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Home , Autotroph, Choanoflagellate, Goniomonas, Habitat, Microbial ecology, Microbial food web

Mechanisms structuring the pelagic microbial web

– Importance of resources and

Kristina Samuelsson

Umeå 2003

Department of and Environmental Science Umeå University SE-901 87 Umeå Sweden

AKADEMISK AVHANDLING

kommer med vederbörlig tillstånd av rektorsämbetet vid Umeå universitet, för erhållande av filosofie doktorsexamen i ekologi, offentligen försvaras torsdagen den 12 Juni 2003, kl. 09.00 i Stora hörsalen, KBC.

Examinator: Professor Lars Ericson

Opponent: Doctor Christiane Lancelot, Ecologie des Systèmes Aquatiques, Université Libre de Bruxelles, Brussels, Belgium

------ISBN 91-7305-464-X ¬ Kristina Samuelsson 2003 Cover: from the Baltic by Kristina Samuelsson Printed by VMC KBC, Umeå University

Organisation Document name Umeå University Doctoral dissertation Dept. of Ecology and Environmental Science

Author Date of issue Kristina Samuelsson June 2003

Title Mechanisms structuring the pelagic microbial – Importance of resources and predation.

Abstract Temporal and spatial variations of pelagic in the northern Baltic Sea were studied, as well as factors influencing their and growth rates. Three main questions were asked 1) How does increased influence the structure of the ? 2) Does predation limitation vary between trophic levels? 3) What is the relative importance of and predation limitation at different trophic levels? A field study in the northern Baltic Sea showed that dominating , flagellates and , increased with increasing primary productivity from north to south. Furthermore, relatively small protozoan cells dominated in the low productive north, while larger cells became more dominant in the south. The relationship between size structure and productivity was further studied in an experimental system. In agreement with present theories regarding status of pelagic food webs, increased productivity caused a lengthening of the as well as a change in plankton size structure. While microplankton dominated in nutrient rich treatments pico- and nanoplankton dominated during nutrient poor treament. The was dominated by a potentially , sp., at low nutrient concentrations. To our knowledge this is the first experimental study showing that Chrysochromulina sp. in resemblance with other is favoured by nutrient poor conditions compared to strict and . During a stratified summer period autotrophic microorganisms in the northern Baltic Sea did not respond to removal of potential predators, indicating that they were primarily limited by inorganic . An exception was small eucaryotic that showed a large response to predator removal. Among the heterotrophic microorganisms direct effect of predation seemed to increase from ciliates, heterotrophic , small heterotrophic flagellates, medium flagellates to large flagellates. No quick indirect effect was observed, but after four days trophic cascades were detected. The relative importance of resource and predation limitation was studied among heterotrophic bacteria, flagellates and ciliates in the northern Baltic Sea. For all these groups, resource limitation seemed to prevail during the summer period. The results also indicated that the relative importance of predation increased with the productivity of the system. To our knowledge there are no earlier measurements on the relative importance of resource and predation limitation for micoorganisms in the pelagic environment.

Key words: pelagic microbial food web, bacteria, flagellates, ciliates, , mixotrophy, nutrient status, predation limitation, resource limitation, Baltic Sea

Language: English ISBN: 91-7305-464-X Number of pages: 129 Signature: Date: 16 May 2003

“As in most sciences, ecologists are concerned with attempting to discern order from the seeming present in natural systems.“

Menge BA 2000

TABLE OF CONTENTS

INTRODUCTION……………………………………………………….…7

The pelagic microbial food web …………………………………….7 Phytoplankton …………………………………………..…………..9 Heterotrophic bacteria……………………………………………..10 Phagotrophic flagellates……………………………….…….……..11 Phagotrophic ciliates…..……...………………….……………...…13 Study area……………………....……………………………...…...14

OBJECTIVES………………………………………………………...…...16

MAJOR RESULTS & DISCUSSION……………………………………16

Importance of resource limitation………………………………….16 Importance of access to attachment sites and metazooplankton grazing…………………………………………...20 Importance of predation limitation……..………………………..…21 Relative importance of resource and predation limitation…….....23

CONCLUSIONS AND FUTURE PERSPECTIVES………………….26

REFERENCES…………………...……………………………………….27

Appendices: Papers I-V

LIST OF PAPERS

I: Samuelsson Kristina, Berglund Johnny, Haecky Pia, Andersson Agneta (2002) Structural changes in an aquatic microbial food web caused by inorganic nutrient addition. Aquatic 29: 29-38.

II: Kuuppo Pirjo, Samuelsson Kristina, Lignell Risto, Seppälä Jorma, Tamminen Timo, Andersson Agneta (2003) Fate of increased production due to nutrient enrichment in late-summer plankton communities of the Baltic Proper. Aquatic Microbial Ecology (in press)

III: Samuelsson Kristina, Andersson Agneta (manuscript) Factors structuring the protozoan community along a productivity gradient.

IV: Samuelsson Kristina, Andersson Agneta (2003) Predation limitation of the pelagic microbial food web in an oligotrophic aquatic system. Aquatic Microbial Ecology 30:239-250

V: Berglund Johnny, Samuelsson Kristina, Müren Umut, Kull Thomas, Andersson Agneta (manuscript) Simultaneous resource and predation limitation in the marine microbial food web.

Papers I and III are reprinted with the kind permission of Inter Research Science Publisher.

INTRODUCTION

In aquatic systems the properties of dominating and food web structure to a large extent determine the flow of material and (Verity and Smetacek 1996). To understand how environmental changes, such as and change, influence the of these systems we thus need to understand the processes determining food web structure. It is known, that the concentrations of nutrients and the process of nutrient cycling greatly influence the dynamics and food web structure within pelagic systems. For example, in nutrient-rich waters the classical or herbivorous food web is predominant, while in nutrient constrained environments the microbial food web is of larger importance (Legendre and Rassoulzadegan 1995). In the classical food web that consists of large phytoplankton, and both resource and predation seems to be important structuring factors (Carpenter et al. 1985, 1987). Less clear is, however, the structure and regulation of the microbial food web. In resemblance to the classical food chain resources and predation are believed to be two of the main structuring factors. In addition it has been shown that other factors such as (e.g. between photosynthetic and heterotrophic organisms), (e.g. Mesodinium rubrum with a cryptophycean symbiont), excretion of nutrients and parasites influence food web structure (Fenchel 1988, Coats et al. 1996). Since microbes constitute more than 50 % of the total plankton (Gasol et al. 1997) and a dominant part of the biosurface in pelagic systems, they are fundamental for the function of these systems (e.g. Azam 1998, del Giorgio and Duarte 2002, Hoppe et al. 2002). However, we still know too little about the mechanisms structuring the pelagic microbial food web to fully understand the spatial and temporal occurrence of different functional groups or , and to predict the response to future environmental changes. It seems as one of the largest challenges in aquatic microbial ecology lies in determining how different food web processes influence structure and functioning (Pace and Cole 1994).

