Seasonal Viral Loop Dynamics in Two Large Ultraoligotrophic Antarctic Freshwater Lakes

Microbial Ecology Seasonal Viral Loop Dynamics in Two Large Ultraoligotrophic Antarctic Freshwater Lakes Christin Sa¨wstro¨m1, M. Alexandre Anesio2, Wilhelm Grane´li3 and Johanna Laybourn-Parry4 (1) (2) (3) (4)

Climate Impacts Research Centre (CIRC), Department of Ecology and Environmental Science, Umea˚ University, SE-901 87 Umea˚, Sweden Institute of Biological Sciences, University of Wales, Aberystwyth, SY23 3DA, UK Department of Limnology, Ecology Building, Lund University, Lund, S-223 62, Sweden Institute for the Environment, Physical Sciences and Applied Mathematics, University of Keele, Staffordshire, ST5 5BG, UK

Received: 16 June 2006 / Accepted: 22 June 2006 / Online publication: 31 October 2006

Abstract

Introduction

The effect of viruses on the microbial loop, with particular emphasis on bacteria, was investigated over an annual cycle in 2003–2004 in Lake Druzhby and Crooked Lake, two large ultraoligotrophic freshwater lakes in the Vestfold Hills, Eastern Antarctica. Viral _ abundance ranged from 0.16 to 1.56  109 particles L 1 and bacterial abundances ranged from 0.10 to 0.24  109 _1 cells L , with the lowest bacterial abundances noted in the winter months. Virus-to-bacteria ratios (VBR) were consistently low in both lakes throughout the season, ranging from 1.2 to 8.4. lysogenic bacteria, determined by induction with mitomycin C, were detected on three sampling occasions out of 10 in both lakes. In Lake Druzhby and Crooked Lake, lysogenic bacteria made up between 18% and 73% of the total bacteria population during the lysogenic events. Bacterial production ranged _ _ from 8.2 to 304.9  106 cells L 1 day 1 and lytic viral production_ ranged_ from 47.5 to 718.4  106 viruslike particles L 1 day 1. When only considering primary production, heterotrophic nanoflagellate (HNF) grazing and viral lysis as the major contributors to the DOC pool (i.e., autochthonous sources), we estimated a high contribution from viruses during the winter months when 960% of the carbon supplied to the DOC pool originated from viral lysis. In contrast, during the summer G20% originated from viral lysis. Our study shows that viral process in ultraoligotrophic Antarctic lakes may be of quantitative significance with respect to carbon flow especially during the dark winter period.

In the extremes of Antarctic freshwaters microbial life is pushed to its limits in regards to temperatures, light, and nutrient levels [26]. These freshwater ecosystems have truncated food chains with no fish, few metazoans, and a dominance of microbial plankton (viruses, bacteria, and protozoa) [26, 39]. In these microbially dominated ecosystems, the major pathway of energy and carbon flow is through the microbial loop where the bacteria play a crucial role as they consume and recycle dissolved organic carbon (DOC) [3]. The bacteria are in turn ingested by protistan grazers, primarily HNFs, but also ciliates. Essentially, the bacteria play an intrinsic part, in continental Antarctic lake food webs, as recyclers of the small organic carbon pool that is largely derived from algal photosynthesis as the allochthonous carbon contribution is assumed to be negligible due to lack of terrestrial input [25, 29, 42]. The freshwater lakes of the Eastern Antarctica, Vestfold Hills, have received considerable attention, and previous seasonal studies have focused on the microbial plankton such as bacterial, phytoplankton, and protozoan dynamics [4, 27–29, 31]. None of the studies noted above have included the virioplankton component and at present there is relatively little information on the seasonal changes in viral abundance and activity in Antarctic lakes. Kepner et al. [23] published the first report on viral abundance and productivity in the lakes situated in the McMurdo Dry Valleys, Antarctica. They revealed high viral production rates in these systems and suggested that the ecological role of viruses in Antarctic lakes may be somewhat greater than in temperate and tropical lakes [23]. Laybourn-Parry et al. [30] gave an account of the viral morphology and abundance in lakes situated in the Vestfold Hills, Eastern Antarctica. However, the above

Correspondence to: Christin Sa¨wstro¨m; E-mail: christin.sawstrom@ emg.umu.se DOI: 10.1007/s00248-006-9146-5 &Volume 53, 1–11 (2007)

&*

Springer Science+Business Media, Inc. 2006

1

2

studies only studied the viral component in the lakes during the austral summer. A recent seasonal study was conducted on the saline lakes situated in the Vestfold Hills and revealed high viral abundance and virus-tobacteria ratios (VBR), thus indicating that viruses played a central role in the saline lakes throughout the season [34]. Nevertheless, none of previous studies on viruses in Antarctic lakes have tried to elucidate the seasonal importance of viruses in regards to carbon cycling in these extreme ecosystems. Viral lysis of bacteria can disrupt the flow of energy and carbon within the aquatic ecosystem by increasing the recycling and respiratory loss in the lower parts of the food web [11, 18, 35–37, 54]. This results in a viral loop where viral lysis transforms particulate organic carbon into DOC, thus making it available for incorporation into new bacterial biomass [48, 49, 54]. It has been suggested that as much as 30% of bacterial production (BP) can be channeled through the viral loop in aquatic systems [10]. The recycled DOC is in turn utilized with less efficiency by the bacterial community [36]. Model food webs have shown that viruses can decrease the biomass transfer to higher trophic levels (i.e., zooplankton), in particular in oligotrophic environments where recycling through the microbial loop prevails [37]. Furthermore, viral-induced mortality is thought to be responsible for 5–50% of bacterial mortality and at times can cause similar or higher bacterial mortality than HNF grazing, depending on the system [6, 12, 17, 18, 51]. A recent study [7], on two temperate lakes differing in nutrient status, indicated that viral-induced bacterial mortality was higher in less productive lake ecosystems. Thus, in Antarctic ultraoligotrophic lakes it could be expected that viruses are important players and may also be responsible for a large proportion of bacterial mortality, as these lake ecosystems are extremely unproductive in regards to microbial activity and nutrient levels. Little is known about the factors that regulate the virus–host interactions in continental Antarctic lakes. One may predict that the unfavorable environmental conditions in Antarctic freshwater lakes, such as low nutrient levels and host availability, may stimulate lysogeny resulting in a large proportion of the bacterial population being lysogenic. Lisle and Priscu [32] estimated high percentage lysogenic bacteria in the lakes of McMurdo Dry Valleys, Antarctica. However, they found that lysogens contributed a very small fraction of the total viral production. Viruses have been explained as catalysts that accelerate the transformation of nutrients from particulate to dissolved form, making them readily available for reassimilation into the microbial biomass [44]. In oligotrophic environments, this viral transformation pathway may be essential in relieving nutrient limitations [19, 36, 37]. The freshwater lakes of the Vestfold Hills,

