ACTA

UNIVERSITATIS

UPSALIENSIS

UPPSALA

2008

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 585

Significance and Diversity of LakeBacteriophages

DAVID LYMER

ISSN 1651-6214ISBN 978-91-554-7371-6urn:nbn:se:uu:diva-9499

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To Gullan and Karin

List of papers

This thesis is based on the following papers which will be referred to by their roman numerals in the summary.

I Lymer, D. & K. Vrede (2006). Nutrient additions resulting in

phage release and formation of non-nucleoid-containing bac-teria. Aquatic Microbial Ecology 43:107-112.

II Lymer, D., E. S. Lindström & K. Vrede (2008). Variable im-portance of viral-induced bacterial mortality along gradients of trophic status and humic content in lakes. Freshwater biol-ogy 53 (6), 1101–1113

III Lymer, D. Logue, J. B. Brussaard, C. P. D. Baudoux, A-C. Vrede, K. and E. S. Lindström (2008). Temporal variation in freshwater viral and bacterial community composition. Freshwater Biology 53 (6), 1163–1175

IV Lymer, D & E. S. Lindström. The importance of nutrient con-centration and prophage induction for freshwater viral com-munity composition. (Manuscript)

Reprinted with the kind permission of the publishers; Inter-Research (Paper I) and Blackwell Publishing (Paper II & III).

Contents

Introduction.....................................................................................................9 Aquatic viral ecology .................................................................................9 Aquatic food webs....................................................................................11 Aquatic bacteria (the predominant host) ..................................................13 Aquatic viral community composition .....................................................13

Aims of this thesis.........................................................................................15

Material and methods....................................................................................16 Environments ...........................................................................................16

Field studies .........................................................................................16 Experiments .........................................................................................17

Methods....................................................................................................17 Epifluorescence microscopy (EM) ......................................................17 Flow cytometry (FCM)........................................................................18 Transmission electron microscopy (TEM) ..........................................18 Pulsed-field gel electrophoresis (PFGE)..............................................19 Terminal restriction-fragment length polymorphism (t-RFLP)...........21 Multivariate statistics...........................................................................22

Summary of the papers and conclusions.......................................................24 Link between bacteria and bacteriophages (Paper I)................................24 Gradients in trophic status and humic content (Paper II) .........................26 Temporal variation in viral community composition (Paper III). ............28 Effects of lysogeny on viral community composition (Paper IV)............31 Overall conclusions and perspectives.......................................................35

Summary in Swedish ....................................................................................38 Bakteriefagers betydelse och diversitet i sjöar .........................................38

Acknowledgements.......................................................................................42

References.....................................................................................................44

Abbreviations

BA Bacterial abundance BGE Bacterial growth efficiency BP Bacterial production BR Bacterial respiration C Carbon CCA Canonical Correspondence Analysis DCA Detrended Correspondence Analysis DNA Deoxyribonucleic acid DOC Dissolved organic carbon FIC Frequency of infected cells FVIC Frequency of visibly infected cells GBM Grazer induced bacterial mortality LB Lysogenic bacteria MDS Multidimensional Scaling N Nitrogen non-NuCC Non-nucleoid-containing cells NuCC Nucleoid-containing cells OTU Operational taxonomic unit P Phosphorus PFGE Pulsed-field gel electrophoresis PGBM Potential grazer induced bacterial mortality Pi Inorganic phosphorus PLS Partial Least Squares Regression RDA Redundancy Analysis RNA Ribonucleic acid rRNA ribosomal RNA Tot-P or TP Total phosphorus t-RFLP Terminal restriction-fragment length polymorphism UV Ultraviolet VA Viral abundance VBM Viral induced bacterial mortality VBR Virus to bacteria ratio

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Introduction

Aquatic viral ecology The first reports of viruses in aquatic environments (Spencer 1955) con-cluded that viruses were sparse. Abundances similar to current numbers were first found in marine environments (Bergh et al. 1989; Proctor and Fuhrman 1990). It has since been concluded that viruses are typically more abundant in freshwater than in marine systems (Maranger and Bird 1995). Typical values are in the order of 1010 l-1 in marine environments (Fuhrman 1999) and 1011 l-1 in freshwater systems (Maranger and Bird 1995). The abundance of planktonic viruses varies both spatially and temporally (Cochlan et al. 1993; Mathias et al. 1995) and generally increases with environmental nutri-ent concentration and productivity (Wommack and Colwell 2000). Further, viral abundance in anoxic water have been found to be as high as, or even higher than in the oxygenated surface layer (Weinbauer and Höfle 1998).

A virus can display either an acute life strategy, lytic, or a persistent life strategy, chronic or lysogenic (Fuhrman 2000). A lysogenic phage is a phage that can exist as a prophage within its host organism (i.e. in the lysogenic life strategy the phage DNA/RNA is incorporated into the bacterial genome and called a prophage) and is also called a temperate phage. It is either integrated into the chromosome of the host bacterium or more rarely exists as a stable plasmid within the host cell. The prophage infection results in the bacteria expressing gene(s) that repress the phage's lytic action, and the phage enters the lytic cycle when this suppression is disrupted (Mukhopadhyay et al. 2003; Snyder and Champness 1997). Alternatively, a lysogen can refer to a bacterial strain that carries a prophage. The lytic life strategy leads to lysis of the host cell and a production of new viral particles (Figure 1).

However, viruses with a lysogenic life strategy can be induced to a lytic life strategy (Figure 1). Hence, they must have the capacity to sense cues to ensure a high probability of transmission by becoming lytic at opportune times (Villarreal 2005). Which environmental cues are important in this con-text in aquatic environments is largely unknown, but UV radiation, tempera-ture, chemicals and nutrients, especially phosphate, may be important (Jiang and Paul 1998; Wilson and Mann 1997).

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Figure 1. Viral lytic or lysogenic life strategy: The two different viral life strategies and the possible connection between viruses and non-nucleoid-containing cells (non-NuCC), bacteria (filled circles).

Most current methods for estimating viral abundance give no information on type of virus and identity of the host. Aquatic bacteria comprise a large pro-portion of the planktonic biomass, especially in oligotrophic and humic lakes (Blomqvist et al. 1995; Hessen et al. 1990; Tranvik 1988). Since viruses have no metabolism of their own and move only passively, the majority of viruses probably infect the organisms they most frequently encounter by random, i.e. the bacteria. Thus, among all the aquatic viruses, bacteriophages are probably the most abundant. Phages, viruses that infect prokaryotes, were described already in the beginning of the 1900s (D'herelle 1917; Twort 1915), and were referred to as phages "indicating something that eats or consumes something specified", since the authors could only see the disap-pearance of bacterial cells in their experiments (this was at a time prior to high magnification microscopes).The total number in the biosphere has been estimated to 1031 phages (Rohwer and Edwards 2002), making them the most abundant biological entities. For comparison, the total number of host organisms, i.e. bacteria, may be between 4 and 6 x1030 (Whitman et al. 1998).

The virus to bacteria ratio is typically higher in freshwater than in marine systems (Maranger and Bird 1995; Weinbauer 2004). Virus to bacteria ratios are higher in more nutrient rich environments, which suggests that bacterio-plankton produce more viruses per bacterium when conditions support fast growth and high productivity (Wommack and Colwell 2000). Accordingly,

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burst size (i.e. the number of viruses produced per bacteria) increases with increasing nutritional status in both freshwater and in marine environments, and is higher in freshwater than in marine environments (Prada et al. 2006).

