantage of this response could be that they remain prepared for immediate utilization of light energy upon its return in the spring. In the Arctic Ocean during the polar night, researchers have found diatoms and dinoflagellates able to immediately begin performing primary production when incubated in the light, which affirms the possibility that some species retain photosynthetic capacity during this period (Berge et al 2015).
34 In contrast with the differences in structural and functional changes in the photosynthetic apparatus, all organisms exhibited significant declines in RubisCO (RbcL) protein abundance during the mimicked winter. This indicates that RubisCO could be a target of degradation during the polar night. High abundances of RubisCO usually imply high C-fixation potential, which is likely less important during extended dark periods (Kong et al. 2012). Reductions in both RbcL protein levels and rbcL transcripts were also observed in transplanted UWO cultures during a polar night transition experiment in Lake Bonney (Morgan-Kiss et al. 2016). UWO possess high levels of RubisCO, which compensate for reduced rates of enzymatic activity at low temperature (Dohli et al. 2013). However, maintenance of high RubisCO levels is a significant allocation of carbon and energy which could be reallocated to other processes during the winter.
5.2 -gam activity and lipid storage during the mimicked polar night
Mixotrophs can rely on alternative sources of energy and nutrients through heterotrophic digestion of bacterial prey and larger particulate carbon. -gam is an enzyme that degrades complex C substances (e.g. chiton and bacteria cell walls) and is an important indicator of heterotrophic activities in diverse protists (Sherr and Sherr 1999, Strojsov and Dyhrman 2008). - gam activity was significantly higher in Isochrysis sp. MDV cultures in the dark vs. light. These results suggest that this mixotroph may supplement energy acquisition in the winter with heterotrophy. In contrast, G. cryophila cells had low -gam activity compared to Isochrysis sp. MDV and did not change in activity upon dark incubation. It is possible that -gam is not as important an enzyme for G. cryophila under the conditions tested. Other metrics of heterotrophic activity, such as bead or heat killed bacteria ingestion may be useful in G. cryophile in future studies.
Microorganisms have a variety of mechanisms for storing excess carbon. Lipid droplets are a common storage product in algae and a previous report noted high oil accumulation in ICE (Li et al. 2016). Neutral lipids were detected in ICE and both mixotroph species. In response to mimicked winter, ICE exhibited significantly greater reductions in neutral lipid content relative to two mixotrophs (Fig. 2.6A and B). These results may indicate that the mixotrophs are less reliant on energy storage products during winter. High amounts of energy storage products, including starch and neutral lipids, have been observed in phototrophs at the onset and during
35 the polar night (Fryxell 1989, Zhang et al. 1998). Thus, it is likely that ICE utilizes energy-rich lipids during the winter as an alternative energy source to maintain low levels of metabolism.
5.3 Cell growth and survival during the mimicked seasonal transitions
All strains underwent a rise in optical density during the period of mimicked fall (days 0- 10, Fig. 2.1A and B), which was followed by a decline throughout the mimicked winter period (days 10-38). However, the strains exhibited different lags in resuming growth following winter. For example, the mixotrophs began increasing in density immediately while the strict photoautotrophs had lag phases of 4-6 days before resuming growth (Table 2.1). This contrast indicates a readiness for growth in the case of the mixotrophs throughout the period of continuous darkness that was activated by the return of light.
The mixotrophic species were likely able to resume immediate growth at the end of the mimicked polar night due to their constitutively active photosynthetic apparatus and physiological processes. Heterotrophic activity through the winter (i.e. higher -gam activity in Isochrysis) could help compensate for the loss of phototrophic abilities and allow the mixotrophs to retain higher respiration during the mimicked polar night than the strict photoautotrophs. These characteristics could give mixotrophic species in polar regions a competitive advantage during the winter and allow them to enter spring with actively growing populations (Laybourn- Parry 2002, Howes et al. 2000). On the other hand, higher photosynthetic capacity among the photosynthetic phytoplankton would allow for higher competition for light energy during the summer months.
The strict photoautotrophs likely took several days of lag time after the dark incubation to upregulate normal metabolic and photosynthetic processes before they were able to resume growth. It is possible that the chlorophytes slowed their metabolic processes during dark incubation and underwent plastid structural changes that could have taken several days from which to recover when returned to the light. Ferroni et al. (2007) conducted detailed investigations into the cellular morphology of another Antarctic chlorophyte subjected to several weeks of darkness and observed extensive chloroplast and mitochondrial changes. These plastid rearrangements were thought to acclimate the cells for a slowed metabolism to better survive the winter (Ferroni et al. 2007). Lag phases observed in K. antarctica after being returned to the light were similar to those displayed by chlorophytes in the present experiment.