The pelagic microbial food web

Within the pelagic system prokaryotic and eukaryotic are the main primary producers. These phytoplankton vary more than 100-times in size from small picoplankton (0.2-2 µm) and nanoplankton (2-20 µm) to large microplankton (20- 200 µm, Fig. 1). The picoplankton fraction mainly consists of (unicellular and ), but also are included. In the nanoplankton fraction flagellates are common, while and usually dominate the microplankton fraction. The main in the pelagic system are and metazooplankton (Valiela 1995). Among the

7

µ Siz e, m

200

20

CO 2 2

0.2 DOC

Fig. 1. The pelagic microbial food web. Pico-, nano- and micro- phytoplankton (unfilled symbols) use dioxide (CO2) during . Phytoplankton are grazed within the phagotrophic food chain represented by flagellates, ciliates and metazooplankton (filled symbols). (DOC) is exudated from cells or produced during “sloppy feeding” and utilized by heterotrophic bacteria. Redrawn from Fenchel 1987 protists, flagellates and ciliates are the most numerous. Flagellates vary in size from about 1 to 20 µm and ciliates from 5 to 100 µm. According to Calbet et al. (2001) there are at least six trophic levels within the phagotrophic food chain (bacteria, <2, 2-3, 3-5, 5-10 and >10 µm heterotrophic protists). However, the number of trophic levels probably varies considerably between systems. A portion of the carbon fixed within the microbial food web is excreted or lost from the phytoplankton as dissolved organic carbon (DOC). In addition DOC is produced due to incomplete ingestion and digestion by metazooplankton and protozoa i.e. sloppy feeding (Fig. 1). Due to their small cell size, osmotrophic bacteria are probably the only heterotrophs that effectively can utilize this carbon source. Depending on food web structure and efficiency the bacterial production either reaches higher trophic levels through the phagotrophic food chain or is respired within the (Azam et al. 1983). A rough estimate is that due to channelling through the microbial loop web only 3-9 % of the reaches higher trophic levels compared to channelling through the classical food chain (Fenchel 1988). The microplankton fraction is usually not grazed within the microbial food web. Instead microplankton influence the food web structure due to release of dissolved organic substances or due to with the smaller cells. They can also be of importance as attachment site for other microorganims.

8 Phytoplankton

Pelagic microbial food web structure and functioning is to a large extent determined by the dominating primary producers. For example, in a system dominated by diatoms a large part of the primary production is channelled through the classical food web or lost from the system through sedimentation (Wassmann 1993; Heiskanen and Kononen 1994). Contrary, in systems dominated by unicellular cyanobacteria a large part of the energy and carbon will be processed within the microbial food web and recycled in the . The fate of the primary production (grazing, exudation, aggregate formation, sedimentation) seems to a large extent depend on cell size. In general high concentrations of micro sized plankton are connected to turbulent waters e.g. areas, fronts or spring and autumn mixed waters, while pico- and nanoplankton dominate in stagnant waters e.g. open sea areas or during summer period stratification (Kiørboe 1993). Well- mixed waters are characterized by high input of “new” nutrients resulting in high nutrient concentrations, while stratified water usually are characterized by regenerated nutrients and a poor nutrient situation (Legendre and Rassoulzadegan 1995). According to Thingstad and Sakshaug (1990) the first algae to be established at low nutrient concentrations are the ones with the lowest value of K/µm. Since small algae generally have a lower half-saturation constant for inorganic nutrients (K) and a higher maximal growth rate (µm) than large cells they will dominate in nutrient poor waters (Malone 1980, Banse 1982, Probyn et al. 1990). If more nutrients are added to the system, the growth rate of small algae will increase until nutrient concentrations equal saturation levels. Still higher nutrient concentrations will allow large cells to be established. At saturated nutrient concentrations uptake is proportional to cell surface and there is no advantage in being small.

The phytoplankton community composition is also influenced by the elementary composition of the water. Species with special requirements such as diatoms need a Si:N relationship of 1:1 (e.g. Harris 1986). A ratio > 25:1 is, however, needed for diatoms to be favoured over other algae (Sommer 1994). Phytoplankton demand for C:N:P is generally 106:16:1 (Redfield ratio). Filamentous cyanobacteria can, however, take up from the and should consequently be favoured in situations were other phytoplankton are nitrogen-limited (Larsson et al. 2001).

When the phytoplankton biomass in a system is high enough herbivores will be established (Thingstad and Sakshaug 1990). Measurements from the northern Baltic Sea indicate that heterotrophic nanoflagellates are the main predators on pico- cyanobacteria, while nano-phytoplankton (2-10 µm) were grazed by ciliates (59%) and by metazooplankton (41%), and micro-phytoplankton were grazed by large

9 dinoflagellates (20%) and by metazooplankton (80%, Uitto et al. 1997). The phytoplankton size structure, thus, seem to have a large impact on the size composition of the community. The consumers also influence the phytoplankton community both due to predation, for example the outcome of competition between small and large phytoplankton cells for inorganic nutrients will change if the small cells are removed through predation (Thingstad and Sakshaug 1990), and because consumers are the main source of regenerated nutrients (Caron and Goldman 1990).

Among the phytoplankton, as well as among other aquatic microorganisms, one common strategy to avoid predation is size. Large cell size can be advantageous both due to size-limited predators and due to slower numerical response of large predators (Kiørboe 1993). oscillations between diatoms and lag about one month, while bacteria and protozoa lag about 2 to 6 days (Fenchel 1982, Kiørboe 1993, Tanaka et al. 1997). Other known strategies among phytoplankton to keep away from the predators are production of toxic substances (Wolfe and Steinke 1996).