C. SA¨WSTRO¨M

ET AL.:

VIRAL LOOP DYNAMICS

IN

ANTARCTIC LAKES

Antarctica, are essentially unsubsidized ecosystems with an extremely small DOC pool with largely unchangeable concentrations throughout the year (unless there is fecal enrichment from birds or seals) [25]. Thus, these lakes offer unique systems to study virioplankton and their role in carbon cycling, as virtually all carbon is derived from autochthonous sources and the major pathway of energy flow is through the microbial loop. In this study, the virio- and bacterioplankton of two ultraoligotrophic Antarctic freshwater lakes were investigated over an annual cycle. The study included measurements of bacterial and viral abundance, bacterial production, lytic and lysogenic viral production and viral-induced bacterial mortality. We estimated the relevance of the viral loop in these extreme ecosystems to gain a better understanding of the viral impact on carbon cycling in the Antarctic microbially dominated systems. Materials and Methods Study Sites and Sampling. The present study was conducted on the two largest freshwater lakes in the Vestfold Hills, eastern Antarctica (Australian Antarctic Territory): Lake Druzhby (68-350 S, 78-200 E) and Crooked Lake (68-370 S, 78-220 E). The lakes were sampled fortnightly over a 12-month period in 2003–2004. Lake Druzhby consists of three basins and has an area of 7 km2 and was sampled at its point of maximum depth (40 m). Crooked Lake consists of one basin and has an area of 9 km2 and a maximum depth of 160 m, and the depth at the sampling site was approximately 60 m. Both Lake Druzhby and Crooked Lake have been previously investigated in terms of their microbial plankton [4, 20, 27–29, 31]. Access in the summer was by helicopter and in the winter by four-wheeled all-terrain vehicles over the sea ice. Due to logistical constraints, we could not collect samples between February and April 2004. The lakes were sampled by drilling a hole in the ice with a jiffy drill (Feldman Engineering, Sheboygan Falls, WI, USA) and water samples were taken with a Kemmerer bottle from discrete depths [0 m (i.e., immediately under the ice), 3, 5, 8, 10, 15, 20, and 30 m]. Lake Druzhby lost its ice cover briefly during the short summer period, and samples were collected from a boat. Crooked Lake kept its ice cover throughout the season with moats only developing around the edges. Vertical profiles of temperature and oxygen saturation (DOsat %) were recorded by using an YSI 6600 sonde (YSI, Marion, MA, USA) during the autumn, winter, and spring. During the summer period, temperature profiles were measured in the field with a digital thermometer. Conductivity and pH were determined on water samples returned to the laboratory. Water samples (2 L) were collected from each depth in acid-washed

C. SA¨WSTRO¨M

ET AL.:

VIRAL LOOP DYNAMICS

IN

ANTARCTIC LAKES

bottles for bacterial and viral abundance, bacterial production, chlorophyll a analysis, inorganic nutrient, and DOC. Measurements of lysogeny were performed on a monthly basis. Samples were brought back to the research laboratory at the Davis station, Australian Antarctic Territory, within 1 h for further processing as outlined below. Bacterial and Viral Abundance. Subsamples (15 mL) were fixed in 0.02-mm filtered glutaraldehyde (final concentration, 2%) for counts of bacteria and viruslike particles (VLP). Bacterial and viral abundances were determined, within 24 h, by using epifluorescence microscopy according to the Noble and Fuhrman [38] method, except that SYBR gold nucleic acid stain was used instead of SYBR green nucleic acid. Induction Assay for Lysogenic Bacteria. Four replicate water samples (15 mL) were either treated with mitomycin C (a potent mutagen for prophage_induction) (Sigma, St. Louis, MO, USA), (1 mg mL 1) or left untreated (controls) [40]. The samples were incubated in the dark at in situ temperature (2–4-C) for 24 h and then fixed with 0.02-mm filtered glutaraldehyde (final concentration, 2%) and stored at 4-C (storage G24 h) before bacterial and viral abundance were determined using SYBR gold staining as described above. Significance of each induction event was determined by comparison of mitomycin C treatment and control levels of viruses by an independent-samples t test. If there was a statistically significant increase in viral abundance in the mitomycin C treatment relative to the control, then this was an indication of the presence of lysogenic bacteria. The fraction of lysogenic bacteria (FLC) was then calculated as: % FLC = [(Vt – Vc)/Bz]/Bc  100, where Vt is the number of viruses enumerated in the mitomycin C treatment at 24 h and Vc is the number of viruses enumerated in the control sample. Bc is the number of bacteria enumerated in the control sample at 24 h and Bz is the burst size. The value for Bz was derived from transmission electron microscopy observation of Lake Druzhby and Crooked Lake samples (Bz = 4; Sa¨wstro¨m et al., unpublished data, 2005). We also used the average Bz of 26 reviewed by Sa¨wstro¨m et al. (unpublished data) for freshwater environments for comparison to the extremely low burst sizes noted in the study lakes. Bacterial Production. Bacterial production was estimated by the incorporation of [3H] thymidine _1 (84.0 Ci mmol ) into the bacterial biomass by the microcentrifuge method as explained by Kirchman [24]. Triplicate 1.7-mL samples were collected into 2-mL microcentrifuge tubes and [3H]thymidine was added to a final concentration of 70 nM. The appropriate isotope concentration was evaluated by saturation experiments

3

and revealed that saturation occurred at 70 nM (results not shown). Duplicate control samples were inactivated with 90 mL of 100% TCA and thymidine label added. The samples were then incubated for 90 min to allow for the label to be incorporated into the bacterial biomass. After incubation, 90 mL of 100% TCA was added to all samples except for the controls. The tubes were then centrifuged at 16,000 g at 4-C for 10 min; after this the supernatant was aspirated and 1.7 mL ice-cold 5% TCA was added to each tube, vortex mixed, and followed by another centrifugation step. The TCA was aspirated and 1.7 mL of ice-cold 80% ethanol was added, followed by another centrifugation step. The supernatant was aspirated and 1 mL of scintillation cocktail (Ecoscint) was added, then the samples were counted by liquid scintillation in a Beckman LS6500 scintillation counter. A conversion factor of 2  _ 1018 cell mol 1 was applied to the incorporation rates of thymidine into DNA [5]. To convert bacterial cell production into carbon production, a conversion factor of _ 28.5 and 17.5 fg C cell 1 was applied for Druzhby and Crooked, respectively. The conversion factor was derived from applying a volume-specific conversion factor of _ 200 fg C mm 3 [9, 47] to the average bacterial volume estimated for each lake in season 1999–2000 [29]. DOC, Inorganic Nutrients, and Chlorophyll a. Samples (õ 50 mL) for DOC were filtered through GF/F filters (preashed for 12 h at 550-C) and immediately transferred into acid-washed bottles and stored at –20-C until analyzed. DOC was analyzed by the Pt-catalyze d (highsensitivity catalyst) high-temperature combustion method using a Shimadzu TOC-5000 total carbon analyzer equipped with an ASI-5000 auto sampler. Inorganic carbon was removed by purging with CO2-free air for 5 min from acidified samples (pH õ2, HCl). Concentrations of soluble reactive phosphorous (PO4-P) and ammonium (NH4-N) were assayed colorimetrically according to the methods of Mackereth et al. [33]. Chlorophyll a was determined according to the protocol described by Jespersen and Christoffersen [21], except that methanol was used rather than ethanol. Briefly, subsamples were filtered through GF/F (47 mm) filters and stored at –80-C. Filters were extracted in 10 mL of 95% methanol at 4-C overnight, and then analyzed with a 10-AU Turner portable fluorometer at the University of Tasmania. Determination of Viral-Induced Bacterial Mortality and The model of Lytic and Lysogenic Viral Production.