36 5.4 Case study of ICE mimicked polar night experiment: bacterial succession and algal viability Cultures of ICE during a 20-day mimicked polar night were determined to have high amounts of stress (i.e. low FV/FM) and reduced cell viability after two weeks of dark incubation (Fig. 2.8A and C). In a similar study, researchers investigating the survival of polar diatoms found that all species tested reached FV/FM of near 0 and dropped drastically in viability after several weeks of dark incubation (Reeves et al. 2011). In parallel with the reduction in ICE culture viability, the relative abundance of bacterial 16S rRNA OTUs increased relative the chloroplast 16S rRNA, suggesting an increase in the relative abundance of Bacteria in the winter (Fig. 2.8C). This may reflect a fraction of non-viable ICE cells leaking organic C and allowing increased bacterial growth during the mimicked polar night. A species of Gammaproteobacteria (uncultured Pseudomonas sp.) was the first to increase in abundance during dark incubation and was followed by a species of Flavobacteriia (Chryseobacterium sp. OV259). The Gammaproteobacteria reached maximum relative abundance on day 21 while the Flavobacteriia did so on day 30. Flavobacteriia and Gammaproteobacteria (along with Roseobacters) are thought to be the dominant groups of bacteria associated with algal blooms (Buchan et al. 2014). Flavobacteriia typically reach their highest abundance during phytoplankton bloom decay and are known to convert high molecular weight compounds into low molecular weight compounds (Buchan et al. 2014). The role of Gammaproteobacteria in algae blooms is less clear, but it is possible that in this case they were more reliant on low molecular weight compounds produced by ICE cultures (Buchan et al. 2014). The polar night darkness period could be an important time for bacteria driven heterotrophic activity in Lake Bonney, but more fieldwork needs to be done in this regard (Vick and Priscu 2012).
6. Conclusions and significance
The experiments of Antarctic strict photoautotrophic and mixotrophic cultures incubated in mimicked polar night darkness indicate two distinct responses. The strict photoautotrophs, which have little means of acquiring energy for their respiration in the absence of light rely on downregulation of their photosynthetic processes and degradation of C storage compounds. The nature of this photosynthetic downregulation differed between the Antarctic and the temperate species of Chlamydomonas. The Antarctic photoautotrophs retained their core photosynthetic apparatus, such as the PSII reaction centers, during dark incubation and increased NPQ processes
37 while the temperate species did not. The mixotrophs, which have expanded nutritional versatility, maintained relatively functional photosynthetic apparatus and stored C levels. Additionally, both mixotrophic algae were observed to undergo immediate growth when transferred back to light incubation, while all of the three photoautotrophic species had lag phases of several days before resuming normal growth.
These differing responses to mimicked polar night conditions represent two different strategies for surviving the long period of darkness in the phytoplankton’s natural environment. The Antarctic strict photoautotrophs retained major photosynthetic apparatus components in a downregulated (i.e. protected) state during the extended darkness. This is likely a strategy to avoid the high cost of rebuilding their photosynthetic apparatus upon the return of light in their cold environment. In contrast, both mixotrophic species responded to dark incubation with little change in photosynthetic or physiological processes other than increasing in heterotrophic activity. This strategy could give these types of phytoplankton an advantage at the start of the summer period with populations able to utilize light and begin growth immediately.
Our understanding of algal physiology and abundance during the polar night both in polar ocean and lake environments has been limited by difficulties in accessing these regions during the heart of darkness. Many researchers have thus turned to mimicking the polar night ex-situ. These studies have often focused on diatoms, with less emphasis on chlorophytes or common mixotrophic taxa such as haptophytes and cryptophytes. The present study broadens our knowledge of the strategies utilized by these other taxa with more diverse trophic modes. These groups are present throughout polar regions and in particular are the dominant primary producers in the unique, extreme MDV lake ecosystems. Hopefully there will come a time when the activities of polar photoautotrophs during this significant portion of the year will be illuminated and we will better understand these vast, vital ecosystems.
38
Figure 2.1. Major cellular and photosynthetic processes in strict photoautotrophic and mixotrophic phytoplankton during the summer and winter. The blue ovals represent PSII reaction centers and crescent shapes represent associated antennae, which likely downregulate in the case of strict photoautotrophs during darkness. These types of organisms also likely degrade storage compounds (POC) during long periods of darkness. In contrast, mixotrophic phytoplankton are expected to increase heterotrophic activity during darkness and undergo less photosynthetic downregulation.
39 Fall Summer 25 Winter
20
15
10 Light period (hours) period Light 5
0
5 10 15 20 25 30 35 40 45 Day number
Figure 2.2. Day number key for the mimicked seasonal photoperiod stimulus. All of the experiments had the same preconditioning phase (i.e. mimicked fall, days 0-10). Durations of continuous dark incubation ranged from 16 to 28 days of darkness depending on the experiment with the maximum duration shown here (days 10-38). Most experiments were ended with a recovery phase of continuous light (i.e. mimicked summer, days 38-50).
