Freshwater Biology (2005) 50, 627–645

doi:10.1111/j.1365-2427.2005.01349.x

Estimates of protozoan- and viral-mediated mortality of bacterioplankton in Lake Bourget (France) S T E´ P H A N J A C Q U E T , * I S A B E L L E D O M A I Z O N , † S E´ B A S T I E N P E R S O N N I C , * A N G I A S R I R A M P R A D E E P R A M , ‡ M I K A L H E D A L , § S O L A N G E D U H A M E L * A N D T E´ L E S P H O R E S I M E - N G A N D O ‡ *UMR CARRTEL, Equipe de Microbiologie Aquatique, Station INRA d’Hydrobiologie Lacustre, Thonon cedex, France † UMR CARRTEL, Equipe de Microbiologie Aquatique, Universite´ de Savoie, Le Bourget-du-Lac cedex, France ‡ Laboratoire de Biologie des Protistes, Universite´ Blaise Pascal (Clermont-Ferrand 2), Campus des Ce´zeaux, Aubie`re cedex, France § Laboratory of Microbiology, University of Bergen, Bergen, Norway

SUMMARY 1. We performed three, 1-week in situ experiments in March-April (expt 1), May (expt 2) and August (expt 3) 2003 in order to assess protozoan and virus-induced mortality of heterotrophic bacteria in a French lake. Viral and bacterial abundances were obtained using flow cytometry (FCM) while protozoa were counted using epifluorescence microscopy (EFM). 2. A dilution approach, applied to pretreated grazer-free samples, allowed us to estimate that viral lysis could be responsible for 60% (expt 1), 35% (expt 2) and 52% (expt 3) of daily heterotrophic bacterial mortality. Flagellate (both mixotrophic and heterotrophic) grazing in untreated samples, was responsible for 56% (expt 1), 63% (expt 2) and 18% (expt 3) of daily heterotrophic bacteria removal. 3. These results therefore suggest that both viral lysis and flagellate grazing had a strong impact on bacterial mortality, and this impact varied seasonally. 4. From parallel transmission electron microscopy (TEM) analysis, we found that the burst size (i.e. the number of viruses potentially released per lysed cell) ranged from nine to 25 (expt 1), 10 to 35 (expt 2) and eight to 25 (expt 3). The percentage of infected heterotrophic bacteria was 5.7% (expt 1), 3.4% (expt 2) and 5.7% (expt 3) so that the calculated percentage of bacterial mortality induced by viruses was 6.3% (expt 1), 3.7% (expt 2) and 6.3% (expt 3). 5. It is clear that the dilution-FCM and TEM methods yielded different estimates of viral impact, although both methods revealed an increased impact of viruses during summer. Keywords: bacteria, lake, mortality, protists, viruses

Introduction Over the past 15 years, it has been realised that viruses are an important component of aquatic microbial food webs. They have been shown to be important controlling agents in planktonic community composition, diversity and succession, playing a key role in cell mortality and nutrient cycles (Bergh Correspondence: Ste´phan Jacquet, UMR CARRTEL, Equipe de Microbiologie Aquatique, Station INRA d’Hydrobiologie Lacustre, 74203 Thonon cedex, France. E-mail: [email protected]
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et al., 1989; Suttle, 1994; Maranger & Bird, 1995; Fuhrman, 1999; Wommack & Colwell, 2000; Weinbauer & Rassoulzadegan, 2004). Of additional ecological significance, the ability of aquatic viruses to transfer genetic material has been demonstrated (Chiura, 1997; Clokie et al., 2003). A large majority of aquatic viral ecological studies have been carried out in seawater (see Wommack & Colwell, 2000; Sime-Ngando et al., 2003; Weinbauer, 2004). Some freshwater systems have also been investigated, although less often, and these include rivers (Mathias, Kirschner & Velmirov, 1995; Farnell-Jackson & Ward, 2003), Antarctic lakes (Kepner, Wharton & Suttle, 627