Binder [8] was used to estimate the fraction of bacterial mortality caused by viral lysis (FMVL): FMVL = FVIB/ [g ln(2) (1 _ e _ FVIB)], where g = 1 (the ratio between the latent period and generation time) and ( = 0.186 (the fraction of the latent period during which viral particles are not yet visible), and FVIB represents the fraction of

4

visibly infected bacterial cells, using FVIB values from Sa¨ wstro¨ m et al. (unpublished data). _ Lytic_ viral production (viruslike particles produced L 1 day 1) was then calculated by multiplying the lysed bacterial production by the average burst size (Bz = 4) from each lake (FMVL  BP  Bz) [51]. The contribution of lysogenic viral production (VL%) to the total viral production was estimated by using the equation of Jiang and Paul [22]: VL% = [(SrGLB)/(DrVt)]  100, where Sr is the _prophage spontaneous induction rate _ (virus bacterium 1 G 1), G is the bacterial generation time per day (bacterial abundance divided by bacterial production), L is the fraction of lysogens in the total bacterial population, B is the total_ bacterial abundance _ (L 1), Dr is the viral decay rate (day 1), and Vt is the total _1 viral abundance (L ). We calculated VL% by using previously published data for Sr and Dr. Prophage _ spontaneous induction rate (Sr) was assumed to be 10 2 [1], and the viral decay rate (Dr) was assumed to be _ 0.3 day 1, as estimated for Lake Hoare in the Dry Valleys, Antarctica by Kepner et al. [23]. Statistical Analysis. Statistical analyses were performed in SPSS (version 11.0.0 for Windows). Data were checked for normal distribution by using the Kolmogorov–Smirnov test. Data with nonnormal distribution were transformed with log transformations. A nonparametric two-way ANOVA test (Scheirer–Ray– Hare) was performed on the seasonal ranked data from each lake to check for depth-related differences in the measured parameters throughout the water column and throughout the season. Because no significant vertical difference was found during the study, an average of the sampled depths at each time point was used in further statistical analyses. Seasonal (data was divided into separate seasons: summer—December, January, February; autumn—March, April, May; winter—June, July, August; spring—September, October, November) variation in the measured parameters were analyzed by using one-way ANOVA for normal distributions and the Tukey post hoc test was used for pairwise comparisons; for nonnormal distributions a nonparametric test (Kruskal–Wallis) was used and the Mann–Whitney U test was used for pairwise comparison. Correlation analyses were determined by using two different tests: Pearson product–moment correlation for normal distributions and Spearman rank–order correlation for nonnormal distributions (number of pairs, 144). Lake comparisons were carried out using one-way ANOVA and the nonparametric Kruskal–Wallis test.

Results

Statistical analyses of the depth profiles of Lake Druzhby and Crooked Lake

Physicochemical Parameters.

C. SA¨WSTRO¨M

ET AL.:

VIRAL LOOP DYNAMICS

IN

ANTARCTIC LAKES

showed that there was no stratification of biological elements, and vertical temperature and dissolved oxygen profiles were constant throughout the season (data not shown). The mean water column temperature in the lakes ranged from 0.9 to 4.4-C (Table 1) with Lake Druzhby having significantly higher temperatures than Crooked lake throughout the season (Kruskal–Wallis, P G 0.001). A slight but significant increase in the water temperature was noted in spring in both lakes (Mann– Whitney U test, P G 0.001). The mean conductivity _ reading was 40.7 and 29.0 mS cm 1 in Lake Druzhby and Crooked Lake, respectively (Table 1). pH values were neutral in both lakes and ranged from 6.7 to 7.3 (Table 1). Spectrophotometric analysis conducted for nutrient concentrations in both lakes indicated low concentrations of both soluble reactive phosphorus (SRP) and ammonium, and on several occasions close to or below the limits of detection (NH4-N, mini_ mum detection level of _4 mg L 1; PO4-P, minimum detection level of 1 mg L 1). SRP concentrations_ ranged between below the detection limit and 5.1 mg L 1 (Table 1). Both lakes had significantly lower levels of SRP concentrations in the summer period relative to autumn, winter, and spring (Mann–Whitney U test, P G 0.001). Ammonium concentrations in both lakes ranged between _ below the detection limit and 61.8 mg L 1 (Table 1). There was no significant variation with seasons in the ammonium levels in Crooked Lake; however, in Lake Druzhby higher ammonium concentrations were noted in winter compared to summer and spring (Mann– Whitney U test, P G 0.001). DOC concentrations were extremely low and in many cases close to or below the resolution of the analytical_ equipment, ranging between undetectable and 1.9 mg L 1 (Table 1). In both lakes, the highest DOC concentrations were noted in summer. Chlorophyll _a concentrations ranged from undetectable to 0.33 mg L 1 in Crooked Lake and from 0.01 to 0.46 mg

Table 1. Average, minimum, and maximum values of the physiochemical parameters in Lake Druzhby and Crooked Lake from December 2003 to November 2004

Druzhby

Crooked

Parameters

Average (min–max)

Average (min–max)

Temperature (C-) pH Conductivity _ (mS cm 1) Soluble reactive phosphorus _ (PO4-P mg L 1) Ammonium _ (NH4-N mg L 1) Dissolved organic _ carbon (mg L 1)

3.0 (2.2–4.4) 7.1 (6.8–7.3) 40.7 (35.9–44.8)

1.3 (0.9–1.9) 7.0 (6.7–7.3) 29.0 (23.1–33.9)

bd: Below detection.