40 Isochrysis OD Geminigera OD C. reinhardtii C. sp UWO-241 C. sp ICE-MDV Isochrysis chl Geminigera chl A 0.8 B 0.7 1.0 Fall Winter Summer Fall Winter Summer
0.7 0.6
)
-1 ) ) 0.8 0.6
0.5
750nm 750nm
0.5 0.6 0.4
0.4 0.3 0.4 0.3 0.2 0.2 0.2
0.1
Culture densityCulture (avg OD Culture densityCulture (avg OD
0.1 chlorophyllCellular chl*cell(pg 0.0 0.0 0 10 20 30 40 50 0 10 20 30 40 50 C D Fall Winter 0.7 Summer 0.7 Fall Winter Summer
0.6 0.6
0.5 0.5
0.4 0.4
M
M
/F
/F
V
V F 0.3 F 0.3
0.2 0.2
0.1 0.1
0.0 0.0 0 10 20 30 40 50 0 10 20 30 40 50 Incubation time (days) Incubation time (days)
Figure 2.3. The physiological indicators: growth (A, B), chlorophyll per cell (B), and FV/FM (maximum quantum yield) (C, D) of the strict photoautotrophic and mixotrophic cultures during mimicked polar night experiments of 28 days continuous darkness (days 10-38). All measurements were done as averages of biological triplicates.
41 C. reinhardtii C. sp UWO-241 C. sp ICE-MDV Isochrysis Geminigera A B Fall Winter Summer Fall Winter Summer 0.6 0.6
0.4 0.4
PSII PSII
0.2 0.2
0.0 0.0 0 10 20 30 40 50 0 10 20 30 40 50 C Fall Winter Summer D Fall Winter Summer
1.5 1.5
1.0 1.0
NPQ NPQ
0.5 0.5
0.0 0.0 0 10 20 30 40 50 0 10 20 30 40 50 Incubation Time (days) Incubation Time (days)
Figure 2.4. The photosynthetic functioning parameters: ΦPSII (effective quantum yield) (A, B) and NPQ (non-photochemical quenching) (C, D) of the strict photoautotrophic and mixotrophic cultures during mimicked polar night experiments of 28 days of continuous darkness. All measurements were done as averages of biological triplicates. These photosynthetic measurements were performed using saturating pulse analysis on a chlorophyll fluorometer.
42 Table 2.1. Relative values for important photosynthetic and growth parameters by the end of the dark incubation (day 38 compared to day 10). Lag time values were calculated as the number of days taken to demonstrate normal growth when cultures were returned to the light (days 38-50). All measurements were done in biological triplicates.
Species Relative culture Lag time before Relative Fv/Fm Relative ΦPSII at density at end of resuming growth by end of dark end of dark dark incubation after return to light incubation incubation (%OD750 d10-38) (days d38-50) (%Fv/Fm d10-38) (%ΦPSII d10-38)
C. sp. ICE- 59.3±4.7 6±0 33.3±6.3 0±0 MDV C. sp. 55.8±8.7 4±1 44.7±1.0 0±0 UWO241 C. reinhardtii 64.9±3.8 4±0 34.1±5.0 0±0
Isochrysis sp. 45.1±7.1 1±0 83.1±6.4 60.0±4.5 MDV G. cryophila 61.4±5.5 1±0 90.4±1.9 57.6±0.9
43
A B
Isochrysis (light) C. sp. UWO-241 (light) Isochrysis (dark) C. sp. UWO-241 (dark)
C D
Fluorescence yield Fluorescence
C. sp. ICE (light) Geminigera (light) C. sp. ICE (dark) Geminigera (dark)
Time
Figure 2.5. Example induction curves recorded for UWO (A), ICE (C), Isochrysis sp. MDV (B), and G. cryophila (D) during mimicked polar night experiments. The light time points were taken at day 5 and the dark time points taken between days 32 and 36. Induction curves were recorded on a dual-PAM 100 chlorophyll fluorometer with 27 µE m-2 s-1 red actinic light at 80C.
44 C. sp UWO-241 C. sp ICE-MDV C. reinhardtii Isochrysis Geminigera 1.5 1.5 A Fall Winter Summer B Fall Winter Summer * ** * * * 1.0 1.0
0.5 0.5
RbcL protein (rel. abundance) (rel. protein RbcL RbcL protein (rel. abundance)RbcL protein(rel.
0.0 0.0 5 11 21 38 43 5 11 21 28
1.5 D C Fall Winter Summer Fall Winter Summer
** * 1.0 1.0
0.5
0.5
PsbA protein (rel. abundance) PsbAprotein(rel. PsbA protein (rel. abundance) PsbAprotein(rel.