A large fraction of the bacterial community is inactive or dormant, and the active fraction varies between different aquatic habitats (Haglund et al. 2002). A large share of the bacteria may contain no visible nucleoid. It has been suggested that these non-nucleoid-containing cells (non-NuCC) might be products of viral lysis or remains of flagellate grazing ("ghost cells", Zweifel and Hagström 1995). However, some of the apparently non-NuCC could be transformed, under appropriate culture conditions, into nucleoid-containing cells (Choi et al. 2003), i.e the cells most probably contained nuclei, although not detectable. The role of viruses in these transformations is still unclear (Figure 1). Therefore it would be most valuable to examine these relationships further.

Aquatic food webs In aquatic food webs there should be large differences in carbon and nutrient fluxes depending on whether the main cause of bacterial mortality is viral infection or grazing (Figure 2). Grazing should result in the bacterial produc-tion being transferred up to higher trophic levels whereas lysis following viral infection should result in a release of organic carbon and nutrients from the lysed bacteria.

The release of organic carbon and nutrients following lysis (Figure 2) can be regarded as a recycling process, whereby new viruses and cellular prod-ucts of the host organisms are returned to dissolved/suspended organic mat-ter phases of the carbon and nutrient cycles (Fisher and Velimirov 2002). The products of cell lysis can then be utilized by other bacteria (Middelboe et al. 1996; Middelboe et al. 2003; Noble and Fuhrman 1999) and for in-stance being an important source of amino acids (Middelboe and Jørgensen 2006). However, bacteria prefer to assimilate inorganic phosphorus (Pi) rather than virally released organic phosphorus, because the bacteria must enzymatically cleave it before use (Gobler et al. 1997). Hence, utilization of organic phosphorus is likely to be most important in oligotrophic systems where phosphorus is in scarce supply and limits bacterial production (Noble and Fuhrman 1999).

Viral infections may enhance bacteria-specific production rates, but the net effect for the ecosystem will be a loss of carbon, since most of the or-ganic carbon will be utilized repeatedly within the bacterial community, resulting in increased losses by respiration (Middelboe and Lyck 2002).

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Figure 2. The microbial food web; the fate of bacteria (filled circles): either bacteria are grazed by flagellates (GBM) or lysed following lytic viral infection (VBM).

The frequency of visibly infected bacterial cells in freshwaters ranges from 0 to 9% (Bettarel et al. 2003; Hennes and Simon 1995; Hofer and Sommaruga 2001; Jacquet S. et al. 2005; Mathias et al. 1995; Vrede et al. 2003; Wein-bauer et al. 2003b; Weinbauer and Höfle 1998), i.e. up to 100% of bacterial production or more (Binder 1999). Thus, viral infection could potentially have large effects on processes involving bacteria.

Viral lysis was found to prevail over heterotrophic nanoflagellate grazing as a cause of bacterial mortality by a factor of 11 in a eutrophic lake (Fisher and Velimirov 2002). It has also been suggested that increased nutritional status of a lake causes an increased importance of phage infection compared to grazing (Weinbauer and Peduzzi 1995). However, Vrede et al (2003) found a high bacterial mortality due to viral cell lysis in oligotrophic lakes, and viral induced bacterial mortality was shown to be relatively more impor-tant in an oligomesotrophic lake compared to an eutrophic lake (Bettarel et al. 2004). Thus, the existing results regarding the relationship between the trophic status of the system and the relative importance of viral lysis versus grazing for bacterial mortality are not consistent, and rely on comparisons of only a few lakes.

Another important characteristic of lakes, besides the trophic status, is the humic content. Food webs in humic lakes are known to function differently compared to those in clearwater lakes (Jansson et al. 1996; Tranvik 1992). The importance of viruses in humic lakes is yet not well known. In a com-bined field and experimental study Anesio et al. (2004) discovered a nega-tive influence of humic matter on viral abundance and the virus to bacteria ratio. Also, Vrede et al. (2003) found viral abundance to be higher in a clearwater lake compared to in a humic lake. However, in the latter study, the frequency of visibly infected cells and burst size did not differ between

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the two lakes, hence, indicating that viral induced bacterial mortality was similar in the two lakes. Thus, the results regarding the importance of colour and humic content on the importance of viral infection of bacteria (compared to grazing on bacteria) are still inconclusive.

Aquatic bacteria (the predominant host) As bacteria are likely to be the predominant host for viruses in aquatic sys-tems, they should have a strong effect on viral parameters. Heterotrophic bacteria use organic carbon as carbon (C) and energy source (Russel and Cook 1995). Bacterial production (BP) in aquatic systems is dependent on the concentration of available organic C and to what extent this organic C can be used for BP and for respiration (BR). In addition, BP is also regulated by inorganic nutrient limitation, and inorganic phosphorus (Pi) is the most frequently reported limiting nutrient for BP (Vadstein 2000). The P content of dissolved organic matter has also been shown to regulate bacterial use of DOC for growth (Lennon and Pfaff 2005).

The physical and chemical environment in temperate lakes shows strong seasonal patterns. Furthermore, different parameters constrain the production in these systems at different times of the year. For instance, temperature and light climate change in a repeatable pattern over the year. Hence, communi-ties of several organism groups are subject to yearly, relatively season-specific changes (e.g. Sommer et al. 1986).

Aquatic viral community composition Differences in aquatic viral community composition over horizontal and vertical spatial scales have been followed in marine ecosystems by using pulsed-field gel electrophoresis (PFGE) (Fuhrman et al. 2002a; Wommack et al. 1999). The studies tracing viral community composition over time are, however, few so far and almost all were performed in marine environments (but see Auguet et al. 2006). Hence, our understanding of changes in viral community composition in lakes over time is far from complete.

Assuming that viruses are host-specific, at least to some extent, it can be expected that changes in the community composition of viruses reflect the seasonal pattern of their host communities. Accordingly, changes in viral communities in association to changes in phytoplankton communities have also been observed (Castberg et al. 2001; Larsen et al. 2001). As described earlier the major host for aquatic viruses is most likely bacteria and most viruses are probably bacteriophages. Therefore, changes in viral community composition should correlate with changes in bacterial community composi-tion.

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In plankton, the composition of bacterial communities varies over the sea-son (Wu and Hahn (2006) and references therein). When a certain bacterial strain increases in abundance, the probability of viral infection and prolifera-tion increases, too, because of increased encounter rates (Thingstad and Lignell 1997), which should lead to seasonal changes also in the viral com-munity composition. Accordingly, Hewson et al. (2006) found bacterial and viral communities to co-vary seasonally in the Gulf of Mexico. In contrast, no effect of experimental viral addition has been observed on marine bacte-rial community composition (Schwalbach et al. 2004). Thus, it is unclear what effect viruses have on bacterial community composition and what fac-tors affect viral community composition.

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Aims of this thesis

The aims of this thesis are: � To establish if there is a link between bacteria without visible nu-

cleoids and bacteriophages, and also if nutrient availability will have an effect on the production of viruses and non-NuCC (Paper I).

� To assess the importance of trophic status of lakes for the relative importance of two bacterial mortality factors, namely virus induced or grazer induced mortality (Paper II).