2.0 (bd–4.6)

2.7 (bd–5.1)

8.9 (bd–61.8)

7.5 (bd–32.7)

0.6 (0.3–1.9)

0.2 (bd–0.9)

C. SA¨WSTRO¨M

ET AL.:

VIRAL LOOP DYNAMICS

IN

5

ANTARCTIC LAKES

_

L 1 in Lake Druzhby (Fig. 1). In Crooked Lake, chlorophyll a concentrations peaked in late spring and showed a positive correlation with the water temperature (Spearman rank–order correlation rs = 0.560, P G 0.001, N = 144). Lake Druzhby showed a peak in chlorophyll a in late autumn and also in early spring. Significantly higher chlorophyll a levels were recorded throughout the season in Lake Druzhby relative to Crooked Lake (Kruskal–Wallis, P G 0.001) (Fig. 1).

viruslike particles L 1 and 0.12 to 0.22  109 bacterial _1 cells L in Lake Druzhby (Figs. 2A, B). The lowest bacterial abundance in the lakes was recorded during the winter months (Fig. 2A). There was a significant increase in both viral and bacterial abundance in Crooked Lake in late autumn when compared to winter, spring, and summer (Mann–Whitney U test, P G 0.001). In contrast, in Lake Druzhby bacterial abundance was significantly higher in late spring than in the winter (Mann–Whitney U test, P G 0.001). However, viral abundance did not follow the same pattern as there was a significant decrease in abundance in spring when compared to summer and winter (Mann–Whitney U test, P G 0.01). Considering the whole seasonal data set, both viral and bacterial abundances were significantly higher in Lake Druzhby than in Crooked Lake (one-way ANOVA; F1,134 = 12.282, P G 0.001 and F1,134 = 15.447, P G 0.001). VBR values were low in both lakes and positively correlated with viruses, ranging between 1.2 and 8.4 (Fig. 2C) (two-tailed Pearson product–moment correlation; Lake Druzhby: r = 0.876, P G 0.01, N =144, Crooked Lake: r = 0.784, P G 0.01, N = 144). Significantly higher VBR values were noted in Lake Druzhby when compared to Crooked Lake (one-way ANOVA; F1,134 = 6.937, P G 0.01) with the highest VBR value noted in Lake Druzhby at the end of May 2004 (Fig. 2C).

Viral and Bacterial Abundance. Viral and bacterial abundances ranged from 0.16 to 0.92  109 viruslike _1 particles L and from 0.10 to 0.24  109 bacterial cells _1 L in Crooked lake, and between 0.30 and 1.56  109

Occurrence of Lysogenic Bacteria. Three significant induction events occurred in Lake Druzhby during season 2003–2004 (Table 2). The calculated fraction of lysogenic bacteria in Lake Druzhby was 22.3% in the

Figure 1. Seasonal changes in chlorophyll a in Crooked Lake and Lake Druzhby. Black dots: Crooked Lake; white dots: Lake Druzhby. Mean T SE (N = 8); letters represent the months. _

Figure 2. Seasonal changes in

the abundance of bacteria (A), abundance of virus like particles (VLP) (B), virus-tobacteria ratio (VBR) (C), and bacterial production (D) in Crooked Lake and Lake Druzhby. Black dots: Crooked Lake; white dots: Lake Druzhby. Mean T SE (N = 8); letters represent the months.

6

C. SA¨WSTRO¨M

ET AL.:

VIRAL LOOP DYNAMICS

IN

ANTARCTIC LAKES

Table 2. Calculations of lysogenic fractions of bacterial populations in Lake Druzhby

Date 12/17/03 01/10/04 05/17/04 06/14/04 07/07/04 08/05/04 09/01/04 09/28/04 10/21/04 11/17/04

_

VLP (control  109 L 1)

Mitomycin C (% of the control)

% FLC (Bz = 4)

% FLC (Bz = 26)

T T T T T T T T T T

122.6* 109.0 NS 156.8 NS 78.4 96.1 91.2 63.9 119.7* 128.8* 103.3 NS

22.3 NA NA NA NA NA NA 20.1 18.2 NA

3.0 NA NA NA NA NA NA 3.1 2.2 NA

0.59 1.11 0.52 0.72 0.48 0.89 0.67 0.81 0.41 0.25

0.03 0.09 0.14 0.02 0.02 0.02 0.05 0.03 0.06 0.03

VLP values are given as mean T SE. **P G 0.01; *P G 0.05. The significance of each induction event was determined by comparison of treatment and control levels of viruses by an independent-samples t test. NA: not applicable; NS: not significant; VLP: viruslike particles; FLC: fraction of lysogenic bacteria.

middle of December (17/12/03), 20.1% in the end of September (28/09/04), and 18.2% in the end of October (21/10/04) using a Bz of 4, or 3.0%, 3.1%, and 2.2% when using a Bz of 26 (Table 2). Three significant induction events also occurred in Crooked Lake during season 2003– 2004 (Table 3). The calculated fraction of lysogenic bacteria in Crooked Lake in August (08/11/04) was 73.0%, 23.6% in the beginning of September (09/06/04), and 43.5% in the beginning of October (10/04/04) using a Bz of 4, or 11.7%, 3.8%, and 5.3% when using a Bz of 26 (Table 3). Bacterial Production, FMVL, and Lytic and Lysogenic Bacterial production ranged from Viral Production. _ _

8.2 to 304.9  106 cells L 1 day_ 1 in _Crooked Lake and from 21.8 to 125.6  106 cells L 1 day 1 in Lake Druzhby (Fig. 2D). In both lakes, peaks in bacterial production were detected in spring and showed a positive correlation with the water temperature (Spearman rank–order correlation; Crooked Lake rs = 0.302, P G 0.05, N = 144; Lake Druzhby rs = 0.251, P G 0.05, N = 144 ). FMVL and viral production was estimated on four sampling occasions: summer, autumn, winter, and spring (Tables 4 and 5). FMVL ranged from a minimum of 38% (autumn) to a maximum of 87% (winter) in Lake Druzhby (Table 4). In Crooked Lake, FMVL was on average higher than in Lake Druzhby, with a minimum

of 46% (summer) to a maximum of 251% (autumn) (Table 5). No lysogenic viral production was detected in Crooked Lake on the four sampling dates. Lytic viral production_rates ranged from 73.3 to 718.4  106 viruslike _1 1 particles L day with the highest rate detected in spring 2004. In Lake Druzhby the contribution of lysogenic viral production was low (around 1%) with a lysogenic viral production of 2.7  106 viruslike particles _1 _1 L day in spring 2004 (Table 3). Lytic viral production in Lake Druzhby ranged from 47.5 to 237.3  106 viruslike _1 _1 particles L day . Discussion