0.0 0.0 5 11 21 38 43 5 11 21 28 Incubation Time (days) Incubation Time (days)
Figure 2.6. Relative photosynthetic protein abundance of rubisco large subunit (RbcL) and PSII reaction center subunit (PsbA) in cultures of chlorophytes (A and C) and mixotrophs (B and D) during mimicked polar night experiments. Relative protein abundance was determined by western blotting with lanes loaded on an equal protein basis. All time points consisted of averaged biological triplicates. Significant differences were determined by t tests.
45
Figure 2.7. Representative western blots used for quantification from UWO (A) and G. cryophila (B) during mimicked polar night experiments. Lanes were loaded on an equal protein basis.
.
46 C. sp. ICE-MDV Isochrysis Geminigera 6 A 1.5x10 B 3x106
Fall Winter Fall Winter Summer
)
)
-1 -1
6 1.0x10 2x106
5
5.0x10 1x106 Neutral lipids (rel. fluorescence lipids(rel. Neutral cell 0.0 fluorescence lipids(rel. Neutral cell 0 5 9 12 15 18 24 30 5 11 15 24 28
C Fall Winter D Fall Winter Summer
) 6
6.0x10 ) 6.0x106
-1 -1
4.0x106 4.0x106
2.0x106 2.0x106
Chlorophyll (rel. fluorescenceChlorophyll (rel. cell Chlorophyll (rel. fluorescenceChlorophyll (rel. cell 0.0 0.0 5 9 12 15 18 24 30 5 11 15 24 28 Incubation time (days) Incubation time (days)
Figure 2.8. Neutral storage lipid and chlorophyll autofluorescence in cultures of ICE (A and C) and the two mixotrophic species (B and D) during mimicked polar night experiments. Cultures of ICE were incubated in continuous darkness for 20 days (days 10-30) and never returned to light incubation. Cultures of the two mixotrophs were incubated in the dark for 16 days (days 10-26) whereupon they were returned to the light for 2 days before the final measurement.
47 C. sp. ICE-MDV 10 Isochrysis Geminigera
8
)
m
6
4
Estimated cell size ( size cell Estimated 2
0 Light (log phase) Dark (16 days)
Figure 2.9. Algal cell diameter during mimicked polar night experiments. Cell size was approximated with the forward scatter measurements on the flow cytometer, which was converted to cell diameter through the use of spherical calibration beads. All measurements were done in biological triplicates.
48 A C. sp. ICE-MDV 1.2 Fall Winter 1.0
0.8
0.6
0.4
0.2
Cell viability (% viable) viability Cell 0.0 B 5 9 12 15 18 24 30
) 3000 0.8
-1 l
Fall Winter 2500 0.6 2000
1500 0.4 M
/F V 1000 0.2 F ICE Fv/Fm 500 ICE culture density
Culture density (cells* density Culture 0.0 0 5 9 12 15 18 21 24 30 Gammaproteobacteria Chloroplast Flavobacteriia Alphaproteobacteria C Fall Winter 100
75
50
25 16S(%) diversity rRNA 0 5 9 12 15 18 21 24 30 Incubation Time (days)
Figure 2.10. Cell viability (A), culture density and FV/FM (B), and 16S rRNA sequencing analysis (C) of ICE cultures during a mimicked polar night experiment with 20 days of continuous darkness (d10-d30). Measurements for the cell viability and FV/FM were done in biological triplicates and the time series sequencing data is an average of biological duplicates.
49 * 8 Iso + 5%CG
) *
-1 Iso + 10%RE 7
Iso (auto) *hr -1 6 Gem (auto)
5 g protein g
4
3
-gam activity * 2
1 (nmolproduct*
0 Light (log phase) Dark (11 days)
Figure 2.11. β-gam activity of the two mixotrophic species under light and dark incubation time points during a mimicked polar night experiment. Isochrysis sp. MDV was cultured either without additional C sources (auto), with 5% cereal grass (CG), and with 10% rice extract (RE). G. cryophila was cultured without any additional C sources (auto). Significance was determined by t tests and was found for all Isochrysis sp. MDV cultures incubated in the dark for 11 days. All measurements were done in biological triplicates.