� To determine whether viral dynamics (infection frequency and rela-tive importance compared to grazing) are different in humic sys-tems compared to less humic systems (Paper II)

� To follow the temporal and spatial variation in viral community composition, and to evaluate possible co-variation with composi-tional changes in bacterial communities (Paper III).

� To investigate how common lysogeny is in lakes and how propha-ges can be induced to become lytic, and if this induction affects vi-ral community composition. (Paper IV).

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Material and methods

Environments

Figure 3. Map of Sweden outlining where the different studies were per-formed. A total of 23 different lakes in 4 distinct regions of Sweden were sampled for the field studies and experiments conducted. The roman numerals correspond to the papers in this thesis. The regions sampled were Uppland (n=12), Bergslagen (n=4), Jämtland (n=1) and the Abisko area (n=6). The lakes in these areas cov-ered environmental gradients as nu-trient status (from oligotrophic to eutrophic) and colour (clear water to highly humic).

Field studies Three field studies where conducted, encompassing gradients in trophic status of lakes in Sweden. The first study compared 21 lakes from three dif-ferent regions of Sweden; Uppland, Bergslagen and Abisko (Paper II). The lakes where sampled once in what was defined as summer for that region.

The second study (Paper III) compared three lakes from two different re-gions in Sweden; Uppland (Lake Erken & Lake Fyrsjön) and Jämtland (Lake Klocktjärn). All three lakes were sampled once each season (four sam-plings). In addition Lake Erken in Uppland was sampled every month in-between the seasonal samplings and two of the lakes were sampled three times during a week.

The third study was performed in Lake Erken in Uppland during the ice-free season (i.e. May – September). Samples were obtained almost biweekly for these 5 months (Paper IV).

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Experiments Four experiments where conducted in the laboratory (Paper I & IV). The experiments where performed as batch cultures under controlled light and temperature and the water used in the experiments was collected from Lake Erken in Uppland.

Methods Standard methods were used for enumeration of bacteria, analyses of chl a and water chemistry (see respective paper). Below are the methods used for viral enumeration (epifluorescence microscopy and flow cytometry), bacte-rial enumeration (flow cytometry), frequency of infected bacterial cells (transmission electron microscopy), viral community composition (pulsed-field gel electrophoresis), bacterial community composition (terminal restric-tion-fragment length polymorphism) and multivariate statistics described.

Epifluorescence microscopy (EM) Epifluorescence microscopy (EM) was used to enumerate viruses in Paper I, II & IV. EM is generally faster and easier than the transmission electron microscopy (TEM), which means that it can be used at sea or at field sta-tions. In this thesis we used the SYBR Green I dye that binds strongly to dsDNA, but also to single-stranded DNA and RNA at lower affinities (Marie et al. 1997). The method is described by Noble & Fuhrman (1998) for viral and bacteria enumeration in marine environments. The water sample, 0.1 ml is enough in normal freshwater conditions (Lymer and Vrede 2006), is fil-tered onto a 0.02 μm filter. Thereafter it is stained with SYBR Green I in darkness and the filter is then mounted on a microscopic slide (Noble & Fuhrman 1998). The samples are exited with blue light (488 nm) and gives a green fluorescence (Marie et al. 1997). Another advantage of EM is the pos-sibility to use different kinds of image analysis to enhance the fluorescent signal. Also automated image analysis protocols can be used in particulate organic matter rich samples to distinguish between free and attached bacte-ria, virus and suspended particles (Shopov et al. 2000). However, it was not used in any of the studies in this thesis.

EM yields higher values than TEM when comparing EM and TEM (Chen et al. 2001; Hennes and Suttle 1995; Noble and Fuhrman 1998; Wein-bauer and Suttle 1997) and also the precision is higher with EM than with TEM (Hennes and Suttle 1995; Weinbauer and Suttle 1997). One possible explanation for these results is the presence of free nucleic acids in the water (Bettarel et al. 2000), i.e. EM overestimates the actual VA as the free nucleic acids (DNA/RNA) will be stained and counted as viral particles. However,

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the higher EM numbers can also be due to an underestimation in TEM (see section Transmission electron microscopy).

Flow cytometry (FCM) In Paper III a relatively recent method in viral and bacterial enumeration was used, namely flow cytometry (FCM) and SYBR Green staining (Marie et al. 1999a; Marie et al. 1999b) using an optimized protocol (Brussard 2004). Briefly, the samples are analyzed in the flow cytometer and the threshold is progressively lowered until viruses are detected (a minimum of 1000 events per second). Results show two, sometimes three or four (Figure 4), distin-guishable viral populations (Chen et al. 2001; Jonsson et al. 2001; Marie et al. 1999a).

In natural samples FCM give up to 1.5 times higher numbers than EM, probably due to difficulties to distinguish between viral and detrital particles (Marie et al. 1999a). By using different dyes the fluorescent signal strength can be doubled (Chen et al. 2001). Furthermore, the signal is more stabile and FCM allows counting of more samples per unit time compared to EM and TEM. Virus-sized particles are close to the detection limit of the appara-tus and the flow rate in the machine has to be stable to give consistent and reliable results. Still, the method has great potential and can be used as a routine method for viral enumeration in most aquatic environments. How-ever for some types of natural samples further development is needed (e.g. lakes with a high humic content, unpublished data).

Figure 4. Flow cytometry of an envi-ronmental sample (Lake Klocktjärn) showing 3 different viral subpopula-tions and different bacterial popula-tions (Paper III).

Transmission electron microscopy (TEM) The electron microscope gives the opportunity to directly study infected cells and this was used in Paper II. The measure obtained from these studies is the frequency of visibly infected cells (FVIC) and the number of viruses within the bacteria corresponds to burst size (BS). To study viral infection in bacteria the direct-centrifugation method can be used, but to see through more dense plankton (e.g. algae) a thin-sectioning method has to be used.

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By using a normal centrifuge with a swing out rotor, bacteria in a water column is directly centrifuged onto an electron microscope grid (Bratbak et al. 1993). The grid is then counterstained and the bacteria are examined in the electron microscope. The basic idea is to see through the bacteria and distinguish the denser viral particles inside the bacteria, which then can be counted (Figure 5). One problem with this method is that particles can block the view (of the bacteria) giving an underestimation of the true numbers of infected cells.

Figure 5. Infected bacterial cell visualized through a transmission electron micros-copy picture. Dense regions within the bacterial cell are viral particles, the total num-ber within each cell is known as the burst size (here ~ 40).

The viral infection is visible by TEM only in the last stage of the infection cycle. Therefore, the number of visibly infected bacteria (FVIC) must be related to the total number of infected cells (FIC). In this thesis we use a model by Binder (1999) to calculate FVIC, although different conversion factors have been used (i.e. Proctor et al. (1993) and Weinbauer et al. (2002b)). The strict definition model was developed by Binder (1999) as-suming that infected and uninfected bacterial cells are grazed at the same rate and that the community is at a steady-state.

FIC = 7.1 FVIC - 22.5 FVIC2

The total number of infected cells (FIC) can then be used to estimate the impact on the whole bacterial community by calculating how much of the bacterial mortality is due to viral infection and lysis. In this thesis we use the term viral induced bacterial mortality (VBM) defined as follows (Binder 1999):

VBM = (FIC+0.6 FIC2)/(1-1.2 FIC)

Pulsed-field gel electrophoresis (PFGE) In Paper III and IV we use pulsed-field gel electrophoresis to examine the viral community composition. The diversity of viruses in aquatic systems was earlier examined by electron microscopy (morphology). This estimate is coarse and most probably underestimates the diversity. To be able to look at viruses and measure differences between individual viruses the high magni-

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fication (~25 000x) in an electron microscope (Proctor 1997) has to be used. However, this method can only be used to obtain a measure of visual differ-ence between viruses (e.g. size, tail length) and hence the genetic difference can not be fully assessed.