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1998; Laybourn-Parry, Ho¨fer & Sommaruga, 2001), oligotrophic lakes (Klut & Stockner, 1990; Tapper & Hicks, 1998; Hoffer & Sommaruga, 2001; Bettarel et al., 2003b, 2004; Vrede, Stensdotter & Lindstro¨m, 2003), mesotrophic lakes (Hennes & Simon, 1995; Maranger & Bird, 1995; Leff et al., 1999; Wilhelm & Smith, 2000), and eutrophic lakes (Sommaruga et al., 1995; Weinbauer & Ho¨fle, 1998; Fischer & Velimirov, 2002; Bettarel et al., 2003a; Weinbauer et al., 2003; Bettarel et al., 2004), as well as lake sediments (Maranger & Bird, 1996). In France, the three largest natural freshwater ecosystems (Lakes Annecy, Bourget and Geneva) have not yet been investigated from this perspective and only the oligotrophic Lake Pavin and the eutrophic Lake Aydat and Sep Reservoir have received recent interest in France (Bettarel et al., 2003a,b, 2004; Pradeep Ram et al., in press). We used a variation of the dilution technique to assess the virus-induced mortality of heterotrophic bacteria in Lake Bourget. The dilution approach initially introduced by Landry & Hassett (1982), and refined by Landry, Kirshtein & Constantinou (1995), has been used routinely as a field technique to quantify grazing of phytoplankton by microzooplankton and to estimate phytoplankton growth rate (Campbell & Carpenter, 1986; Weisse & Scheffel-Mo¨ser, 1990; Landry, Monger & Selph, 1993; Calbet & Landry, 2004). Briefly, the technique consists of incubations of water samples after dilution at different levels of the original sample to reduce the abundance of algal and/or bacterial predators and thus to render less likely contact and interactions between predator and prey species. The net growth rate of the prey can therefore be expected to be highest in the most diluted fractions. Overall, the method allows an estimate of the grazing impact of zooplankton. To the best of our knowledge, this technique has been used to study the impact of viruses on microorganisms on very few occasions, for both heterotrophic and autotrophic groups. Wilhelm, Brigden & Suttle (2002) studied the ‘rebound’ in virus numbers following dilution in virus free water. Evans et al. (2003) estimated virus-induced mortality in a coastal and marine phytoplankter. Our objective was to assess the impact of viruses on the heterotrophic bacterial communities at different periods of the year in Lake Bourget and then to compare these findings with those obtained in various other European lakes. Parallel grazing experiments were also performed in order to estimate the potential

impact of protozoan predators on the bacterial community and to permit a comparison of bacterial mortality induced by viral lysis and protozoan predation. The grazing data were also compared with those from various other European freshwater ecosystems.

Methods Study site Lake Bourget (4544¢N, 0551¢W, 231 m altitude), on the western edge of the Alps, is the largest natural lake in France. It is an elongated and north–south orientated lake (length, 18 km; width, 3.5 km; area, 44 · 106 m2; volume, 3.5 · 109 m3; maximum depth, 145 m; mean depth, 80 m; residence time, 10 years) and is warm and meromictic. The catchment is about 560 km2, with maximum and average altitudes of 1845 and 700 m, respectively. More details (including a map of the lake) are available in Jacquet et al. (in press).

Assessment of in situ microbial community dynamics In situ dynamics of the microbial community (i.e. viruses, heterotrophic bacteria and picocyanobacteria) were assessed using flow cytometry (FCM) on samples of water from the reference station, known as ‘B’, which is located in the middle and deepest part of the northern basin. This station is more than 1.5 km from each bank and more than 5 and 10 km from the Sierroz and Leysse rivers (the two main freshwater inputs in the lake), respectively. Cell or particle concentrations for the different assemblages were measured at seven different depths between 0 and 50 m (2, 6, 10, 15, 20, 30 and 50 m) and sampled on average every 2 weeks between March and September 2003. In addition, water temperature, transparency, and nitrate and phosphate concentrations were assessed on the same sampling occasions.