50 Chapter 3 - Physiological characterization of Isochrysis sp. MDV and Geminigera cryophila
1. Introduction Protists have wide ranging photosynthetic and heterotrophic abilities. The term “mixotrophy” refers to those protists which combine photosynthesis and phagotrophy, and it includes the major constituents: phagotrophic phytoflagellates, photosynthetic ciliates, and dinoflagellates. Mixotrophic protists have been discovered to be major components of freshwater and marine plankton and are important for C cycling on a global scale (Zubkov and Tarran 2008). They are studied as living examples of the process of endosymbiosis and plastid formation, which are foundational processes for understanding eukaryotic cells (e.g. Oakley and Taylor 1978). Recently, it has even been observed that mixotrophic dinoflagellates form large, harmful algal blooms in coastal marine environments (Jeong et al. 2010). Mixotrophs are understudied despite their significance, and a better understanding of their ecology and physiology is currently needed (Sanders 2011). Phagotrophic phytoflagellates ingest particulate organic matter for a variety of reasons, and they can be classified into the categories of obligate phototroph and obligate heterotroph based on which type of energy generation is more essential for their way of life. An example of an obligate phototroph is the chrysophyte, Dinobryon cylindricum, which is reliant on light to the extent that the species is unable to grow and stops bacterivory altogether when placed in darkness (Caron et al. 1992). An example of an obligate heterotroph is the chrysophyte, Poterioochromonas malhamensis, which was found to be reliant on phagotrophy at all times and phototrophy was induced only during periods of low bacteria concentration (Sanders et al. 1990). Reasons for having mixotrophic adaptations are thought to be related to acquiring enough macronutrients, micronutrients, or phospholipids to help compensate for the stress of living under oligotrophic conditions, or to supplement energy acquisition under conditions of insufficient light (Sanders 2011). The present experiments focused on characterizing the phototrophic and heterotrophic abilities of Gemingiera cryophila and Isochrysis sp. MDV, two phytoflagellates that are of significance to the MDV lake ecosystems. Isochrysis sp. MDV is an abundant phytoplankton in Lake Bonney especially at the chemocline (Kong et al. 2012). It possesses an acidic food vacuole as well as two chloroplasts, but the extent of its mixotrophic abilities are still largely unknown (Li
51 et al. 2016). Geminigera cryophila was isolated from the Antarctic, Ross Sea and is the closest known relative to a cryptophyte species in Lake Bonney (Li et al. 2016). Mixotrophic cryptophytes are often the dominant phytoplankton in MDV lakes (Laybourn-Parry 2002). G. cryophila ingests bacteria at rates similar to those in MDV lakes and was found to increase ingestion rates in response to dark incubation (McKie-Krisberg et al. 2015). As cryptophytes are difficult to isolate, G. cryophila remains one of the best representatives of this important group of algae in MDV lake ecosystems. 2. Methods The effects of incubation under varied light intensity on ICE, Isochrysis sp. MDV, and G. cryophila were studied in multi-cultivator MC 1000 (Photon Systems Instruments) photo- bioreactors. All cultures were started at the same culture density (measured spectrophotometrically) and were grown in duplicates under white LED lights at intensities 20, 50, 100, and 250 µmol photons m-2 s-1. Fluorescent parameters measured on the flow cytometer included neutral lipids, cell counts, and chlorophyll autofluorescence. Details for these methods are given in chapter 2. Chlorophyll fluorescence parameters were measured using the Dual-PAM 100 (Waltz) as described in chapter 2. These parameters were measured during late log phase growth.
Isochrysis sp. MDV cultures were incubated in tubes under different nutrient conditions to assess β-N-Acetyl-glucosaminidase (β-gam) activity. The media used for the nutrient incubations included full strength F/2 (36.3 mM NaH2PO4 and 880 mM NaNO3), 5% nitrogen F/2
(40 mM NaNO3), and 5% phosphorous F/2 (2 mM NaH2PO4). Prior to incubation in these different media, cultures were centrifuged gently (1,000 rpm for 15 min at 40C) and resuspended in sterile saline to avoid the addition of excess nutrients. Cultures were incubated for a week in their respective media, all with a starting OD750 of 0.180 before measurements were taken. Optical density and chlorophyll fluorometry were performed to assess culture health and growth (data not shown) prior to β-gam enzyme assays.
Cultures of Isochrysis sp. MDV and G. cryophila were incubated under a variety of added C sources to assess their growth and photochemistry under these conditions. All of these experiments were done without replicates, in flasks, at 20 µmol photons m-2 s-1 and 40C. Cereal grass media and rice extract were the two most commonly used organic C sources. Other C sources tested included 20 mM glycerol and 10 mM glucose. All C supplements were filter
52 sterilized using a 0.2 μm filter. The concentration of stock rice extract was 20 g/L, which was prepared by boiling rice for 20 minutes. Chlorophyll fluorescence parameters, chlorophyll concentrations, and optical density were measured on these cultures.
3. Results
3.1 Effects of variable irradiance on Isochrysis sp. MDV, G. cryophila, and ICE
The goal of this experiment was to determine how each mixotrophic species would respond to variable light intensity. Cell counts were conducted using the flow cytometer during late log phase growth to assess differences in growth due to light intensity. ICE cultures showed similar densities of ~1,400 cells*µl-1 at intensities 50 to 250 µmol photons m-2 s-1 but ~0.67-fold lower culture densities at 20 µmol photons m-2 s-1 (Fig. 3.1C). Isochrysis sp. MDV densities were highest at 3,046 cells*µl-1 under 20 µmol photons m-2 s-1 and ~0.7 fold lower at 350 µmol photons m-2 s-1. G. cryophila cultures were similar in density (~275 cells*µl-1) at 20 and 50 µmol photons m- 2 s-1, but began to decline at 100 µmol photons m-2 s-1 and fell to just 116 cells*µl-1 by 250 µmol photons m-2 s-1.