Heterotrophic bacteria

Resource level is also an important factor controlling the bacterial biomass and production (Gasol and Duarte 2000). Bacteria utilize dissolved organic carbon (DOC) produced within or outside the system. Of the primary production within aquatic systems about 40-45% is generally assumed to be consumed by the bacteria (Larsson and Hagström 1979, White et al. 1991). If the nutrient composition of the resource is poor, bacteria will also take up dissolved inorganic nutrients. During these situations bacteria will compete with phytoplankton for nutrients. Since bacteria have a higher affinity for nutrients than phytoplankton and due to higher surface to volume ratio, they should out compete the phytoplankton. At too low phytoplankton concentration, however, the bacteria will become carbon limited, since often the main source of carbon is derived from the phytoplankton. This situation allows co-existence between the two competitors (Thingstad and Lignell 1997). Carbon limitation of bacteria is implied from field observations where higher primary production or chl a level often is followed by higher bacterial production (Cole et al. 1988, Dufour and Torréton 1996). Another mechanism allowing co-existence between bacteria and phytoplankton is bacterivory, keeping the biomass of the competing bacteria low. In this situation surplus nutrients are taken up by phytoplankton, and bacteria can become nutrient limited (Thingstad and Lignell 1997).

1 0 The main grazer on bacteria is thought to be nanoplanktonic protists (Fenchel 1982, Wikner and Hagström 1988, Sherr et al. 1989). The importance of these predators for the regulation of bacterial abundance seems, however, to differ between systems (Billen et al. 1990, Pace and Cole 1994, Dufour and Torréton 1996). A study from the Baltic Sea showed that most of the intra-annual variability in bacterial production was due to a variation in the specific growth rate, controlled by environmental factors such as , substrate quality and competition (Wikner and Hagström 1999). In other studies heterotrophic flagellates have been found to control bacterial numbers, production and population structure (Kuuppo- Leiniki 1990, Gasol et al. 2002). Another important mortality factor for bacteria is viral lysis (Fuhrman 1999, Tuomi and Kuuppo 1999, Bettarel et al. 2003). The phage- interaction has high species specificity and in a theoretical model controlled the diversity more than the actual numbers (Thingstad and Lignell 1997). The importance of viral lysis compared to grazing have been compared in several studies. The results vary from viral lysis being more than 11 times higher to 30 times lower than flagellate grazing (Guixa-Boixarey 1996, Fischer and Velmirov 2002, Bettarel et al. 2003). In general the importance of viral lysis increase with enrichment.

Phagotrophic flagellates

Phagotrophic flagellates utilize energy and substrate from particulate organic material. The most common obligate phagotrophic flagellates are found within taxa (chrysomonads and bicosoecids), dinophycean and (Boenigk and Arndt 2002). In addition a large group of known species have not been possible to characterize and are classified in an group (Vørs 1992). Flagellates are versatile in their choice and they are common both as free-living and attached forms. In general about half of the cells are free swimming and the rest attached or temporarily attached (Boenigk and Arndt 2002). The attached forms are found on phytoplankton such as diatoms and filamentous cyanobacteria or on aggregates of bacteria or (Leadbeater 1991, Carrias et al. 1998). Bacterivory seems to be the main energy source among the smallest flagellates (<5µm) and choanoflagellates, while algivory, detrivory and seems to be important in larger heterotrophic flagellates (Sherr 1988, Simek et al. 1997, Sleigh 2000). Viruses can also make up a large pool of the ingested carbon (9%, Gonzalez and Suttle 1993). The flagellates show at least three different feeding mechanisms: filter feeding, direct interception and raptorial feeding (Fenchel 1987). Filter feeding organisms produce a feeding current through a filtration structure for sieving the water for food particles, examples are helioflagellates and choanoflagellates. Interception feeders also produce a feeding

1 1 current but the prey is taken up by direct interception, chrysophyceans are interception feeders. In contradiction to filter and interception feeders, raptorial feeders are actively searching for the prey that is taken up by direct interception, examples are sp. and sp. (Boenigk and Arndt 2000a). A comparison of the handling time between these strategies showed that a filter feeder (Monosiga ovata) had much longer handling time (~ 300 s) than interception and raptorial feeders (3.7-95 s, Boenigk and Arndt 2000a,b). The long handling time is, however, compensated by the capacity to handle several preys at a time. Due to morphological limitations of the different feeding apparatuses there seems to be a passive selection for prey of a special size. Filter feeders generally take up smaller cells than interception and raptorial feeders (Boenigk and Arndt 2000). In addition to passive food selection there is evidence of active food selection due to differences in the size, swimming speed and the biochemical surface composition of the prey (Matz et al. 2002). The existence of specific predation pressure on different bacteria indicates that flagellate feeding should be an important factor structuring the bacterial community composition (Boenigk and Arndt 2000a).

In natural aquatic environments the concentration of bacteria vary from 105 to 107 ml-1. Threshold bacteria concentrations supporting flagellate growth have been reported to be in the range of 105–106 bacteria ml-1 (Eccleston-Parry and Leadbeater 1994). Maximum growth rates are possibly reached at concentrations of 1-2 * 107 bacteria ml-1 (Boenigk and Arndt 2002). This comparison indicates that flagellates probably are resource limited in many aquatic systems. A frequently used indicator for resource limitation of flagellates is a positive correlation to their main prey, the bacteria. Using this criterion, both evidence for and against prey limitation of flagellates have been suggested (Sherr 1984, McManus and Fuhrman 1990, Berninger 1991, Sanders et al. 1992, Weisse 1997). In general bacterial numbers increase with nutrient status of a system and the importance of resource limitation for the flagellates should consequently decrease. However, this increase in bacteria could be due to inedible forms, known to increase with nutrient enrichment (Gasol et al. 2002). By using simple models Sanders et al. (1992) and Gasol (1994) suggested that resource limitation of flagellate decrease with increased nutrient status.

Absence of a positive relationship between bacteria and flagellates could be due to alternative prey or high predation control of the flagellates. In many aquatic environments flagellate density varies in a rather narrow range because of tight constraints imposed by different kind of predators (Weisse 1991; ciliates, Dolan and Gallegos 1991; rotifers, Kuparinen and Bjørnsen 1992; dinoflagellates). Despite this high predation pressure few predation resistant forms of flagellates have been found and it seems like heterotrophic flagellates have little potential for shifting to forms

1 2 resistant to predation. High predation pressure might, however, favour species with high growth rates. Small fast growing heterotrophic flagellates can probably compensate predation losses faster than larger forms, which have a slower growth and (Pace at al. 1998, Auer and Arndt 2001). Little is, however, known about the growth rates and variation in growth rate of heterotrophic flagellates in their natural (Weisse 1997). Another strategy to avoid predation may be to attach to particles (Jürgens et al. 1996, Carrias et al. 1998).

