Psychrophilic Diatoms in Ice-Covered Lake Erie

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PSYCHROPHILIC DIATOMS IN ICE-COVERED LAKE ERIE

Nigel A. D'souza

A Dissertation

Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

May 2012

Committee:

Dr. Robert Michael McKay, Advisor

Dr. Jeffrey A. Snyder Graduate Faculty Representative

Dr. George S. Bullerjahn

Dr. Scott O. Rogers

Dr. Rex L. Lowe

Nigel A. D’souza

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ABSTRACT

Robert Michael McKay, Advisor

Winter surveys across Lake Erie between 2007 to 2011 documented diatom assemblages associated with extensive ice cover. This study aims to characterize these winter assemblages in terms of their community structure, their interactions, adaptations to a psychrophylic lifestyle and their role in the biogeochemistry of the lake. The winter assemblage was dominated by diatoms, specifically by Aulacoseira islandica (O. Müller)

Simonsen (≈80%) and Stephanodiscus binderanus (Kützing) Krieger (≈19%), with other taxa like Fragilaria spp., Cyclotella spp., Asterionella spp. and Tabellaria spp. making up less than 1% of the assemblage. The detailed morphology of the two dominant taxa observed are described in this study, and the presence of resting cells and auxospores in filaments of A. islandica, but not in the other taxa are documented. The study compares different sample processing techniques for scanning electron microscopy (SEM), and describes a rapid and effective technique, substituting critical point drying (CPD) with hexa-methyl-di-silazane (HMDS) treatment, that allows viewing of fine details including the epiphytic colonization of the diatoms by flagellated rod-shaped bacteria embedded in an extracellular matrix. The study also describes ice nucleating activity associated with assemblages of filamentous diatoms sampled during winter and early spring across the

Laurentian Great Lakes. The ability to promote ice formation offers a previously undescribed mechanism by which non-motile phytoplankton can attach to overlying ice and, thereby, maintain a favorable position in the photic zone. The ice nucleating activity

is attributed to bacteria. Bacteria isolated from the phytoplankton showed high

temperatures of crystallization (Tc) to -3°C. Ice-nucleating active (INA) isolates were identified as belonging to the genus Pseudomonas. Whereas INA bacteria have been isolated from lakes and streams, their presence in these environments is attributed primarily to runoff and atmospheric deposition as rain or snow consistent with their proposed role as biological ice nuclei in clouds. Far from a passive existence in the aquatic milieu, the INA microbes associated with winter diatoms in the Great Lakes may possess a role in promoting the formation of ice during winter and in so doing, promote the growth of their diatom hosts under ice. The novel mechanism presented may be relevant to temperate and polar ecosystems beyond the Great Lakes including coastal oceans. Finally, photosynthetic O2 evolution rates measured in winter support characterization of the assemblage as a photosynthetically robust population, with rates of primary production in winter higher than those measured in spring and comparable to those reported in summer. Results further suggest that the winter diatom assemblage may play an important role in the export of carbon to the benthos, thereby potentially driving the hypolimnetic oxygen deficits observed months later in summer.

ACKNOWLEDGMENTS

First of all, I want to thank my advisor Dr. R. Michael McKay for his support,

guidance and patience during the course of my Ph.D. program here at Bowling Green

State University. It was a privilege to work under his guidance, and this has given me a

great opportunity to grow both intellectually and personally. I would also like to thank

the members of my Ph.D. committee, Dr. George Bullerjahn, Dr. Scott Rogers, Dr. Rex

Lowe and Dr. Jeffrey Snyder for their support and advice on this project.

I would like to thank Dr. Benjamin Beall, Dr. Paul Morris, Dr. Vipaporn Phuntumart, Dr.

Marilyn Cayer, Dr. Paula Furey, Yury Shtarkman, Robyn Edgar and Wakanene Kamau

from BGSU for their assistance with different aspects of my work. I would also like to acknowledge the efforts of all members of the McKay and Bullerjahn labs for their constant intellectual and moral support through the course of my program.

In addition, I would like to acknowledge the efforts of Dr. Richard Lee from Miami

University, OH and his students Yuta Kawarasaki and Nicholas Levis for their help with work on the ice nucleating activity experiments. I would also like to thank collaborators

M. Saxton, D. Smith, S. Wilhelm, M. Twiss, R. Smith, H. Carrick, and R. Bourbonniere for their assistance with fieldwork. I would like to acknowledge the the support of the captains and crews of the CCGS Griffon, CCGS Limnos, R/V Lake Guardian, USCGC

Alder and USCGC Neah Bay for their assistance with the lake surveys. In kind support from the Canadian Coast Guard, EPA-Great Lakes National Program Office and the U.S.

Coast Guard is acknowledged as are the efforts of Technical Operations personnel from

Environment Canada and EPA-GLNPO.

TABLE OF CONTENTS

Page

CHAPTER I: INTRODUCTION ...... 1

Diatoms ...... 1

Diatom blooms ...... 3

The role of light in bloom dynamics ...... 5

The role of nutrients in bloom dynamics ...... 7

Seasonal succession ...... 10

Background to the Lake Erie Winter Study ...... 11

Aulacoseira blooms in Lake Baikal ...... 14

Objectives of the current study ...... 17

Why is this work necessary ...... 18

REFERENCES ...... 18

CHAPTER II: EXAMINATION OF THE WINTER DIATOM ASSEMBLAGE IN

LAKE ERIE: IDENTIFICATION, MORPHOLOGY AND ECOLOGY BASED ON

TECHNIQUES IN LIGHT- AND SCANNING ELECTRON MICROSCOPY ...... 27

ABSTRACT ...... 27

INTRODUCTION ...... 28

METHODS ...... 31

Sampling sites and sample collection ...... 31

Cultures ...... 32

Light- and epifluorescence microscopy ...... 33

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Scanning electron microscopy:

Conventional sample preparation ...... 34

Modified sample preparations ...... 35

Air drying samples ...... 35

Using liquid CO2 for drying ...... 35

Using HMDS for drying ...... 36

RESULTS ...... 36

Morphology of Aulacoseira islandica ...... 36

Morphology of Stephanodiscus binderanus ...... 42

Other diatoms observed ...... 42

Comparison of sample processing techniques for SEM ...... 47

Diatom cultures ...... 48

DISCUSSION ...... 56

Comparison of sample processing techniques for SEM ...... 56

Morphology of diatoms ...... 58

EPS production ...... 64

Diatom – Bacteria associations ...... 66

REFERENCES ...... 67

CHAPTER III: BACTERIAL EPIPHYTES OF DIATOMS PROMOTE ICE

NUCLEATION IN LARGE LAKES ...... 74

ABSTRACT ...... 74

INTRODUCTION ...... 75

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METHODS ...... 79

Sampling Sites ...... 79

Collection and maintenance of samples ...... 79

Determination of Temperature of crystallization ...... 82

Tc as effected by sample manipulation ...... 82

Effect of dilution ...... 82

Effect of sample heating ...... 83

Scanning electron microscopy ...... 83

Isolation of diatoms ...... 83

Isolation and identification of bacteria ...... 83

Biochemical tests for identification of bacterial isolates ...... 85

Growth on KBC medium ...... 85

Oxidase test ...... 85

Nitrate reduction test ...... 85

Fermentation of Carbohydrates ...... 86

Gelatin Liquefaction ...... 86

RESULTS ...... 87

Temperature of crystallization ...... 87

Effect of dilution on the Tc of phytoplankton ...... 87

Effect of temperature on the Tc of phytoplankton ...... 88

Scanning electron microscopy ...... 88

Isolation, identification and ice nucleation activity of bacteria ...... 88

DISCUSSION ...... 97

Ice nucleation in phytoplankton assemblages ...... 97

Bacterial epiphytes of diatoms promote ice nucleation ...... 98

Ice formation influenced by INA microbes ...... 99

REFERENCES ...... 102

CHAPTER IV. PRIMARY PRODUCTION IN ICE COVERED LAKE ERIE ...... 107

ABSTRACT ...... 107

INTRODUCTION ...... 108

Terms involved in photosynthesis ...... 108

Photosynthetically active radiation (PAR) ...... 110

Photosynthesis versus Irradiance (P versus E) curves ...... 111

Causes of variation in photosynthetic parameters ...... 112

Methods used in estimating rates of primary production ...... 113

Summer Hypoxia: Causes and knowledge gap ...... 115

METHODS ...... 116

Study sites ...... 116

Sample collection and handling ...... 117

Chlorophyll-a analysis ...... 117

Primary production ...... 117

RESULTS ...... 121

Photosynthesis rate ...... 121

Light Saturation index (Ek) ...... 121

Samples from CACHE sites and from melted ice ...... 122

Chlorophyll-a concentrations ...... 122

Calculating rates of primary production in terms of C uptake ...... 122

DISCUSSION ...... 129

Primary production rates in winter ...... 129

Converting O2 evolved to C uptake ...... 131

Si:C ratios for Lake Erie winter assemblages ...... 134

REFERENCES ...... 136

CONCLUSION ...... 142

REFERENCES ...... 143

LIST OF FIGURES

Figure Page

CHAPTER II: EXAMINATION OF THE WINTER DIATOM ASSEMBLAGE IN

LAKE ERIE: IDENTIFICATION, MORPHOLOGY AND ECOLOGY BASED ON

TECHNIQUES IN LIGHT- AND SCANNING ELECTRON MICROSCOPY

Fig. 1: Stations sampled each year in February from 2009–11 across Lake Erie. 32

Fig. 2. The filamentous diatom Aulacoseira islandica ...... 38

Fig. 3. Scanning electron micrographs of frustules of A. islandica...... 39

Fig. 4. Frustules of A. islandica seen cracking under a coverslip...... 40

Fig. 5. Resting cells of the filamentous diatom A. islandica...... 41

Fig. 6. Auxospore formation in filaments of A. islandica...... 41

Fig. 7. The filamentous diatom S. binderanus...... 43

Fig. 8. Scanning electron micrographs of frustules of the diatom S. binderansus. 44

Fig. 9. Image of a filament of the diatom Fragilaria sp ...... 45

Fig. 10. Other diatoms observed in the winter phytoplankton assemblage ...... 46

Fig. 11. A comparison of the different techniques used in sample processing for

SEM ...... 49

Fig. 12 The presence of EPS on filaments of S. binderanus ...... 50

Fig. 13. SEM micrographs showing filaments of the diatom A. islandica with the

presence of EPS that is colonized by flagellated rod-shaped bacteria...... 51

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Fig. 14. SEM micrographs showing EPS on filaments of the diatom S.

binderanus and colonization by flagellated rod-shaped bacteria ...... 52

Fig. 15. Filaments of the diatom Fragilaria spp ...... 53

Fig. 16. Flasks containing (non axenic) cultures of filamentous diatoms

Fragilaria spp. (A), S. binderanus (B), and. A. islandica (C) ...... 54

Fig. 17. DAPI stained filaments of diatoms ...... 55

CHAPTER III: BACTERIAL EPIPHYTES OF DIATOMS PROMOTE ICE

NUCLEATION IN LARGE LAKES

Fig. 1. A Moderate Resolution Imaging Spectroradiometer (MODIS) image taken

on February 16th, 2008 shows the extent of ice cover across Lake Erie ...... 77

Fig 2. An example of the extent of ice cover across the eastern Great Lakes on

February 18th 2008 obtained from the Daily Ice Analysis Charts from the

Canadian Ice Service...... 78

Fig 3. A: Stations sampled across Lake Erie during winter, spring and summer...... 80

Fig 3. B: Nearshore stations sampled in Lake Superior during March 2011 ...... 81

Fig 3. C: Stations sampled across Lake Ontario in spring 2010-11 ...... 81

Fig. 4. Temperature of crystallization (Tc) for phytoplankton samples collected

by net during winter and spring 2010-11 in the Laurentian Great Lakes ...... 90

Fig. 5. The effect of dilution on the ice nucleating activity of seston collected

from Lake Erie in 2010...... 91

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Fig. 6. The effect of heat treatment on temperature of crystallization of

phytoplankton samples collected by net during spring 2010 in Lake Erie ...... 92

Fig. 7. SEM micrographs showing diatom EPS being colonized by bacteria ...... 93

Fig. 8. Temperature of crystallization analysis comparing bacterial and diatom

isolates from Lake Erie established in 2010 and 2011 ...... 94

Fig. 9. Phylogenetic analysis of bacterial isolates by maximum likelihood

inference………...... 95

Figure 10: Temporal schematic representation of ice formation influenced by INA

microbes ...... 101

CHAPTER IV. PRIMARY PRODUCTION IN ICE COVERED LAKE ERIE

Fig. 1. A typical PI curve as described in Forget et. al ...... 111

Fig. 2: Stations sampled across Lake Erie during winter, spring and summer ...... 119

Fig. 3: The O2 electrode setup ...... 120

Fig. 4. Spatial distribution of primary production rates (Pmax) for plankton samples

collected by vertical net tows during winter and spring from different sites across

Lake Erie ...... 125

Fig. 5 Spatial distribution of Ik values for plankton samples collected by vertical

net tows during winter and spring from different sites across Lake Erie ...... 126

Fig. 6. Chlorophyll-a concentrations (µgL-1) of the different size fractions of

phytoplankton in surface waters of Lake Erie...... 127

Fig. 7. Si deposition in filamentous diatom A. islandica...... 135

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LIST OF TABLES

Table Page

CHAPTER III: BACTERIAL EPIPHYTES OF DIATOMS PROMOTE ICE

NUCLEATION IN LARGE LAKES

Table 1: Biochemical properties of ice nucleating active (INA+) isolates collected from

Lake Erie in winter 2011 compared with INA+ strains of Pseudomonas and Erwinia...... 96

CHAPTER IV. PRIMARY PRODUCTION IN ICE COVERED LAKE ERIE

Table 1: Pmax values observed across Lake Erie during winter, spring and summer

sampling ...... 123

Table 2: Ik values observed across Lake Erie during winter, spring and summer sampling ...... 124

Table 3. Primary production (Pmax) rates and light saturation index (Ik) for plankton

samples in CACHE sites and from melted ice collected in the central basin of Lake Erie

during winter ...... 128

Table 4. Primary production in terms of µmol C µg Chl a -1d-1, calculated using previously documented PQ values ranging from 0.67 and 7.12 respectively ...... 128

Table 5. Volumetric estimates of carbon incorporation into Lake Erie phytoplankton across seasons (2008 – 2011) ...... 133

CHAPTER I

INTRODUCTION

The term “plankton” refers to a group of organisms that are adapted to spend their life cycles in apparent suspension in the open waters of seas, lakes, ponds and rivers (Reynolds, 2006). The definition essentially excludes all non living suspended matter, and includes only organisms that are not active swimmers. The term “phytoplankton” refers to photoautotrophic plankton.

Suspended particulate matter in either marine or freshwater is often found to contain zooplankton and phytoplankton. Obvious representatives of phytoplankton in both marine- and freshwater samples are picocyanobacteria, diatoms, and dinoflagellates (Boney, 1989) Phytoplankton are the major primary producers of organic carbon in the pelagic waters of seas and of inland waters.

Identification of planktonic organisms is usually based on certain cell characteristics. These can include cell shape, cell dimensions, cell walls, mucilage layers, chloroplasts, flagella, reserve substances and other unique cellular features they may possess.

Diatoms - Diatoms are one of the most common types of phytoplankton in marine and freshwater ecosystems and are a globally important group of eukaryotic algae. Diatoms are classified as:

Domain: Eukaryota

Kingdom: Chromalveolata

Phylum: Heterokontophyta

Class: Bacillariophyceae

Order(s): Centrales and Pennales 2

Vegetative cells of Bacillariophytes are either circular, elongate or multipolar, lack flagella, and are encased in silicified cell walls (Adl et al., 2005). A characteristic feature in diatoms are cell walls made of silica (hydrated silicon dioxide) called frustules (Round et al., 1990, Adl et al.,

2005). These cell walls usually consist of two asymmetrical valves, the epitheca and the hypotheca, that typically overlap each other in the girdle region like the two halves of a petri dish, with the epitheca overlapping the hypotheca. The valves are linked either by siliceous bands or by the presence of small teeth, fringing the inner edge of one girdle. The frustules show a wide diversity in valve shape (contour) as well as in ornamentation, and can be used to identify different species (Round et al., 1990, Du Buf et al., 1999). The silica in the cell walls is synthesized by intracellular polymerization of silicic acid monomers, which is then transported to the exterior of the cell and incorporated into the cell wall. During cell division, each daughter cell formed retains one of the frustules from the parent and develops a smaller frustule within.

This results in a decline in mean cell size in a population, which continues for a few generations.

Cell size is restored via an auxospore, a specialized cell known to develop and expand in a controlled manner before producing a new frustule (Round et al., 1990). Auxospore formation results from gamete fusion and is involved in the sexual reproduction of these organisms.

Chloroplasts, bounded by four membranes, and with lamellae of three thylakoids and a ring nucleoid are observed in cells (Adl et al., 2005). Lobed or discoid chloroplasts observed in diatoms are yellow-brown colored in planktonic species and dark brown in sessile forms.

Reserve substances like lipids are commonly observed in these cells (Round et al., 1990). Two major orders of diatoms are observed - the Centrales and the Pennales. Centrales or centric diatoms have radial symmetry (circular or triangular) whereas Pennales or pennate diatoms show bilateral symmetry (Boney, 1989). A more recent classification divides the diatoms into three 3

classes: centric diatoms (Coscinodiscophyceae), pennate diatoms without a raphe

(Fragilariophyceae), and pennate diatoms with a raphe ( Bacillariophyceae) (Round et al., 1990).

Recent studies based on molecular phylogeny have grouped diatoms into the group

Stramenopiles, a group that also includes brown algae, many zoosporic fungi, and the opalinids amongst others). The Stramenopiles along with the Alveolates, Haptophytes and Cryptophytes together were grouped under the super-group Chromalveolata (Adl et al., 2005).

Chromalveolates are argued to have evolved from an endosymbiotic event involving a phagotrophic heterotrophic eukaryote with a photosynthetic red alga eukaryote (Keeling, 2003,

Patron et al., 2004, Armbrust, 2009). Molecular evidence recognize centrics as paraphyletic

(Kooistra et al., 2003, Medlin and Kaczmarska, 2004), but this relationship is still unclear.

Several major molecular clades are cryptic, with morphological and life history traits unable to provide compelling evidence to categorize them as synapomorphies (Adl et al., 2005).

Diatom blooms - A major requirement of any organism is to increase and multiply its kind, in numbers large enough for a suitable number of progeny to survive long enough to form the next generation. For aquatic photoautotrophs like diatoms, this implies the ability to fix sufficient carbon and thus build enough biomass to develop into the next generation before being lost to grazers or any other potential fate. Life as a planktonic form results in difficulties like having to obtain nutrients from potentially highly dilute solutions as well as obtaining enough light to facilitate net photosynthetic carbon fixation. Attenuation of photosynthetically active radiation

(PAR) by water implies that the survival of a phytoplankter depends on its ability to remain in the upper layers of water for at least a part of its life cycle (Reynolds, 2006). 4

Diatoms are known to exhibit a “bloom and bust” life cycle. As a classic “r-selected” taxon

(Kilham and Hecky, 1988), diatoms are able to dominate phytoplankton communities under favorable nutrient conditions. Over time, as nutrients are depleted, diatom production is exported to the benthos. The sinking is induced by loss of buoyancy (Richardson and Cullen, 1995), production of heavy resting spores (Jewson et al., 2008) or synthesis of mucilage that binds cells together (Thornton, 2002). Once in deeper waters, or on the seafloor or lake bed, the cells remain static until conditions are favorable again for growth. Under suitable conditions, these cells emerge from the benthos and enter the surface water via vertical mixing, which is accompanied by nutrient flux into the upper water column (Jewson et al., 2008). The sinking of diatoms has been proposed to be an important part of their survival strategy, as a transition from a planktonic growing stage to a benthic resting stage (Smetacek, 1985).