The development of molecular methods has made it possible to more pre-cisely identify the members of a viral community. The challenge with mo-lecular studies of aquatic viruses is that there is no common gene sequence that exist in all viral types, which is the case for bacteria (Fuhrman et al. 2002b; Steward 2001). Hence, specific primers would have to be developed for different types of viruses and, thus, no general surveys can be done.

Pulsed-field gel electrophoresis is a general fingerprinting approach that gives a measure of the composition of viruses having different length of the genome (number of base pairs). Hence, it does not measure individual viral species diversity but instead the size distribution of components of the viral community.

Water samples are pre-filtered to remove bacteria and viruses are concen-trated by ultrafiltration. Thereafter the viral capsids are destabilized to re-lease the viral DNA (Steward 2001; Wommack and Colwell 2000). The in-tact viral genomes are separated in an agarose gel using PFGE with different pulses. Longer pulses will give higher resolution for higher molecular weight material but too long pulses will cause loss of resolution for the low molecu-lar weight material (Steward 2001).

Figure 6. Pulsed-field gel electrophoresis (PFGE), left panel, showing six samples from Lake Erken in 2004 (se Paper III for details) and a standard (far left). In the right panel the relative abundance (right) of one of the lanes is displayed.

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The gel is stained with a fluorescent DNA dye (Fuhrman et al. 2002b; Wommack and Colwell 2000). Thereafter the gel is digitally scanned using a fluoroimager. The individual bands are then compared to migration rate and fluorescence intensity of DNA standards to get a measurement of molecular weight and the number of identical genome copies in each band and molecu-lar weight size range (Steward 2001). Because there is no amplification step involved in PFGE the intensity of the bands is directly related to the relative abundance (Figure 6) of differently sized genomes (Steward 2001). Different gels can be compared to give a measure of the similarity between different samples (Wommack and Colwell 2000). One possible source of error associ-ated with this method and specifically with the collection of DNA from the environment is the introduction of non viral DNA into the samples. How-ever, uncoated DNA (viral DNA is coated with protein) would probably rapidly disappear from the environment due to decay by sunlight (Noble and Fuhrman 1997).

Terminal restriction-fragment length polymorphism (t-RFLP) In Paper III we used terminal restriction-fragment length polymorphism (t-RFLP) to estimate the bacterial community composition. T-RFLP is a method that uses the 16S rRNA gene from bacteria in environmental sam-ples. DNA is extracted from filters and then amplified using PCR amplifica-tion. The resulting fluorescently labelled products are digested with three different restriction enzymes BstU I, Hinf I and Msp I. The resulting termi-nal restriction fragments are separated and detected by a capillary sequencer. t-RFLP peaks are determined including fragments with lengths of 50-500 bp with an initial peak detection threshold of 80 RFU (relative fluorescent units).

Peaks less than 0.5 bases apart from each other are merged. As applying different thresholds when distinguishing t-RFLP-peaks from background noise can give quite different results (Abdo et al. 2006; Dunbar et al. 2001; Osborne et al. 2006) we apply two different thresholds to the t-RFLP data. The data set is either subjected to a second peak detection threshold of 0.5% of the total lane fluorescence intensity or not (0%). Only peaks present in both samples are included in the final t-RFLP-data sets. The three different datasets obtained from the three different restriction enzymes are stacked to form one dataset used for statistical analysis. Our data set might have been skewed by the choice of restriction enzymes, such that some taxa were dis-criminated by all enzymes, while others may have been targeted in only one or two data sets (e.g. Engebretson and Moyer 2003). The biases inherent in the use of fingerprinting techniques, such as t-RFLP, to analyse bacterial community composition are extensively described elsewhere (e.g. Forney et al. 2004; Fuhrman et al. 2002b; Hewson et al. 2006). In order to minimize

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quantification biases introduced by the PCR-step, only the presence-absence data are used in the analysis.

Multivariate statistics Multivariate statistics is becoming increasingly used in microbial ecology, however mostly for exploratory purposes (e.g. Multidimensional scaling , MDS) and rarely with hypothesis driven techniques as redundancy analysis (RDA), canonical correspondence analysis (CCA) and mantel tests (Ramette 2007). In this thesis we applied both explanatory and hypothesis driven techniques.

Multidimensional scaling (MDS) was used in Paper III & IV to detect prominent patterns that could explain the observed similarities or dissimilari-ties (distances) among samples (Ramette 2007). MDS uses an algorithm evaluating different configurations with the goal of maximizing the goodness of fit. How well (or poorly) a particular configuration reproduces the ob-served distance matrix (i.e. goodness of fit) can be interpreted by means of the stress value. The lower the stress value the better the goodness of fit. The number of dimensions applied in the analysis is determined as to obtain a d^ stress value lower than 0.15. Basically, the closer two samples are, the more similar, and the opposite, two distant samples are more dissimilar.

Partial Least Squares Regression (PLS) is used in Paper II to check which environmental variables are important in predicting response variables (VA, FVIC, VA, Burst size). All variables are checked for normal distribution and log(1+x) transformed to receive normal distribution. All models are con-structed with the autofit function (of Simca-P 9.0. software) in order to ob-tain the highest predictive (Q2) value. A Q2 value above 0.4 is considered as good, but irrelevant parameters and outliers will lower the Q2 value (Eriksson et al. 2001). Effects for a large number of predictors of any type on a large number of dependent (response) variables can be analyzed with PLS. To determine the importance of X variables in predicting Y variables the variable influence on projection (VIP) parameter is used. A VIP value over 1.0 is considered to be highly influential whereas a lower value is con-sidered to be of less influence (Eriksson et al. 2001).

To examine which environmental factors are most significant in explain-ing the variation in viral and bacterial community composition direct gradi-ent analysis methods are used, either Redundancy Analysis (RDA) or Ca-nonical Correspondence Analysis (CCA). The choice of method is based upon a pretest with a Detrended Correspondence Analysis (DCA) to exam-ine if a unimodal or linear model would fit the data best. In DCA the length of the resulting first ordination axis is considered. If the length of the axis is

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Redundancy analysis (RDA) is used to determine which variables are im-portant in explaining variation in viral and bacterial community composition (Paper III). Canonical Correspondence Analysis (CCA) is used in Paper IV to explore the variables important in predicting changes in viral community composition following the procedure by Blanchet et al. (2008).

The Mantel test is used in Paper III and IV to test if bacterial species and viral types datasets (community composition) are significantly related (Ramette 2007). The test is used either in comparing viral and bacterial community composition or viral – viral community composition. The Mantel test calculates the correlation coefficient between corresponding positions in similarity or distance matrices and then assesses the significance using per-mutations.

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Summary of the papers and conclusions

To examine the role and importance of aquatic viruses in general and bacte-riophages in particular in freshwater systems we conducted four experiments (Papers I & IV) and three field studies (Papers II, III & IV).