In addition to obligate phagotrophs some flagellates are capable of both phagotrophic heterotrophy and phototrophic autotrophy, these flagellates are called mixotrophs. Mixotrophy has developed independently in several taxonomic groups and is found mainly among the dinophyceans, prymnesiophyceans and chrysophyceans (Jones 1994, Jones 2000). Mixotrophs includes nutritional strategies from species being almost 100% autotrophic to almost 100% heterotrophic (Jones 1994). For the former, mixotrophy can be a strategy to survive during nutrient poor situations. In this situation a mixotrophic flagellate has an advantage compared to an obligate due to the capacity to take up nutrients through bacterivory. In addition this behaviour involves eating the main competitor for inorganic nutrients during nutrient poor conditions (Thingstad et al. 1996). Phagotrophy can also be a strategy to supplement growth during limitation, to survive during darkness or to aquire essential substances for growth e.g. micronutrients, vitamins or phospholipids (Kimura and Ishida 1989, Maranger et al. 1998). For mainly phagotrophic flagellates mixotrophy can be beneficial during situations with low prey concentrations. The importance of mixotrophs as compared to heterotrophs has been reported to vary from 0 to 86 % in aquatic systems (McManus and Fuhrman 1986, Havskum and Riemann 1996).

Phagotrophic ciliates

The ciliates are a heterogenous group consisting of about 3000 free-living species. Cilia are used for feeding or motion. Nutrient strategies include both mixotrophy and heterotrophy. Small prey particles are taken up through filtration feeding, while raptorial feeding is used for uptake of larger particles. Ciliates are thought to be the main predator on nanoplankton (2-20 µm), but are also known to feed on bacteria (Sherr and Sherr 1987, Sherr et al. 1989). Due to sensory and behavioural capacities ciliates can locate and exploit food patches (Jonsson and Johansson 1997). Increased swimming under starvation can therefore increase survival. Metazooplankton seems to be the main predator on ciliates and to be the main limiting factor in some areas (Kivi et al. 1996). Other studies imply that resource limitation is the main limiting factor (Johansson et al. 2002). Ciliates are known to

1 3 escape predation due to behavioural escape mechanisms (Jonsson and Tiselius 1990, Stoecker and Capuzzo 1990, Broglio et al. 2001). The hard of tintinnids is also suggested to be a protection against predation (Fenchel 1987, Kivi et al. 1996). In a study comparing these two strategies it was shown that the swimming behaviour was of a larger importance to avoid predation than hard covering (Broglio et al. 2001).

All these species-specific differences in nutritional demands, prey selection, habitat choice and susceptibility to predation imply that the species composition of microorganisms varies both temporally and spatially depending on factors such as nutritional status, access to attachment sites, prey community composition and type of predators.

The study area

The field sampling in this work was performed in the Baltic Sea. The three main basins within this sea are the Bothnian Bay in the north, Bothnian Sea and Baltic Proper in the south (Fig. 2). The Baltic Sea is an almost enclosed brackish sea area with an average depth of only 52m (Niilonen 2002). Due to high rate of freshwater input in the north and inflowing saltwater in the south the surface water salinity varies from 1-3 PSU in the Bothnian Bay, 3-7 PSU in the Bothnian Sea to 6-8 PSU in the Baltic Proper (Wulff et al. 1990). The inflow of saltwater to the deeper parts creates a strong permanent halocline in the Baltic Proper. In the two northern basins the halocline is weaker but a strong thermocline is developed during the summer. Ice is normally covering the Bothnian Bay and Bothnian Sea in winter, while the Baltic Proper is only partially covered. Nitrogen and phosphorous are mainly discharged to the southern parts (Wulff et al. 1990). enters the Baltic Sea with freshwater running into the northern parts. This freshwater input also imports humic substances into the Baltic Sea. This inflow is largest in the Bothnian Bay with about twice the inflow compared to the Baltic Sea (Mikulski and Falkenberg 1986). There is a gradient in mineral nutrients with relatively high nitrogen and low phosphorous concentration in the north compared to the south. The elemental ratio (SI:N:P) in the Bothnian Bay are in average 98:60:1 and 19:4:1 in the Baltic Proper (Wulff et al. 1990). Since phytoplankton requires an elemental composition of 16:16:1 (Redfield et al. 1963) phosphorous limitation is dominating in the north and nitrogen limitation in the south. Low phosphorous concentrations in the Botnian Bay probably cause the low primary production, which is ten times lower than in the Baltic Proper (Andersson et al. 1996, III).

1 4 In the Baltic Sea area there are large seasonal variations in light and temperature as well as in stability and nutrient concentrations (Andersson et al. 1994). These changes influence the growth of the phytoplankton community, and different phytoplankton groups will show maximal growth rates during different times of the year, resulting in a seasonal succession of phytoplankton. In general microplankton e.g. diatoms and dinoflagellates are dominating during spring, while nano and picoplankton eg. flagellates and unicellular cyanobacteria are dominating during summer and autumn. During spring the Peridiniella catenata, and diatoms of the genera and Thalassiosira are dominating (Andersson et al. 1996). After the the chlorophyte Monoraphidium contortum shows high values. In the nano-fraction Pyramimonas spp. and Cryptophyceans are important. Unicellular cyanobacteria often peak in August. During late summer and autumn blooms of filamentous cyanobacteria are common. These blooms often consists of Aphanizomenon sp., Nodularia spumigena and Anabaena sp.. In the Baltic Proper there can be a second peak of microplankton in autumn.

I, II, IV, V

II

Fig. 2. Map of the Baltic Sea showing sampling stations for experiments performed in paper I, II, III, IV and V. In the field study six stations were sampled located in the Bothnian Bay (BB1, BB2), the Bothnian Sea (BS1, BS2) and the Baltic Proper (BP1, BP2).

1 5

In this study we have performed four experiments with water sampled from a coastal station in the northern Bothnian Sea (I, III, IV, V) and one experiment with water from the Gotland Basin (II, Fig. 2). All these experiment were performed during the summer when regenerated production was dominating. In addition we performed a field survey studying the seasonal succession of protozoa (III). This study was performed at two stations in each of the three basins (Fig. 2).

OBJECTIVES

The aim of this thesis was to investigate the importance of resources and predation in structuring the pelagic microbial food web. Questions asked were:

- How does increased productivity influence the structure of the pelagic microbial food web?

- Does predation limitation vary between trophic levels?