Natural water bodies are rarely static. Warming and cooling of water, gravity, wind stress, and mechanical disturbances all result in convection with vertical and horizontal displacements. As photosynthetic organisms, it is imperative that diatoms remain in suspension in the upper water column in order to grow. Certain aspects like size, shape, and density interact to play an important role to entrain these organisms in the upper layers of the water column. While the necessity to stay in suspension remains one of the constraints on determining the size and shape of a phytoplankter, the need to take up inorganic nutrients from a dilute medium surrounding the cell places additional restraints on cell size and shape. Wind-aided mixing can help to maintain cells in suspension. Deviation of cell shape from the basic “sphere” results in a greater surface area-to-volume ratio and can increase drag. Features that increase “drag” include adopting a colonial morphology (e.g. Aulacoseira spp., Fragilaria spp., Asterionella spp.) and appendages such as spines and setae (e.g. Chaetoceros spp.). The ability of a cell to sink or float facilitates a 5

certain degree of movement through the water and probably enhanced nutrient sequestration as

the medium (water) flows over the cell surface, a consequence of the vertical and horizontal

displacements of the cell in the water column. Embedding vegetative cells in mucilage on the

other hand results in creation of an aggregate with a relatively low surface area-to-volume ratio, but could act as a defense against fungal attack, grazers, digestion and metal toxicity. In diatoms, extracellular polysaccharides are secreted by the cell from specialized structures of the frustule. These function in attachment and movement on a substrate (Higgins et al., 2003). In centric diatoms, these substances act to regulate buoyancy. Further, studies on the extracellular polysaccharides have shown them to act as defense mechanisms against grazers (Malej and

Harrison, 1993). However, a study examining suspension of the diatom Aulacoseira baicalensis in Lake Baikal found that convective velocities were more than enough to suspend the cells, suggesting that variations in light transmission through the ice probably influence the intensity of the algal blooms in the lakes (Kelley, 1997). Opacity of ice would probably be the most important factor in under-ice convection and would play an important role in influencing the diatom blooms.

The role of light in bloom dynamics - The effect of light on planktonic populations depends on the intensity of the incident light, the immediate changes in light on passing from air into water, the extent to which light can penetrate a given depth and the means by which phytoplankton cells utilize radiant energy (Boney, 1989).

Considered to be the most accepted model until recently, Sverdrup’s “critical depth” hypothesis states that the onset of stratification in spring acts as a switch from insufficient light to light sufficiency (Sverdrup, 1953). Thus, at conditions of identical water transparency and surface 6

irradiance, phytoplankton circulating through a shallow mixed surface layer experience more

light than phytoplankton circulating through a deeper mixed water layer, thus resulting in net

growth and phytoplankton blooms. According to this hypothesis, there exists at any given point

in location and time in the ocean, a depth known as the critical depth possessing a light fluence rate at which growth of phytoplankton is matched by respiratory losses of phytoplankton biomass. The vernal bloom is initiated as a consequence of mixed layer shoaling that causes phytoplankton growth rates (µ) to exceed losses (l), resulting in net growth (r). The hypothesis assumes that (l) is constant and independent of (µ). Three flaws in the analysis have been noted; namely the 2 day lag between mixed layer shoaling and occurrence of the bloom, the correlation between phytoplankton growth rates and grazing rates (g) and the apparent absence of water column stratification during the spring bloom formation at some locations.

A recent study on North Atlantic phytoplankton blooms provided fresh insights into the issue, and resulted in the development of the “Dilution – Recoupling Hypothesis” (Behrenfeld, 2010).

Behrenfeld concluded that the North Atlantic phytoplankton blooms result from the decoupling between phytoplankton growth and losses. The underlying concept of this hypothesis is that the complex heterotrophic food web of planktonic ecosystems allows a constant tight coupling between phytoplankton growth and losses, but seasonal mixed layer deepening has the potential to slightly decouple growth rates and grazing rates by influencing prey-predator interactions via dilution of both groups. In late spring, surface nutrient depletion or overgrazing terminates the

North Atlantic phytoplankton blooms. Through the summer, losses exceed growth rates, and biomass levels decrease. Deepening of the mixed layer and an eventual return to isothermal conditions in autumn replenishes nutrients regenerated from depth and dilutes phytoplankton and grazer populations. The lowered predator-prey concentrations during this “dilution” phase 7

weaken the link between (µ) and (g). Net growth (r) is thus unaltered. However as the mixed layer penetrates deeper to entrain plankton-free waters from lower depths, an increase in net growth rate is observed due to the “decoupling” between growth rates (µ) and grazing rates (g).

This results in the initiation of the bloom. Once the mixed layer stops deepening, grazing pressure increases, but the light driven increases in (µ) allows for (r) to remain high. In the

“recoupling” phase, (g) increases relative to (µ) and a balance between (µ) and (l) is favored.

Both the above models assume that turbulence within the mixed layer is capable of overcoming processes that result in phytoplankton accumulation at a specific depth and is also capable of mixing the phytoplankton cells evenly through the mixed layer. Recent studies however, have shown that atmospheric forces (cooling, wind, etc.) required to trigger blooms are very weak, and the onset of blooms can also be triggered by reduced air-sea fluxes at the end of winter

(Taylor and Ferrari, 2011). The study uses numerical models to show that reduced forcing at the end of winter results in decreased turbulent mixing, allowing retention of phytoplankton cells in the euphotic zone, resulting ultimately into the development of a bloom. Satellite based observations of blooms coupled with meteorological data support the numerical simulations contributing to this hypothesis (Taylor and Ferrari, 2011).

The role of nutrients in bloom dynamics - Cells of planktonic algae conform to a basic model similar to eukaryotic plants with similar cellular structures. Both marine and freshwater phytoplankton are characterized by a predictable blend of elemental constituents and requirements. The elemental composition of phytoplankton normally occurs in stable relative proportions (Reynolds, 2006), with the dry mass amounting to a probabilistic atomic ratio of 8

106C:16N:1P, a proportion very similar to the Redfield ratio for particulate matter in oceans.

Amounts of silica, hydrogen, oxygen, sulfur and iron also show similar constant values.

Phosphorus functions not only in the storage and transfer of a cell’s energy but also in its genetic

system (Dyhrman et al., 2007). Phosphate groups occur in the universal energy carrier ATP

(adenosine triphosphate) as well as in nucleotides making it an essential element. The abundance

of phosphorus on the surface of the earth is about 1% by weight and occurs primarily as

3- orthophosphate (PO4 ) that results from the weathering of rocks and sediments, and which is eventually taken up rapidly by cells. Decay and mineralization of biomass is usually the primary source of phosphorus to the biotic components of ecosystems. Bacteria regenerate organic molecular phosphorus to inorganic orthophosphate which can be assimilated. Cells of marine phytoplankters show changes in protein, carbohydrates, lipids and certain fatty acids under P limitation (Harrison et al., 1990). P limitation could result in a decrease in long chain polyunsaturated fatty acids associated with the phospholipid component of cellular membranes.

(Harrison et al., 1990) and can also induce sporulation in diatoms (Jewson et al., 2008).

With nitrogen accounting for up to 12% dry weight in a typical bacterial cell, it is the second most abundant element in cells after carbon (Madigan et al., 2009). Nitrogen is an important component in proteins, nucleic acids and a number of cellular components. Other than elemental nitrogen in the atmosphere, ammonia, nitrates and other oxides of nitrogen are found in abundance in a variety of sources. Reduction of fixed nitrogen compounds to N2 gas decreases

the total fixed nitrogen in a system, thus impacting the nitrogen budget of ecosystems.

Denitrification is most commonly carried out by facultatively anaerobic heterotrophic bacteria

- - + - that reduce NO3 to NO2 , NO, N2O and N2. Conversion of NH4 to NO2 and N2 also occurs via 9

anammox (Ward et al., 2007). Ammonia oxidizing bacteria (AOB) oxidize ammonium to nitrite,

Nitrite oxidizing bacteria oxidize nitrite to nitrate, whereas ammonia oxidizing archaea oxidize ammonium to nitrite (Ward et al., 2007). Only a few prokaryotes can convert elemental nitrogen from the atmosphere into compounds that can be useful to a cell. Nitrogen fixation (reduction of

N2 gas to bioavailable ammonium) in aquatic systems is observed in certain cyanobacterial

species and some alpha- and gamma proteobacteria. N limitation in phytoplankton can result in changes in protein, carbohydrates, lipids and certain fatty acids (Harrison et al., 1990)

Diatoms use silica to build their frustules, making this an essential requirement for diatom growth and development. Weathering of feldspar rocks is the main source of silica in fresh- and marine waters where concentrations range from 2 to 25 mg L-1. There exists an inverse ratio

between the diatom crop and soluble silica in water (Lund, 1965), with some diatom blooms

capable of depleting silica concentrations to deficiency levels (Munawar and Munawar, 1986).

Declines in diatom populations in spring and summer are accompanied by a slow rise in reactive

soluble silica. Sometimes it may be important to consider the Si:P molar ratio rather than just

silica concentrations in evaluating planktonic populations. Phosphorus is known to stimulate

growth of diatoms (which effectively compete with other organisms by their ability to utilize

relatively small concentrations of phosphates) which utilize silica in the water column as the

populations increase. Once silica levels are depleted, other non-siliceous algae then compete

successfully with the diatoms and eventually replace them. Aulacoseira skvortzowii was found to

use environmental cues like decreases in phosphate levels to initiate development of resting

spores (Jewson et al., 2008). 10

In addition to macronutrients like nitrogen, phosphorus, and silica, compounds like iron, zinc, copper, manganese and molybdenum are also required although in extremely small amounts

(reviewed in McKay et al., 2001). Compounds like boron, cobalt, iodine, sodium and vanadium are needed by some but not all algae. Iron is an important and abundant trace metal associated with numerous enzymes in cells. Iron is found in 2 states, the insoluble oxidized ferric state

(Fe3+) and the soluble reduced ferrous state (Fe2+). Manganese is important as an activator of

certain enzyme pathways. Copper and zinc are somewhat similar to iron and manganese. These

are heavy metals, insoluble in oxidized states and in intensely reduced states where they form

sulfides. Elements like vanadium, chromium and selenium are soluble when oxidized. Both,

essential and non essential heavy metals are toxic at high concentrations and usually exert their

action on algal enzyme systems including protein synthesis, photosynthesis and respiration.

Seasonal succession - Seasonal succession in phytoplankton communities in temperate oceans

and lakes occurs in a well known pattern (Sommer and Lengfellner, 2008). The succession has

been ascribed to depletion of Si or P or both. A transition to a phytoplankton community

dominated by heterocystous cyanobacteria has been linked to low molar N:P ratios (Smith,

1983). Phosphate limitation can induce sporulation in diatoms as seen in the 11 year study on A.

skvortzowii in Lake Baikal where a decline in phosphate concentrations acted as the primary cue

to induce sporulation in these cells (Jewson et al., 2008). These spores sink, and rest on coastal

sediments at depths up to 500 m. Once resuspended by water currents and vertical mixing, these

spores germinate under favorable conditions of light and nutrient availability and initiate the next

“bloom”. Coastal sediments along the extensive shoreline of the lake were considered as ideal

sites of a “seed bank” for recurring populations. The resting spores thus allow A. skvortzowii to survive periods of relative high temperatures and low nutrients in summer. 11

Diatoms use silica to build their frustules, making this an essential requirement for diatom

growth and development. A decline in diatom populations in spring and summer was found to

be accompanied by a slow rise in reactive soluble silica in the water. Sometimes it may be

important to consider the Si:N or Si:P ratio rather than just silica concentrations in evaluating

planktonic populations (Turner et al. 2003). Phosphorus is known to stimulate growth of diatoms

which utilize silica in the water column as the populations increase. Once silica levels are

depleted, other non-siliceous algae then compete successfully with the diatoms and eventually

replace them.

Background to the Lake Erie Winter Study - Most of the existing knowledge about the

biological limnology of the Laurentian Great Lakes is based on data that has been collected

between spring and early autumn. Whereas analysis of the ice cover across the lakes has been

carried out, research in winter limnology has been rare. Existing literature describing winter

phenomena in Lake Erie is limited with the most extensive studies being the surveys carried out in the western basin by David Chandler at the Ohio State University’s Stone Lab from 1938-

1942 (Chandler, 1940, Chandler, 1942a, Chandler, 1942b, Chandler, 1944, Chandler and Weeks,

1945). Arguably the biggest reason for the lack of information from winter surveys is related to inherent logistical difficulties in winter sampling. In addition, a generalized assumption that the lakes are less productive in winter may have contributed to the sampling bias.

Whereas studies of winter limnology in the Laurentian Great Lakes are relatively few, a common feature of these studies is the observation of phytoplankton, specifically diatoms, associated with ice cover. While not highlighted in his studies, Chandler documented blooms of diatoms in mid- winter under the ice in the western basin of Lake Erie (Chandler, 1940, Chandler and Weeks, 12

1945). Likewise, Wallen and colleagues identified diatoms (mainly Fragilaria spp.) associated with ice cover in Lake St. Clair, the smallest of the Laurentian Great Lakes where they found nutrient concentrations beneath the ice to be high for several weeks after ice formation and high again in the spring with mid-winter declines (Wallen and Tuppling, 1977). Soluble reactive silica

appeared to influence primary production and chlorophyll (Chl) a biomass accumulation.

Further, data from three ice-covered stations in Lake St. Clair indicated that the ice-bound

phytoplankton were adapted to low irradiation associated with ice-cover (Wallen, 1977).

A few centimeters of snow on the ice can reduce photosynthetically active radiation (PAR) by

90% (Bolsenga et al., 1988, Bolsenga and Vanderploeg, 1992). Deposition of snow on ice, which

results in the greatest attenuation of light, is usually followed by the replacement of phototrophic

phytoplankton by heterotrophic flagellates (Rodhe, 1955, Allen, 1969). An under-ice ecology

program conducted by NOAA-GLERL in the east arm of Grand Traverse Bay in Lake Michigan

in 1986 looked at a potential link between phytoplankton and copepods in the bay as potentially

influential factors in recruitment of larval whitefish during spring (Vanderploeg et al., 1992).

High wind activity resulted in snow-free ice cover that allowed for higher levels of PAR

penetrating the ice. This resulted in a bloom of phytoplankton dominated by the diatoms

Fragilaria crotonensis and Tabellaria spp. as well as the cryptomonad Cryptomonas erosa that

collectively augmented reproductive output of copepods. Wind mixing and late ice formation at

the east arm of Grand Traverse Bay in Lake Michigan during the winter of 1986 was found to

result in an isothermal water column with an algal bloom documented under the ice

(Vanderploeg et al., 1992). The photosynthetic characteristics of this assemblage were found to

be similar to that of the spring phytoplankton assemblage that occurred later that year at the same 13

site. Both assemblages were adapted to low light levels, and most of the photosynthate was

incorporated into polysaccharides and small molecular weight compounds.

Other episodic monitoring efforts carried out in winter have shown a high degree of variability in

biological and chemical properties (Stewart, 1973, Glooschenko et al., 1974, Burns et al., 1978,

Rockwell et al., 1989). These studies, however, have lacked the consistency and scope of

sampling to link this variability to features of Lake Erie in winter.

In response to the need for more hibernal limnology on the Great Lakes, members of the ad hoc

research network MELEE (Microbial Ecology of Lake Erie Ecosystems) conducted a one week

expedition in February 2007 across Lake Erie. The team sought to answer the question:

“What microbes are in the lake in the dead of winter and what are they doing?”

The group surveyed the length of the lake during which they observed discrete phytoplankton

blooms located just below the ice, with chl a levels > 10 µg L-1 and dominated by the

filamentous centric diatom, Aulacoseira islandica (O. Müller) Simonsen (McKay et al., 2011,

Twiss et al., 2012). Viable filaments were also observed embedded in the ice. Other diatoms

including Stephanodiscus binderanus (Kützing) Krieger, Stephanodiscus niagarae Ehrenberg,

Asterionella formosa Hassall, Tabellaria spp. and Cyclotella spp. were observed, albeit in much smaller numbers. Autotrophic picoplankton (0.2 – 2 µm) and nanoplankton (2 – 20 µm) densities were lower than found in typical summer assemblages. Plankton were nutrient (N, P and Si) replete. Zooplankton densities were low and dominated by copepods (≈ 92%) and cladocerans.

The assemblages were termed CACHE’s (Concentrated Algal Community and Heterotrophic

Ecosystems). The CACHE’s were dispersed throughout the lake, and had chl a levels far greater 14

than levels observed in the spring and summer. The water column in the lake was found to be

isothermal (or alternatively, with modest inverse stratification), with temperatures just above freezing and oxygen concentrations at supersaturated levels. High turbidity resulted in low light penetration indicating a planktonic assemblage adapted to low-light conditions.

Aulacoseira blooms in Lake Baikal – Comparison to Lake Erie - Of particular interest to this

study, is the similarity of a phenomenon that occurs in Lake Baikal, Russia. Located in a rift

valley between 51 and 56 °N, Lake Baikal is the oldest and deepest lake in the world (Kozhov,

1963). The spatial and temporal variability of phytoplankton in Lake Baikal are well described

(Goldman et al., 1996). Intra-annual differences in temperature and irradiances result in changes

in the hydrodynamics of the lake and thereby expose the phytoplankton to variations in their

environment (temperature, irradiance and nutrient availability) (Kozhov, 1963, Goldman et al.,

1996). Lake Baikal phytoplankton populations in winter are dominated by the diatoms A.

baicalensis (K. Meyer) Simonsen and A. skvortzowii Edlund, Stoermer & Taylor, while the

phytoplankton population in summer is dominated by the cyanobacterium Synechocystis

limnetica Popovskaja (Richardson et al., 2000). Insights into the diatom bloom in Lake Erie

dominated by A. islandica, may be gained from existing knowledge on its congeners in Lake

Baikal. Under conditions of clear ice in winter, a combination of relatively high light and low

temperatures favors the growth of A. baicalensis. As the seasons progress into summer, high

surface irradiation, warm temperatures, summer stratification and a shallower mixing layer

(resulting in greater light exposure of the phytoplankton) results in a shift in the phytoplankton

community to one dominated by the cyanobacterium S. limnetica. Temperature changes were of

primary importance in this transition rather than light, with the results agreeing with a previous

study on the seasonal succession of algal populations in the Barents Sea (Andersson et al., 1994). 15

Lake Baikal experiences brief, but warm summers and very cold winters resulting in the freezing

of the entire lake each winter. In spite of water temperatures of 0°C below the ice, endemic

psychrophilic diatom species like A. baicalensis, Cyclotella baicalensis and Cyclotella minuta

are known to grow well (Kozhov, 1963). Highly transparent ice can result in irradiances of

around 450 µmol m-2 s-1 at the ice – water interface (Richardson et al., 2000). Periodic shifts of

snow cover by wind and variations in incident solar radiation can cause considerable fluctuations

in these irradiance levels. The combination of relatively high light and low temperatures

probably results in blooms of the A. baicalensis in surface waters under ice, and years of

especially high growth are known as “harvest years” or Melosira years1 (Kozhov, 1963,

Bondarenko et al., 1996).

A high degree of penetration of incident light results in warming of waters just below the ice that

causes convective mixing in the upper water column extending down to 50 m (under clear ice)

(Jewson et al., 2009) where cells of A. baicalensis are found to be most abundant (Bondarenko et

al., 1996, Bengtsson, 1996). A. baicalensis was found to have a still-water sinking rate of 4 m d-1, a value that falls within the range of values reported for marine- (Smayda and Bienfang, 1983)

______

1While the genus Aulacoseira had been established, many species in the genus today were historically placed in the genus Meloseira (Krammer, 1991). Simonsen in 1979, moved the species of Meloseira that possessed a collum into the genus Aulacoseira (Siver and Kling, 1997)

While most recent taxonomic studies recognize the genus Aulacoseira, some may refer to the taxa under the genus Meloseira or Melosira (Round et al., 1990, Krammer, 1991, Siver and

Kling, 1997, Kooistra et al., 2003, Adl et al., 2005). 16

and freshwater algae (Reynolds, 1984). Suspension of these diatoms in the upper layer of the

lake would probably be due to penetrative convection resulting in lift. A numerical model for this

phenomenon was provided by Kelley (1997).

Aulacoseira baicalensis was found to grow fastest at 2 - 3 °C with specific rates increasing as

temperatures decreased (Richardson et al., 2000). No growth was observed above 8 °C consistent

with the the psychrophilic nature of this diatom. Maximum growth rates were observed at relatively low irradiances between 25 - 41 µmol m-2 s-1, with a decrease in growth rates in response to increasing irradiances. Temperature was established as a major driving force in the seasonal succession of species in the lake.