In all the papers we have focused our research on bacteriophages and the interactions with their host – bacteria. However, several of the methods used do not distinguish between bacteriophages and other types of viruses (e.g. cyanophages and algal viruses). Hence, although the main focus is on bacte-riophages, some of the data and analyses presented in this thesis may more generally reflect aquatic viruses.

Link between bacteria and bacteriophages (Paper I) In two experiments we investigated in what way aquatic bacteriophages re-sponded to phosphorus and nitrogen additions. We used batch cultures of water collected from the mesotrophic Lake Erken (Sweden) in late fall. The responses were examined by following the changes in the abundance of vi-ruses and bacteria using epifluorescence microscopy (Noble and Fuhrman 1998). Furthermore, the bacterial abundance was divided into nucleoid-containing bacterial cells and non-nucleoid-containing bacterial cells (Choi et al. 2003; Zweifel and Hagström 1995). Possible connections between the abundance of non-nucleoid-containing bacterial cells and viral abundance were also examined.

The main difference between the two experiments was that the cultures were pre-incubated into either phosphorus-limitation or phosphorus- and nitrogen-limitation. In both experiments nutrient addition resulted in in-creases in viral abundance and non-nucleoid-containing bacterial cells. In the phosphorus and nitrogen limited cultures, in the cultures receiving nitrogen additions, increases in viral abundance were accompanied by increases in the abundance of both bacteria and non-nucleoid-containing cells. In contrast, phosphorus addition resulted in increases in viral abundance and non-nucleoid-containing bacterial cells, but in no increase in bacterial abundance. (Figure 7).

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Hence, the following conclusions can be drawn: 1. Viral infection and subsequent lysis of the host cell can partly ex-

plain the formation of non-nucleoid-containing bacteria. 2. Viral abundances can increase due to increases in phosphorus con-

centrations without any net increase in bacterial abundance.

Figure 7. Changes in the abundance of viruses (VA); bacteria (BA); nucleoid-containing bac-teria (NuCCA); and non-nucleoid-containing bac-teria (non-NuCCA) in response to nutrient addi-tions in the N/P exp. at time 1 (from Papr I).

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Gradients in trophic status and humic content (Paper II) The goal of the field study was to evaluate which lake characters favour viral induced bacterial mortality as opposed to grazer induced mortality. To do this we investigated the epi- and hypolimnion of 21 lakes differing in trophic status and humic content. Samples were obtained for viral (Noble and Fuhr-man 1998) and bacterial abundance (Hobbie et al. 1977), the frequency of visibly infected bacterial cells, flagellate grazer abundance (Jansson et al. 1996; Olrik et al. 1998) and water chemistry.

Potential grazing of bacteria was calculated using flagellate and ciliate abundance and clearance rates. Three different clearance rates were obtained by taking the average and the mean of both minimum and maximum pub-lished clearance rates for flagellates (Bergström et al. 2000; Carrias et al. 1996; Cleven and Weisse 2001; Comte et al. 2006; Domaizon et al. 2003; Jonsson et al. 2001; Kisand and Zingel 2000; Tranvik 1989; Vrede et al. 1999) and likewise for ciliates (Carrias et al. 1996; Comte et al. 2006; Kisand and Zingel 2000; Tranvik 1989). From the frequency of visibly in-fected bacterial cells and flagellate abundance the potential importance of viral induced bacterial mortality (VBM) (Binder 1999; Weinbauer et al. 2002b) in relation to grazer induced bacterial mortality (GBM) was calcu-lated.

Partial Least Squares Regression analysis showed that viral abundance, as well as the ratio between viral abundance and bacterial abundance, is posi-tively correlated to inorganic nutrients, dissolved organic carbon, chloro-phyll a and bacterial production. In 25 out of the 38 samples grazer induced bacterial mortality appeared to be dominating over viral induced bacterial mortality (Figure 8. ratio < 1). The statistical model moreover revealed that the frequency of visibly infected bacterial cells decreased with increasing trophic status. Further, grazing is positively correlated with increasing tro-phic status and decreasing humic content.

Table 1. Differences between viral induced bacterial mortality (VBM) and either: average; minimum; or maximum estimates of potential grazer induced bacterial mortality (PGBM) in the epilimnion and hypolimnion (p value of Wilcox matched-pairs test test). n.s : not significant. Data set PGBMaverage PGBMmin PGBMmax

Epilimnion

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factors in hypolimnion (Table 1). Also, the ratio between potential viral in-duced bacterial mortality and potential grazer induced bacterial mortality is significantly higher in hypolimnion. Consequently, even though potential viral induced bacterial mortality is not significantly different between epilimnion and hypolimnion, viral infection appeared relatively more impor-tant, as compared to grazing, in hypolimnion since grazers are fewer in hypolimnion.

The theoretical clearance rates used for calculation of grazing in this study may result in an inaccurate estimation of grazer induced bacterial mor-tality under certain conditions. Also, the limit, of 6 distinguishable viral par-ticles within a bacterial cell, for calculating the frequency of infected cells, may underestimate viral induced bacterial mortality. Hence, the exact num-bers of the proportions between grazer induced bacterial mortality and viral induced bacterial mortality could not be determined. Nonetheless, we con-clude that the trends should be representative and can serve as a good meas-ure of under which conditions the relative impact of viral induced mortality is greatest.

0

1

2

3

1 10 100 1000

TP (μg L-1)

DO

C-s

peci

fic a

bs. (

L m

-1 m

g-1 )

Hypolimnion

Epilimnion

Figure 8. Bubble plot for the VBM/GBM ratio in relation to Tot-P and DOC-specific absorbance. The axes are log scaled. Dark grey circles represent epilimnion and light grey circles hypolimnion. Symbol area represents the VBM/GBM ratio. The area inside the circles in the legend represents a VBM/GBM ratio of one (from Paper II)

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Hence, the following conclusions can be drawn: 3. Generally, a larger part of bacterial production in lakes appeared

to be grazed by flagellates than lysed by viruses. 4. A larger fraction of the total bacterial mortality could be attributed

to viruses in hypolimnion compared to in epilimnion. 5. The largest relative impact of viruses on bacterial mortality is in

humic lakes, which had a relatively high frequency of visibly in-fected bacterial cells, but low flagellate abundance (Figure 7).

Temporal variation in viral community composition (Paper III). This paper investigates which factors could potentially regulate or influence viral community composition in freshwater aquatic systems.

The sampled viral communities composed of viruses with both smaller and larger genomes. Both the larger and smaller genome sized viruses dis-played a seasonal pattern in community composition. The similar communi-ties found in July 2004 and 2005 in both Lakes Erken and Klocktjärn suggest that the seasonal pattern we observed might be recurring (Figure 9). Bacte-rial community composition in Lake Erken also displayed a seasonal pattern with a high similarity between samples taken a year apart. Further, Lake Klocktjärn differed from the other two lakes in community composition of both bacteria and viruses.

Our hypothesis was that the community composition of viruses follows that of their hosts, i.e. that the PFGE pattern from the community of viruses with genomes 100 kb, since many of these viruses may have been specific for certain phytoplankton species (Steward et al. 2000). If, in addition, the composition of the DOC pool changed as a conse-

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quence of changes in the phytoplankton community, different bacteria could have been selected at different times of the year, thus causing simultaneous changes in bacterial community composition (Kritzberg et al. 2006).

Bacterial community composition also varied over the season and among lakes. The most important factors co-varying with bacterial community composition, as determined by multivariate analysis, were temperature and DOC concentration.