Environmental variables A conductivity-temperature-depth measuring device (CTD SBE 19 Seacat profiler, Seabird, SBE, Bellevue, WA, U.S.A.) was used to obtain vertical profiles (from the surface to the bottom) of water temperature, and to make sure that in each of the three experiments we sampled in the mixed surface layer and never below the epilimnion (see below). Nutrient
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Bacterial mortality in Lake Bourget concentrations (total phosphorus, P-PO4, N-NO3, N-NH4 and Si-SiO2) were measured at the Institut National de la Recherche Agronomique (INRA) Hydrobiological Station chemistry laboratory (details available at http://www.thonon-inra-chimie.net/ pages/public/analyses.asp). Water transparency was measured using a Secchi disk, before and after the experiments, and during routine surveys of the lake, and all measurements were performed by the same person. The underwater light intensity was measured using a LI-1400 current meter and data logger combined with a spherical quantum sensor LI-193SA (LI-COR, Lincoln, NE, U.S.A.).

Experimental set-up for estimating the impact of viruses on bacteria Three similar experiments were conducted from 31 March to 4 April (expt 1), 19–23 May (expt 2) and 18– 22 August (expt 3), which correspond to distinct periods in terms of the microbial planktonic dynamics and diversity in surface waters of the lake (Comte et al., in press; Jacquet et al., in press). For each period, an integrated >60-L sample was taken in the 0–10 m surface layer of the second reference station (called A) located in the middle and deepest part (100 m) of the southern basin of the lake, located at a distance >1 km from each bank and 6 km from B. The decision to use water of station A, rather than B, was mainly on the grounds of convenience, as the former was nearer than B, where the incubations were carried out, but also because we demonstrated in a previous study that communities and dynamics were similar between stations A and B (Comte et al., in press). The water sample was first filtered through a series of 200- and 20-lm mesh filters (NYCOM, Buisine, France) and then through a 2-lm filter (Nuclepore, Whatman, San Diego, CA, U.S.A.) twice, which theoretically should eliminate all the bacterivores (i.e. nanoflagellates and ciliates). However, as some flagellates may not have been entirely removed by filtration, phagotrophic protists were counted in less than the 2 lm fraction using a Nikon TE 200 epifluorescence microscopy (EFM). A fraction of this 2-lm filtered water was subjected to tangential ultra-filtration using a mini-Ultrasette with a 100 kDa cut-off membrane (Vivaflow, Vivasciences, Hannover, Germany) in order to eliminate all organisms (including viruses). The purity of the water was checked using both EFM
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and FCM. The two fractions were mixed in order to obtain percentages of the initial 2-lm filtered whole water of ca 20, 40, 70 and 100%. Dilution reduced the background of free viruses by adding virus-free water, thus reducing the amount of contact between viruses and bacteria. A control bottle containing none of the original 2-lm filtered water was also prepared for each experiment, in order to confirm the absence of background contamination. For each level of dilution, duplicates were prepared in acid-washed, water rinsed and autoclaved 250 mL polycarbonate bottles (Nalgene, Bioblock, Illkirch, France). Immediately after preparation, the bottles were attached to the side of a bridge in the eastern part of the lake and then incubated at a depth of 1 m, for 5 days. Population dynamics were monitored during the whole week but the time of incubation for assessing the viral impact was limited to the first 24 h, assuming that there were no nutrient limitation and/or confinement effects during the first day of incubation. All bottles were sampled early each morning, and samples were subsequently prepared for FCM analysis.

Flow cytometry sample analysis We used a FACSCalibur cytometer (Becton Dickinson, Franklin Lakes, NJ, U.S.A.) equipped with an aircooled laser providing 15 mW at 488 nm with the standard filter set-up. One millilitre samples were analysed without adding any fixative or dye to analyse the picocyanobacterial community dynamics and also to check for the absence/presence of eukaryotic autotrophic organism in the