Lake Baikal is one of the few places known in the world where diatom resting spores are found in an open lake environment (Edlund et al., 1996, Likhoshway et al., 1996). In 1996, it was

proposed that Lake Baikal’s spore forming diatom was morphologically distinct from the more

common A. islandica (O. Müller) Simonsen, and was henceforth to be considered a separate

species, which was named A. skvortzowii Edlund, Stoermer & Taylor (Edlund et al., 1996).

Recent molecular studies have suggested that A. skvortzowii and A. baicalensis (Meyer)

Wislouch (Popovskaya, 2000), are the closest among living forms to extinct taxa of the genus and may have diverged from each other within the lifetime of the lake (i.e., less than 25 million yr) (Sherbakova et al., 1998).

Blooms of A. skvortzowii develop at temperatures below 4 °C with increasing mortality at temperatures above 6.5 °C (Jewson et al., 2008). These cells were found to use environmental phosphate levels as cues to produce resting spores before the temperature rise associated with summer stratification. The spores were capable of surviving in coastal sediments for up to a year. 17

Under favorable conditions of light, temperature and nutrient availability, the spores would be

resuspended in the water column, resulting in the next round of the diatom bloom (Jewson et al.,

2008).

Objectives of the current study - The focus of this project was to understand the dynamics of

the winter assemblage in Lake Erie, analyze its biochemical and physiological state and to study

the physical and biological interactions within the community. Specific objectives include:

a) Assessment of community structure and bloom dynamics of winter diatoms in Lake

Erie - This includes identifying the components of the CACHE and looking at the physical

and biological interactions among the components of the CACHE.

b) Measure rates of primary production in the diatom communities - Dual proxies will be

used to measure rates of primary production at the different sampling points across the lake.

14 These are incorporation of [ C]-NaHCO3 and O2 evolution, respectively by the biomass in

response to varying light intensities. c) Study details of the interaction between the phytoplankton and ice cover – This includes

assessment of the physical nature of the association between the biological components with

the ice and identification of the mechanisms involved. d) Characterization of the dominant species in the assemblage - Individual components of

the assemblage, (primarily the two dominant diatoms A. islandica and S. binderanus) will be

isolated and cultured in the lab using multiple approaches. Once pure cultures of these

diatoms are obtained, the strains will be characterized in terms of their biochemical and

physiological status, in an attempt to understand their role in the initiation, development and

fate of the CACHE’s. 18

Why is this work necessary? Recurrent hypolimnetic hypoxia observed in the central basin of

Lake Erie is well documented. Internal loading of nutrients into the lake due to the hypoxic

waters could result in the development and proliferation of harmful algal blooms. The hypoxia

could further result in a loss of habitat to benthic macrofauna and fish, and if persistent, could

lead to noxious gas emissions. Phytoplankton biomass, nutrient geochemistry and plankton

production in winter could have potential impacts on, or may even drive the development of the

summer hypoxia. Climate change is predicted to impact the extent of ice cover on the Great

Lakes. Reduced ice cover could result into greater wind-driven mixing in the water column. The

declining ice cover could result in shifts in the under-ice diatom community structure, thus influencing the amount of organic carbon production in the lake, and subsequently influencing events in spring and summer. Predicting these impacts would require a better understanding of the lake ecosystem in winter. Current mechanistic models of lake ecosystem function have relied on either assumptions or omissions of state- and rate variables for winter, resulting in biased predictive capabilities. A clear understanding of the lake ecosystem in winter would allow for the development of more accurate models, and could also enable better lake management decisions.

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CHAPTER II

EXAMINATION OF THE WINTER DIATOM ASSEMBLAGE IN

LAKE ERIE: IDENTIFICATION, MORPHOLOGY AND

ECOLOGY BASED ON TECHNIQUES IN LIGHT- AND

SCANNING ELECTRON MICROSCOPY.

ABSTRACT

Winter surveys across Lake Erie between 2007 to 2011 documented diatom assemblages associated with extensive ice cover (Twiss et al., 2012, McKay et al., 2011). The microplankton assemblages were dominated by Aulacoseira islandica (O. Müller) Simonsen (≈80%) and

Stephanodiscus binderanus (Kützing) Krieger (≈19%), with other taxa like Fragilaria spp.,

Cyclotella spp., Asterionella spp. and Tabellaria spp. making up less than 1% of the assemblage.

In this study we describe the detailed morphology of the two dominant taxa observed. We documented the presence of resting cells and auxospores in filaments of A. islandica, but not in the other taxa. We compared various sample processing techniques for scanning electron microscopy (SEM) to determine the best method for observation of diatoms and their interactions with other components of the plankton. We describe here, a rapid and effective technique for SEM; namely, substituting critical point drying (CPD) with hexa-methyl-di- silazane (HMDS) treatment. Among all methods tested, the HMDS treatment resulted in well preserved cells with the least morphological distortion of the organic components on the cell surfaces. Of notable interest to the study was the secretion of extracellular polymeric substances 28

(EPS) on the surface of A. islandica and S. binderanus, the two dominant taxa in the winter

blooms, but not on the other taxa observed. The new sample processing method of treatment

with HMDS also revealed extensive colonization of the diatom EPS by flagellated, rod-shaped

bacteria. The observations, when coupled with data obtained from parallel research on this

phenomenon, are shedding new light on the ecological dynamics of the winter phytoplankton in the lake.

INTRODUCTION

Initial observations using light microscopy indentified diatoms as the key component of the

winter phytoplankton assemblage associated with ice cover in Lake Erie. The blooms were

dominated by Aulacoseira islandica (O. Müller) Simonsen comprising about ~80% of the cells

and Stephanodiscus binderanus (Kützing) Krieger making up around ~18%, with other diatoms

(Fragilaria spp., Asteronella spp. Cyclotella spp. and Tabellaria spp.) together accounting for the remaining 1-2 % (Twiss et al., 2012).

In this study, we sought to elaborate on the morphology of the diatoms, linking morphological characteristics to the ecology of the diatoms in the lake. To accomplish this, we relied on techniques in light- and scanning electron microscopy (SEM) to document detailed morphological features of the diatoms. Here we describe in detail the morphological characteristics of the two main taxa, Aulacoseira islandica and Stephanodiscus binderanus. The

presence of resting stages and auxospores in cells A. islandica, are highlighted. We also 29

compared various sample processing techniques used in SEM and determined the procedure best suited for observations of diatoms nearest to their native state.

Diatoms are unicellular algae that possess a cell wall composed of silica (SiO2) referred to as the frustule. The frustules are made up of two valves, with one valve fitting into the other, similar to the two parts of a petri dish. Diatoms probably evolved from a chrysophyte ancestor in silica replete oceans, with two major lineages: the centric diatoms and the pennate diatoms (Round et al., 1990). Centric diatoms are usually radially symmetrical with oogamous sexual reproduction.

Examples relevant to this study are A. islandica and S. binderanus. Pennate diatoms show bilateral symmetry with amoeboid gametes. Examples relevant to this study are Fragilaria spp.,

Tabellaria spp. and Asterionella spp.

Identification of diatom species relies heavily on characteristics such as dimensions of mantle height, valve diameters, orientation of the areolae, details of the spines and morphology of valve faces ( Likhoshway et al., 1992, Babanazarova et al., 1996, Le Cohu, 1996, Siver and Kling,

1997, Crawford and Likhoshway, 1999). The relative low degree of resolution obtained in even the best light microscopes makes accurate identification of diatoms challenging. Thus, identification of diatoms to the species level often relies on SEM analysis of cleaned frustules, an approach involving removal of organic material from samples using a strong acid, followed by repeated rinses with water. The resulting cleaned frustules can then be air dried onto cover slips, coated and viewed by SEM. Detailed observations and measurements of morphological components of the frustules are then used to identify the diatom.

In addition to studying cleaned frustules, other common methods of studying diatoms using SEM involve direct observation of air dried specimens, and filtering fixed specimens onto 30

polycarbonate membranes of a fixed pore size followed by dehydration and drying of the specimen. Samples are then coated with a layer of a conducting material for viewing by SEM.

Whereas critical point drying (CPD) is the most commonly used procedure for drying, other methods involving the use of chemicals like Peldri II and hexa-methyl-di-silazane (HMDS) have also been employed (Nation, 1983, Braet et al., 1997, Araujo et al., 2003, Jung et al., 2010,). In this study, three techniques, viz. air drying, critical point drying and HMDS treatment were used so as to discern the best approach towards studying the morphological characteristics of the diatoms involved in winter bloom formation and their interactions with other members of the plankton community.

As diatom cells divide, each daughter cell retains one valve from the parent cell resulting in one of the two cells being slightly smaller than the parent. With successive vegetative divisions, there is a progressive decrease in the size of cells. Once cell dimensions are reduced below a certain threshold and if presented with suitable environmental conditions, gametogenesis is triggered.

Successful fusion of gametes results in a zygote, termed the auxospore. The auxospore expands in volume, forming a new vegetative cell. The cell at this point is the largest cell in the life cycle of the diatom, and allows for a restoration of cell size in the population (Round et al., 1990).

Certain features of the fine structure of auxospores are used in determining phylogeny at higher taxonomic levels (Medlin and Kaczmarska, 2004). Resting stages are common across many groups of algae and include zygotes, akinetes, cysts, spores and modified vegetative cells (Davis,

1972, McQuoid and Hobson, 1996). Resting stages appear to have many roles, but their primary function serves to allow survival through periods of adverse conditions that could span annual- and sometimes even decadal scales (McQuoid and Hobson, 1996). 31

Diatoms are known to produce and secrete extracellular polymeric substances (EPS). These can

include a wide variety of forms, and have been observed in sessile and planktonic diatoms as

stalks, tubes, apical pads, adhering films, fibrils and cell coatings (Hoagland et al., 1993,

Thornton, 2002). Diatom EPS has been implicated in a variety of functions including motility,

adhesion, microhabitat stabilization, colony formation and use as anti-dessicants (Hoagland et al., 1993). Bacteria are also capable of EPS secretions (Decho, 1990, Decho, 2000, Bhaskar and

Bhosle, 2006) that allow for adsorption onto surfaces. Bacterial EPS has been proposed to provide a microenvironment to the bacteria embedded in it. The matrix can provide protection from physical and chemical stresses, and could also facilitate cell-cell communication and genetic transfer between cells (Krembs et al., 2002, Collins and Deming, 2011, Krembs et al.,

2011). Understanding how nutrient and light limitation influence EPS production would allow in situ predictions of bloom aggregation, and perhaps help understand the question as to why diatoms aggregate (Thornton, 2002). The diatom EPS also provides a complex matrix that can be colonized by many populations of heterotrophic bacteria. Here we show the presence of EPS on the cells of the dominant diatom taxa, and subsequent colonization of this EPS by bacterial epiphytes. The implications of EPS production and association with bacterial epiphytes are discussed.

METHODS

Sampling sites and sample collection - Sites in each of the three basins of Lake Erie were sampled on surveys during February 2009 - 2011 (Fig. 1). Vertical net tows were collected at each site using a 154 µm pore size plankton net deployed to depths of 10 – 15 m in open water. 32

Samples for culturing were diluted with filtered lake water and collected in opaque amber bottles and placed at 4°C in the dark. Samples to be used for microscopy were fixed by addition of glutaraldehyde (final concentration 2% v/v) and placed at 4°C in the dark until used for experiments. All samples were transported to the lab at BGSU in insulated coolers. Samples collected in February 2010 were used for SEM and light microscopy whereas samples collected in both 2010 and 2011 were used to establish diatom cultures. Samples from these cultures were also used for light microscopy.

Figure 1: Stations sampled each year in February from 2009 – 2011 across Lake Erie. Stations were selected so as to include sites from all three basins in the lake.

Cultures - Surface water was collected at each station using a submersible pump deployed 1 m below the surface. This water was filtered through a 0.2-µm capsule filter and used for dilutions 33

and media preparation. The filtered lake water was amended with nutrients and used as culturing

+ medium (referred to as FLW medium). This medium contained 50µM Na2SiO3×9H2O, 61µM

Ca(NO3)2×4H2O, 14µM K2HPO4, 25µM MgSO4×7H2O, 2.5µM Fe-EDTA (FeCl3 + Na2EDTA),

-1 -1 -1 48µM Na2CO3, 5µg L Vitamin B12, 1 µg L Biotin, 200 µg L Thiamine HCl, 0.03µM

Na2EDTA, 2.5µM H3BO3, 0.02µM CuSO4×5H2O, 0.04µM ZnSO4×7H2O, 0.7µM CoCl2×6H2O,

0.02µM MnCl2×4H2O, and 0.01µM Na2MoO4×2H2O.

Sterile FLW+ medium was amended with the antibiotics streptomycin (50 mg L-1),

chloramphenicol (50 mg L-1) and ampicillin (160 mg L-1). Seston (~ 500 µL) from each station

was inoculated into glass flasks containing 80 ml of this medium. In addition, the medium was

distributed into 12-well polystyrene tissue culture plates, and each well was inoculated with a

single diatom filament. The flasks and plates were incubated at 4°C, under low light

(approximately 60µM quanta m-2 s-1) set at 11:13 h day: night cycles, with periodic replacement of medium.

Light- and epifluorescence microscopy - Samples fixed with glutaraldehyde (2% final conc. v/v) were used for light microscopy. Cells were viewed under 40× magnification using an

Olympus EX51 light microscope. Images were acquired using SPOT imaging software (version

4.0.1, Diagnostic Instruments Inc.). Cells of the filamentous diatom A. islandica were identified based on morphological descriptions provided in previous studies (Sicko-Goad et al., 1989).

Fully expanded cells, with cytoplasmic components at the periphery of the cell, a central cytoplasmic bridge and well defined vacuolar areas on either side of the cytoplasmic bridge were used as identifying characteristics of vegetative cells. Cells with a condensed cytoplasmic mass 34

with rounded chloroplasts were identified as resting cells. Cells completely devoid of cytoplasm

were classified as dead cells.

The presence of epiphytes on the diatom filaments was checked by staining samples with the

blue fluorescent dye 4’,6-diamidino-2-phenylindole (DAPI) that preferentially binds to regions

of double-stranded DNA rich in A-T bases. Stained samples were then observed using

epifluorescence microscopy. Samples of diatoms from cultures maintained in the lab were

filtered through a 0.2µm membrane and treated with a solution of 1 µg L-1 (final conc. v/v) DAPI

for 3 min. The filters were then placed on glass microscope slides. A drop of mounting medium

(AF1, Citifluor, Electron Microscopy Sciences) was placed on the sample followed by placing a

cover glass on the filter. The slides were placed at -20°C in the dark until ready for imaging.

Samples were viewed at 100× magnification under a Zeiss Axioskop epifluorescence

microscope.

Scanning electron microscopy: Conventional sample preparation - We followed the protocol

of Hasle and Fryxell (1970). Samples of diatom seston collected by net were treated with equal

volumes of concentrated nitric acid, and boiled until the solution attained half its original

volume. Once cooled, an equal volume of ultrapure water (Milli-Q; >18 MΩ cm -1) was added to the container, and the material was allowed to settle for up to 8 h. Once the frustules had settled, the overlying liquid was removed and replaced with Milli-Q. The samples were once again

allowed to settle for up to 8 h. The procedure was repeated up to 10 times, or until the solution

attained neutral pH. Frustules thus obtained were air dried onto glass cover slips in a dust-free

environment. The cover slips were attached to SEM stubs, and coated with a 10 nm Au-Pd layer

using a sputter coater (Hummer VI-A) before viewing with a Hitachi S2700 SEM. 35

Scanning electron microscopy: Modified sample preparation

Air drying samples - An aliquot of the fresh plankton material was allowed to settle in a glass

vial following which the overlying liquid was removed and replaced with sterile 0.1M sodium

phosphate buffer (pH 7.2). After rinsing the material by gentle agitation, the sample was allowed

to settle and the overlying liquid was again replaced with buffer. An aliquot of the above rinsed

plankton sample was transferred to a clean sterile glass tube and glutaraldehyde was added to to

a final concentration of 2% (v/v). The sample was gently mixed and placed at 4°C for 2 h

followed by rinsing with sodium phosphate buffer. Both unfixed- and glutaraldehyde-fixed

samples were air-dried on clean glass coverslips in a dust free chamber. The cover slips were

then attached to SEM stubs, and coated with a 10 nm Au-Pd layer using a sputter coater

(Hummer VI-A) before viewing with a Hitachi S2700 SEM.

Using liquid CO2 for drying - An aliquot of the fixed plankton material was allowed to settle in a glass vial. The overlying liquid was removed and replaced with a solution of 2% glutaraldehyde (v/v) in sterile 0.1M sodium phosphate buffer (pH 7.2). The plankton samples were then filtered through a 0.2µm polycarbonate filter. Samples retained on filters were rinsed with 0.1M sodium phosphate buffer (pH 7.2) and the samples were dehydrated through a graded series of ethanol to 100%. Dehydration in 100% ethanol was repeated three times. Critical point drying (CPD) was carried out by flooding with liquid CO2 and then raising the temperature to the critical point. The filters were then trimmed to appropriate dimensions and mounted on SEM stubs. The samples were sputter-coated with a 10 nm Au-Pd layer using a sputter coater

(Hummer VI-A) before viewing with a Hitachi S2700 SEM. 36

Using HMDS for drying - An aliquot of the fixed plankton material was allowed to settle in a

glass vial. The overlying liquid was removed and replaced with a solution of 2% glutaraldehyde

(v/v) in sterile 0.1M sodium phosphate buffer (pH 7.2). Unbound glutaraldehyde was then gently

removed by allowing the sample to settle followed by replacement of the overlying liquid with

fresh buffer. This was done up to four times. Samples were then dehydrated through a graded

series of ethanol to 100%. Dehydration in 100% ethanol was repeated three times. Critical point

drying (CPD) was replaced by treatment with a solution of hexa-methyl-di-silazane (HMDS) for

5 min following which the sample was air dried onto cover slips in a dry, dust free environment.

The cover slips were then attached to SEM stubs, and sputter-coated with a 10 nm Au-Pd layer using a sputter coater (Hummer VI-A) before viewing with a Hitachi S2700 SEM.

RESULTS

Morphology of Aulacoseira islandica - A. islandica was observed at all sites across Lake Erie with cells linked together by spines to form long filaments of up to 12 cells (Fig. 2). Cells were

approximately 12 - 18 µm in diameter and 18 - 22 µm in length. More detailed observation of the

fine structure of the frustules (Figs. 3A, B) reveals cells in girdle view, with a deep valve mantle.

Cells are attached by linking spines which were spatulate in shape (Fig. 3A-b), consistent with

previous observations (Babanazarova et al., 1996, Siver and Kling, 1997). Rimoportulae, while

common in centric diatoms (Round et al., 1990) were not clearly visible in micrographs of A.

islandica in this study. Cells possess an unornamented area called a collum at the edge of the

valve mantle (Fig. 3A-c). Mantle areolae (Fig. 3A-a) were small and positioned parallel to the

pervalvar axis. Aerolae were polymorphic, and were either circular or consisting of two sets of 37

pores, with both types sometimes seen in the same filament. Stepped valves were commonly observed. Frustules of A. islandica appeared to be thinner and more delicate when collected at

Eastern basin stations (Figs. 4 A, B), and were prone to breaking in spite of special handling.

Resting spores were not observed in any of the samples. Occasionally, resting cells, characterized by rounded plastids were observed (Fig. 5 A-D). Cells from the Western basin (St

341; Fig. 5 A), Central basin (St 1290 and St 880; Fig. 5 B, C) and eastern Central basin (St

1053; Fig. 5 D) appear to have condensed cytoplasm and rounded plastids, and were identified as resting cells. Resting cells were not observed in samples collected in the Eastern basin (St 452).

Auxospore formation was observed across different sites throughout the Central- and Eastern basins in Lake Erie (Fig. 6 A-D). Filaments in these observations typically ranged from 10 to 11

µm in diameter. Auxospores were around 26 to 31 µm in diameter and were always observed in a terminal position of the filament. 38

Figure 2. The filamentous diatom A. islandica from St 1290 (A) and St 880 (B) of Lake Erie.

Scale bar: 10 µm.