The following conclusions can be drawn from this study: 6. Viruses displayed a seasonal pattern in community composition,

and the similar communities found in July 2004 and 2005 in both Lakes Erken and Klocktjärn suggest that the seasonal pattern we observed might be recurring.

7. Community composition of bacteria and viruses were weakly cou-pled in the different aquatic systems tested.

8. The most important factors for predicting viral community com-position are temperature and concentrations of dissolved organic carbon (DOC), total phosphorus and soluble reactive phosphorus.

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Figure 9. Multidimensional scaling (MDS) of viral community composition as de-termined by PFGE based on Jaccard’ similarity index: (a and b) low setting (stress value = 0.120) and (c and d) high setting (stress value = 0.090). The filled (black) symbol indicates the first sampling date for the respective lake and filled (grey) symbols indicates samples taken days apart (from Paper III).

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Effects of lysogeny on viral community composition (Paper IV) Our understanding of changes in viral community composition and the fac-tors influencing the composition of viral communities in lakes over time is far from complete (but see previous chapter).

As described in the introduction, whether the virus enters the acute life strategy (lytic) or a persistent life strategy (chronic, lysogenic) should be of great importance. As viruses with a lysogenic life strategy can be induced to a lytic life strategy they must be able to sense cues to ensure a high probabil-ity of transmission (Villarreal 2005). It has been suggested that the environ-mental cues that are important in this context in marine environments in-clude UV radiation, temperature, chemicals and nutrients, especially phos-phate (Jiang and Paul 1998; Wilson and Mann 1997).

In a wide range of freshwater environments the percentage of natural bac-terial communities containing prophages (LB) can vary between 0.1 – 62.5, with an average value of 17 % (Anesio et al. 2004; Bettarel et al. 2006; Co-lombet et al. 2006; Lisle and Priscu 2004; Säwström et al. 2008; Tapper and Hicks 1998). Using isolates, 50% of the freshwater heterotrophic bacterial strains were induced by mitomycin C and 84% of the cyanobacterial strains tested (Dillon and Parry 2008). Further, the LB is negatively correlated to the frequency of infected cells (Colombet et al. 2006) and LB is not corre-lated to DOC concentrations in freshwaters (Anesio et al. 2004).

In marine environments, where the most extensive studies performed so far have been done, an average value of 12 (+-7) % of the natural bacterial community containing inducible prophages have been found (Cochran and Paul 1998; Jiang and Paul 1996; Weinbauer et al. 2003a; Weinbauer et al. 2002a; Williamson et al. 2002). Using isolates, again the figure is much higher with an average of 46 % of bacterial strains lytic phase is induced with mitomycin C (Jiang and Paul 1994; Jiang and Paul 1998; Stopar et al. 2004). Also cyanobacterial strains lytic phase is induced with mitomycin C (Mcdaniel et al. 2006). In some cases prophage induction does not seem to be stimulated by inorganic and organic nutrient additions (Hewson et al. 2006), whereas it is also reported that prophage induction may occasionally be phosphate limited or respond to increases in phosphate concentration (Williamson et al. 2002). However, using single phage-host systems it was shown that conditions that stimulated growth resulted in greater lytic phage production, whereas slow growth favoured lysogeny (Williamson and Paul 2006). Additionally, LB has been found to be negatively correlated to viral and bacterial abundance and bacterial production (Weinbauer et al. 2003a) and lysogeny in cyanobacteria is favoured at low host growth (Mcdaniel and Paul 2005)

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Figure 10. Results from the spring and summer experiments for viral abundance (VA), bacterial abundance (BA), lysogenic bacteria (LB), bacterial production (BP) and nutrient concentrations (PO4-P and NH4-N). Data are mean values of the dupli-cate treatments from the end of experiment; error bars denote standard deviation (from Paper IV).

If the large part of the bacterial community containing prophages (lysogene-gic life strategy) were to be induced to shift into lytic viral production, it could possibly alter viral community composition. Additionally, it has been shown that prophages (temperate viruses) induced from bacterioplankton isolates can be detected using PFGE (Leitet et al. 2006). The effect of ly-sogens on viral diversity was recently highlighted as one of the major open questions in freshwater aquatic ecology (Middelboe et al. 2008).

To examine the role of lysogenic life strategy in freshwater viruses, we conducted several experiments and a field study in Lake Erken to identify; how the viral community composition changes over the ice-free season, whether these changes can be induced by nutrient additions (specifically N and P), and whether these seasonal patterns can partly be explained by in-

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duction of lysogenic bacteria and the subsequent production of temperate phages.

The following conclusions can be drawn from this study: 9. The viral community composition changed over the season and

temperate phages could be detected by incubations with mitomy-cin C (see Figure 11). A large fraction of the OTUs detected ap-peared to be temperate phages (19 %).

10. The most important environmental factor co-varying with viral community composition, as determined by multivariate analysis, was the concentration of Tot-P.

11. Addition of PO4-P can induce temperate phages to become lytic (see Figure 10), and cause subsequent changes in community structure that are as large as changes observed in Lake Erken on biweekly to monthly scales or in incubations where mitomycin C is used to induce prophages.

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Figure 11. Schematic representation of results from pulsed-field gel electrophoresis (PFGE) of samples from in Lake Erken taken at different dates. The samples are presented in panels as follows: a) field samples; b) Induced genome size samples i.e. bands showing a positive change when incubated with mitomycin C compared to control. Each dot represents one band on the gel and areas inside the circles corre-spond to the abundance of that particular genome size (log scale). Grey circles area corresponds to: 1, 5, 10 and 100 x106 viruses mL-1. (From Paper IV)

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Overall conclusions and perspectives The conclusions drawn in this thesis are as follows: Viral infection and sub-sequent lysis of the host cell can partly explain the formation of non-nucleoid-containing bacteria. Virus can respond to increases in phosphorus concentrations without any net increase in bacterial abundance.

Generally, a larger part of bacterial production in lakes appeared to be grazed by flagellates than lysed by viruses. A larger fraction of the total bac-terial mortality could be attributed to viruses in hypolimnion compared to in epilimnion. The largest impact of viruses on bacterial production may be in humic lakes, which have a relatively high frequency of visibly infected bac-terial cells, but low flagellate abundance (Figure 8).

Community composition of bacteria and viruses are only weakly coupled in our studied systems. The most important factors for predicting viral com-munity composition are temperature and concentrations of dissolved organic carbon (DOC), total phosphorus and soluble reactive phosphorus.

The viral community composition changes over the season and temperate phages can be detected by incubations with mitomycin C. A large fraction of the OTUs detected appeared to be temperate phages (19 %). The most im-portant environmental factor co-varying with viral community composition, as determined by multivariate analysis, is concentrations of Tot-P. The PO4-P additions in experiments induced temperate phages to enter a lytic phase, which resulted in changes in viral community structure that are as large as changes observed in Lake Erken on biweekly to monthly scales.

Putting the results from the four papers together a few overarching con-clusions can be made. There is a consistency between the papers that phos-phorus is important for the regulation of viral infection and production and possibly community composition. Further, in both the field and in confined culture, viruses have a large impact on bacterial communities especially through infection and lysis but also through effects on bacterial community composition. Hence, when examining freshwater microbial carbon flow, the viral component has to be included. Also, we show that viral abundance and the virus to bacteria ratio are not positively correlated to the frequency of infected cells (Figure 12). Hence, viral abundance alone is not a good meas-ure of what impact viruses have on bacterial communities at a specific time. To summarize, bacteriophages, as a bacterial mortality factor, are important in freshwater microbial food webs and phosphorus supply has a potential central function in the regulation of the importance of bacteriophages and their diversity.