The physiochemical parameters in the study lakes were fairly stable over the annual cycle and there was no evident vertical temperature stratification in the lakes and the water temperature never exceeded 5-C. In general, inorganic nutrients were low in both lakes, and on several occasions below the detection limit, which is in concordance with previous data reported from these lakes [4, 20, 27–29, 31]. The phytoplankton community should play an important role in supplying DOC into these systems, because the watersheds of these lakes are basically barren with no terrestrial influence and contribution from

Table 3. Calculations of lysogenic fractions of bacterial populations in Crooked Lake

Date 12/11/03 01/20/04 05/10/04 06/07/04 07/13/04 08/11/04 09/06/04 10/04/04 10/27/04 11/15/04

_

VLP (control  109 L 1)

Mitomycin C (% of the control)

% FLC (Bz = 4)

% FLC (Bz = 26)

T T T T T T T T T T

68.5 101.6 NS 95.2 109.7 NS 90.5 206.1** 149.3* 152.9** 52.7 163.1 NS

NA NA NA NA NA 73.0 23.6 43.5 NA NA

NA NA NA NA NA 11.7 3.8 5.3 NA NA

0.50 0.12 0.75 0.11 0.19 0.41 0.27 0.37 0.62 0.22

0.03 0.03 0.13 0.08 0.05 0.10 0.06 0.03 0.08 0.04

VLP values are given as mean T SE. **P G 0.01; *P G 0.05. The significance of each induction event was determined by comparison of treatment and control levels of viruses by an independent-samples t test. NA: not applicable; NS: not significant; VLP: viruslike particles; FLC: fraction of lysogenic bacteria.

C. SA¨WSTRO¨M

ET AL.:

VIRAL LOOP DYNAMICS

IN

7

ANTARCTIC LAKES

Table 4. Estimated impact of viruses on bacteria in Lake Druzhby on four sampling occasions in 2004 9

_1

VLP (10 L ) FMVL (%) _ _ Lytic viral production (106 L 1 day 1) FLC (%) _ _ Lysogenic viral production (106 L 1 day 1) (VL%  lytic viral production) _ Bacteria (109 L 1) _ _ Bacterial cell production (106 L 1 day 1) _1 _ Bacterial carbon production (mg C L day 1)

Summer (01/10/04)

Autumn (05/17/04)

Winter (07/07/04)

Spring (10/21/04)

1.56 70 175.6 – –

0.30 38 47.5 – –

0.64 87 237.3 – –

0.32 43 91.3 18.2 2.7

0.21 63.2 1.80

0.19 31.0 0.88

0.13 68.6 1.95

0.22 53.1 1.51

FMVL = FVIB/[0.693  (0.814 – FVIB)]. Lytic viral production = FMVL  bacterial cell production  4. Lysogenic viral production = [10–2  (bacteria/ bacterial cell production)  FLC  bacteria]/[0.3  VLP]  100. Values are averages of the water column including eight different depths. FMVL: fraction of bacterial mortality caused by viral lysis; FVIB: fraction of visibly infected bacterial cells; FLC: fraction of lysogens.

benthic algal mats and mosses would be expected to be small because they are very sparse in both lakes [15]. Chlorophyll a levels were low throughout the year, with peaks detected twice in Lake Druzhby, whereas in Crooked Lake only a small phytoplankton biomass increase was noted during early summer. In Lake Druzhby, the phototrophic plankton is dominated by a small rod-shaped mucous-producing cyanobacterium [27]. These cells were noted in late autumn and also in early spring in Lake Druzhby and coincided with the peaks in chlorophyll a. The differences in chlorophyll a levels between the lakes are probably related to the variations in light transmission and water temperatures. Previous studies have shown that light can penetrate into the water column even through 2-m-thick ice, with 74% of the surface photosynthetically active radiation (PAR) penetrating to 25 m depth in Lake Druzhby, whereas in Crooked Lake only about 40% penetrated to 25 m depth [27]. Lake Druzhby is a shallower system and subjected to larger temperature variations and also significantly higher water temperatures than Crooked Lake. Lake Druzhby also became completely ice-free during summer, whereas Crooked Lake maintained its ice cover and only developed moats around the edges. Bacterial abundance and activity was low in both lakes and within the previously reported range for these systems [29]. Both lakes showed a decrease in bacterial

abundance in the winter months. Throughout the year, Lake Druzhby had overall higher bacterial abundance, activity, and DOC concentrations than Crooked Lake. With the onset of slightly warmer water temperatures during spring, bacterial production increased in both lakes. The viral abundance observed in our study—the first seasonal data provided for Antarctic freshwater lakes—is in the lower range from previously reported data [55]. The viral abundance was approximately 10 times lower than previously reported data for saline and freshwater lakes from the continental Antarctic [23, 30, 34]. Low viral abundance is often found in unproductive and nutrient-poor environments [55], which our results corroborate as both study lakes are classed as ultraoligotrophic. Viral abundance exceeded bacterial abundance in both lakes during the entire season. However, the VBR values were low throughout the season in both the lakes. The mitomycin C induction experiments indicated that lysogenic bacteria were uncommon in both lakes throughout the season. A larger proportion of lysogenic bacteria was detected in Crooked Lake relative to Druzhby Lake. A high percentage of bacterioplankton were lysogenically infected in winter in Crooked Lake, indicating that during periods with decreased bacterial abundance, as was noted for winter, lysogeny may be a

Table 5. Estimated impact of viruses on bacteria in Crooked Lake on four sampling occasions in 2004 _

VLP (109 L 1) FMVL (%) _ _ Lytic viral production (106 L 1 day 1) _ Bacteria (109 L 1) _ _ Bacterial cell production (106 L 1 day_ 1) _ Bacterial carbon production (mg C L 1 day 1)

Summer (10/21/04)

Autumn (05/10/04)

Winter (07/10/04)

Spring (10/27/04)

0.16 46 73.3 0.13 40.2 0.70

0.91 251 98.4 0.20 9.8 0.17

0.26 120 400.6 0.15 83.7 1.46

0.69 129 718.4 0.14 139.6 2.44

FMVL = FVIB/[0.693  (0.814 – FVIB)]. Lytic viral production = FMVL  bacterial cell production  4. Values are averages of the water column including eight different depths. FMVL: fraction of bacterial mortality caused by viral lysis; FVIB: fraction of visibly infected bacterial cells; FLC: fraction of lysogens.