Figure 3. SEM micrographs of frustules of A. islandica collected in Lake Erie in Feb 2009, showing morphological fine structures including areolae (a), linking spines (b) and the collum

(c). Scale bar: 5 µm (A) 10 µm (B). 40

Figure 4. Frustules of A. islandica seen cracking under a coverslip. Cracks in the frustules are indicated by arrows. The samples were from St 949 (A) and St 452 (B) in the Eastern basin of

Lake Erie. Scale bars: 10 µm. 41

Figure 5. Resting cells of the filamentous diatom A. islandica from Western basin St 341 (A), and Central basin St 1290 (B), St 880 (C) and St 1053(D) Scale bars: 10 µm.

Figure 6. Auxospore formation in filaments of A. islandica observed in February 2011 from

Central Basin St 1290 (A and B), St 1053 (C) and Eastern Basin St 452 (D). Scale bars: 10 µm. 42

Morphology of Stephanodiscus binderanus - S. binderanus was observed at all sites across

Lake Erie with cells linked by spines similar to A. islandica to form long filaments of up to 24 cells (Fig. 7A-E). Cells were approximately 12 - 15 µm in diameter and 14 - 20 µm in length.

SEM fine structure analysis of S. binderanus frustules (Figs. 8A, B) revealed cells in girdle view

with a distinct set of linking spines (Fig. 8A-a) connecting the cells. Fultopartulae (Fig. 8A-b)

were present on the valve margins and areolae (Fig. 8A-c) were visible along the ends of the

cells along the valve margins.

Other diatoms observed - Fragilaria spp. filaments were observed in low numbers at multiple

stations across the lake. Fig. 9A shows a typical filament viewed with a light microscope. Cells

were approximately 1.5 - 3 µm wide and 33 - 51 µm in length. Lanceolate frustules of the diatom

were connected to each other by small spines (Fig. 9B) to form the ribbonlike colony shown in

Fig 9A. Fig. 10A shows a valve view of a single Cyclotella sp. cell adjacent to a filament of S.

binderanus. The cell is approximately 32 µm in diameter. A girdle view of Tabellaria sp. (Fig

10B) shows an elongated valve with capitate ends. The valve is wider in the center. Cells were

approximately 2.6 µm wide at the ends, 6.5 µm at the center and 42 µm in length. No distinct

rimoportulae were seen. In a few other light microscope observations (images not shown),

Tabellaria cells were observed attached end to end by mucilage pads to form a characteristic zig-

zag pattern. A girdle view of Asterionella sp. (Fig. 10C) shows linear- to lanceolate valves with capitate ends attached to each other at their basal ends by mucilage pads to form the characteristic stellate colonies. Cells were approximately 2 µm wide and 43 µm in length. The cells were rare in samples collected in winter, and only 1 - 2 instances of the taxon was observed.

Figure 7. The filamentous diatom S. binderanus from Western basin St 341 (A), Central basin St

1290 (B), St 880 (C), St 1053 (D), and Eastern basin St 452 (E) in Lake Erie in February 2010.

Scale bars: 10 um.

Figure 8. SEM micrographs of frustules of the diatom S. binderansus from Lake Erie in

February 2009, showing morphological fine structures including linking spines (a), fultopartulae

(b) and areolae (c). Scale bars: 5 µm 45

Figure 9. Image of a filament of the diatom Fragilaria sp. (Scale bar: 10 um) and SEM micrograph of frustules of the same taxon. (Scale bar: 5 µm)

Figure 10. Other diatoms observed in the winter phytoplankton assemblage in 2009 and 2011:

Cyclotella sp. (A) (Scale bar: 10 µm), Tabellaria sp. (B) (Scale bar: 10 µm) and Asterionella sp.

Comparison of sample processing techniques for SEM - A comparison of the different sample

processing techniques is shown in Fig. 11. Unfixed samples air dried in water (Fig. 11 - row 1)

showed structural damage with all diatoms appearing to have fractures. Samples fixed with

glutaraldehyde and air dried (Fig. 11 - row 2) looked considerably different from specimens in

the unfixed air dried samples. A striking difference was the presence of EPS on filaments of A.

islandica and S. binderanus. Fixed samples dried using CPD (Fig. 11 - row 3) showed reasonable clarity in cell surface details, with the notable absence of the EPS shown in the fixed air dried samples. Samples treated by replacing CPD with HMDS treatment (Fig. 11 – row 4) showed cells with the fewest morphological distortions. EPS on the cells was clearly seen in

filaments of A. islandica and S. binderanus. EPS was not observed on filaments of Fragilaria sp. using any of the methods. In some cases, copious amounts of EPS resulted in attachment of filaments of S. binderanus via “sticky” bridge – like connections (Fig. 12). This characteristic

was corroborated by observations of slimy, sticky aggregates of filaments in senescent diatom

blooms sampled in April 2008 and April 2010 (not shown).

The micrographs also showed in clear detail, flagellated rod-shaped bacteria colonizing the EPS on the filaments of A. islandica and S. binderanus (Figs. 11, 13 and 14). This detail was not clear on filaments using any of the sample processing techniques other than HMDS. Bacterial epiphytes were not observed on filaments of Fragilaria spp. (Figs. 11, 15), Asterionella spp. (not shown ) and Tabellaria spp. (not shown). Distribution of the EPS and bacteria across the cells was uneven, making it difficult to enumerate the number of bacteria per diatom cell. In addition, the 3- dimensional nature of the EPS matrix makes it difficult to enumerate the number of bacteria embedded inside the matrix. Bacterial colonization of A. islandica and S. binderanus 48

was observed around the connecting spines (Figs. 13A, 14A) as well as in the girdle region (Figs.

13B, 14B) of the cells.

Diatom cultures - Cultures of diatoms were prepared by inoculation of a single diatom filament

into a flask containing FLW+ mediaum. Resulting cultures were non axenic. Attempts to remove

the bacterial component associated with the filaments by means of treatment with antibiotics

were unsuccessful. Cultures were slow growing and took up to 2 months to form visible clumps

of filaments (Fig. 16, A-C). Fragilaria spp. was the most successful taxon to grow under lab

conditions. Although slow growing, the cultures formed visible clumps of long filaments

measuring up to a couple of centimeters in length (Fig. 16A). Some success was observed with

filaments of S. binderanus (Fig. 16B), whereas the least success was observed with A. islandica

cultures (Fig. 16C). In both these cases, the filaments were much shorter than with Fragilaria

spp., measuring only a few mm. Cultures of A. islandica and S. binderanus, but not Fragilaria sp. when stained with DAPI showed extensive colonization by bacteria (Fig. 17), seen as small blue specs, embedded in an EPS matrix. The images were taken under 100× magnification, and thus reveal only a single plane of focus, making enumeration of the bacteria in the matrix extremely challenging.

Aulacoseira islandica Stephanodiscus binderanus Fragilaria sp.

Row 1

(Not Observed) Row 2

Row 3

Row 4

Figure 11. A comparison of the different techniques used in sample processing for SEM. Row

1: Air dried samples. Row 2: Samples fixed with glutaraldehyde and air dried. Row 3: Samples

fixed with glutaraldehyde, filtered onto a polycarbonate membrane, dehydrated through a graded

series of ethanol, and dried by CPD. Row 4: Samples fixed with glutaraldehyde, dehydrated

through a graded series of ethanol, treated with HMDS and air dried. 50

Figure 12. The presence of EPS on filaments of S. binderanus and the consequent aggregation of the filaments.

Figure 13. SEM micrographs showing filaments of the diatom A. islandica with the presence of

EPS that is colonized by flagellated rod-shaped bacteria (panels A and B). Scale bars: 5 µm 52

Figure 14. SEM micrographs showing EPS on filaments of the diatom S. binderanus and colonization by flagellated rod-shaped bacteria (Panels A and B). Scale bars: 2 µm (A), 5 µm

(B). 53

Figure 15. SEM microgtaphs of filaments of the diatom Fragilaria spp. (Panels A and B).

Unlike the dominant taxa, A. islandica and S. binderanus, this taxon was found in lower numbers, with the absence of EPS’s or bacterial colonization. Scale bars: 10 µm.

Figure 16. Flasks containing (non axenic) cultures of filamentous diatoms Fragilaria spp. (A),

S. binderanus (B), and. A. islandica (C). Cultures of Aulacoseira shown here are mixed cultures that include filaments of Stephanodiscus and Fragilaria.

Aulacoseira islandica Stephanodiscus binderansus Fragilaria sp

(Not Observed)

Figure 17. DAPI stained filaments of diatoms. Filaments of A. islandica, S. binderanus and

Fragilaria sp. isolated from plankton samples collected from St 880 (row 1), St C23 (row 2) and

St 84C (row 3) in February 2011, were grown in nutrient amended lakewater in the lab.

Filaments from these cultures were stained with DAPI to reveal epiphytic colonization on filaments of Aulacoseira and Stephanodiscus, but not on filaments of Fragilaria. 56

DISCUSSION

Comparison of sample processing techniques for SEM - In addition to studying cleaned

frustules, a common method of studying diatoms using SEM involves direct observation of air

dried specimens coated with a layer of a conducting material. Air drying samples in water,

however, could result in loss of structural rigidity of some features due to the destructive action

of the meniscus during drying. Other methods involve filtering fixed specimens onto

polycarbonate membranes of a fixed pore size (Paerl and Shimp, 1973) followed by treatment

through a graded series of ethanol or acetone. These samples are then dried before being coated

for viewing by SEM. Physical stresses incurred during transfer of samples through different

solutions used in the dehydration and drying procedures may result in the loss of structural

integrity of the samples. Processing samples on filters may also result in the sample being

washed off the filter if subjected to rough handling.

Of the many alternatives for drying biological specimens in preparation for SEM, the most

commonly used procedure involves critical point drying (CPD). The procedure removes liquids

from a specimen by adjusting the temperature and pressure so that the liquid and gas phases of

the sample are in equilibrium. The CPD technique, however, requires a specific apparatus, and

the sample needs to be fixed well onto a surface to avoid being washed off due to shear pressures

caused by the filling and draining of the chamber. Delicate associations between cells and finer

structures that may be loosely attached on cell surfaces can be lost in the process. CPD had in

some studies been shown to cause cell shrinkage as well ( Braet et al., 1997, Araujo et al., 2003).

Other techniques for drying specimens include use of chemicals like Peldri II and Hexa-methyl- di-silazane (HMDS) to remove liquids from soft tissues without inducing additional artifacts and 57

distortions (Nation, 1983, Braet et al., 1997, Araujo et al., 2003, Jung et al., 2010) . HMDS

cross-links protein and adds structural rigidity to the sample, thus preventing fracturing and

collapsing of the specimen. It reduces the surface tension and distortions in the sample during air

drying. Unlike CPD, the procedure is cost effective, simple, and eliminates excessive handling

of the specimens. Studies comparing the effects of CPD and HMDS found little to no differences

in quality of images from either technique (Braet et al., 1997, Araujo et al., 2003, Jung et al.,

2010).

Our results showed that samples fixed with glutaraldehyde, dehydrated through an ethanol

gradient and dried with HMDS provided samples with the least morphological distortion and

preserved the most fine structural detail. Samples air dried in water exhibited the greatest

damage with visibly dry EPS coating the cells. The EPS in these samples appeared more like a

thin dry film rather than the gelatinous matrix seen in samples processed using other techniques.

Samples fixed with glutaraldehyde and air dried showed slightly better retention of morphology

than the non-fixed samples. Samples dried using CPD had to be filtered on a 0.2µm

polycarbonate membrane and placed in a chamber that was flooded with ethanol followed by

treatment with liquid CO2. The cells in these samples may have been subjected to shear forces resulting in the removal of the EPS and associated material. Cells treated with HMDS, on the other hand, were treated gently and allowed to settle before replacing the overlying liquid.

Surface tension forces generated during HMDS evaporation did not appear to have any distorting effect on the diatoms, the EPS or the bacterial epiphytes colonizing diatoms. Whereas both

CPD- and HMDS-treated samples followed the same regimen of fixation and dehydration , the

drying step for HMDS-treated samples took only 5 min compared to 2 h in case of the CPD

treatment. 58

Previous work comparing sample treatment techniques have reported little to no differences in samples dried using CPD and HMDS (Braet et al., 1997, Araujo et al., 2003, Jung et al., 2010), arguing that the HMDS treatment only provided benefits in terms of simplicity and time. Here we argue that the gentle nature of the treatment with HMDS coupled with reduced handling of the sample contributes to greater sample integrity and greater detail in sample observations.

Morphology of diatoms - This study describes several morphological details of the vegetative, resting and auxospore cells of the centric filamentous diatom Aulacoseira islandica that forms part of the winter-spring plankton assemblage in Lake Erie. Diatoms of the taxon Aulacoseira are some of the most commonly occurring freshwater diatoms, and their morphology has been extensively characterized (Babanazarova et al., 1996, Edlund et al., 1996, Siver and Kling, 1997,

O'Farrell et al., 2001, Jewson et al., 2008). Features of Aulacoseira commonly used in identifying the species include height of the mantle, diameter of the valve, number of aerolar rows per 10µm length, height of the step (corresponds to the thickness of the girdle bands), linking spines, ratio of the length of a spine to its width, and type of aerolae (Babanazarova et al.,

1996). Morphological characteristics of the frustules of A. islandica in Lake Erie were similar to previously published reports on characteristics of the taxon based on samples collected in North

America and in Lake Baikal, Russia (Babanazarova et al., 1996, Siver and Kling, 1997).

Frustules of A. islandica did not possess long separating spines located on the separating valve.

These spines are assumed to restrict filament length and their absence may explain the occurrence of long chains of the diatoms observed in field samples. There appears to be a certain degree of polymorphism in the type of aerolae (the perforations in the valve) on the frustules with two main types observed, either circular, or consisting of two sets of pores sometimes on 59

the same filament, an observation consistent with previous descriptions of the species (Siver and

Kling, 1997).

Diatoms comprising the winter assemblage in Lake Erie, when cultured in the lab, did not grow

at temperatures above 8°C. While Lake Erie surface water temperatures in winter are close to

0°C (Burns et al., 2005, Twiss et al., 2012), water temperatures begin to rise in early April (early spring), with temperatures reaching up to 10°C by mid May (Burns et al., 2005). It is likely that the warming of Lake Erie surface waters through spring leads to the eventual demise of the diatom population that blooms in winter. Nutrient levels in the waters are also reduced, presumably due to a drawdown caused by the winter bloom itself (Twiss et al., 2012). In

response to adverse thermal and nutrient regimes, we posit that cells of A. islandica are exported

from the surface mixed layer. Formation of a resting stage would not only promote export to

deeper water but could also help A. islandica survive long periods of adverse physico-chemical

conditions.

Resting stages are common across many groups of algae and include zygotes, akinetes, cysts,

spores and modified vegetative cells. (Davis, 1972, McQuoid and Hobson, 1996). Resting stages

appear to have many roles, but their primary function serves to allow survival through periods of

adverse conditions that could span annual- and sometimes even decadal scales (McQuoid and

Hobson, 1996). In diatoms, resting spores and specialized resting cells are the predominant forms of resting cells observed (McQuoid and Hobson, 1996). While the stages may be part of the diatom life cycle, in most cases they appear to be coupled with senescence in populations and the onset of unfavorable conditions. 60

Resting spores are morphologically and physiologically different from vegetative cells, the former having thicker frustules, with a rounded shape and less elaborate surface pattern. Resting cells on the other hand appear morphologically similar to vegetative cells (Sicko‐Goad et al.,

1986). Unlike resting spores, the resting cells show no observable change to the cell wall or visible accumulation of storage products. Both resting stages are physiologically different from vegetative cells and are characterized by dense, dark cytoplasmic matter and rounded plastids

(Sicko-Goad et al., 1989, Gibson and Fitzsimons, 1990, Round et al., 1990). Other differences from vegetative cells include higher C:N ratios, higher chl a levels, condensed organelles, storage vesicles, and low sugar phosphate content. Resting cells have lower respiration and photosynthetic rates than vegetative cells. (Kuwata, 1993, McQuoid and Hobson, 1996).

Sporulation tends to be more common in marine species whereas freshwater diatoms and pennate taxa tend to form resting cells (Sicko‐Goad et al., 1986, McQuoid and Hobson, 1996) Resting spores in A. skvortzowii can develop when cells sink to the aphotic zone (Jewson et al., 2008).

Spore formation in these diatoms usually followed the end of an exponential growth phase, and could also indicate phosphate limitation (Jewson et al., 2008). The time required for the process of sporulation to complete can take weeks. Successive sporulation before the onset of adverse environmental conditions would therefore require an early cue to prompt cells to prepare for dormancy. Formation of resting spores does, however, require larger amounts of silica in order to develop the highly silicified casing of the spore (Kuwata, 1993).

Development of resting cells, characterized by rounding of chloroplasts was observed in cultures of A. subarctica in response to low illumination (Gibson and Fitzsimons, 1990). Their study documented physiological responses of the cells incubated in the dark, and found a progressive 61

decrease in cellular carbohydrate and lipid reserves as well as a decrease in photosynthetic

capacity accompanied by rounding of chloroplasts (Gibson and Fitzsimons, 1990). The diatom A. islandica has been shown to produce resting cells, but not resting spores (Sicko‐Goad et al.,

1986, Sicko-Goad, 1986, McQuoid and Hobson, 1996). Export of resting stages to the sediments in lakes requires that they sink out of the photic zone. Migration of cells to greater depths into the aphotic zone can trigger formation of resting cells. It has been suggested that factors associated with resting stage initiation are similar to those involved in enhanced sinking rates

(Sicko‐Goad et al., 1986, Sicko-Goad, 1986, McQuoid and Hobson, 1996). These include aging, temperature, decreasing light and nutrient limitation. EPS production in senescent populations can increase sinking rates and has also been linked to spore formation (Myklestad and Haug,

1972). Resting cells are usually formed when cells are light limited, and usually indicate a change in environmental conditions. Resting spores on the other hand rely on a cue preceding an environmental change.

Once removed from the photic zone, both, resting cells and resting spores can remain viable in cold, nutrient limited, dark environments for long periods (McQuoid and Hobson, 1996). Resting cells carry out respiration and photosynthesis at very low rates. Resting cells of the freshwater diatom A. granulata were revived after having been in anoxic sediments for over 20 years

(Sicko‐Goad et al., 1986), a phenomenon found to occur across other taxa as well (Nipkow,

1950, Stockner and Lund, 1970). Survival of resting cells is contigent upon factors of time, temperature, light and nutrient availability (McQuoid and Hobson, 1996, Jewson et al., 2008).

Under favorable environmental conditions, spores and cells can germinate rapidly. Changes that occurred during sporulation are reversed rapidly including proliferation of the cytoplasm and 62

organelles, changes in reserve bodies, commencement of cell division, increase in storage

product and increased photosynthetic capacity (Sicko‐Goad et al., 1986, Sicko-Goad, 1986,

McQuoid and Hobson, 1996). Retention or discarding of the spore valve is taxon dependant.

Unlike spores, resting cells do not begin growing in high nutrient waters if light levels are low,

giving the cells an advantage in short term adaptability. Resting cells can initiate growth faster

than resting spores when exposed to ideal conditions, but are unable to survive for as long as

resting spores. Studies on A. subarctica have shown rapid rejuvenation of resting cells exposed to light, with photosynthetic competence being restored in as little as 8 h (Lund, 1954).

Cells in resting stages can lie dormant in sediments for extended periods of time. Resuspension of these cells to the photic zone coupled with the right environmental conditions could result in germination of the cells and eventually to the formation of diatom blooms. Another role for the

formation of resting cells is to prevent a population crash after a bloom (McQuoid and Hobson,

1996). Large blooms could result in depletion of nutrients in surrounding waters. The nutrient

depleted conditions coupled with high light could cause photooxidation and metabolic

imbalances in cells that could destroy a population. Entering a resting stage, and sinking out of

the euphotic zone may thus be essential for ensuring survival of the population. This is consistent

with findings that show greater occurrence of spore formation in bloom forming taxa (Garrison,

1981), and lesser occurrence in polar waters where nutrients are rarely depleted (Hargraves and

French, 1983, McQuoid and Hobson, 1996).