Relative levels of neutral lipids per cell were determined with BODIPY staining and measured on a flow cytometer. All three species of algae were observed to increase levels of neutral storage lipids as light intensity increased with the highest levels for each species observed at 250 µmol photons m-2 s-1 (Fig. 3.1A). Over the light range tested (20 to 250 µmol photons m-2 s- 1), G. cryophila increased lipids 2.2-fold, Isochrysis sp. MDV 2.2-fold, and ICE from 2.8-fold.
Cellular chlorophyll followed the opposite pattern as neutral lipids with highest values occurring at 20 µE m-2s-1 for both mixotrophic species (Fig. 3.1B). From 20 to 250 µmol photons m- 2 s-1, G. cryophila cells decreased in chlorophyll from 3.6x106 to 1.66x106 RFU and Isochrysis sp. MDV decreased from 2x106 to 9.4x105 RFU. ICE cells exhibited no significant changes in cellular chlorophyll in response to light intensity.
FV/FM values were used to assess relative amounts of stress in algae cultures under the range of light intensities tested. ICE cultures showed high FV/FM values at all light intensities, staying above 0.40 (Fig. 3.1D). Isochrysis sp. MDV cultures exhibited a 68% decline in FV/FM over
53 the range of light intensities. FV/FM values for G. cryophila declined further over the same range, going from 0.54 at 20 µmol photons m-2 s-1 to nearly 0 at 250 µmol photons m-2 s-1.
3.2 Effects of varied macronutrients on β-gam enzyme activity in Isochrysis sp. MDV
The goal of the nutrient experiment was to determine if Isochrysis sp. MDV showed changes in the nutrient scavenging enzyme, β-gam under conditions of low inorganic N or P macronutrients. Cultures of Isochrysis sp. MDV grown under conditions of reduced N and P exhibited reduced growth and photosynthetic stress (i.e. reduced Fv/Fm) after a week of incubation (data not shown). β-gam activity of Isochrysis sp. MDV exhibited a 3-fold increase under conditions of low N relative to replete. In contrast low P incubation did not significantly impact β-gam activity (Fig. 3.2).
3.3 Effects of various organic C sources on mixotroph species growth and photochemistry
The goal of these experiments was to investigate the growth and photochemistry of the two mixotrophic species in mixotrophic or heterotrophic culture conditions (e.g. with added organic C source in the presence or absence of light). Isochrysis sp. MDV was generally found to grow better under all mixotrophic conditions tested than it did under purely autotrophic or heterotrophic conditions (Table 3.1). Maximum OD750 values observed for 5% cereal grass and 5% rice extract were above 1.0 while autotrophic growth never exceeded 0.5. Heterotrophic growth
(i.e. without light) was found to be 0 OD750 day-1 for Isochrysis sp. MDV cultures with the organic
C sources tested. FV/FM values were consistently high in all of the conditions tested besides cultures grown in darkness. ΦPSII values were found to be higher in the mixotrophic media than autotrophic with values near 0.5 in mixotrophic media and near 0.3 in autotrophic.
G. cryophila cultures grew to higher yields and had higher photosynthetic efficiencies in autotrophic media compared to the mixotrophic media tested (Table 3.2). G. cryophila cultures were also found to not grow without the presence of light. Cultures grown in the presence of glucose and glycerol were observed to have extremely high amounts of bacterial growth. Photochemistry in G. cryophila cultures was similar under all conditions except those cultures grown in the dark.
54 4. Discussion
4.1 Acclimation of ICE, Isochrysis sp. MDV, and G. cryophila to varied light intensities G. cryophila and Isochrysis sp. MDV grew optimally under low light intensities, suggesting that both mixotrophs are adapted to shade conditions. In contrast, ICE exhibited comparable growth under the range of light levels, suggesting that this strain has a broader range of light tolerance. . G. cryophila appears to be the most light-sensitive: FV/FM values declined drastically at irradiances at or above 100 µmol photons m-2 s-1 (Fig. 3.1D). Despite this significant loss of photosynthetic ability, cultures exhibited relatively consistent growth at 100 µmol photons m-2 s- 1 (Fig. 3.1C). Isochrysis sp. MDV cultures followed a similar pattern to G. cryophila with less drastic declines in FV/FM between 20 and 100 µmol photons m-2 s-1. At light intensities of 250 µmol photons m-2 s-1 however, low FV/FM and culture density values indicated high amounts of light induced stress. ICE cultures were able to grow to their highest densities and maintain high FV/FM between 50 and 250 µmol photons m-2 s-1, indicating a much higher tolerance of high light intensities in this chlorophyte compared to both mixotroph species.