8

strategy for viruses to ensure survival throughout the harsh winter months. However, the estimated percentage lysogens were about 6 times lower when estimated with an average Bz of 26. This emphasizes the importance of determining accurate Bz for each lake ecosystem as it has a big knock-on effect on the estimations of viral proliferation and viral-induced bacterial mortality. Furthermore, a recent review on viral burst sizes in aquatic systems revealed a large variation in freshwater burst sizes, ranging from 4 to 140 depending on the investigated environment [41]. Mitomycin C has been used in several studies [22, 32, 46, 50, 52, 53] as a prophage induction agent; however, it should be noted that its effectiveness as an induction agent for natural freshwater bacterioplankton communities is still largely unknown. It is possible that the percentage of lysogens was underestimated, as the inductant may not have affected the entire lysogenic bacterial community. However, compared to other studies that used a similar approach to estimate lysogeny [22, 32, 46, 50, 52, 53], our findings show that lysogenic viral production was probably of minor importance in both Lake Druzhby and Crooked Lake. Only about 1% of the total viral production came from spontaneous prophage induction events in Lake Druzhby. The lytic cycle seems to predominate in these ultraoligotrophic Antarctic lakes. Lytic viral production rates in Lake Druzhby and Crooked lake were similar to previously reported rates from temperate environments but significantly lower than the rates detected in Lake Hoare, McMurdo Dry Valleys, Antarctica [23, 55]. Sa¨wstro¨m et al. (unpublished data) showed that there was a large proportion of visibly infected bacterial cells in these lakes. Using previously reported FVIB values from Lake Druzhby and Crooked Lake and the model of Binder [8], we estimated the FMVL and found that a very large proportion of bacterial mortality was attributable to viral lysis. Our results add to a growing set of evidence that the greatest viral impact occurs in low productivity lake ecosystems, ranging from temperate to polar environments [7, 37]. In both Lake Druzhby and Crooked Lake, bacterioplankton are primarily dependent on autochthonous carbon sources with negligible input from allochthonous carbon sources [25]. Thus, DOC supplied from in situ microbial activity, such as viral lysis, heterotrophic flagellate (HNF) grazing, and primary production (PP), should hypothetically meet the bacterial carbon demand within these lakes. To see if this hypothesis held true, four sampling occasions (summer, autumn, winter, and spring) were investigated from each lake and the potential flow of carbon through the viral loop was estimated. The carbon input from viral lysis was calculated by multiplying FMVL by bacterial carbon production rates. Primary production rates were obtained from a previous study on the lakes conducted

C. SA¨WSTRO¨M

ET AL.:

VIRAL LOOP DYNAMICS

IN

ANTARCTIC LAKES

in 1999 by Henshaw and Laybourn-Parry [20]. It was assumed that all the carbon produced from phytoplankton growth was available for bacterial usage and that the entire DOC pool was accessible for bacterial carbon production. The percentage bacterial biomass removed by HNF per day was obtained from previous studies conducted on the lakes [27, 28]. These studies estimated a low impact of HNF grazing with 2% of the bacterial carbon pool being removed per day in Lake Druzhby and a maximum of 10% of the bacterial production being removed per day in Crooked Lake. As the removal of bacteria by HNF grazing is relatively low, it was assumed that all grazed bacterial biomass was transformed into DOC, which was then readily available for bacterial usage. This will of course substantially overestimate the contribution of DOC from grazing, as in reality only a small part of the bacterial biomass would directly be turned into DOC via sloppy feeding. The above assumptions may not be completely accurate as there are several uncertainties involved with our estimations; data for primary production and HNF grazing are from previous seasons, all PP and HNF grazing is assumed to directly contribute to the DOC pool, the entire DOC pool is available for bacterial use, bacterial growth efficiency (BGE) and bacterial respiration (BR) are estimated from BP using models based on mainly temperate aquatic environments, and additional sources of DOC are not included. Even though our estimates of planktonic DOC production will substantially overestimate the potential autochthonous DOC input in the lakes, especially regarding the PP and the grazing contribution, we believe that our estimations can add important information on the relevance of the viral loop in unproductive Antarctic lakes and any seasonal changes occurring. BGE was estimated by applying the measured BP values to the hyperbolic relationship between BP and BGE found by del Giorgio and Cole ([16]; BGE = 0.037 + 0.65  BP/(1.8 + BP)). The calculated BGE in the two lakes varied from 2% to 5%. With such low assimilation efficiencies we estimated that the net DOC requirement for bacterial production (BP/BGE) would vary between _ _ _ 26.6_ to 40.9 mg C L 1 day 1 and from 7.4 to 45.0 mg C L 1 1 day in Lake Druzhby and Crooked Lake, respectively. The potential DOC input from viral lysis, HNF grazing, and primary production varied from 1.2 to _7.6 mg C _ _ _ L 1 day 1 and from 1.4 to 38.9 mg C L 1 day 1 in Lake Druzhby and Crooked Lake, respectively. With low BGE values, we expected high BR values and a large loss of organic carbon from the lakes. We used BP and BGE values to estimate BR by the following equation ([16]; _ BP), and it ranged between 25.7 and BR = BP/BGE _1 _ _ 38.9_ mg C L day 1, and between 7.3 and 42.5 mg C L 1 1 day in Lake Druzhby and Crooked Lake, respectively. In Lake Druzhby, the bacterial carbon demand (BP/ BGE) was up to 30 times higher than the estimated total

C. SA¨WSTRO¨M

ET AL.:

VIRAL LOOP DYNAMICS

IN

9

ANTARCTIC LAKES

DOC input (viral lysis + HNF grazing + PP) throughout the season, whereas in Crooked Lake the bacterial carbon demand was up to 5 times higher than the estimated DOC supply in autumn and spring and 14 times higher in the winter. However, during summer in Crooked Lake high primary production provided a considerable pulse of carbon into the DOC pool, which resulted in balanced bacterial carbon demand and organic input. It is not uncommon to find that bacterial carbon demand exceeds the organic inputs in lakes as organic carbon can be reassimilated and recycled by other consumers in the ecosystem [43]. However, in the study lakes the low estimated BGE restricts the amount of carbon that can be reassimilated by the bacterial biomass. We found that BR exceeded the estimated organic input throughout the season, in Lake Druzhby, whereas in Crooked Lake the estimated organic input in the summer was sufficient to_ sustain the estimated BR rate _1 1 (BR = 22.2 mg C L day ; potential DOC input = 38.9 _1 _1 mg C L day ). Several studies have revealed that oligotrophic lakes are often net heterotrophic ecosystems, i.e., more carbon consumed than is being produced [13, 14]; interestingly, this also seems to be the scenario in the ultraoligotrophic Antarctic lake ecosystems as the lakes seemed to have a tendency toward net heterotrophy, indicating that there may be some carbon input from allochthonous sources. The estimated DOC supply to the lakes may have been subsidized by additional carbon sources (autochthonous and/or allochthonous sources) that were not included in our calculations, which could have resulted in the imbalance in bacterial carbon demand and supply. Studies conducted in the permanently icecovered Lake Fryxell, McMurdo Dry Valleys, Antarctica, showed that the major processes controlling the DOC concentrations were active leaching of biomass in glacial meltwater streams and degradation of organic matter in the sediments and relict organic matter that diffuses into the water column [2]. It is possible that these processes also take place in our study lakes, thus contributing to the DOC pool. Another possible explanation for the high BR could be the high contribution of viral activity to the DOC pool of Antarctic lakes. Studies have shown that viral lysis releases nutrients and DOC, which can stimulate growth of the uninfected bacterial community that has the net effect of lowering the BGE and increasing the BR in the system [19, 35, 36]. The importance of viral lysis as a contributor to the DOC pool throughout the season was estimated as a percentage of the total potential autochthonous DOC input ([FMVL  BP]/[(FMVL  BP) + HNF grazing + PP]  100) and varied from 5% to 62% and from 0.8% to 69% per day in Lake Druzhby and Crooked Lake, respectively. This suggests that viral-induced carbon transfer could play a significant role in these lakes, especially during the winter months, where over 60% of the carbon