Initiation of resting stages can likewise result from environmental cues. Earlier studies have

reported a number of environmental cues required for successful sporulation, in particular, low

light and low nutrient levels (McQuoid and Hobson, 1996). A study on the winter diatoms in 63

Lake Baikal implicated low phosphate levels as the primary trigger for sporulation (Jewson et al.,

2008). Studies of nutrient concentrations across Lake Erie in winter however indicate N and P replete conditions (Twiss et al., 2012), and the exact trigger for resting state development in the winter assemblages remains to be determined. Decreased silica levels were recorded in the

Eastern basin of the lake as early as mid- winter (Twiss et al., 2012), presumably a result of silica depletion by the extensive diatom blooms in the Western and Central basins of the lake. Resting spores have highly silicified frustules. Thus, lowered silica levels through late winter and spring could be a reason for the lack of resting spore formation. By contrast, resting cells possess frustules identical to vegetative cells, and are formed by changes in the cellular organization of the cell (Sicko‐Goad et al., 1986, Sicko-Goad, 1986). Formation of the resting cells may be followed by their subsequent export to deeper water.

Export of resting cells has also been linked to senescent populations of diatoms. These diatoms are usually at the end of a bloom phase, where nutrients have been exhausted. High light in nutrient deficient environments could result into photooxidation and production of other damaging metabolites. Cells in these situations typically tend to aggregate, usually by production of EPS that makes them “sticky”. The aggregates tend to possess greater settling velocities, and can thus move out of the photic zone (Smetacek, 1985, Thornton, 2002). This mechanism also protects cells from pelagic grazers, and is coupled with the formation of resting stages. Lower temperatures and no light could allow the resting cells to remain dormant for extended periods of time that could span months, years and even decades. Resting cells of Aulacoseira have been recovered from lake sediments and rejuvenated after extended periods. In Lake Baikal, resting spores have been found at depths of 10-25 m, where wind mixing can cause resuspension of sediment (Jewson et al., 2008). Investigations of Lake Erie sediment samples in 2007-08 found 64

an average of 66.2% of cells in the benthos were vegetative resting cells (Lashaway and Carrick,

2010). The cells were primarily composed of three taxa, A. islandica, S. niagarae and S.

binderanus.

The relatively shallow maximum depths of Lake Erie (10 m in the Western basin, 24 m in the

Central basin and 64 m in the Eastern basin) would aid both the deposition (A. baikalensis has sinking rates of 4m d-1(Kelley, 1997)) and resuspension of diatoms cells or spores. Resuspension

events in late fall, and interactions with ice in early winter (refer to Chapter 3 – Bacterial

epiphytes of diatoms promote ice nucleation in large lakes) could then allow the cells to move to

the photic zone in winter, where under conditions of nutrient sufficiency and adequate light

availability, they could germinate and result in an eventual bloom. Resting cells have been

shown to resume rapid growth when exposed to nutrient rich conditions under adequate lighting.

In addition, resting cells, unlike resting spores germinate faster, giving the population an

advantage in bloom initiation.

Our previous work has shown that nutrient levels in the water column in winter are high (Twiss

et al., 2012) and the endemic diatoms appear well-suited to low light expected for life under ice

(refer to Chapter 4 – Primary Production), thus providing optimal conditions for the initiation of

a bloom. Settling and resuspension of cells in sediments is common across lake systems (Lund,

1955), and a benthic resting phase in the life cycle of diatoms could provide an advantage to the

survival and recruitment of a species (McQuoid and Godhe, 2004).

EPS production - EPS was observed on filaments of A. islandica and S. binderanus, the two dominant taxa prevalent in the winter phytoplankton assemblage in Lake Erie. Other taxa present in smaller numbers did not possess this EPS on the cells. The scope of the current research described here, makes it difficult to attribute the production of the EPS to either the diatoms or 65

the bacteria associated with it. EPS may be defined as any macromolecule secreted external to the plasma membrane (Hoagland et al., 1993). These can include a wide variety of forms including crystalline, rigid fibrils to highly hydrated mucilaginous capsules (Hoagland et al.,

1993). EPS secretion has been observed in sessile and planktonic diatoms as stalks, tubes, apical pads, adhering films, fibrils and cell coatings (Hoagland et al., 1993, Thornton, 2002).

Polysaccharides are usually the primary component of diatom EPS (Hoagland et al., 1993).

Bacterial EPS exist as part of the dissolved organic matter in aquatic environments (Lignell,

1990), and as organic particulate matter in the form of marine snow, microbial mats and biofilms

(Decho, 1990). Production of EPS leads to diatom stickiness and subsequent aggregation in both planktonic and benthic habitats (Thornton, 2002). Diatom EPS has been implicated in a variety of functions including motility, microhabitat stabilization, colony formation and protection against desiccation (Hoagland et al., 1993). Diatom aggregation via EPS and subsequent vertical migration to the benthos is suggested to result in a rapid export of particulate organic matter to the benthos (Smetacek, 1985). It has also been suggested that diatom EPS may act as a sink for excess photosynthetic production (Staats et al., 2000).

EPS may also confer protection against grazers. It has been suggested that selective grazing pressure could account for the extinction of populations of certain taxa in phytoplankton blooms

(Hansen et al., 1995). Some supporting evidence for this is indicated in our laboratory diatom cultures where, under identical conditions of temperature, light and nutrients, A. islandica and S. binderanus grew slowly while producing copious amounts of EPS that was colonized by bacteria. By contrast, Fragilaria spp. grew rapidly forming long filaments with no EPS production or bacterial colonization. Phytoplankton cultures containing a mix of the three filamentous diatoms showed rapid growth of Fragilaria spp., with little to no increase in 66

numbers of A. islandica and S. binderanus. These cultures eventually became overrun by

Fragilaria spp. We hypothesize here that grazing pressure dictates the composition of the winter

phytoplankton blooms with non-EPS-producing diatoms being grazed, allowing the EPS producing diatoms to flourish.

Continuous culture techniques could enable testing the response of the diatoms to different limiting factors, and measurement of amounts and composition of EPS could establish the correlation between nutrient and light limitation on EPS production. Additional testing with detailed grazing assays, and corresponding cell counts would be needed, however, to confirm the grazing preference hypothesis.

Diatom – Bacteria associations - The EPS provides a complex matrix that can be colonized by heterotrophic bacteria (Alldredge and Gotschalk, 1990, Hoagland et al., 1993, Krembs et al.,

2002). Analysis of bacterial numbers and production in diatom aggregates found larger and more metabolically active bacteria in the EPS matrix than observed in the surrounding water column

(Alldredge and Gotschalk, 1990). Bacteria are also capable of EPS secretions (Decho, 1990,

Decho, 2000, Bhaskar and Bhosle, 2006) that allow for adsorption onto surfaces. Bacterial EPS has been proposed to provide a microenvironment to the bacteria embedded in it. The matrix can provide protection from physical and chemical stresses. The close vicinity of cells in this matrix has also been shown to facilitate cell-cell communication and genetic transfer between cells

(Krembs et al., 2002, Collins and Deming, 2011, Krembs et al., 2011). High resolving power using SEM, coupled with careful sample preparation techniques allowed for the observation of bacteria colonizing the surface of the filamentous diatoms. The techniques make it possible to study the distribution, numbers, and morphology of populations of bacteria associated with the 67

diatom filaments. Our work on interactions between the phytoplankton and ice in winter has

identified one of the bacteria colonizing the EPS on the diatoms as Pseudomonas fluorescens

(refer to Chapter 3 – Bacterial epiphytes of diatoms promote ice nucleation in large lakes). This

bacterium was subsequently implicated in the ice nucleating activity of the phytoplankton, and is

hypothesized to aid attachment of the diatoms to the bottom of the lake ice, thereby ensuring a

favorable position in the euphotic zone of the lake.

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CHAPTER III

BACTERIAL EPIPHYTES OF DIATOMS PROMOTE ICE

NUCLEATION IN LARGE LAKES

ABSTRACT

Sea ice supports diverse assemblages of planktonic microbes in polar waters. Freshwater ice, in contrast, is believed devoid of the network of channels used by marine plankton as microhabitat.

In cases where plankton are associated with ice cover on lakes, their recruitment is often attributed to atmospheric deposition. Here we document ice nucleating activity associated with assemblages of filamentous diatoms sampled from the ice-covered Laurentian Great Lakes. The ability to promote ice formation offers a previously undescribed mechanism by which these non- motile phytoplankton can attach to overlying ice and, thereby, maintain a favorable position in the photic zone. We attribute the ice nucleating activity to bacteria; Scanning Electron

Microscopy revealed associations of bacterial epiphytes with the dominant diatoms of the phytoplankton assemblage and bacteria isolated from the phytoplankton showed high temperatures of crystallization (Tc) to -3°C. Ice-nucleating active (INA) isolates were identified

as belonging to the genus Pseudomonas. Whereas INA bacteria have been isolated from lakes

and streams, their presence in these environments is attributed primarily to runoff and

atmospheric deposition as rain or snow consistent with their proposed role as biological ice

nuclei in clouds. Far from a passive existence in the aquatic milieu, the INA microbes associated

with winter diatoms in the Great Lakes may possess a role in promoting the formation of ice

during winter and in so doing, promote the growth of their diatom hosts under ice. The novel 75

mechanism presented may be relevant to temperate and polar ecosystems beyond the Great

Lakes including coastal oceans.

INTRODUCTION

Winter presents a logistical obstacle to our understanding of lake ecosystems, yet current

warming trends in global climate resulting in declining ice coverage (Assel et al., 2003) provide

cause for establishing in-depth baseline measurements during winter. Our recent collaboration with the Canadian- and U.S. Coast Guards and their icebreaking programs has facilitated winter surveys of Lake Erie since 2007 (McKay et al., 2011, Twiss et al., 2012). Conducted during

times of expansive ice cover, these surveys have documented high phytoplankton biomass, often

in discrete formations termed ‘CACHES’ and dominated by a physiologically robust,

filamentous centric diatom, Aulacoseira islandica (O. Müller) Simonsen (Twiss et al., 2012).

The apparent success with which diatoms are able to colonize the surface waters of Lake

Erie during winter is puzzling. Whereas psychrophilic microbes are widespread throughout polar

and even some temperate environments, as photoautotrophs, diatoms need light. Yet Lake Erie is

predominantly ice-covered in winter; Moderate Resolution Imaging Spectroradiometer (MODIS)

images have demonstrated the lake to be >80% ice-covered during our mid-February surveys

conducted since 2007 (Fig. 1), an observation supported by Daily Ice Analysis Charts from the

Canadian Ice Service (http://www.ice-glaces.ec.gc.ca/) (Fig. 2). Depending on snow cover, light

attenuation through ice can be >90% (Bolsenga and Vanderploeg, 1992).

With potentially extensive ice cover across polar and temperate lakes, light attenuation due to the

ice and accumulated snow is expected to play an important role in shaping adaptive mechanisms 76

of phytoplankton found below the ice at that time. Proposed mechanisms for keeping organisms suspended in the photic zone near the surface include wind driven mixing, (Stewart, 1973) convective mixing (Bondarenko et al., 1996, Bengtsson, 1996), and adhesion to frazil ice that could trap larger particles that eventually get incorporated into ice cover (Eicken, 1992, Barnes et al., 1994, Kempema et al., 2001). In a low light environment such as ice- covered Lake Erie, it is imperative that the winter diatoms occupy a position near the ice cover – itself a challenge given that ice cover negates wind-driven physical mixing and diatoms are non-motile plankton lacking flagella.

During an early survey, we observed ice formation in samples of filtered lake water to which we had added some of the diatom-dominated seston collected by net tow and had left an insulated cooler on the ship’s deck overnight. Adjacent samples containing only filtered (< 0.45 μm) lakewater did not freeze. Biological ice nucleating components associated with the diatom seston offered a logical explanation for the discrepancy in freezing parameters.

Here we ask the question whether freshwater phytoplankton - particularly diatoms - possess the means to anchor themselves into the overlying lake ice, thereby placing themselves in the photic zone of the water column. In the process, could the diatoms promote the formation of lake ice, and influence the extent of ice cover?

Figure 1. A Moderate Resolution Imaging Spectroradiometer (MODIS) image taken on

February 16th, 2008 shows the extent of ice cover across Lake Erie. (Great Lakes CoastWatch,

NOAA-GLERL)

Figure 2. An example of the extent of ice cover across the eastern Great Lakes on February 18th

2008, obtained from the Daily Ice Analysis Charts from the Canadian Ice Service.

METHODS

Sampling Sites - Sites in each of the three basins in Lake Erie were sampled on surveys onboard

the light icebreaker CCGS Griffon during February and April in 2010-11. Sites in ice-covered

western Lake Superior were sampled using USCGC ALDER as a platform in March 2011

whereas sampling in Lake Ontario was conducted from RV Lake Guardian in April 2011 (Fig.

3).

Collection and maintenance of samples - Vertical net tows were collected using a 154-µm pore

size plankton net deployed to depths of 10 – 15 m in open water. Chlorophyll (chl) a

concentrations for each of these samples were measured by fluorometry following extraction in

90% (v/v) acetone at -20 °C (Welschmeyer, 1994) and were used as a proxy for biomass

estimates. In Lake Erie, surface water was collected at each station using a submersible pump

deployed 1 m below the surface. This water was filtered through a 0.2-µm capsule filter and used

for dilutions and media preparation. FLW+ media used to maintain samples in the lab is described in Chapter 2. Samples in the lab were maintained at 4°C, under low light

(approximately 60 µmol quanta m-2 s-1) set at 11:13 day: night cycles, with periodic replacement

of media. 80

Figure 3. A: Stations sampled across Lake Erie during winter ( ; 341, 1290, 880, 1053, 452), spring ( ; ER91M, ER78M, ER43,

ER09, ER15M) and summer ( ; 880, 849, 33, 1078).Stations were selected so as to include representative stations across the

Western-, Central- and Eastern basins of the lake. Each season included stations that were similar to or near stations selected during previous seasons; however the selection was guided by the cruise plan of each survey. 81

Figure 3. B: Nearshore stations sampled in ice-covered western Lake Superior during March

2011.

Figure 3. C: Stations sampled across Lake Ontario in spring 2010-11 82

Determination of Temperature of crystallization (Tc) - Temperature of crystallization (Tc),

was determined using approximately 30 µl of the sample in an 80 µl capillary tube (Fisherbrand

Micro-Hematocrit) to which a thermocouple was attached. Chl a biomass of phytoplankton

samples loaded into capillary tubes ranged from 0.5 – 3 μg chl a ml-1. The capillary tube was

placed in a foam plugged glass test tube and suspended in a cooling bath initially set at 4°C. A

thermologger recorded the temperature at 30 s intervals. Following equilibration to 5°C for 10

min, the temperature of the cooling bath was decreased at a controlled rate of 0.3°C min-1. The

Tc was recorded as the highest temperature at which ice crystals began to form, as indicated by

the release of latent heat of crystallization.

One-factor ANOVA’s were conducted on samples collected during each field season, from each

set of cultures, and during the heat denaturation experiment. Homogeneity of variance was tested

for each ANOVA by the Levene test with an α = 0.05, and normality was checked using normal

Q-Q plots. A log(-Tc) transformation was required for homogeneity of variances in the

denaturation experiment. Tukey’s HSD was used, with α = 0.05, to compare Tc of samples and cultures against controls. All statistics were performed using the stats and car packages in R

(version 2.12.2, www.r-project.org).

Tc as effected by sample manipulation

Effect of dilution - A plankton sample collected in spring (April) 2010 from the central basin in

Lake Erie was serially diluted to 0.1×, 0.05×, 0.01×, 0.005×, 0.002×, 0.001× and 0.0001× with minimal medium prior to assay of Tc. Chl a concentration of the original sample was estimated

as a proxy for biomass in the sample. 83

Effect of sample heating - Two plankton samples collected in spring (April) 2010 from the

western basin and eastern basin of Lake Erie, were subjected to heat treatments of 45 °C, 65 °C,

95 °C and autoclaving (western basin sample only) for 2 h prior to assay of Tc.

Scanning electron microscopy - For analysis by SEM, plankton were transferred to a glass vial

and fixed by addition of glutaraldehyde to a final concentration of 2.5% (v/v) in 0.1M sodium

phosphate buffer (pH 7.2). Following two rinses with this buffer, samples were dehydrated

through a graded series of ethanol to 100%. Critical point drying was replaced by treatment with a solution of hexa-methyl-di-silazane (HMDS) for 5 min and then air dried onto cover slips and

coated with a 10 nm Au-Pd layer using a sputter coater (Hummer VI-A) before viewing with a

Hitachi S2700 SEM.

Isolation of diatoms - Individual filaments of diatoms were isolated using a fine tipped Pasteur

pipette. The filaments were washed in clean medium before being transferred to a glass flask

containing sterile FLW+ medium. The flasks were placed at 4°C, under low light (approximately

60 µmol quanta m-2 s-1) set at 11:13 light:dark cycles, with periodic replacement of media.

Isolation and identification of bacteria - Seston collected using vertical net tows in April 2010

was streaked on Nutrient Broth agar plates and incubated at 4°C in the dark for 72 h. Colonies

from the plate were selected based on differences in colony morphology, and repeatedly

subcultured so as to ensure purity of the final isolates. Isolates thus obtained were assayed for ice

nucleating activity. A similar procedure was used for samples collected in April 2011, with a

selective bias towards colonies similar in morphology to the INA isolates from 2010. 84

16S rRNA sequences were amplified by colony-PCR for all isolates using the primers 8F (5’ -

AGAGTTTGATCMTGGCTCAG - 3’) and 1492R (5’ – GGYTACCTTGTTACGACTT - 3’).

The resulting PCR products were purified using a Qiagen PCR purification kit. Purified fragments were sequenced by the DNA sequencing facility at the Cancer Research Institute

(Chicago, IL) using standard methods. 16S rDNA sequences for the environmental bacterial isolates have been deposited to GenBank under accession numbers: JN201533- JN201573. From these sequences, 23 sequences representing best matches were retrieved and together with 16S rRNA sequences of other pseudomonads and E. coli (Anzai et al., 2000) were aligned using

Clustal-W. A 336 nucleotide sequence with a high degree of similarity across these taxa was selected to generate a phylogenetic tree. The sequences were processed using MEGA5(Tamura

et al., 2011). A tree was generated using the maximum likelihood method (Hasegawa et al.,

1985) with the bootstrap consensus tree inferred from 1000 replicates (Felsenstein, 1985) taken

to represent the evolutionary history of the taxa analyzed.

Bacterial isolates and INA+ control strains (Pseudomonas fluorescens, P. putida, P. syringae and

Erwinia herbicola – Erwinia ananas) were plated on KBC medium (Mohan and Schaad, 1987,

Riffaud and Morris, 2002), a modification of Kings medium B (King, 1954) supplemented with

cephalexin and boric acid. Production of fluorescent pigment on KBC was checked at 2 d after

incubation. Isolates were tested for cytochrome c oxidase production, ability to utilize carbohydrates glucose and maltose, nitrate reduction and gelatin liquefaction as described below.

Biochemical tests for identification of bacterial isolates:

Growth on KBC medium - KBC medium (Mohan and Schaad, 1987, Riffaud and Morris,

2002) was prepared by adding 10 g peptone (2% final conc.), 7.5 ml glycerol (1% final conc.),

0.75 g K2HPO4 (0.15% final conc.), 0.75 g MgSO4×7H2O (0.15% final conc.) and 10 g agar to

500 ml Milli-Q water. Components were solubilized with gentle heating followed by autoclaving

at 121°C for 30 min. The solution was cooled to 50°C at which point 50 ml of a preheated 1.5%

boric acid solution (1.5 mg ml-1 final conc.), 4 ml of cyclohexamide solution (200 µg ml-1 final

conc.) and 4 ml of cephalexin solution (80 µg ml-1 final conc.) were added. The contents were mixed well and poured into petri dishes. The plates were allowed to stand overnight, following which they were placed at 4°C. Isolates were spot inoculated onto the plates and incubated at

4°C in the dark for 48 h. Presence of a blue-green pigment in the colonies indicated pyocyanin production, whereas blue fluorescence under UV light indicated fluorescein production.

Oxidase test (Gaby and Hadley, 1957, Bergey and Boone, 2009) - A single drop of fresh oxidase

reagent (BD oxidase reagent kit) was placed on a clean filter paper. A small amount of cells from

an actively growing colony (taken from the KBC plates) was then placed on this drop.