- What is the relative importance of resource limitation and predation limitation at different trophic levels?

MAJOR RESULTS AND DISCUSSION

Importance of resources

In a theoretical linear food chain with homogenous trophic levels the top level (n) and level n-2, n-4 etc. will increase in biomass with nutrient concentration while level n-1, n-3 etc. will remain at constant biomass at equilibrium (Oksanen et al. 1981, Thingstad and Sakshaug 1990). When food supply is sufficient to balance loss rates a new will be established. In a more complex food web the outcome is not as obvious. Several characteristics of the organisms in natural systems could cause deviations from the predictions made from a simple linear food chain. One example is, if the different prey types on a particular level are not substitutable resources (Leibold 1989, McCauley and Murdoch 1990, Leibold and Wilbur 1992, Steiner 2001), or that the prey develops antipredator behaviour (Abrams 1992). In addition the theoretical model assumes that all consumers should have linear functional responses (Akçakaya et al. 1995) and that the predators are homogenously distributed (Arditi and Saiah 1992). Further, omnivory i.e. a high

1 6 frequency of predation on more than one trophic level is not allowed (Abrams 1993).

To study how the structure of a natural aquatic food web was affected by nutrient addition we performed an experiment using seawater from the northern Baltic Sea (I). The organisms included were divided into three main autotrophic and heterotrophic groups based on assumed trophic position. Autotrophs where divided into bacteria, flagellates and diatoms and heterotrophs into bacteria, flagellates and ciliates representing pico- nano- and microplankton, respectively (Fig. 1). Addition of nutrients resulted in an increased biomass of all trophic levels and a changed size-structure of the community. Among the osmotrophs (bacteria and phytoplankton) diatoms dominated in the enriched system while bacteria and nanoflagellates dominated the treatment without added nutrients. This change in size-structure is in agreement with both theoretical studies and earlier empirical studies (Thingstad and Sakshaug 1990, Kivi et al. 1996, Duarte et al. 2000). Due to higher biovolume-specific uptake small cells should out compete larger forms in nutrient poor conditions (Legendre and Rassoulzadegan 1995). First when small forms are saturated or limited by predation larger forms can establish (Thingstad and Sakshaug 1990).

The increased energy input into the enriched system also resulted in a lengthening of the food chain, in the form of a community. Establishment of new trophic levels with higher productivity has been shown both theoretically and in experimental microbial food chains (Oksanen et al. 1981, Kaunzinger and Morin 1998). The occurrence of inedible or toxic forms can stop this transport of material to higher trophic levels. In experimental systems with bacteria and bacterivorous flagellates, inedible forms of bacteria are known to dominate (Pernthaler et al. 1997, Hahn and Höfle 1999, Jürgens and Sala 2000). These inedible bacteria are either too small or too large for protozoan ingestion. In the study from the northern Baltic Sea we did not find any inedible bacteria, at least no large filamentous forms. Nor did we find any known toxic phytoplankton. One explanation to the absence of inedible forms could be that the duration of the experiment was too short. An earlier study with metazooplankton as the top predator showed, however, that the ciliates and phytoplankton could change their taxonomic composition within less than 6 days (Kivi et al. 1996).

In the nutrient poor treatment the flagellate community was dominated by a potentially mixotrophic flagellate, while nutrient addition decreased the importance of this mixotrophic flagellate compared to specialised autotrophic and heterotrophic flagellates (I). In the nutrient limited situation bacteria were probably acting as a sink for inorganic nutrients, and due to their higher affinity for inorganic nutrients

1 7 and small size, bacteria almost out competed the strict autotrophs. During this situation low bacterial concentrations also made it impossible for the phagotrophic flagellates to establish a high biomass. The mixotrophs, on the other hand, had an advantage in being able to utilize both inorganic and organic carbon and nutrient sources. In theory predation by mixotrophs on bacteria may allow an autotroph community to establish (Thingstad et al. 1996). In our study autotrophs were, however, not increasing (I). The reason for that was most likely that the mixotrophic grazing did not decrease the bacterial number enough to allow establishments of autotrophs.

The superiority of a generalist in front of a specialist in severe condition are known in theory and have earlier been shown experimentally for some species. For example, Ochromonas sp. and a mixotrophic dinoflagellate increased during poor nutrient conditions (Rothaupt 1996, Havskum and Hansen 1997). No earlier results are, however, presented on mixotrophic prymnesiophyceans. In our experiment the potentially mixotrophic flagellate was the prymnesiophycean Chrysochromulina sp. This species is common in the Baltic Sea and known to occur after periods with low levels of inorganic phosphorus (Hajdu et al. 1996). In agreement with the results from the presented experiment a mesocosm study with water from the northern Baltic Sea showed a decrease in Chrysochromulina sp. with enrichment. In a four- level nutrient gradient Chrysochromulina decreased with increasing nutrient concentrations from 84, 32, 22, to 6 % of the phytoplankton community (Samuelsson et al. manuscript). It seems that higher nutrients concentrations generally decrease the importance of mixotrophs compared to strictly (Arenovski 1995, Havskum and Hansen 1997). The picture is, however, not clear since a comparison of a high productive coastal environment and an oligotrophic oceanic ecosystem showed that the relative importance of mixotrophy did not differ (Sanders et al. 2000). In our study the establishment of a mixotrophic flagellate in nutrient poor treatment probably allowed a third trophic level, the ciliates, to be at least temporally established despite low concentrations of strict autotrophs and heterotrophic flagellates. Mixotrophs have traditionally not been included in food web models. However, they are most likely of large importance, especially in low productive aquatic environments.

The response of the microbial food web to nutrient addition was also studied in a situation with high concentrations of filamentous cyanobacteria (II). In late summer potentially nitrogen fixating cyanobacteria, Aphanizomen sp. and Nodularia spumigena Mertens, often form dense blooms in the central Baltic Proper. The development of these blooms is probably favoured by nitrogen-limited conditions (Larsson et al. 2001). To study how filamentous cyanobacteria influence resource limitation of primary producers as well as bacteria, nutrient enrichment experiments

1 8 were performed in the Gotland Basin (II). These blooms might influence the microbial community due to e.g. an increased N:P ratio (Larsson et al. 2001, Rydin et al. 2002). Granéli et al. (1990) also showed that during these blooms the primary production was P limited in otherwise N limited areas. Furthermore, the nutrient enrichment experiments were used to study the channelling of an eventual increased production in the pelagic food web. Additions of inorganic nutrients (N,P) and liable organic carbon were made to both seawater pre-filtered through a 40 µm filter to exclude filamentous cyanobacteria (<40 µm treatment) and to seawater with a 13-fold concentration of filamentous cyanobacteria (>90 µm treatment). Of the single additions N gave the highest response in primary and bacterial production in the <40 µm treatment. In the treatment with addition of filamentous cyanobacteria (>90 µm) P seemed to be the main limiting nutrient for both bacteria and phytoplankton. The result indicates that by the cyanobacteria decreased N limitation of other phytoplankton and bacteria. The response to single additions was, however, small. In general it seemed as phytoplankton primary production was limited by both nitrogen and phosphorous and bacterial production co-limited by carbon, nitrogen and phosphorous in both treatments.