G. cryophila and Isochrysis sp. MDV were able to photoacclimate to the range of light intensities tested, and all three species increased their production of neutral storage lipids at higher light intensities. Both mixotrophic species exhibited reductions in their relative cellular chlorophyll concentrations ~2 fold as light intensity increased to minimize the absorption of excess light, which indicates a strong ability to photoacclimate to the light intensities tested (Fig. 3.1B). All three Antarctic species of algae increased their relative cellular neutral lipid concentrations as light intensity increased, indicating that these neutral lipids are a C sink for excess light energy (Fig. 3.1A).
4.2 Isochrysis sp. MDV -gam activity under low nutrient conditions
Isochrysis sp. MDV cultures exhibited an increase in -gam activity under low N conditions, indicating the importance of this enzyme as a nutrient scavenging mechanism for this organism. Isochrysis sp. MDV increased -gam activity three-fold in response to low N (Fig. 3.2). -gam could be produced in response to low N to aid in degradation of complex, organic N compounds such as glycoproteins and chitin. Strojsova and Dyhrman (2008) found that among 10 species which showed -gam activity, 5 were found to increase under conditions of low N, and 5 increased under conditions of low P, which highlights this enzyme’s variable use under
55 conditions of nutrient stress. Additionally, the closest known relative of Isochrysis sp. MDV, Isochrysis galbana is not mixotrophic and was not found to produce -gam, which could suggest that Isochrysis sp. MDV acquired mixotrophic adaptations for living in the oligotrophic, Lake Bonney (Strojsova and Dyhrman 2008).
4.3 Growth and photochemistry under mixotrophic and heterotrophic conditions Isochrysis sp. MDV appears to be an obligate phototroph and but G. cryophila was not observed to increase in growth under any of the mixotrophic or heterotrophic conditions tested. Isochrysis sp. MDV was found to double in maximum yield and growth rate under mixotrophic growth conditions with media containing small amounts of cereal grass and rice extract as compared to autotrophic media (Table 3.1). When grown under these same conditions in the dark however, no growth was detected. Isochrysis sp. MDV was also found to increase chlorophyll and photosynthetic efficiencies with small amounts of these two C sources added, which is further evidence that Isochrysis sp. MDV may be an obligate phototroph with a constitutive investment in its photosynthetic apparatus.
G. cryophila cultures were not observed to increase in maximum yield or growth rate under any mixotrophic conditions tested as compared with autotrophic media (Table 3.2). As this species is known to phagocytize bacteria is it likely that the right conditions for optimum growth have not been found in the present experiment and its categorization as an obligate phototroph or heterotroph remains uncertain. Out of all of the psychrophiles cultured, G. cryophila is also the most temperature sensitive algae, which was commonly observed to die within minutes of room temperature incubation (data not shown).
5. Conclusions
Isochrysis sp. MDV appears to be a shade adapted, obligate phototroph that increased its reliance on heterotrophic abilities during conditions of extremely low light or nutrients. The alga was found to not only grow, but perform photosynthesis best under mixotrophic growth conditions, which indicates a constitutive investment in photosynthesis. These characteristics indicate that the phytoplankton is well suited for life under the low blue glow of Lake Bonney’s oligotrophic water column where any opportunity to acquire energy is likely important to capitalize on. G. cryophila was found to be even more shade adapted than Isochrysis sp. MDV, but
56 the extent of its heterotrophic abilities is still largely unknown. At light intensities of just 100 µmol photons m-2 s-1, G. cryophila showed signs of extreme light stress, high production of neutral lipids, and underwent large changes in chlorophyll concentrations to photoacclimate. It would be desirable to repeat these experiments with more replicates in the future before reliable conclusions could be drawn about the physiologies of these two interesting algae.
57
Figure 3.1. Relative amounts of neutral storage lipids (A), chlorophyll autofluorescence (B), culture density (C), and FV/FM (D) results from a light intensity experiment where ICE, Isochrysis sp. MDV, and G. cryophila were incubated for 7, 10, and 11 days respectively. All measurements were done in biological duplicates.
58 3.5 Iso (auto)
3.0
) -1
2.5
*hr -1
2.0
1.5
g protein g
1.0
-gam activity 0.5
(nM product* (nM 0.0 Replete Low N Low P
Figure 3.2. β-gam activity of Isochrysis sp. MDV cultures incubated under conditions of replete, low N, and low P for one week. All measurements were done in technical triplicate. A significant difference was determined between the low N and replete conditions using a t test.
59 Table 3.1. Growth and photochemistry of Isochrysis sp. MDV during C source experiments in flasks under low light. All measurements were done without replicates.