Development of a blackish-purple color indicated a positive reaction.

Nitrate reduction test (Bergey and Boone, 2009) - Medium containing 25 g peptone (5% final

conc.), 1.5 g meat extract (Difco; 0.3% final conc.), and 0.5 g potassium nitrate (0.1% final

conc.) dissolved in 500 ml Milli-Q water was dispensed into glass culture tubes and autoclaved.

The tubes were allowed to cool and then placed at 4°C. Isolates were inoculated into the tubes

and incubated at 4°C for 72 h. Development of a red color after addition of a few drops of α-

naphthalamine (6 g of N,N-Dimethyl-1-naphthylamine dissolved in 1 liter 5 N acetic acid) and 86

sulfanilic acid (8 g of sulfanilic acid dissolved in 1 liter 5 N acetic acid) to the cultures indicated

reduction of nitrates to nitrites. Absence of color indicated possible reduction of nitrates to

ammonia or nitrogen, whereas absence of color even after addition of a small amount of zinc

powder indicated no nitrate reduction.

Fermentation of Carbohydrates (Molin and Ternström, 1982, Bergey and Boone, 2009) -

Medium was prepared by dissolving 5 g of casein peptone (1% final conc.), 2.5 g of sodium

chloride (0.5% final conc.), and 0.01 g phenol red (0.0001% final conc.) in 500 ml Milli-Q

water. To this, 5 g of the carbohydrate to be tested (glucose and maltose respectively; 1% final

concentration) were added. The medium was thoroughly mixed, and dispensed into glass tubes

into which inverted Durhams tubes had been placed. The tubes were then capped and autoclaved

for 20 min at 121°C. Following autoclaving, the tubes were gradually cooled to 4°C and

inoculated with the bacterial isolates. Samples were placed at 4°C, and observations were

recorded at 12, 24, 48 and 72 h. Acid production was indicated by a change in the color of the

medium from reddish-orange to yellow whereas gas production was determined based on the

formation of a gas bubble in the inverted Durham tube.

Gelatin Liquefaction (Molin and Ternström, 1982, Bergey and Boone, 2009) - Medium was prepared by adding 8 g Nutrient Broth (Difco) to 1000 ml Milli-Q water. Once mixed and heated, 150 g gelatin (15% final conc.) was added. Once the gelatin was completely dissolved,

10 ml aliquots were added to glass tubes. The tubes were then capped and autoclaved for 20 min at 121°C. Following autoclaving the tubes were gradually cooled to 4°C. Tubes were inoculated by stabbing the medium to the bottom of the tube. Samples were placed at 4°C, and observations were recorded at 12, 24, 48 and 72 h. Non-hydrolysed gelatin was observed as a white opaque 87

precipitate, whereas liquefied gelatin was characterized by a clear colourless viscous liquid

around regions of bacterial growth.

RESULTS

Temperature of crystallization (Tc) for phytoplankton samples collected by net during

winter and spring 2010-11 in the Laurentian Great Lakes - Samples from winter and spring

phytoplankton assemblages in Lake Erie collected in 2010 and 2011 were analyzed for potential

ice nucleating activity based on the Tc values of these samples. Seston mean Tc values for the

winter and spring assemblages were significantly higher than filtered lake water or deionized

water controls (Fig. 4). All seston samples from Lake Erie were significantly different than the

controls of filtered (< 0.45 μm) lake water and purified (MQ; >18 MΩ cm) water (one-factor

ANOVA for each sampling effort; FApr10, 5, 30 = 23.6, pApr10 < 0.001; FFeb11, 7, 16 = 11.0, pFeb11 <

0.001; FApr11, 5, 12 = 34.8, pApr11 < 0.001; post-hoc comparison to control Tukey’s HSD, α = 0.05).

Likewise, winter and spring assemblages from two other Great Lakes, Lakes Superior and

Ontario, also showed elevated mean Tc values that were significantly different than their controls

(one factor ANOVA; FLO, 4, 10 = 27.8, pLO < 0.001; FLS, 7, 16 = 20.0, pLS < 0.001; post-hoc

comparison to control Tukey’s HSD, α = 0.05).

Effect of dilution on the Tc of phytoplankton - A seston sample was serially diluted with

culture medium (CHU-10) from 10× to 1000× with each dilution tested for ice nucleation

activity (Fig. 5). Only the Tc of undiluted seston and the first two dilution steps (10× and 20×) were significantly different than the culture medium control (Fig. 4; Kruskal-Wallis test H9 = 88

62.5, p < 0.001; Nemenyi-Damico-Wolfe-Dunn posthoc test (Hollander and Wolfe, 1999), α =

0.05).

Effect of temperature on the Tc of phytoplankton - Incubating samples at high temperatures for 2 h reduced the ice nucleating activity of samples collected from sites in both the western-

(A) and eastern basins (B) of Lake Erie (Fig. 6); one factor ANOVA on loge(-Tc); F9,107 = 158.3,

p < 0.001). Samples incubated at 95°C showed Tc values similar to the controls. No freezing exotherms were observed in the samples incubated at 4°C, and no change in the Tc before and

after the incubation was observed (p > 0.5).While autoclaving a sample resulted in low Tc values,

some replicates showed Tc values as high as -3.6°C, indicating the presence of some ice

nucleating active component even in the treated samples.

Scanning electron microscopy - SEM analysis of diatoms growing in association with ice cover

showed a close association of flagellated rod-shaped bacteria with the EPS secreted along

filaments of A. islandica (Fig. 7 A,B) and S. binderanus (Kützing) Krieger (Fig. 7C), another

abundant diatom found in the CACHE formations. Other diatom taxa like Fragilaria sp.

enumerated at much lower densities in the CACHE formations had few bacterial epiphytes (Fig.

7D).

Isolation, identification and ice nucleation activity of bacteria - Attempts to isolate bacteria

from the net tow seston yielded 40 discrete colonies of which 21 isolates exhibited Tc values significantly higher than their growth medium, and similar to those from freshly collected diatom material (Fig. 7). INA-positive cultures had Tc significantly different from the Tc of the culturing

medium (one-factor ANOVA for each set of cultures; FB2010,20,39 = 36.7, pB2010 < 0.001; FD2010,3,8

= 0.8, pD2010 = 0.53; FB2011,15,32 = 24.8, pB2011 < 0.001; INA-positive cultures determined post-hoc 89

by Tukey’s HSD test, α = 0.05). The range of Tc values of the INA-positive bacterial isolates were similar to those seen in phytoplankton samples collected in winter and spring of 2010-11

(Fig. 3). By contrast, cultures of A. islandica that had been maintained in an enriched lake water medium at 4ºC over a period of several months showed significantly lower Tc values when compared with the original phytoplankton sample from which they were isolated, and not significantly different than the Tc values for their growth medium. Likewise, assay of cleaned A. islandica frustules tested negative for enhanced ice nucleating activity (not shown).

Analysis of 16S ribosomal DNA sequences identified these isolates as members of the genus

Pseudomonas with high identity (> 99%) to congeners, P. fluorescens, P. syringae and P. fragi

(Fig. 8). Whereas all isolates tested positive for growth on KBC media, production of fluoroscein, and gelatin liquefaction, they tested negative for cytochrome oxidase production, glucose utilization, maltose utilization, and nitrate reduction; identifying the isolates as P. fluorescens (Table 1).

Figure 4. Temperature of crystallization (Tc) for phytoplankton samples collected by net during winter and spring 2010-11 in the Laurentian Great Lakes. Each point represents the mean Tc for

seston from a sampling location, with the range of observed Tc shown as whiskers. The number

of replicate Tc measures for each seston sample was 3, except for the April 2010 samples from

Lake Erie that ranged from 4 to 8. Controls consisted of purified water (MQ; >18 MΩ cm) and

filtered (< 0.45 μm) lake water (FLW).

Figure 5. The effect of dilution on the ice nucleating activity of seston collected from Lake Erie in 2010. The seston sample was serially diluted with culture medium (CHU-10) from 10× to

1000×. Symbols indicate means with the range of 8 replicate observations. Controls consisted of purified water (MQ; >18 MΩ cm), filtered (< 0.45 μm) lake water (FLW) and CHU-10 minimal medium.

Figure 6. The effect of heat treatment on temperature of crystallization of phytoplankton samples collected by net during spring 2010 in Lake Erie. Data are presented as box and whisker plots showing median Tc values. Vertical boxes around each median show the upper and lower quartiles whereas whiskers extend from the 10th to 90th percentile. Potential outliers are shown as discrete points. Incubating samples at high temperatures for 2 h reduced the ice nucleating activity of samples collected from sites in both the western- (A; ER91M) and eastern basins (B;

ER15M) of Lake Erie (one factor ANOVA on loge(-Tc); F9,107 = 158.3, p < 0.001). Each plot represents a minimum of 6 replicate samples. Letters beside each box denote significant differences according to post-hoc Tukey’s HSD test (α = 0.05) indicating significantly elevated

Tc values for samples incubated at 4°C, 45°C, and 65°C respectively compared to the controls. .

Controls consisted of purified water (MQ; >18 MΩ cm) and filtered (< 0.45 μm) lake water

(FLW). 93

Figure 7. Samples processed using a modified SEM processing technique show colonization by flagellated rod-shaped bacteria on the extracellular polymeric substance (EPS) found on

filaments of A. islandica (A, B) and S. binderanus (C). Other diatoms in the assemblage did not

exhibit this association with bacteria, as seen on filaments of Fragilaria (D). Scale bars: 5 µm

(A,B) 2 µm (C) and 10 µm (D). 94

Figure 8. Temperature of crystallization analysis comparing bacterial and diatom isolates from

Lake Erie established in 2010 and 2011. Each point represents the mean Tc (± range, n = 3) for

each cultured microorganism. The area delineated by the dashed lines shows the range of Tc

values observed for seston from the same locations as the isolates. Media controls consisted of

nutrient-enriched filtered (< 0.45 μm) lake water (diatom cultures) and Nutrient Broth (bacterial

cultures). 95

Figure. 9. Phylogenetic analysis of bacterial isolates by maximum likelihood inference. Branches corresponding to partitions

reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered

together in the bootstrap test (1000 replicates) is shown next to the branches. Initial tree(s) for the heuristic search were obtained

automatically as follows: when the number of common sites was 100 or less than one fourth of the total number of sites, the maximum

parsimony method was used; otherwise BIONJ with MCL distance matrix was used. The tree is drawn to scale, with branch lengths

measured in the number of substitutions per site. All positions containing gaps and missing data were eliminated. There were a total of

336 positions in the final dataset.

Table 1: Biochemical properties of ice nucleating active (INA+) isolates collected from Lake Erie in winter 2011 compared with INA+ strains of Pseudomonas and Erwinia.

KBC Nitrate Isolate Oxidase Glucose Maltose Fluroscen Pyocyanin Gelatinase Final ID medium Reduction

INA+ Isolates + + - - + - - + P. fluorescens

P. fluorescens + + - - + - - + P. fluorescens

P. putida + + - - + - - - P. putida

P. fragi + + - - + - NA - P. fragi

P. syringae + - - - + - NA - P. syringae

Erwinia sp. + - + + - - - - Erwinia sp.

DISCUSSION

Ice nucleation in phytoplankton assemblages - We analyzed samples from winter and spring

phytoplankton assemblages in Lake Erie collected in 2010-11 for potential ice nucleating activity

based on the Tc values of these samples. Seston mean Tc values for the winter and spring assemblages were significantly higher than filtered lake water or deionized water controls.

Likewise, winter and spring assemblages from two other Great Lakes, Lakes Superior and

Ontario, also showed elevated mean Tc values.

Diatoms are the most abundant phytoplankton in sea ice and have been implicated in ice-

structuring activities that may promote their growth in this extreme environment (Raymond et

al., 1994, Krembs et al., 2011). Extracellular polymeric substances (EPS) produced by sea ice

diatoms can alter the structure of ice pores enhancing habitability. Susceptibility of the activity to

heat treatment as well as inhibition by glycosidase supports involvement of an ice-binding

glycoprotein in the ice-structuring activity (Krembs et al., 2011). These proteins are thought to

adsorb to the face of ice crystals, inhibiting their growth and further acting to structure the ice

surface to promote light scattering for the associated algal cells (Raymond et al., 1994). Despite

this ability to structure ice, the ability to promote ice formation has not been specifically linked

to diatoms. Indeed, to our knowledge, ice nucleating activity associated with phytoplankton has

been documented only twice prior to this report (Schnell and Vali, 1975, Schnell, 1975). In the

former study, ice nucleating activity was not widespread with only phytoplankton collected from

high latitude coastal waters of the North Atlantic Ocean testing positive. This was consistent

with observations documenting bands of airborne ice nuclei at high latitudes between 40 to 55°

(Bigg, 1973). Indeed, Schnell and Vali (Schnell and Vali, 1975) hypothesized that the airborne 98

ice nuclei might be biogenic in origin and derived from the ocean, a concept recently validated by the demonstration that diatoms can serve as ice nuclei (active only at -23°C or lower) under typical tropospheric conditions (Knopf et al., 2010).

Bacterial epiphytes of diatoms promote ice nucleation - Bacteria commonly colonize diatom cells, particularly during blooms and accompanying senescence phases (Staley and Gosink,

1999). SEM analysis of diatoms growing in association with ice cover showed a close association of flagellated rod-shaped bacteria with the EPS found on filaments of A. islandica and S. binderanus, another abundant diatom found in the ‘CACHE’ formation (McKay et al.,

2011, Twiss et al., 2012). Other diatom taxa enumerated at much lower densities in the

‘CACHE’ formations had few bacterial epiphytes.

Attempts to isolate bacteria from the net tow seston yielded 40 discrete colonies of which 21 isolates exhibited Tc values significantly higher than their growth medium, and similar to those from freshly collected diatom material. These results strongly implicate the bacterial component in the ice nucleating activity seen in the winter and spring diatom assemblages in the Great Lakes and is consistent with previous studies implicating bacteria as the INA component of phytoplankton samples (Schnell and Vali, 1975, Fall and Schnell, 1985).

Bacteria are the most active ice nucleators in the environment (Christner et al., 2008), capable of catalyzing ice nucleation at temperatures as high as -2°C (Lindow, 1983). INA bacteria were discovered in the 1970’s and include members of the gram negative bacterial genera Erwinia,

Pseudomonas, and Xanthomonas (Schnell and Vali, 1972, Maki et al., 1974, Lindow et al.,

1978). Whereas INA bacteria exhibit robust growth in culture, a common life strategy among these strains is their association either as epiphytes on plants (Lindow, 1983) or commensals in 99

gut microflora of ectotherms (Lee and Costanzo, 1998). Analysis of 16S ribosomal DNA

sequences identified the isolates from our study as members of the genus Pseudomonas with

high identity to congeners, P. fluorescens, P. syringae and P. fragi. Biochemical tests further

identified the isolates as likely P. fluorescens. Among INA pseudomonads, ice nucleating

activity resides in ina genes coding for ice nucleating proteins localized on the outer membrane

of the bacterium (Orser et al., 1985). When Lake Erie phytoplankton samples were exposed to

heat for 2 h, Tc decreased significantly suggesting at least some denaturation of the ice

nucleating proteins of bacterial epiphytes. Not all ice nucleating activity was lost as samples

showed elevated Tc values compared to controls even after exposure to 45°C, 65°C, and 95°C.

Ice formation influenced by INA microbes - With the reduced radiant flux that accompanies

winter at temperate- and high latitudes coupled with expansive ice cover on lakes in these

regions, reduced light availability is expected to play an important role in shaping adaptive

mechanisms of phytoplankton located below the ice at this time. A few centimeters of snow

accumulated on ice can result in 90% attenuation of photosynthetically active radiation

(Bolsenga and Vanderploeg, 1992). As non-motile phytoplankton, diatoms are reliant on physical mixing to remain suspended in the photic zone. With ice cover presenting a barrier to wind-aided mixing, convective mixing has been proposed to maintain diatoms suspended near the water-ice interface in Lake Baikal (Kelley, 1997). Vegetative resting cells of A. islandica are

found in Great Lakes surface sediments (Sicko-Goad et al., 1989, Lashaway and Carrick, 2010).

Their potential to seed seasonal diatom blooms has been demonstrated via sediment resuspension

assays, the success of which is affected by light, temperature and nutrient conditions (Sicko-

Goad et al., 1989, Lashaway and Carrick, 2010). During cold nights in winter, supercooled (<

0°C) water can be mixed to the sediments. These supercooled conditions promote the formation 100

of frazil ice and ultimately anchor ice in the Great Lakes (Kempema et al., 2001, Daly and

Ettema, 2006) and elsewhere. Much of the frazil- and anchor ice formed as a result of supercooling events floats towards the surface and may be incorporated into floating ice cover.

Adherence of diatoms to frazil crystals and sediment-scouring anchor ice may thus provide a means of recruitment to the photic zone for these freshwater phytoplankton. However, rather than relying solely on passive recruitment, INA bacteria and their diatom hosts may actively promote the formation of attached frazil ice, both creating more frazil ice and providing themselves with a buoyant ice raft for their recruitment to the photic zone (Fig. 10). This, in turn facilitates growth of the diatoms during winter. For Pseudomonas spp., overwintering in association with actively growing diatoms is expected to increase fitness of the bacteria via enhanced nutrient acquisition and ready dissemination to land following ice out. 101

Figure 10: Temporal schematic representation of ice formation influenced by INA microbes. (1) Open water with moderate

winds during clear, cold winter nights is conducive to the formation and mixing of supercooled water through the water column to

depths up to 30 m (Kempema et al., 2001, Daly and Ettema, 2006). Frazil ice forms in supercooled water and on the sediment surface;

INA-bacteria associated with diatom filaments likewise induce frazil ice formation. (2) Anchor ice forms on the sediment surface from aggregation of frazil ice, and entrains sediment and diatom filaments. (3) As water warms to freezing, anchor ice detaches from the sediment resulting in rafting of ice and ice-attached organisms to the surface or to overlying ice cover (4). (Credit: Benjamin Beall) 102

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CHAPTER IV

PRIMARY PRODUCTION IN ICE-COVERED LAKE ERIE

ABSTRACT

Recent investigations across Lake Erie through winter have documented the occurrence of large and abundant phytoplankton communities throughout the lake. The communities were dominated by diatoms, with the filamentous centric diatoms Aulacoseira islandica (O. Müller) Simonsen

and Stephanodiscus binderanus (Kützing) Krieger making up the dominant taxa. Photosynthetic

O2 evolution rates of phytoplankton seston collected by net in response to varying light intensities were measured using a Clark-type electrode in a sealed cuvette and used as a proxy to determine rates of photosynthesis for communities across the lake during winter (2008 – 11), spring (2008 and 2010) and summer (2010) seasons. Photosynthesis rates were coupled with previously determined photosynthetic quotient (PQ) values to extrapolate rates of carbon incorporation by the phytoplankton communities. We also used Si incorporation rates to determine diatom-driven production rates based on the Si:C molar ratios. Our results support a photosynthetically robust winter assemblage, with production rates higher than those measured in spring and comparable to those reported in summer. Phytoplankton embedded in lake-ice was also found to be photosynthetically active. Our results suggest that the winter diatom assemblage may play an important role in the carbon export to the benthos, thereby potentially driving the hypolimnetic oxygen deficits observed months later in summer.

108

INTRODUCTION

Photosynthesis involves the biological conversion of light energy to chemical energy which is

stored as organic carbon compounds. Whereas plankton account for only around 1 – 2 % of the

total global biomass, they are responsible for 30 – 60 % of the global annual fixation of carbon

on Earth (Falkowski, 1994, Sakshaug et al., 1997). In aquatic systems, photosynthesis supplies

the primary source of organic matter for most trophic levels, and rates of photosynthesis

therefore places an upper limit to the overall biomass and productivity of ecosystems. In its

simplest form, photosynthesis can be written as an oxidation-reduction reaction in the general

form:

2H2A + CO2 + Light → (CH2O) + 2H2O + 2A

While oxygenic photosynthesis can be modified to:

2H2O + CO2 + Light → (CH2O) + 2H2O + O2

The process involves using light energy to oxidize water:

+ - 2H2O + Light (Chl-a) → 4H + 4e + O2

and the reduction of CO2:

+ - CO2 + 4H + 4e → CH2O + H2O

Terms involved in photosynthesis – Gross photosynthesis is defined as the rate of electron equivalents photochemically extracted from the oxidation of water. Assuming the absence of any respiratory losses, this corresponds to the gross oxygen evolution rate (Sakshaug et al., 1997).