The increased production by heterotrophic bacteria due to enrichment did not result in higher bacterial biomass (II). This was probably a result of grazing control by protozoa (flagellates and ciliates), which increased in the enriched treatments. Increased primary production, however, resulted in increased biomass, especially of picocyanobacteria. The lower loss rate of phytoplankton are in agreement with other studies showing low grazing rates on phytoplankton, especially picocyanobacteria (Kuuppo et al. 1998, Boenigk et al. 2001, IV). The larger response in higher trophic levels due to increased bacterial production compared to primary production indicated a tighter link on the heterotrophic part of the microbial food web.

The three main basins in the Baltic Sea, Bothnian Bay in the north, Bothnian Sea and Baltic Proper in the south form a primary production gradient with highest production in the south. To study the effect of increased production on the protozoa community, the spatial and temporal variation in the community composition was studied during one year (III). The results showed that the biomass of flagellates and ciliates increased with primary production. Furthermore, relatively small protozoan cells dominated in the low productive north, while larger cells became more dominant in the south. Our results are in agreement with studies in showing a positive influence of trophy on flagellate biomass (Sanders et al. 1992, Mathes and Arndt 1994, Gasol et al. 1995). The effect of enrichment on the species composition seems to be less clear. One tendency is that Chrysomonads and Bodonoids increase with trophic status, while choanoflagellates decrease (Auer and

1 9 Arndt 2001). In contradiction, the results from our study showed that the relative importance of Chrysomonads decreased with primary productivity from north to south. In the north high input of allochtonous carbon allow a high bacterial production despite low primary produciton. This allochtonous based bacterial production probably explained the relatively high abundance of the bacterivorous chrysophyceans (Andersson et al. 1989, Rothhaupt 1996).

In lake systems the seasonal dynamics of the protozoan community has been observed to depend on nutrient status. While oligotrophic lakes have the highest protozoa concentrations in summer, highest concentrations are reached already in spring in more eutrophic systems (Laybourn-Parry and Rogerson 1993, Auer and Arndt 2001). This pattern was also found in the Baltic Sea, with high summer concentrations in the Bothnian Bay and high spring concentrations in the Bothnian Sea and Baltic Proper (III). At all stations the concentration of small flagellates seemed to follow the dynamics of heterotrophic bacteria, while large flagellates and ciliates showed a positive relation to the concentration of chl a. These results indicate that resource was an important limiting factor for protozoa in the entire Baltic Sea.

In conclusion resource had a large influence on food web structure. Addition of nutrients resulted in a lengthening of the food web according to traditional models (Oksanen et al. 1981, Thingstad and Sakshaug 1990). In addition higher nutrient levels resulted in larger autotrophs indicating that small phytoplankton are better competitors than large cells (I). In agreement, heterotrophs increased in size with productivity of the system (I, V). Mixotrophs seemed to have a large advantage in nutrient poor treatments and might explain the temporal establishment of phagotrophic ciliates at a low nutrient level (I).

Importance of access to attachment sites and metazooplankton grazing

A large part of microorganisms in the aquatic system are adapted to live on surfaces. Surface attached forms are found among bacteria, flagellates and ciliates. It has been shown that attachment to surfaces is the most favourable situation for suspension feeding protozoa (Carrias et al. 1996). The abundance of suitable attachment sites most likely influences the of these forms. In an experiment we tested if access to attachment sites was an important limiting factor for protozoa in the northern Baltic Sea (III). The results showed that increased access to surface promoted the choanoflagellates, while other nanoflagellates were not promoted by surface alone. In a field study in the Baltic Sea, we observed that while other bacterivorous flagellates peaked during summer,

2 0 loricated choanoflagellates showed highest concentrations in spring and autumn (III). The inverse relationship found between the loricated choanoflagellates and other nanoflagellates in the field study, was thus probably explained by seasonal changes in the access to surface attachment sites. In our study in the Baltic Sea the spring peak of choanoflagellates actually coincided with a peak in diatoms, while the autumn peak coincided with the autumn re-suspension of detritus material (III). During both periods, thus, possible attachment sites were enhanced.

Like other heterotrophic flagellates choanoflagellates are thought to be mainly bacterivorous (Leadbeater 1991, Carrias et al. 1996). In addition some species have the capacity to ingest pico- and nanoautotrophs, detritus as well as dissolved organic carbon (Sherr 1988, Buck et al. 1991, Marchant and Scott 1993). Loricated choanoflagellates, however, differ morphological from other flagellates due to that the lorica increase its size at least to the double. An explanation to the dynamical difference between the two groups of flagellates could, thus, also be variation in vulnerability to predation. Metazoplankton is known to show high densities during the summer in the Baltic Sea (Johansson et al. 2002). To study the effect of metazooplankton on the flagellate community we performed an in situ experiment where metazooplankton were added in different concentrations to a natural flagellate community (III). In the treatment with highest metazooplankton concentration the number of loricated choanoflagellates decreased. In the same treatment the concentration of ciliates decreased while the concentrations of small flagellates increased. The increase of small flagellates was probably a consequence of low ciliate concentrations. The results showed that metazooplankton had the capacity to graze choanoflagellates. Thus, they probably have a negative effect on the community during periods of high metazooplankton abundance, such as the summer. This result also shows that choanoflagellates are a direct link between bacteria and metazooplankton.

The inverse relationship found between loricated choanoflagellates and small heterotoprhic flagellates in the field study, was thus probably explained by both seasonal changes in the predator community and seasonal changes in the access to surface attachment sites.

Importance of predation

Although grazing rates on several groups within the microbial food web have been measured. Predation limitation in these groups is poorly understood. Predation limitation has been defined as the degree to which per capita growth rate is decreased by predation (Osenberg and Mittelbach 1996).