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Figure 12. Viral abundance (VA) and frequency of visibly infected cells (FVIC) in epilimnion of 21 lakes, sampled in July and August 2003 (data from Paper II)

In this thesis we examine the response and regulation of communities of virus and bacteria. The focus in the three first papers is on the lytic pathway of viral infection, but it is impossible to say if the infected bacterial cells detected in the field studies (Paper II & III) are a result of lytic viruses or induced lysogenic viruses. Neither is it possible to say that the nutrient addi-tions in the experiments (Paper I) did not induce lysogenic viruses as in the P-treatment in the other experiments (Paper IV). Hence, there is always an element of uncertainty when considering viral production and replication, i.e. changes in viral abundance or the frequency of infected cells. Therefore, we addressed the following question in paper IV: � How common lysogeny is in temperate lakes?; � What environmental cues induce the switch from a lysogenic life strategy

to a lytic life strategy?; and � If induction of a lytic life strategy affects viral community structure? The answer seems to be that lysogeny is widespread in lakes and that the temperate phages can be induced to switch life strategy. In addition, these switches can be detected in viral community composition and hence, can be said to affect the overall diversity of the viral community.

Some initial steps have been taken to examine the diversity of viruses in freshwater systems in this thesis. However, many areas of viral aquatic ecol-ogy are still not thoroughly examined. Even though aquatic virology is clearly an expanding field of research with advances at many fronts (Raven

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2006) most of this research has been, and continues to be, performed in ma-rine environments. A recent compilation of the unaddressed or only margin-ally research areas in freshwater viral ecology listed the following: a) com-plex interactions among organic matter and diverse groups of bacteria, vi-ruses and higher trophic levels in aquatic food webs; b) extent and ecological consequences of viral genetic diversity; c) metagenomic studies; d) influence of viral activity on host diversity (e.g. by selective mortality, lysogeniza-tion); and e) influence of viral activity on biogeochemical cycles (Middelboe et al. 2008). Further, the authors concluded about the research field freshwa-ter viral ecology “..we are still far from a deep understanding of the mecha-nisms underlying the influence of viruses on microbial diversification and biogeochemical processes.”. From that perspective it is inspirational that this thesis has, at least partly and most certainly incompletely, but nevertheless, addressed some, if not most, of the issues raised by the authors.

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Summary in Swedish

Bakteriefagers betydelse och diversitet i sjöar Virus är den vanligaste och antagligen den mest artrika gruppen på vår jord, och de infekterar alla andra typer av organismer, t.ex. bakterier, alger, fåglar och däggdjur. I akvatiska miljöer, som sjöar, hav och floder, finns det väldigt många virus. Generellt brukar man säga att det finns runt tio miljoner virus per milliliter vatten i dessa miljöer. Man tror att den största delen av dessa virus är bakteriefager, alltså virus som infekterar bakterier. En anledning till att man tror detta är att bakterier, näst virus, är den vanligaste organismgrup-pen. Virus har ingen möjlighet att förflytta sig själva och flyter omkring med vattenströmmar. Alltså är det troligast att ett virus stöter på en bakterie när den flyter omkring i vattnet. I en kallsup (1 ml) får du alltså i dig upp till tio miljoner virus och en miljon bakterier, detta kan ju dock variera något bero-ende på kallsupens storlek.

En virusinfektion hos en bakterie kan antingen vara akut eller dold. Den akuta infektionen (lytisk) gör så att bakterien börjar massproducera nya virus tills den sprängs. Vid den dolda infektionen (lysogen) inkorporeras viruset i bakteriens arvsmassa och följer med då bakerien delar sig till nya generatio-ner av bakterier. Den lysogena infektionen kan sen bli lytisk om värden ut-sätts för olika typer av variationer i miljön (t.ex. ultraviolett ljus). Man har också sett att variationer i koncentrationen av olika typer av näringsämnen (speciellt fosfor) kan vara en viktig faktor för att omvandla den lysogena infektionen till en lytisk infektion.

Kol och näringsämnen, såsom kväve och fosfor, är tillsammans med sol-ljus grunden för att det finns liv på vår planet. I akvatiska system transporte-ras kol och näringsämnen från växtplankton och bakterier uppåt till högre trofiska nivåer (vattenloppor, småfiskar till rovfiskar). Denna näringstrans-port till högre trofiska nivåer påverkas av omsättnings-hastigheten och till-gängligheten av näring i vävens bas. Att virus är viktiga för denna närings-omsättning har man först på senare år börjat undersöka. Virus infekterar bakterier och större organismer och förvandlar därigenom dessa till mindre delar, också kallade lysat. Resterna av de lyserade organismerna (lysatet) används av bakterier och man kan alltså säga att en viss mängd av det orga-niska kolet och näringsämnena transporteras i en cirkel vid näringsvävens

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botten. Summan av detta blir att, med ett högre antal infekterade bakterier, så ökar denna cirkulation och mindre näring når upp till de högre trofinivåerna. Det övergripande målet med denna avhandling var att undersöka betydelsen och effekten av virus infektioner av bakterier och diversiteten hos virussam-hällen i olika typer av sjöar. De mer specifika målen var följande:

� Undersöka om det finns en länk mellan bakterier utan synlig cell-kärna och bakteriefager, vidare om näringstillgänglighet har en ef-fekt på produktionen av virus och bakterier utan synlig cellkärna (Paper I).

� Bedöma betydelsen av sjöars trofiska nivå för den relativa betydel-sen av två mortalitetsfaktorer hos bakterier, nämligen virusinduce-rad eller betarinducerad mortalitet (Paper II).

� Bestämma om virusdynamiken (infektionsfrekvens och den relativa betydelsen jämfört med betning) är annorlunda i humösa sjöar jäm-fört med mindre humösa sjöar (Paper II).

� Följa rums- och tidsvariationer i artsammansättningen hos virus-samhällen och utvärdera den möjliga samvariationen med bakterie-samhällen (Paper III).

� Undersöka hur vanliga de lysogena infektioner är i sjöar och hur dessa virus kan induceras att bli lytiska, vidare också om detta kan ha en effekt på artsammansättningen hos virussamhällen (Paper IV).

Då vissa bakterier har visat sig vara tomma skal spekulerade vi i att dessa skulle kunna vara resterna av en virusinfektion (Paper I). För att undersöka detta nogrannare så samlade vi sjövatten och tillsatte sedan antingen fosfor eller kväve för att se hur många av dessa tomma skal som producerades och hur många nya virus detta gav upphov till.

Det visade sig att det var när vi tillsatte fosfor som vi fick den största ef-fekten. Andelen tomma skal ökade och mängden nyproducerade virus var också högre än då vi tillsatte kväve. Slutsaten är att det finns ett samband mellan virusproduktion och dessa tomma skal och att fosfor kan spela en viktig roll i virusproduktionen. I vissa typer av sjöar står bakterier för en större del av biomasseproduktionen än i andra sjöar (Paper II). I exempelvis humösa, brunfärgade, och närings-fattiga sjöar är de väldigt viktiga ur denna synvinkel. En viktig undersökning är därför att se om andelen synligt infekterade bakterier skiljer sig mellan olika typer av sjöar.