Gross carbon uptake, is the rate of carbon fixation, irrespective of the fate of the incorporated carbon (i.e. either incorporated into the organism, or secreted into the environment), and is lower than the gross oxygen evolution rate (Williams, 1993, Sakshaug et al., 1997). The molar ratio of 109

O2 evolved to CO2 fixed on a molar basis is called the photosynthetic quotient (PQ). While most of the energy captured by photosystems is used in C fixation, a small fraction may be used in nitrate and sulfate reduction, resulting in PQ values greater than 1 (i.e. the rate of O2 evolution is greater than the rate of CO2 fixation) (Falkowski and Raven, 1997). Net photosynthesis on the

other hand, corresponds to the net evolution of oxygen following all autotrophic respiratory

costs. As in this study, when working with environmental samples of a mixed community,

respiration rates reflect community metabolism, making the differentiation between autotrophic

respiration and total respiration impossible to determine (Williams, 1993). Estimates of

production in this study therefore reflect gross production rates.

Net carbon uptake, is the carbon uptake rate following all losses of CO2 due to oxidation of

organic carbon of cells in daylight. Net rates of O2 evolution and C uptake should therefore be

the same. Gross primary productivity thus usually refers to the gross C uptake rate over a 24 h

time period, whereas net primary productivity refers to the net C uptake rate over 24 h following

all day and night time respiratory losses. A “compensation depth” exists in aquatic environments,

where gross photosynthesis and (autotroph) respiration losses are equal. The depth separates

zones of net positive production (above the compensation depth) and net negative rates of

production (below the compensation depth).

Chl a is the terminal photosynthetic pigment in light absorption and is commonly used as a proxy

for photosynthetically active phytoplankton biomass. Productivity can therefore be normalized to

-1 Chl a. In this study, O2 evolution is expressed in mol units for convenience (µmol O2 µg Chl a min-1) and using known PQ values from literature, can be expressed in terms of C uptake (µmol 110

C µg Chl a-1 min-1) and used thus in comparisons with existing information of the lake in spring

and summer seasons. Radiance has been expressed as µmol photons m-2 s-1.

Photosynthetically active radiation (PAR) - A photochemical charge separation may be induced by an absorbed photon with a wavelength in the range 350 – 700 nm, making it convenient to express the amount of energy fuelling photosynthesis in terms of photons with a specified wavelength or frequency. Photosynthetically active radiation (PAR) is the solar radiation in the spectral range of 400 to 700 nm that photosynthetic organisms use in photosynthesis. The near-UV domain (350 – 400 nm) accounts for 5 – 7 % of the incident radiation, and neglecting it therefore does not entail any significant error. Irradiance is a measure of the amount of radiant energy incident per unit of time and unit of area. In this study, irradiance

-2 -1 is expressed as µmol photons m s , and is denoted by the symbol Io.

The effect of light on planktonic populations depends on the intensity of the incident light, the

immediate changes in light on passing from air into water, the extent to which light can penetrate

a given depth and the means by which phytoplankton cells utilize radiant energy (Boney, 1989).

In all current hypotheses involving the formation of winter/spring blooms (Sverdrup, 1953,

Behrenfeld, 2010, Taylor and Ferrari, 2011) entrainment of phytoplankton in the euphotic zone is

ultimately the cause of the spring blooms. Ice cover and overlying snow on the ice cover can

cause up to 90% attenuation in incident light intensity (Richardson et al., 2000). Measurements

of secchi depths and light extinction coefficients of PAR during winter sampling across Lake

Erie indicated highly turbid waters suggesting a shallow photic zone (Twiss et al., 2012). We

therefore hypothesized that phytoplankton adapted to growth under these conditions are low-

light adapted organisms. 111

Photosynthesis versus Irradiance (P versus I) curves - The non linear relationship between photosynthetic rates and irradiance is described by P versus I (or PI) curves (Fig 1 (Forget et al.,

2007)). At low irradiances, photosynthetic rates increase with increasing irradiance in a linear

fashion, indicating that the absorption of photons is slower than the capacity rate of steady state

electron transport from water to CO2. As irradiances increase, the photosynthetic rates become

non linear, and attain a saturation level, indicating that, the rate of photon absorption exceeds the

rate of steady state electron transport from water to CO2. At high irradiances, photoinhibition may occur, with photosynthetic rates dropping (relative to the saturation point).

Ik (I)

Figure 1. A typical PI curve as described in Forget et al. (2007).

One of many P versus I curve equations may be applied to the data to generate parameters used to characterize the photosynthesis in organisms. The parameters relevant to this study are:

α , the initial slope of the PE curve and ϕm, the maximum quantum yield - At low levels,

evolution of oxygen is approximately a linear function of irradiance. α is the ratio between the 112

photosynthesis and irradiance in this part of the curve.The parameters α and ϕm are related, but not the same. α is defined in terms of ambient light, whereas ϕm is defined in terms of light absorbed by the phytoplankton. ϕm is thus variable, and is a small fraction of the absorbed light in water. Dividing α by the Chl a specific absorption coefficient of the phytoplankton (aϕ) would yield the maximum quantum yield.

Pmax : The maximum photosynthetic rate – At a certain irradiance, photosynthesis rates

plateau off, at a rate designated as Pmax. Pmax is independent of the absorption cross section of the photosynthetic apparatus, and is therefore not spectrally dependant. Pmax at steady state is related

to the number of photosynthetic units and the minimum turnover time for electrons.

Ik (or Ek) : The light saturation index - Ik is the intercept between the initial slope of the PE

curve (α), and the maximum photosynthetic rate (Pmax). It indicates the irradiance at which

control of photosynthesis passes from light absorption and photochemical energy conversion to

reductant utilization, and could in principle be used as an indicator of the photoacclimational

status of phytoplankton.

β : The photoinhibition parameter – At higher irradiances, photosynthetic rates decline relative

to Pmax. at a rate designated as β, the photoinhibition parameter.

Photosynthesis rates in deeper waters with low light levels are determined largely by α, and in high light impacted surface waters by Pmax. Ik represents the transition between the two light

regimes.

Causes of variation in photosynthetic parameters – Multiple proxies like O2 evolution rates and C uptake rates may be used in estimating rates of photosynthesis. With the two estimates not 113

being identical as explained above, variation in estimated rates may be possible. ϕm values are

generally lower for C uptake (0.06-0.08) compared to those for O2 evolution (0.10-0.12),

reflecting a PQ value greater than 1. Consequently, other parameters like α, β and Pmax are also lower in case of C uptake. Non photosynthetic pigments (often produced at high irradiances during nutrient deprivation), could dissipate absorbed energy as heat rather than transferring it to the photosynthetic reaction centers, lowering the cross section yield of the photosystem (σPS and

σPSII). The initial slope (α) is reduced, and the light saturation index (Ik) increases. The number of

photosynthetic units (n) is high in nutrient replete cells. Under nutrient limitation, a decrease in n

could lower α and Pmax. At high irradiances, cyclic electron flow around PSII could bypass the

oxidation of the water splitting complex. In cyanobacteria, cyclic electron flow around PSI is an

essential metabolic process capable of generating ATP. In such situations, the utilization of

photons without a reduction of CO2 or oxidation of the water splitting complex, manifests as an

overall reduced photosynthetic quantum yield, with lower values of ϕm and α, and increased Ik

values. At elevated concentrations of ambient O2 , rubisco can utilize O2 as a substrate to form 2

carbon molecules like glycolate. Values of α and Pmax could be lowered for C uptake experiments. Packaging of pigments in cells could reduce the absorption efficiency of the pigments. Parameters normalized to Chl a therefore may be influenced by this. Low-light acclimated cells could absorb more light per cell than high-light acclimated cells.

Methods used in estimating rates of primary production - One of the most common methods

14 to evaluate rates of photosynthesis involves incubating samples with [ C]-NaHCO3, under a

given light intensity for a fixed amount of time, and evaluating the amount of 14C incorporated into the cells (Steemann-Nielsen, 1952, Steemann-Nielsen and Hansen, 1959, Bender et al.,

1987, Grande et al., 1989b). Drawbacks of this method include adhesion of bacteria or 114

phytoplankton to the inner walls of the bottle, lack of mixing, exclusion of grazers, loss of 14C as

CO2 or dissolved organic carbon (DOC), and assimilation of unlabeled respired CO2.

Methodological constraints result in rates that fall between the gross and net production rates.

A second method involves incubating samples in air tight bottles under light and dark conditions, followed by evaluating the concentration of O2 in the bottles at the end of the specified incubation period (Bryan et al., 1976, Bender et al., 1987, Grande et al., 1989b, Williams, 1993).

Assuming that O2 loss in the dark corresponds to the O2 loss in light, rates of photosynthesis can

be calculated. While this method has the advantage of not losing the radiolabel, it still suffers

from the some of the same drawbacks mentioned in the previous method involving incubating

samples in bottles. In addition, certain cellular functions like photorespiration, and increased

mitochondrial respiration in the presence of light could result in variation in respiration rates by

up to a factor of 10.

A third method involves incubating samples with [18O] enriched water (Bender et al., 1987,

Bender and Grande, 1987, Grande et al., 1989a, Grande et al., 1989b, Williams, 1993). While it

suffers from the same “bottle effects” mentioned for the 14C method, the method measures production of O2 by photosystem II, making it a better estimate of gross primary production.

This study employs a different approach; we incubated a small volume of the sample in an air-

tight cuvette and exposed it to increasing light intensity over time. Photosynthetic O2 produced, was measured by a Clark-type oxygen electrode located at the bottom of the cuvette. The shorter incubation time and the use of a stirrer in the chamber minimized the “bottle effects” commonly observed in the other techniques. In addition, by comparing the rates of O2 utilization in the dark,

we can expect a good estimate of the gross photosynthesis rates in the samples. We can then 115

reference these rates with rates estimated by other methods for the same sample to determine differences in estimates.

Summer Hypoxia: Causes and knowledge gap - Hypolimnetic oxygen deficits in summer are well documented in Lake Erie (Charlton, 1980, Dybas, 2005), with nearly the entire Central basin becoming hypoxic in 2005 (Hawley et al., 2006). Effects of this event range from proliferation of harmful algal blooms (HAB’s) due to nutrient loading in hypoxic waters to loss of habitat for benthic micro- and macrofauna. Whereas other events of hypolimnetic hypoxia have be predictably modeled using morphometric and primary productivity data, these models have failed to explain the extent of hypoxia in the Central basin of Lake Erie (Charlton, 1980).

Diatoms have been recognized as the primary component of the spring phytoplankton assemblage in Lake Erie (Barbiero and Tuchman, 2001). While diatoms dominate the spring assemblage, silica, an essential requirement for diatoms, was found to be depleted in the water column as early as April/May, suggesting that the bloom had already occurred (Hartig and

Wallen, 1984). In addition, studies on the spring blooms were unable to couple the hypolimnetic hypoxia observed in the Central basin in summer to phytoplankton biomass observed through spring and summer (Charlton, 1980, Hawley et al., 2006).

Research conducted by our group across Lake Erie during winter and spring seasons from 2007 to 2011 indicate that a diatom bloom occurs in winter, when the lake is 80 – 90% ice covered

(McKay et al., 2011, Twiss et al., 2012). The studies document high phytoplankton biomass with

Chl a concentrations at some locations higher than those recorded in spring or summer (McKay et al., 2011). The winter assemblage is dominated by the diatoms Aulacoseira islandica (O. 116

Müller) Simonsen (≈80%) and Stephanodiscus binderanus (Kützing) Krieger (≈19%) (McKay et

al., 2011, Twiss et al., 2012).

Diatoms are known to have higher sinking rates than most other phytoplankton, primarily due to

the high density of their silica frustules (Gibson, 1984, Smetacek, 1985). Export of biomass

(organic carbon) to the benthos is well studied in marine systems (Smetacek, 1985, Smetacek,

2000). The role of diatoms in the export of carbon to the benthos has also been documented in

regions experiencing hypolimnetic hypoxia like the Gulf of Mexico, and Lake Erie (Hawley et

al., 2006, Diaz and Rosenberg, 2008, Rao et al., 2008). Respiration of the exported carbon in the

benthos could result in the hypoxia observed.

In this study, O2 evolution rates in response to varying light intensities were measured using a

Clark-type electrode in a sealed cuvette, and rates were used to generate PE curves. O2 evolution was used as a proxy to determine rates of photosynthesis for communities across the lake during winter (2008 – 11), spring (2008 and 2010) and summer (2010) seasons. Photosynthesis rates were coupled with previously determined photosynthetic quotient (PQ) values to determine rates of carbon incorporation by the phytoplankton communities. We also used Si incorporation to determine diatom-driven production rates based on the Si:C molar ratios.

METHODS

Study sites – Sites in each of the three basins in Lake Erie were sampled on surveys during winter (February) and spring (April) seasons from 2008 to 2011. Additional sampling was carried out in January 2009, and in summer (July) 2010 (Fig. 2). 117

Sample collection and handling – Vertical net tows were collected at each site using a 154-µm

pore size plankton net deployed to depths of 10 – 15 m. Plankton collected was then placed into

clean opaque plastic bottles, and placed in incubators set at temperatures corresponding to the

surface water temperatures (4°C for samples collected in winter and spring, and 22°C for

samples collected in summer). Aliquots of the sample were filtered through a 0.2µm

polycarbonate filter and used for Chl-a estimates. All samples were used within 2-3 h of

collection (a constraint imposed by the design of the O2 electrode setup and the experimental

procedure involved). Surface water was collected at each station using a submersible pump

deployed 1 m below the surface. This water was filtered through a 0.2-µm capsule filter and used

for dilutions in subsequent experiments.

Chlorophyll-a analysis - Chlorophyll (chl) a concentrations for each of these samples were

measured by fluorometry following extraction in 90% (v/v) acetone at -20 °C (Welschmeyer,

1994) and were used as a proxy for biomass estimates.

Primary production - O2 evolution method – Photosynthesis rates in the phytoplankton

assemblages were measured using a temperature controlled Qubit Systems Dissolved Oxygen

sensor (Fig. 3) to measure photosynthetic oxygen evolution of plankton acquired from net tows

in response to varying light intensities. Material placed into a temperature controlled cuvette (a),

was exposed to a given light intensity (c) during which the oxygen evolved in the chamber was

measured by a Clark-type electrode at the bottom of the cuvette (d). The material was exposed to

light intensities ranging from 5 to 400 µmol photons m-2 s-1. PE curves and associated parameters

(Pmax and Ik values) were generated by using a non-linear curve fitting function (Platt et al.,

1980) in SigmaPlot software. Pmax and Ik values were calculated for samples collected at each 118

station during each survey. Samples from all sites were assayed in duplicate (or triplicate when

possible). Data sets yielding poor PI curve fits were discarded from any further analysis. For

statistical analysis, data was pooled together based on location (Western, Central or Eastern

Basin) and season (winter or spring). Satellite images (MODIS) and meteorological data indicated that sampling efforts during winter and spring were carried out under similar conditions for each respective season, we therefore did not consider the year as a variable in our statistical analysis for this study. Station locations for winter and spring surveys were similar, but not identical and therefore, while differences at specific sites could not be estimated, differences within each basin were estimated for winter and spring samples. 119

Figure 2. Stations sampled across Lake Erie during winter ( ; 341, 1290, 880, 1053, 452), spring ( ; ER91M, ER78M,

ER43, ER09, ER15M) and summer ( ; 880, 849, 33, 1078). Stations were selected so as to include representative stations across the Western-, Central- and Eastern basins of the lake. Each season included stations that were similar to or near stations

selected during previous seasons; however the selection was guided by the survey plan of each respective ship. 120

Figure 3. The temperature controlled Qubit Systems Dissolved Oxygen sensor used in this study to measure photosynthetic oxygen evolution of plankton. Picture shows a cuvette (a) with an outer jacket (b) connected to a circulating water bath to regulate temperature. The sample was subjected to increasing light intensities by fiber-optic light sources (c), and a Clark – type electrode at the base of the cuvette (d) measured dissolved O2 levels in the sample.

121

RESULTS

Photosynthesis rates - Table 1 summarizes Pmax values observed during the different surveys.

Primary production rates in winter and spring across the different basins in Lake Erie are shown

in Figure 4. Each data point in the figure corresponds to the average Pmax value for a specific station during a specific sampling effort, with blue symbols indicating samples from winter assemblages and red symbols indicating samples from spring assemblages. Primary production

rates in spring were similar across sites within basins, as well as across the three basins, with

little variability in samples tested. With the exception of one station in the Western Basin, winter

and spring primary production rates in the Western and Eastern Basins of Lake Erie were similar

(p ˃ 0.05). Winter primary production rates in the Central Basin showed high degree of

variability within sites, with rates that were either similar to or greater than spring primary

production rates in the Central Basin. While winter production rates did appear visibly higher

than spring production rates across the Central Basin, no significant differences were found

between the median values of the production rates of the two seasons (Mann-Whitney Rank Sum

Test , p = 0.063). While our sampling efforts in summer were limited for this study (with data for

only two stations), we see that the high rates recorded in the winter samples were much higher

than even those measured in summer (Table 1).

Light Saturation index (Ik) - Table 2 summarizes Ik values observed during the different

surveys. The light saturation index (Ik) values for winter and spring across the different basins in

Lake Erie are shown in Figure 5. Each data point in the figure corresponds to the average Pmax value for a specific station during a specific sampling effort, with blue symbols indicating samples from winter assemblages and red symbols indicating samples from spring assemblages. 122

No clear differences were observed in Ik values between winter and spring assemblages (t-test, p

> 0.05 for winter and spring assemblages in respective basins). Assemblages from both seasons

-2 -1 showed low Ik values (< 100 µmol photons·m ·s ), indicating acclimation to low light

conditions.

Samples from CACHE sites and from melted Ice – Table 3 summarizes Pmax and Ik values for

samples collected from CACHE sites and from melted ice. Samples collected from CACHE sites

-1 -1 in the central basin showed production rates ranging from 0.0035 µmol O2µg Chl a min to

-1 -1 0.0384 µmol O2µg Chl a min . Ik values for the samples ranged from 52.29 to 92.74 µmol

photons m-2 s-1. Lake ice samples collected from the vicinity of Central Basin site St 880 were

found to contain pockets of biomass and sediment within it. Ice samples were thawed at 4°C and

the resulting meltwater was tested. Rates of primary production in these samples ranged from

-1 -1 0.0043 to 0.037 µmol O2 µg Chl a min . Ik values for these samples ranged from 30.59 to

91.78 µmol photons m-2 s-1.

Chlorophyll-a concentrations – Chl-a concentrations were high for all size fractions, indicating

biomass dominated by larger cells (Fig. 6). Chl-a levels for the > 20µm fraction ranged from

0.310 to 56 µgL-1. Concentrations in the > 2µm fraction and > 0.2µm fraction by comparison

ranged from 0.045 – 61.65 µgL-1, and 0.33 – 70.3 µgL-1 respectively. The > 20µm fraction

accounted for a large percentage of the total biomass in the phytoplankton population assessed.

Calculating rates of primary production in terms of C uptake – Table 4 summarizes rates of

primary production µmol C µg Chl a -1 d -1, calculated using previously documented PQ values of 0.67 and 7.12 respectively (Ostrom et al., 2005). Although variable, rates in winter are much higher than rates observed during spring, and are comparable to rates observed in summer. 123

Table 1. Pmax values observed across Lake Erie during winter, spring and summer sampling. Values shown indicate station means ± standard deviation, number of replicates is indicated in parenthesis.