2 1

A truncation experiment were performed to measure predation limitation within the microbial food web (III). The main questions asked were if predation limitation differs at varying trophic levels or in different functional groups, i.e. autotrophs and heterotrophs. Potential predators of different size were removed and the response on both autotrophs and heterotrophs was studied. Heterotrophic bacteria, heterotrophic flagellates and autotrophic increased when potential predators were removed, indicating predation limitation of these groups. Other phytoplankton and ciliates did not respond to predator removal, showing that predation was not a limiting factor for these organisms. These results are in agreement with our nutrient addition experiment (II) showing a higher transport within heterotrophs than between autotrophs and heterotrophs. Low predation limitation of phytoplankton during summer has been observed earlier in the Baltic Sea (Lignell et al. 1992, Kivi et al 1993). Microprotozoans have, however, been observed to effectively graze autotrophic nanoplankton (Kivi et al. 1993). Only a few measurement of grazing on picoautotrophs have been performed. In agreement with our result these showed that picoeukaryotes are limited by predation (e.g. Pernthaler et al. 1996, Reckerman and Veldhuis 1997). Most likely, the autotrophs in this study were mainly resource limited.

The appearance of trophic cascades has been used to study the structure of the microbial food web (e.g. Wikner and Hagström 1988, Calbet et al. 2001). Alternating positive and negative responses of the bacterial community due to removal of predators of different sizes have been explained by quick trophic cascades (within 24 h). We did not find such a quick indirect response to removal of predators. After about 3-6 days, however, trophic cascades were observed (II,III,V). Earlier studies have shown both the occurrence and absence of trophic cascades within the pelagic microbial food web (Güde 1988, Dolan and Gallegos 1991, Reckermann and Veldhuis 1997, Kuuppo et al. 1998, Caron et al. 1999, Calbet et al. 2001). Several explanations to lack of trophic cascades within microbial communities have been suggested. For example, that removal of a predator or prey is quickly compensated due to high turnover rates of the microbes (Pace et al. 1998, Mikola and Setälä 1998), that indirect trophic links such as nutrient cycling makes the identification of top-down and bottom-up forces harder (Mikola and Setälä 1998) or due to predator avoidance in the form of chemical, morphological, behavioural or history defence (Verity and Smetacek 1996). There is also a reason to believe that cascades are less likely in oligotrophic environments, due to strong resource control and larger importance of mixotrophy. The theory also assumes a linear food web (Morin and Lawler 1995, Polis and Strong 1996). One explanation for weak importance of trophic cascades may, thus, be omnivory. In the northern Baltic Sea the main links between

2 2 the heterotrophs seemed to be from bacteria to small heterotrophic flagellates, from medium heterotrophic flagellates and small ciliates to large flagellates and ciliates (IV). The results, however, also indicated omnivory. About 30 % of the predation on bacteria and small flagellates was due to grazing by a non-adjacent trophic level .

Relative importance of resource and predation limitation

Both resource and predation limitation seemed to be important factors structuring the microbial food web. A comparison of their relative importance requires simultaneous measurement of these two limiting factors (Osenberg and Mittelbach 1996). Some theories hypothesize that predation and resource limitation alternate between adjacent trophic levels, while others assume simultaneous effects of resource and predation (Hairston et al. 1960, Oksanen et al. 1981). Others suggest that top-down regulation is dominating in the top of a food web and bottom-up regulation at the bottom of a food web (McQueen et al. 1986). To our knowledge the relative importance of resource and predation limitation of the main microbial heterotrophs in the pelagic system, bacteria, flagellates and ciliates, have not been quantified before.

To study the relative importance of resource and predation we used a cross factorial experimental design were resource and predator level were varied (V). Resource was added in the form of the bacterium Pseudomonas fluorescence and potential predators were removed by filtration. The relative importance of resource limitation for the population growth rate was on average 62% in June and 74% in September. Thus, during both experiments the heterotrophic flagellates were mainly resource limited. Unfortunately we did not perform any simultaneous measurements on the relative importance of resource and predation for bacteria and ciliates. From other studies we can, however, calculate the relative importance of these two factors for bacteria and ciliates. In the nutrient addition experiment performed at the Gotland Basin in 1997 (II), bacteria increased their specific growth rate from – 0.29 d-1 to 0.18 d-1 when predators larger than 0.8 µm were removed. After adding carbon, nitrogen and phosphorous the specific growth rate increased further to 0.25 d-1. The relative importance of resource limitation was thus 14%. In the truncation experiment in the northern Baltic Sea in 1998, the importance of predation on bacteria was measured (IV). In addition, the treatment with only bacteria was diluted ten times with free water to reduce resource limitation. By removing predators the growth rate increased from 0 d-1 to 0.49 d-1 and by reducing resource limitation the growth rate increased to 2.29 d-1. In this case the relative importance of resource limitation was 79%. In the same experiment removal of potential predators on ciliates did not affect the specific growth rate of the ciliates. After an

2 3 increase of their main prey, heterotrophic flagellates, the specific growth rate of the ciliates increased to 0.80 d-1. Ciliates were, thus, mainly resource limited. These calculations show that in the Bothnian Sea all three adjacent trophic levels where mainly resource limited during summer (Fig. 3). In the Baltic Proper, however, it seemed, that at least the bacteria were mainly predation limited. A rough estimation of the response of heterotrophic flagellates and ciliates from the experiment in the Gotland Basin 1998 indicates that also these were mainly predation limited. Our results indicate that the importance of predation varies at different trophic levels, but generally the importance of predation limitation within the microbial food web seems to increase with productivity of the system. This is in agreement with earlier hypotheses that heterotrophic flagellates should be mainly resource limited in nutrient poor environments (Sanders et al. 1992, Gasol 1994). The importance of predation limitation for bacteria has been suggested both to decrease and increase with system productivity (Billen et al. 1990, Sanders et al. 1992, Gasol 1994, Gasol et al. 2002). In addition Fenchel (1986) suggested that grazing by heterotrophic nanoplankton maintained bacteria just below their in oligotrophic , but in eutrophic systems the mean number of bacteria were kept well below the carrying capacity by heterotrophic flagellate grazing.