supplied to the DOC pool originated from viral lysis. The viral loop seemed to be of less importance during summer when only 0.8% of DOC came from viral lysis in Crooked Lake and 17% in Lake Druzhby. It seems plausible that occasionally peaks in phytoplankton growth in the summer periods can supply sufficient DOC for the bacterial carbon demand. In the unproductive winter, bacterial production may be sustained by recycling of viral lysates (high viral activity in the winter months), which causes an increase in bacterial respiration. However, as BR is higher than the carbon supply, this must be compensated through something, for instance, in situ lake processes such as DOC flux from sediments or allochthonous input in the form of meltwater from the Sørsdal glacier or the plateau. In partial carbon budgets created for the McMurdo Dry Valley lakes, the bacterial carbon demand and BR also exceeded the measured organic inputs [45]. The authors suggested that the missing carbon might be derived from viral lysis and grazing; however, they were unable to estimate the DOC contribution of these groups to the food web. In the present study, we have estimated the significance of viral activity in Lake Druzhby and Crooked Lake, showing that the viral loop could play an important role in these systems as it allows for the flow and reassimilation of organic carbon from lysed bacterial cells into the biomass of the uninfected bacterial community.

Acknowledgments

This work was funded by a Marie Curie Scholarship, the Australian Antarctic Division and VR the Swedish Research Council. We thank the crew at Davis Station, Antarctica 2003/2004 for logistical support and field assistance.

References 1. Ackermann, HW, Dubow, MS (1987) Viruses of prokaryotes, Vol. 1, In: General Properties of Bacteriophages. CRC Press, Boca Raton 2. Aiken, G, McKnight, D, Harnish, R, Wershaw, R (1996) Geochemistry of aquatic humic substances in the Lake Fryxell Basin, Antarctica. Biogeochemistry 34: 157–188 3. Azam, F, Fenchel, T, Field, JG, Gray, JS, Meyer-Reil, LA, Thingstad, TF (1983) The ecological role of water-column microbes in the sea. Mar Ecol Prog Ser 10: 257–263 4. Bayliss, P, Ellis-Evans, JC, Laybourn-Parry, J (1997) Temporal patterns of primary production in a large ultraoligotrophic Antarctic freshwater lake. Polar Biol 18: 363–370 5. Bell, RT (1993) Estimating production of hetertrophic bacterioplankton via the incorporation of tritiated thymidine. In: Kemp, BF, Sherr, EB, Cole, JJ (Eds.) Handbook of Methods in Aquatic Microbial Ecology, CRC Press, Boca Raton, pp 495–503

10

6. Bettarel, Y, Amblard, C, Sime-Ngando, T, Carrias, J-F, Sargos, D, Garabe´tian, F, Lavandier, P (2003) Viral lysis, flagellate grazing potential and bacterial production in Lake Pavin. Microb Ecol 45: 119–127 7. Bettarel, Y, Sime-Ngando, T, Amblard, C, Dolan, C (2004) Viral activity in two contrasting lake ecosystems. Appl Environ Microbiol 70: 2941–2951 8. Binder, B (1999) Reconsidering the relationship between virally induced bacterial mortality and frequency of infected cells. Aquat Microb Ecol 18: 207–215 9. Bratbak, G, Dundas, I (1984) Bacterial dry matter content and biomass estimations. Appl Environ Microbiol 48: 755–757 10. Bratbak, G, Heldal, M (2000) Viruses rule the waves—the smallest and most abundant members of marine ecosystems. Microbiol Today 27: 171–173 11. Bratbak, G, Heldal, M, Thingstad, TF, Riemann, B, Haslund, OH (1992) Incorporation of viruses into the budget of microbial Ctransfer. A first approach. Mar Ecol Prog Ser 83: 273–280 12. Choi, DH, Hwang, CY, Byung, CC (2003) Comparision of virusand bacteriovory-induced bacterial mortality in the eutrophic Masan Bay, Korea. Aquat Microb Ecol 30: 117–125 13. Cole, JJ, Carpenter, SR, Kitchell, JF, Pace, ML (2002) Pathways of organic carbon utilization in small lakes: results from whole-lake 13C addition and coupled model. Limnol Oceanogr 47: 1664–1675 14. Cole, JJ (1999) Aquatic microbiology for ecosystem scientists: new and recycled paradigms in ecological microbiology. Ecosystems 2: 215–225 15. Dartnall, HJG (2000) A limnological reconnaissance of the Vestfold Hills. ANARE Rep 141: 57 16. del Giorgio, P, Cole, JJ (1998) Bacterial growth efficiency in natural aquatic systems. Annu Rev Ecol Syst 29: 503–541 17. Fischer, UR, Velimirov, B (2002) High control of bacterial production by viruses in a eutrophic oxbow lake. Aquat Microb Ecol 27: 1–12 18. Fuhrman, JA (1999) Marine viruses and their biogeochemical and ecological effects. Nature 399: 541–548 19. Gobler, CJ, Hutchins, DA, Fisher, NS, Cosper, EM, San˜udoWilhelmy, SA (1997) Release and bioavailability of C, N, P, Se and Fe following viral lysis of a marine chrysophyte. Limnol Oceanogr 42: 1492–1504 20. Henshaw, T, Laybourn-Parry, J (2002) The annual patterns of photosynthesis in two large freshwater, ultra-oligotrophic Antarctic lakes. Polar Biol 25: 744–752 21. Jespersen, AM, Christoffersen, K (1987) Measurements of chlorophyll a from phytoplankton using ethanol as extraction solvent. Arch Hydrobiol 109: 445–454 22. Jiang, SC, Paul, JH (1998) Significance of lysogeny in the marine environment: studies with isolates and a model of lysogenic phage production. Microb Ecol 35: 235–243 23. Kepner, RL, Wharton, RA, Suttle, CA (1998) Viruses in Antarctic lakes. Limnol Oceanogr 43: 1754–1761 24. Kirchman, D (2001) Measuring bacterial biomass production and growth rates from leucine incorporation in natural aquatic environments. In: Paul, JH (Ed.) Marine Microbiology—Methods in Microbiology, Academic Press, London, pp 225–237 25. Laybourn-Parry, J (1997) The microbial loop in the Antarctic lakes. In: Lyons, WB, Howard-Williams, C, Hawes, I (Eds.) Ecosystem Processes in Antarctic Ice-Free Landscapes, Balkema, Rotterdam, Netherlands, pp 231–240 26. Laybourn-Parry, J (2002) Survival mechanisms in Antarctic lakes. Philos Trans R Soc Lond B 357: 863–869 27. Laybourn-Parry, J, Bayliss, P (1996) Seasonal dynamics of the planktonic community in Lake Druzhby. Princess Elizabeth Land, Eastern Antarctica. Freshw Biol 35: 57–67