340 84 CACHE 84 CACHE 2 949 1026 2008 February 0.04275 ± 0.01337 (2) 0.02510 ± 0.03457 (3) 0.00348 (1) 0.03836 (1) 0.00328 ± 0.00107 (3) 0.00167 ± 0.00099 (2)

ER42 ER43 ER78M ER32 ER30 ER15M 2008 April 0.00368 ± 0.00109 (2) 0.00411 (1) 0.00360 ± 0.00032 (2) 0.00481 ± 0.00205 (2) 0.00652 ± 0.00140 (2) 0.00691 ± 0.00038 (2)

St 357 St 341 St 84 St 452 2009 January NA 0.00338 ± 0.00139 (3) 0.00695 (1) 0.00370 ± 0.00039 (2)

ST 357 ST 341 ST 1053 Ice Block 1 Ice Block 2 2009 February NA 0.00035 ± 0.00007 (3) 0.00322 ± 0.00105 (2) 0.02069 ± 0.02311 (2) 0.00967 ± 0.00069 (2)

St 452 St 1023 St 84 St 341 St CCB 2010 February 0.00299 ± 0.00100 (2) 0.02321 ± 0.00847 (2) 0.02265 ± 0.00441 (2) 0.00667 ± (1) 0.00545 ± 0.00091 (2)

ER 91M ER 43 ER 78M ER 15M 2010 April 0.00832 ± 0.00135 (2) 0.00275 (1) 0.00502 ± 0.00015 (2) 0.00324 (1)

St 849 St 33 St 880 St 1078 2010 July 0.01268 ± 0.00162 (2) NA NA 0.02101 (1)

St 341 St 880 St 1290 St 1053 St 452 2011 February 0.00985 ± 0.00023 (2) 0.01168 ± 0.00803 (2) 0.00791 ± 0.00436 (2) 0.00462 ± 0.00033 (2) 0.00799 (1)

124

Table 2. Ik values observed across Lake Erie during winter, spring and summer sampling. Values shown indicate station means ± standard deviation, number of replicates is indicated in parenthesis.

340 84 CACHE 84 CACHE 2 949 1026 2008 February 41.87 ± 32.37 (2) 74.28 ± 11.32 (3) 52.29 ± (1) 92.74 ± (1) 58.51 ± 15.71 69.83 ± 1.99 (2)

ER42 ER43 ER78M ER32 ER30 ER15M 2008 April 14.82 ± 3.65 (2) 43.81 (1) 73.07 ± 5.45 (2) 71.58 ± 6.19 (2) 58.79 ± 0.22 (2) 67.14 ± 0.29 (2)

St 357 St 341 St 84 St 452 2009 January NA 32.04 ± 10.47 (3) 9.79 (1) 37.63 ± 5.03 (2)

ST 357 ST 341 ST 1053 Ice Block 1 Ice Block 2 2009 February NA 22.20 ± 1.79 (3) 34.56 ± 20.89 (2) 80.70 ± 15.67 (2) 32.74 ± 3.04 (2)

St 452 St 1023 St 84 St 341 St CCB 2010 February 23.03 ± 0.74 (2) 18.20 ± 10.33 (2) 26.04 ± 1.65 (2) 23.10 (1) 21.55 ± 1.58 (2)

ER 91M ER 43 ER 78M ER 15M 2010 April 30.81 ± 3.33 (2) 41.05 (1) 25.41 ± 0.95 (2) 32.78 (1)

St 849 St 33 St 880 St 1078 2010 July 36.40 ± 7.21 (2) NA NA 15.73 (1)

St 341 St 880 St 1290 St 1053 St 452 2011 February 26.75 ± 1.14 (2) 30.93 ± 2.25 (2) 37.26 ± 1.88 (2) 25.82 ± 1.96 (2) 39.26 (1)

125

0.07 Winter 0.06 Spring ) -1

0.05 min -1

0.04 Chla

0.03 µgm 2 0.02

(µmol O 0.01 max P 0.00 Western Basin Central Basin Eastern Basin

Figure 4. Spatial distribution of primary production rates (Pmax) for plankton samples collected by vertical net tows during winter

(blue symbols) and spring (red symbols) from different sites across Lake Erie. Error bars indicate the range of values observed.

126

100 Winter Spring 80 -1

s 60 -2

µmol photons µmol m 20

0 Western Basin Central Basin Eastern Basin

Figure 5. Spatial distribution of Ik values for plankton samples collected by vertical net tows during winter (blue symbols) and spring

(red symbols) from different sites across Lake Erie. Error bars indicate the range of values observed 127

80 Central Basin A Western Basin 2008 Feb Eastern Basin 2009 Jan 2009 Feb 2010 Feb 2011 Feb 2008 April 2010 April 60 20 (µg·L-1) Chlorophyll - concentrationa Chlorophyll

0 83 82 81 80

B Western Basin Central Basin 2008 Feb Eastern Basin 2009 Jan 2009 Feb 2010 Feb 2011 Feb 2008 April 2010 April 60 20 (µg·L-1) Chlorophyll - concentrationa Chlorophyll

0 83 82 81 80

80 C Western Basin Central Basin 2008 Feb Eastern Basin 2009 Jan 2009 Feb 2010 Feb 2011 Feb 2008 April 2010 April 2010 July 60 20 (µg·L-1) Chlorophyll - concentrationa Chlorophyll

0 83 82 81 80 Longitude

Figure 6. Chlorophyll-a concentrations (µg·L-1) of the >20µm (A), >2µm (B) and >0.2µm (C) size fractions of phytoplankton in surface waters of Lake Erie at stations where production rates were estimated. Error bars represent standard deviations and are included where available. 128

Table 3. Primary production (Pmax) rates and light saturation index (Ik) for plankton samples in

CACHE sites and from melted ice collected in the central basin of Lake Erie during winter.

P (µmol O µgm Chl a -1 min-1) I (µmol photons m-2 s-1) max 2 k

CACHE A 0.0035 52.29 CACHE B 0.0384 92.74

Ice 1 0.0370 69.62 Ice 2 0.0043 91.78 Ice 3 0.0102 30.59 Ice 4 0.0092 34.89

Table 4. Primary production in terms of µmol C µg Chl a -1d-1, calculated using previously documented PQ values ranging from 0.67 and 7.12 respectively (Ostrom et al., 2005)

P (µmol C µg Chl a -1 d-1) PQ = 0.67 max Number of samples (n) Mean ± SD Max Min

Winter 45 23.88 ± 30.56 139.59 0.66 Spring 17 11.10 ± 4.14 15.44 5.9 Summer 3 33.22 ± 10.63 45.16 24.78

P (µmol C µg Chl a -1 d-1) PQ = 7.12 max Number of samples (n) Mean ± SD Max Min

Winter 45 2.25 ± 2.88 13.14 0.06 Spring 17 1.04 ± 0.39 1.88 0.56 Summer 3 3.13 ± 1.00 4.25 2.33

129

DISCUSSION

Primary production rates in winter - This study was initiated by earlier observations of

phytoplankton assemblages across Lake Erie during the winter of 2007 (McKay et al., 2011,

Twiss et al., 2012). The studies revealed discrete blooms of phytoplankton dominated by the

filamentous diatom A. islandica (≈80%) and S. binderanus (≈19%). In the absence of winter

sampling across the ice covered lake, earlier studies had documented phytoplankton in spring,

which was considered to be part of a bloom assumed to have been initiated in late winter/early

spring, coinciding with warmer temperatures and a thawing ice cover. Export of the spring and

summer phytoplankton biomass to the benthos, and its subsequent respiration were assumed to

be responsible for the hypolimnetic oxygen deficits observed in the Central basin. However, the

extent of the hypoxia could not be coupled to the amount of biomass observed in spring and

summer. The presence of a highly productive winter assemblage could thus prove to be a missing

parameter in explaining the summer hypoxia occurring months later. In the current study we

estimate rates of primary production based on rates of O2 evolution by phytoplankton in response to varying light intensities.

Chl-a levels across the central basin of the lake were high during all winter surveys from 2007-

2011 (Fig. 6). Size fractionated Chl-a assays indicated that most of the observed Chl-a could be

attributed to cells larger than 20 µm in size (Fig. 6). Production rates in winter and spring

therefore were estimated for phytoplankton collected via vertical net tows, and included

primarily cells larger than 154 µm (the mesh size of the net used). Observations using light

microscopy confirmed that these cells were predominantly the filamentous diatoms that

dominated the winter assemblages. Care should be taken in interpreting the results from these 130

experiments, as they do not account for photosynthesis rates that could be attributed to plankton smaller than 154 µm in diameter. The study does however yield information regarding photosynthesis rates for the larger filamentous diatoms, and allows for estimations of diatom- specific production rates.

Primary production rates in spring were similar across sites within basins, as well as across the three basins, with little variability in samples tested. With the exception of one station in the

Western Basin, no significant differences were observed between winter and spring primary production rates in the Western and Eastern Basins of Lake Erie (p ˃ 0.05). Winter primary production rates in the Central Basin showed considerable variation across sites, with rates that were either similar to or greater than spring primary production rates in the Central Basin. While winter production rates at certain sites did appear visibly higher than spring production rates across the Central Basin, no significant differences were found between the median values of the production rates of the two seasons (Mann-Whitney Rank Sum Test , p = 0.063). Variation across sites in the Central Basin could be attributed to the nature of the winter assemblages, that occur as discrete blooms ranging from 10 – 2000 m2 in size (McKay et al., 2011, Twiss et al.,

2012).While our sampling efforts in summer were limited for this study (with data for only two

stations), primary production rates at some sites in winter were higher than even those measured

in summer (Table 1).

No clear differences were observed in Ik values between winter and spring assemblages (t-test, p

> 0.05 for winter and spring assemblages in respective basins). Both winter and spring

-2 -1 assemblages showed low Ik values (< 100 µmol photons·m ·s ), indicating assemblages that are adapted to low light conditions. Light attenuation by ice (and snow on the ice) in winter results in 131

over 90% attenuation of incident light (Bolsenga et al., 1988, Bolsenga and Vanderploeg, 1992),

and could explain the low light adapted assemblage in winter. A closer analysis of the P vs I

curves (not shown) indicated no significant photoinhibition in most samples, even at light intensities exceeding 300 µmol photons·m-2·s-1. Wind-driven mixing during spring results in an

isothermal, well mixed water column, and diatoms with high sinking rates would mix through

the water column. Diatom cells could be exposed to strong vertical gradients of spectral quality

and quantity of radiation, with the integrated light exposure dependant on the rate and depth of

mixing as well as the transparency of the water column (Franks, 1994; Smith, R.E.H., 1999).

The vertical migrations of the diatoms would thus result in exposure to varying light intensities

over time, with the integrated light exposure for each cell being lower than that of cells

suspended at the surface. The lack of visible photoinhibition observed in the PI curves at

intensities exceeding 300 µmol photons·m-2·s-1, could explain the ability of the diatoms to

withstand high photon fluxes that cells may encounter in surface waters.

Converting O2 evolved to C uptake – Ostrom et al. (Ostrom et al., 2005) described a range of

PQ values estimated for Lake Erie based on multiple proxies. The PQ values were generated

14 18 based on [ C]-NaHCO3 uptake versus the light-dark bottle method and [ O]-H2O method respectively. PQ values reported in this study ranged from 0.67 to 7.12. A second study by

(Depew et al., 2006) describing production rates in Lake Erie reported PQ values ranging from

0.97±0.33 for net photosynthesis and 1.29±0.48 for gross photosynthesis. However this study was carried out in the oligotrophic Eastern basin of Lake Erie, and was not necessarily representative of photosynthetic rates in the Central basin of the lake. We consider the light-dark

bottle method described by Ostrom et al. (Ostrom et al., 2005) to be a nearer approximation of 132

the O2 electrode method, and we therefore use PQ values generated for these methods to calculate estimates of C uptake in our samples.

Our results indicated high production rates in winter, with average rates significantly higher than those observed in spring. While the range of values observed in winter was extensive (0.66 –

139.59 µmol C µg Chl a -1 d -1), the differences may have been due to the discrete nature of the biomass accumulations observed across the lake (McKay et al., 2011, Twiss et al., 2012). In contrast, samples collected and assayed in spring showed lower rates of production with little variation among samples across the lake. Spring assemblages did not appear to aggregate in discrete clumps like the winter assemblages, and the increased homogeneity of the spring samples could explain the uniformity in rates. While our efforts in summer sampling were limited for the purposes of this study, we did document higher mean rates of production in summer than in spring (Fig’s. 4 and 5; Table 2), with mean rates of production higher than that observed in winter and spring.

Production rates across Lake Erie between May through August have been previously estimated at around 10.51 µmol C µg Chl a -1 d -1 for the Western basin, 8.07 µmol C µg Chl a -1 d -1 for the Central basin and 7.79 µmol C µg Chl a -1 d -1 for the Eastern basin of the lake (Smith et al.,

2005). Production rates calculated for spring and summer in this study (Table 2) approximate these findings.

Other studies also describe volumetric or areal production rates for whole water samples (Ostrom et al., 2005, Smith et al., 2005, Depew et al., 2006). Our samples are based on values observed in concentrated phytoplankton assemblages, and are therefore normalized to Chl-a concentrations rather than volumetric units. Conversion of these rates to volumetric units requires either 133

information regarding the amount of water passing through the plankton nets for each net tow or integrated Chl-a values through the water column at each of the sites sampled. In the absence of this information, our estimates are restricted to production rates normalized to Chl-a levels.

If we use surface Chl-a concentrations (as a proxy for integrated water column Chl-a) to estimate rates of carbon incorporation into phytoplankton in surface waters, we obtain rates as seen in

Table 5. The table summarizes carbon incorporation rates across Lake Erie for the >20µm fraction in winter and spring and for the >0.2µm fraction in summer.

Table 5. Volumetric estimates of carbon incorporation into Lake Erie phytoplankton across seasons (2008 – 2011).

µmol C µg Chl a -1 d -1 (PQ = 0.67) Chl a (µgL-1) g C m -3 d-1

Winter 23.88 ± 30.56 5.19 0.0552

Spring 11.10 ± 4.14 2.39 0.0557

Summer 33.22 ± 10.63 1.45 0.2749

µmol C µg Chl a -1 d -1 (PQ = 7.12) Chl a (µg L-1) g C m -3 d -1

Winter 2.25 ± 2.88 5.19 0.0052

Spring 1.04 ± 0.39 2.39 0.0052

Summer 3.13 ± 1.00 1.45 0.0259

134

Depending on the PQ used to generate the production rates, we notice differences of up to an order of magnitude for samples in each season (Table 5). Further work is required to calculate

14 exact PQ values by comparing rates of O2 evolution with C uptake in the same samples. The resulting PQ values could then provide a better estimate of carbon incorporation into the lake.

The above values should thus be interpreted with caution.

Si:C ratios for Lake Erie winter assemblages - Silica (Si) is an essential requirement for diatoms, and is known to control productivity and distribution of diatoms. Silica incorporation into diatoms is indicative of actively growing cells, and can be studied using the fluorescent dye

PDMPO (2-(4-pyridyl)-5((4-dimethylaminoethyl-aminocarbamoyl) methoxy) phenyl) oxazole) that selectively binds to polymerizing silica and emits an intense fluorescence under ultraviolet

(UV) light wherever newly formed Si is deposited (Shimizu et al., 2001, Leblanc and Hutchins,

2005). Rates of silica incorporation when coupled with Si:C (molar) ratios allow for estimates of diatom-specific primary production (Leblanc and Hutchins, 2005). Experiments carried out in winter and summer of 2009 found actively growing diatom communities in the lake in winter, with >90% cells showing Si incorporation (Fig. 7) (Saxton et al., 2012). Si:C ratios in the summer assemblage ranged from 0.002 – 0.007, while ratios in winter ranged from 0.0036 to

0.0254 (Saxton et al., 2012). Previous work on Si:C ratios in 27 species of marine diatoms yielded Si:C ratios of 0.13 ± 0.04 (ranging from 0.04 to 0.36) (Brzezinski, 1985). The lower Si:C ratios for the Lake Erie winter assemblage could be attributed to high rates of primary production, and therefore high C uptake rates. While Si incorporation and C fixation rates were higher in summer, the Si:C ratios were higher for the winter assemblage. The high Si:C ratios have been attributed to high cellular Si:C ratios of A. islandica (Sicko-Goad et al., 1984), as well as due to non-diatom production in the summer assemblage. The Si:C ratios for the winter 135

assemblage indicated a diatom-dominated community disposed to higher sinking rates. With filamentous diatoms exhibiting high Si:C ratios, and sinking rates, the winter assemblage in Lake

Erie, being dominated by these diatoms, may be an efficient shuttle of both, carbon and silica to the benthos.

Figure 7: Si deposition in filamentous diatom A. islandica. Newly incorporated silica (and

PDMPO) in the frustules of cells glow blue under UV light (Saxton et al., 2012).

The most conservative estimates of diatom production (Table 5) if extrapolated to the surface 1m of the maximum observed hypoxic area in Lake Erie’s Central basin (approximately 10,000 km2)

(Hawley et al., 2006) indicate an estimated 520,201 kg C·d-1 during winter. The proportion of 136

this carbon that may be respired back as CO2 and/or transferred to the next trophic level remains to be estimated. Nonetheless, the example illustrates the magnitude of the winter diatom productivity.

The study highlights some important points. The winter diatom assemblage in Lake Erie is a photosynthetically robust assemblage, with winter production rates similar to (and sometimes higher than) rates seen in spring and summer. Photosynthetically active diatoms are present in the water column and are incorporated into the ice cover as well. While comparisons of O2 evolution rates with C uptake rates in order to generate accurate PQ values are still pending, trends in production rates across seasons can still be established. Production rates in winter are higher than those observed in spring, and comparable to rates observed in summer. While mean production rates in winter are lower than rates observed in summer, a high degree of variation was observed in winter samples with some samples showing production rates significantly higher than those seen in summer. High rates of production, silica incorporation and high Si:C ratios implicate the winter assemblage as a major player in the export of carbon and silica to the benthos, thus potentially driving the summer hypoxia occurring months later.

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142

CONCLUSION

Surveys across ice covered Lake Erie during winter revealed discrete phytoplankton assemblages

throughout the Western and Central basins of the Lake, with biomass levels frequently in excess of 50 µg Chl a L-1 (McKay et al., 2011, Twiss et al., 2012). The assemblages were dominated by

a psychrophilic, low light-adapted, filamentous centric diatom, Aulacoseira islandica (O. Müller)

Simonsen. While A. islandica dominated the bloom comprising ~ 80% of the assemblage, the

filamentous centric diatom, Stephanodiscus binderanus (Kützing) Krieger made up around 19% of the bloom, and other diatoms including Fragilaria spp. Cyclotella spp. Asteronella spp. and

Tabelaria spp. collectively accounted for around 1% of the assemblage. The blooms are thought to form sometime during early winter, coinciding with the onset of ice cover on the lake, and persist through early spring.

A. islandica filaments collected in winter showed auxospore formation, indicative of an actively growing population. Photosynthetic O2 evolution rates in the phytoplankton collected from the water column as well as from ice revealed a photosynthetically robust assemblage, with winter production rates higher than production rates observed in spring assemblages, and in some cases, higher than rates observed even in summer assemblages. High Si:C molar ratios in the assemblage indicated an actively growing diatom population, with the potential for the assemblage to be a driving factor in carbon and silica export to the benthos (Saxton et al., 2012).

A modified SEM technique allowed for observation in fine detail, the presence of extracellular polymeric substances on frustules of A. islandica and S. binderanus but not other taxa. EPS contributed by diatoms acts as a sink for excess photosynthetic production (Staats et al., 2000), and also contributes to diatom aggregation, resulting in export of the cells and associated organic 143

matter to the benthos (Smetacek, 1985). Our observations show resting cells in filaments of A.

islandica, which have also been documented in surface sediments in the Great Lakes (Sicko-

Goad et al., 1989, Lashaway and Carrick, 2010). Respiration of the organic matter exported to the benthos via the winter assemblage, could result in depletion of oxygen in the hyplimnion during warmer summer months, and eventually result in the formation of the “dead zone” in the

Central basin of the Lake.

We documented ice nucleating activity in the phytoplankton; a phenomenon we attribute to the epiphytic bacteria seen colonizing the EPS secreted on diatom frustules. Molecular and biochemical analyses identified the bacteria as Pseudomonas fluorescens. The ice nucleating bacteria could actively promote ice crystal formation around the cells, providing themselves with a buoyant ice raft for their recruitment to the lake ice, thereby placing the cells in the photic zone. This facilitates growth of the diatoms during winter, initiating the winter bloom. The association with actively growing diatoms would in turn increase the fitness of the ice nucleating pseudomonads via enhanced nutrient acquisition and ready dissemination to land following ice out.

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