C source Growth rate Max OD750 FV/FM ΦPSII Total chl/OD (OD/day-1) (µg*L-1*OD750-1) Autotrophic 0.019 0.485 0.576 0.255 3.991 5% Cereal grass 0.038 1.068 0.624 0.497 6.223 5% Rice extract 0.036 1.07 0.636 0.509 7.642 10% Rice extract 0.036 0.957 0.631 0.511 6.432 20mM Glycerol 0.034 0.651 0.518 0.401 10mM Glucose 0.029 0.577 0.515 0.378 Dark (any C source) 0 same as 0.348 0 inoculum
60 Table 3.2 Growth and photochemistry of G. cryophila during C source experiments in flasks under low light. All measurements were done without replicates.
C source Growth rate Max OD750 FV/FM ΦPSII Total chl/OD (OD*day-1) (µg*L-1*OD750-1) Autotrophic 0.013 0.257 0.597 0.441 6.778 5% Cereal grass 0.003 0.164 0.576 0.35 N.D. 5% Rice extract 0.012 0.231 0.584 0.419 5.253 10% Rice extract 0.008 0.196 0.603 0.378 7.163 20mM Glycerol 0.01 0.255 0.398 10mM Glucose 0.008 0.253 0.355 Dark (any C source) 0 same as 0.281 0 inoculum
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68 Appendix
10000 Isochrysis Geminigera Light Dark Light
8000
)
-1
l
6000
4000
2000 Bacteria density (cells* density Bacteria
0 5 11 15 24 28 Day number
Figure 5.1. Bacterial density associated with mixotrophic cultures during a mimicked polar night experiment. Bacterial density was determined for mixotrophic cultures by staining with the nucleic acid stain, sybr green, and counted through flow cytometry. All measurements were done in biological triplicates.
69 3.0 2.0
Geminigera 1.8 )
Isochrysis )
-1 2.5
Std enzyme 1.6 -1
*hr -1 1.4 2.0
1.2
g protein g
M product*hr M 1.5 1.0
0.8 1.0 0.6
0.4 0.5
0.2
std. enzyme activity ( activity enzyme std. B-Gam activity (nmol* activity B-Gam 0.0 0.0 5 10 15 20 25 Temperature (oC)
Figure 5.2. β-gam activity of the two mixotrophic species with enzyme incubation at various temperatures as compared to the standard enzyme isolated from jack beans over the same temperature range. All measurements were done in technical triplicates.
70 Table 5.1. Chlorophyll and protein measurements from ICE culture, rapid thylakoid extractions during the light intensity gradient experiment.
Light intensity Chl a/b ratio Chl/protein (µmol photons m-2 s-1) (µg Chl*µg protein-1) 20 2.74 ± 0.01 756.28 ± 195.8
50 2.90 ± 0.09 505.60 ± 160.8
100 3.35 ± 0.03 439.6 ± 9.6
250 4.26 ± 0.01 371.62 ± 69.9
71 C. sp. ICE-MDV C. sp. UWO-241 C. reinhardtii Light Dark
700
) -1
750 600
500
400
300
200
100 ATP concentration (nM ATP*OD (nM concentration ATP 0 4 8 12 18 Day number
Figure 5.3. ATP concentrations measured in all three chlorophyte species during a short mimicked polar night experiment. ATP concentrations were determined by CellTiter-Glo® 2.0 Assay and were normalized to culture OD750. All measurements were done in biological triplicates.
72 Fluorescence Yield Fluorescence
C. rein (light, -4) C. rein (dark, 24)
Time
Figure 5.4. Example chlorophyll fluorescence yield from saturating pulse analysis of C. reinhardtii cultures incubated under declining light and 24 days of continuous darkness.
73 A 4000 B 3000
Isochrysis 3500 2500 ICE Adj. R-Squ 0.8411
Adj. R-Square 0.94701 ) ) 168.15172
Intercept -214.85578 -1
-1 Intercept l
l 3000 Slope 4036.7064 2000 Slope 3059.88319
2500 1500
2000 1000
1500
500 Culture densityCulture (algae* Culture densityCulture (algae* 1000 0 500 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.0 0.2 0.4 0.6 0.8 1.0 700 Optical density (OD ) Optical density (OD ) C 750nm 750nm
600 Geminigera Adj. R-Squa 0.88133
) Intercept 33.97448 -1 l 2341.8210 500 Slope
400
300
200 Culture densityCulture (algae* 100
0.05 0.10 0.15 0.20 0.25
Optical density (OD750nm)
Figure 5.5. Cell counts and optical density measurements were compared on a variety of samples of -1 Isochrysis (A), Gemingiera (C), and ICE (B) to determine their relationship between OD750 and cells*µl . Culture conditions included samples grown in low versus high light and at various stages of growth, such as lag phase or clumping during log phase. Cell counts were conducted on a flow cytometer equipped with a peristaltic pump to ensure accurate recording of flow rates.
74