The observation that resource was the main limiting factor at adjacent trophic levels are not in agreement with the EEH model by Oksanen et al. (1981), predicting that

Bothnian Sea Baltic Proper

100

100

) 80 80 % (

n 60 60 o i t a t

i 40 40 m i

L 20 20

0 0 June Bact June Flag Sept Flag June Ciliates Aug Bact

Fig. 3. Resource (grey/black bar) and predation (white bar) limitation of heterotrophic bacteria (Bact), heterotrophic flagellates (Flag) and phagotrophic ciliates (Ciliates) in the Bothnian Sea in June and September. Limitation of heterotrophic bacteria in the Baltic Proper were measured in August.

2 4 the importance of resource and predation alternates between adjacent trophic levels. Several factors have been presented to cause the discrepancy between the behaviour of natural food webs and the EEH model (Polis and Strong 1996, Mikola and Setälä 1998, Steiner 2001, see above). Some of these factors may be important in the pelagic microbial food web. Heterogenous trophic levels is one of these factors Leibold 1989, McCauley and Murdoch 1990, Leibold and Wilbur 1992, Steiner 2001). For example, at resource addition the increase of the basal level, phytoplankton or bacteria do not necessarily have to affect primary consumers, due to the establishment of inedible forms. This is well known for heterotrophic bacteria, which can escape into size refuges, cells that are too small or too large for the predator (Simek et al. 1997, Hahn and Höfle1999, Jürgens and Sala 2000). of the top predator (Arditi and Saiah 1992) can also result in resource limitation of adjacent trophic levels. Ciliates and flagellates are known to aggregate around food patches (Fenchel and Blackburn 1999).

According to a model by Gasol (1994) the relationship between abundance of heterotrophic bacteria and heterotrophic flagellates can be used to evaluate if flagellates are bottom-up or top-down regulated. We compared the results from this model with the results from our empirical measurements of resource and predation limitation of flagellates. Within this model the maximum attainable abundance (MAA) describes the linear relationship between bacteria and the maximum abundance of flagellates attained at equilibrium. The line corresponds to the prey- predator zero-isocline in a Lotka-Volterra model. A second line, mean realized abundance (MRA), describes the relationship between the mean abundance of flagellates and bacteria from measurements in aquatic systems. This line is assumed to average the effect of both top-down and bottom-up regulation over the year, and is used as a reference line to interpret the relative importance of bottom-up or top- down factors regulating heterotrophic nanoflagellates abundance. If the data points lies above the MRA-line the flagellate community is thought to be bottom-up regulated, and if the data ends up below the MRA-line they are thought to be top- down regulated. According to this model the flagellates in our resource-predation experiment were bottom-up regulated in June but top-down regulated in September. It seemed, thus, that the model worked well in June but not in September. Two assumptions are made in the model. The first is that flagellates are only eating heterotrophic bacteria and the other that all bacteria are edible. The relatively low abundance of flagellates compared to the number of bacteria could in September be explained by that a large part of the bacterial community was inedible. One criterion for edibility of bacteria is the size. Assuming that only bacteria in the range of 0.7 µm3 to 1 µm3 are edible (Andersson et al. 1986, Gonzalez et al. 1990, Hahn and Höfle 1999) the September point move towards the bottom-up regulated end of the scale. One years data from a station in the Bothnian Bay and one station

2 5 in the Bothnian Sea were also plotted in the model. The analysis indicated that all over the year the flagellates were mainly resource-limited in these two basins (V). Our data thus fits with earlier hypothesis that flagellates are mainly resource limited in oligotrophic systems (Sanders et al. 1992, Gasol 1994).

CONCLUSIONS AND FUTURE PERSPECTIVES A field study in the northern Baltic Sea showed that dominating protozoa, flagellates and ciliates, increased with increasing primary productivity from north to south. Furthermore, relatively small protozoan cells dominated in the low productive north, while larger cells became more dominant in the south. The relationship between plankton size structure and productivity was further studied in an experimental system. In agreement with present theories regarding nutrient status of pelagic food webs, increased productivity caused a lengthening of the food chain as well as a change in size structure in an experimental system. While microplankton dominated in nutrient rich treatments pico- and nanoplankton dominated during nutrient poor treament. The flagellate community was dominated by a potentially mixotroph, Chrysochromulina sp., during low nutrient concentrations. This is the first study showing that Chrysochromulina sp. in resemblance with other mixotrophs seems to be favoured in nutrient poor situations compared to strict autotrophs and heterotrophs.

During a stratified summer period autotrophic microorganisms in the northern Baltic Sea did not respond to removal of potential predators, indicating that they were primarily limited by inorganic nutrients. An exception was small eucaryotic picoplankton that showed a large response to predator removal. Among the heterotrophic microorganisms direct effect of predation seemed to increase from ciliates, heterotrophic bacteria, small heterotrophic flagellates, medium flagellates to large flagellates. No quick indirect effect was observed, but after four days trophic cascades were detected.

The relative importance of resource and predation limitation was studied among heterotrophic bacteria, flagellates and ciliates in the northern Baltic Sea. For all these groups, resource limitation seemed to prevail during the summer period. The results also indicated that the relative importance of predation increased with the productivity of the system. To our knowledge there are no earlier measurements on the relative importance of resource and predation limitation for micoorganisms in the pelagic environment.

2 6 To verify the pattern found in this study, concerning the importance of productivity and trophic level for the relative importance of resource and predation limitation, similar questions have to be asked across a variety of aquatic systems. This also requires careful observation of long time series of bacteria, flagellates, ciliates and metazooplankton.

In addition to field studies, it is necessary to perform manipulation experiments of simple culture systems and from these develop model systems. It is necessary to examine mechanisms structuring the microbial environment such as antipredator behaviour, behavioural of the predators and the importance of patchiness for the dynamics within the microbial community. Studies of impact of protistan predation on the community structure and of chemical communications between predator and prey in microbial food webs are also lacking. In agreement with the suggestion of Kaunzinger (1998) future studies should vary trophic complexity and omnivory to determine how these aspects of natural systems alter responses to changes in productivity.

Another unanswered question is the abundance of inedible bacteria in the . Is this an important mechanism for the dynamic of bacteria? Which are the processes selecting for inedibility? Is the occurrence of inedible bacteria connected to predation pressure from flagellates? In addition to bacteria are there inedible forms of flagellates? The lorica of choanoflagellates could be a strategy to escape predation from ciliates. There are examples of behavioural antipredator strategies among ciliates. Can a similar strategy be found for flagellates and perhaps also for bacteria?

ACKNOWLEDGMENTS: I want to thank Agneta Andersson, Johnny Berglund and Pia Haecky for comments on earlier versions of this thesis.

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