C. SA¨WSTRO¨M

ET AL.:

VIRAL LOOP DYNAMICS

IN

ANTARCTIC LAKES

28. Laybourn-Parry, J, Bayliss, P, Ellis-Evans, JC (1995) The dynamics of heterotrophic nanoflagellates in a large ultra-oligotrophic Antarctic lake. J Plank Res 17: 1835–1850 29. Laybourn-Parry, J, Henshaw, T, Jones, DJ, Quayle, W (2004) Bacterioplankton production in freshwater Antarctic lakes. Freshw Biol 49: 735–744 30. Laybourn-Parry, J, Hofer, JS, Sommaruga, R (2001) Viruses in the plankton of freshwater and saline antarctic lakes. Freshw Biol 46: 1279–1287 31. Laybourn-Parry, J, Marchant, HJ, Brown, PE (1992) Seasonal cycle of the microbial plankton in Crooked Lake, Antarctica. Polar Biol 12: 411–416 32. Lisle, JT, Priscu, JC (2004) The occurrence of lysogenic bacteria and microbial aggregates in the lakes of the McMurdo Dry valleys, Antarctica. Microb Ecol 47: 427–439 33. Mackereth, FJH, Heron, J, Talling, JF (1989) Water analysis. Freshwater Biological Association UK, Publ No 36 34. Madan, NJ, Marshall, WA, Laybourn-Parry, J (2005) Virus and microbial loop dynamics over an annual cycle in three contrasting Antarctic lakes. Freshw Biol 50: 1291–1300 35. Middelboe, M, Jørgensen, NOG, Kroer, N (1996) Effects of viruses on nutrient turnover and growth efficiency of noninfected marine bacterioplankton. Appl Environ Microbiol 62: 1991–1997 36. Middelboe, M, Lyck, PG (2002) Regeneration of dissolved organic matter by viral lysis in marine microbial communities. Aquat Microb Ecol 27: 187–194 37. Murray, AG, Eldridge, PM (1994) Marine viral ecology: incorporation of bacteriophage into the microbial planktonic food web paradigm. J Plank Res 16: 627–641 38. Noble, RT, Fuhrman, JA (1998) Use of SYBR Green I for rapid epifluorescence counts of marine viruses and bacteria. Aquat Microb Ecol 14: 113–118 39. Parker, BC, Simmons, GM (1985) Paucity of nutrient cycling and absence of food chains in the unique lakes of southern Victoria Land. In: Sigfried, WR, Condy, RR, Laws, RM, (Eds.) Antarctic Nutrient Cycles and Food Webs, Springer-Verlag, Berlin, pp 238–244 40. Paul, JH, Jiang, SC (2001) Lysogeny and transduction. In: Paul, JH (Ed.) Marine Microbiology—Methods in Microbiology, Academic Press, London, pp 105–125 41. Parada, V, Herndl, GJ, Weinbauer, MG (2006) Viral burst size of heterotrophic prokaryotes in aquatic systems. J Mar Biol Assoc UK 86: 613–621 42. Priscu, JC, Wolf, CF, Takacs, CD, Fritsen, CH, Laybourn-Parry, J, Roberts, EC, Sattler, B, Lyons, WB (1999) Carbon transformations in a perennially ice-covered Antarctic lake. BioScience 49: 997– 1008 43. Strayer, D (1988) On the limits to secondary production. Limnol Oceanogr 33: 1217–1220 44. Suttle, CA (2005) Viruses in the sea. Nature 437: 356–361 45. Takacs, CD, Priscu, JC, McKnight, DM (2001) Bacterial dissolved organic carbon demand in McMurdo Dry Valley Lakes, Antarctica. Limnol Oceanogr 46: 1189–1194 46. Tapper, MA, Randall, EH (1998) Temperate viruses and lysogeny in lake superior bacterioplankton. Limnol Oceanogr 43: 95–103 47. Theil-Nielsen, J, Søndergaard, M (1998) Bacterial carbon biomass calculated from biovolumes. Arch Hydrobiol 141: 195–207 48. Thingstad, TF (2000) Elements of a theory for the mechanisms controlling abundance, diversity, and biogeochemical role of lytic controlling abundance, diversity, and biogeochemical role of lytic bacterial viruses in aquatic systems. Limnol Oceanogr 45: 1320– 1328 49. Thingstad, TF, Heldal, M, Bratbak, G, Dundas, I (1993) Are viruses important partners in pelagic food webs? TREE 8: 209–212 50. Weinbauer, MG, Brettar, I, Ho¨fle, MG (2003) Lysogeny and virus-

C. SA¨WSTRO¨M

ET AL.:

VIRAL LOOP DYNAMICS

IN

ANTARCTIC LAKES

induced mortality of bacetrioplankton in surface, deep, and anoxic marine waters. Limnol Oceanogr 48: 1457–1465 51. Weinbauer, MG, Ho¨fle, MG (1998) Significance of viral lysis and flagellate grazing as factors controlling bacterioplankton production in a Eutrophic lake. Appl Environ Microbiol 64: 431–438 52. Weinbauer, MG, Suttle, CA (1999) Lysogeny and prophage induction in coastal and offshore bacterial communities. Aquat Microb Ecol 18: 217–225

11

53. Williamson, SJ, Houchin, LA, McDaniel, L, Paul, JH (2002) Seasonal variation in lysogeny as depicted by prophage induction in Tampa Bay, Florida. Appl Environ Microbiol 68: 4307– 4314 54. Wilhelm, SW, Suttle, CA (1999) Viruses and nutrient cycles in the sea. BioSci 49: 781–788 55. Wommack, KE, Colwell, RR (2000) Virioplankton: viruses in aquatic ecosystems. Microbiol Mol Biol Rev 64: 69–114