I denna undersökning kom vi fram till att andelen infekterade bakterier är högre i näringsfattiga sjöar, med låg fosforhalt, än i näringsrika sjöar samti-digt som det omvända sambandet gäller för antalet virus. Det innebär att färre virus ger upphov till fler infektioner i näringsfattiga miljöer. Andelen

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infekterade bakterier är också ungefär lika högt i humösa sjöar som i de när-ingsfattiga sjöarna.

För att kunna dra slutsatser om vilken effekt den varierande andelen in-fekterade bakterier skulle kunna tänkas ha så uppskattade vi också andelen bakterier som blev konsumerade av större organismer som flagellater och ciliater. Då andelen bakterier som blev konsumerade av större organismer var högre i näringsfattiga (oligotrofa) miljöer än i humösa miljöer är vår slutsats att den största effekten av virusinfektioner på näringsflöden troligtvis är i de humösa systemen. En annan viktig komponent för att studera effekten av virus på den mikrobi-ella näringsväven är att se vilka virus som finns i olika typer av sjöar (Paper III). Det är också intressant att se hur sammansättningen av virustyper för-ändras under året och om det är samma sorts virus som infekterar samma bakterie år efter år. För bakterier har man sett att fosfor är väldigt viktigt för tillväxten och produktionen och det verkar inte otänkbart att även virus till stor del styrs av tillgången på fosfor. Kanske är det så att fosfor styr vilka virus som infekterar vilken värd.

I denna studie undersökte vi artsammansättningen hos virus i olika sjöar under ett år. Det egentliga måttet vi använde var sammansättningen av ge-nomstorlekar, då man tänker sig att olika virus har olika stora genom. Vi undersökte också hur artsammansättningen hos virus och bakterier samvarie-rade.

Vi kom fram till att artsammanssättningen varierar i sjöarna under året och att skillnaderna mellan sjöar också är stor. En intressant iakttagelse är att förändringen i sammansättningen kan vara lika stor på ett par dagar som det kan vara på ett par månader. Vi hittar dock inget starkt samband mellan vi-rus- och bakteriesammansättningen. I och med att viruset följer med värden över generationer i den lysogena infektionen kan det ju till slut bli ganska många bakterier som är infekterade av detta virus (Paper IV). Man kan tänka sig att samma typ av virus kan fås att övergå i den lytiska infektionen om den utsätts för en variation i sin miljö. I dessa fall borde man kunna få situationer med massdöd av en den virusspe-cifika värdarten vid dessa plötsliga förändringar i miljön, som till exempel vid vår- och höstcirculationen i sjöar då temperatur, syrgas och koncentra-tioner av näringsämnen snabbt förändras.

I miljöer som är föränderliga, t.ex. sjöar som är isbelagda halva året, bor-de det vara en fördel att kunna övervintra som en lysogen infektion och sen övergå till en lytisk infektion när livsbetingelserna blir bättre. Detta fenomen studerade vi genom att följa artsammansättningen av virus under den del av året då vår valda sjö inte var isbelagd. Dessutom undersökte vi med tillsats av en kemikalie (mitomycin C), som får de lysogena infektionerna att bli aktiva och producera nya virus (i.e. lytiska), om detta har en effekt på art-

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sammansättningen av virus. Vi fann i experiment att tillsats av näring får de lysogena infektionerna att bli aktiva och vidare i fältstudier att de lysogena infektionerna blir vanligare under året. Vidare observerade vi att fosfor har en viktig roll för den totala artsammansättningen av virus och för att induce-ra de lysogena infektionerna. Den övergripande slutsen är att även fast virus är alldeles för lite studerat i sjöar så kan vi genom upptäckterna i denna avhandling säga att virus är en väldigt viktig komponent i sjöekosystem. Vidare finner vi att fosfor spelar en betydelsefull roll för virus i sjöar, både i samband med virusproduktion och artsammansättning av virus. En ytterligare viktig slutsats är betydelsen av virus inte enbart kan mätas i form av hur vanlig de är (antal virus per voylm-senhet) utan även infektionsfrekvensen borde uppskattas för att få ett rätt-visande mått på dess betydelse i akvatiska mikrobiella system, såsom i sjöar.

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Acknowledgements

I would first like to say – thank you. If you read this you have probably in some way, close or distant, been involved in my quest for knowledge about the extremely important viruses in lakes (to learn more about this: see this thesis).

Especially, I would like to thank my supervisors. Thank you – Katarina, for starting the project and giving me the opportunity to realize it. I also ap-preciate that you have taken the time, even after you went on for bigger and better, to provide comments and direction. Thank you – Lars, for stepping in and trying to steer me in the right direction. It is probably not easy to sud-denly be asked to give expert advice on viral ecology but you did a good job. Finally; thank you – Eva for putting all the pieces together. Thanks also for a nice time in Jämtland and for helping me tremendously in the final stages of the production of this thesis; it could not have been done without you.

Credit goes to all my roommates during the years, Jonas, Eva, Brian & Katie, and Jürg for the great company and occasional escapes from academ-ics. Special recognition to colleagues making the time at the department fly by: Jens, Henrik, Eddie & Irina. Further I thank all my great teaching part-ners and the teaching boss himself Anders B, thanks for letting me stay on the ice at negative 29°C with the students, while you did the hard work - like buying new batteries. Thanks to the people at Lake Erken for great company during the “water days” and sampling trips: Maria, Kurt, Björn and Helena

Appreciation also to everybody else I worked with during my years at the Limnology department, for making these years fun and rewarding – Tobias, Stina, Stefan, Silke, Sebastian, Richard, Rahmat, Peter, Monica, Ingemar, Helmut, Hampus, Gunnel, Göran, Emil, Cristian, Cecilia, Ann-Louise, Anna and Alex.

A big part of the work in this thesis has been spent outside of the lim-nologcal department in Uppsala. Several people deserve particular recogni-tion for helping me during my travels. Thanks to the people in Abisko, Mats, Janne, Grete, and Peter for teaching my how to catch fish the hardest way possible (fly-fishing) and also a piece or two about bacteria and arctic envi-ronments. Special gratitude goes to Anne-Claire and Corina for your wel-come and help during my trips to Texel, Netherlands. Your efforts were highly appreciated and beyond what one would expect and your teachings about PFGE have been successfully implemented.

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My most frequent travel and sampling companion, Ulf -thank you, for let-ting me win in cards and catching the biggest fish; carrying the heaviest buckets and for giving me the laugh of a lifetime when watching you try to escape from a mouse attack still in your sleeping bag. Thanks also to Sophia and Maria for help with field sampling in Jämtland.

Thanks to Janne for helping me with countless of analyses and always explaining the times I actually had to do them myself.

This could not have been done without the understanding and help of all my great friends. Especially I wish to thank those who adjusted their holiday schedule to fit my field samplings – Mattias, Johanna, Andreas, Emma, Mat-tias (twice) and Sofie. Also acknowledged for good company in Uppsala is Philip and I am sure you helped somehow.

My family has to be acknowledged for being perfect at just that, being family. Thanks to the person who invented weekends and evenings and in connection to that and my recent work on completing this thesis I have to say that I will make up all those lost to us, my most precious two – Ellen and Birgitta.

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