Viral impacts on bacterial communities in Arctic cryoconite

This study addressed viral dynamics in cryoconite holes over two consecutive field seasons in Svalbard and Greenland. The Svalbard study centred on two valley glaciers, Midtre Lovénbreen (ML) and Austre Brøggerbreen (AB), near Ny-Ålesund (78°55'N 11°55'W, figure 1). Virus and bacterial production was measured in incubation experiments on the 25 July 2009 on sediment sampled from cryoconite holes at point ML, and on the 4 August 2009 from site AB (figure 1). Between June and August 2010, sampling and activity measurements were conducted on the western margin of the Greenland ice sheet (GrIS) around Kangerlussuaq. The study sites for activity measurements were approximately 1 km from the ice margin on the Russell Glacier (RG) at approximately 67°9'39.7''N,50°0'52.7''W (figure 1). Viral production was measured in two independent cryoconite holes at RG1 on 15 June 2010 and three independent cryoconite holes on 27 July 2010 at RG2 (figure 1 and table 1). A transect was also conducted via helicopter on 2 August 2010 to measure virus and bacterial abundances throughout the entire ablation zone of the GrIS (sample points 5–42 km); (figure 1). These points represented elevations of ∼550 m above sea level at point RG1 to 1186 m above sea level at 42 km.

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For enumeration of viruses and bacteria, 1 ml of cryoconite was sampled with a large pipette and transferred into a sterile 15 ml centrifuge tube. The sample was mixed with 9 ml of 30 kDa glacial ultrafiltrate which had a virus concentration of at least four orders of magnitude lower than the sediment and is hence referred to as virus-free water. This was shaken vigorously for 2 min. Fifteen seconds after cessation of shaking, 100 μl of supernatant was sampled and fixed with glutaraldehyde (2% final conc.), which allowed us to detect viruses at concentrations of 10⁶ VLP g⁻¹ or greater in this instance. Samples were filtered onto 0.02 μm Anodisc filters (Whatman) within 24 h, stained with SYBR gold (Invitrogen) and counted using epifluorescence microscopy (Anesio et al 2007). Bacterial biovolume was also calculated by measuring approximately 2000 individual bacteria from six cryoconite holes (three from RG1, two from ML, one from AB) using an F-view II CCD (Olympus) and digitally measuring the length (L) and width (W) using Cell^∧f software (Olympus) of bacteria in three fields of view per cryoconite. Cell biovolume (V) was calculated by using the formula $V=(\pi /4){W}^{2}\hspace{0.167em} \left (L-\frac{W}{3}\right )$. The mean biovolume from the six cryoconites was used in further calculations. Biovolume (μm³) was subsequently converted to carbon content (CC; fg cell⁻¹) by using a range of suitable conversion factors in the literature. Based on a review by Posch et al (2001) we chose CC conversion factors that are in common use in the literature and had been derived from freshwater bacteria and tested on the size range of bacteria present in cryoconite holes. This created a range of conversion factors from CC = 105V (Theil-Nielsen and Søndergaard 1998) to CC = 218V^0.86 (Loferer-Krößbacher et al 1998).

1 ml of cryoconite from six cryoconite holes (three from RG1, two from ML, one from AB) was diluted with 9 ml of extraction buffer (2% glutaraldehyde, 10 mM sodium pyrophosphate and Milli-Q™ water to 9 ml). The mixture was subjected to three cycles of vortexing and sonication for one minute, before it was passed through a 3 μm mixed cellulose ester filter (SSWP Millipore). To further separate the bacteria from the sediment matrix, a 200 μl aliquot of the filtered sediment extract was centrifuged onto a 70% sucrose cushion at 10 000 × g for 10 min. The top layer of this mixture, plus 100 μl from the top of the sucrose cushion, were removed and made up to 2 ml with 2% glutaraldehyde. This mixture was then pelleted at 10 000 × g for 5 min. The supernatant was removed and discarded before the pellet was resuspended in 1 ml 2% glutaraldehyde. The dilution step and further pelleting served to reduce the background level of viruses which remained in the supernatant. 5 μl of each sample was placed onto a carbon coated 400-mesh Cu grid resting on filter paper. Grids were left overnight to dry, before being negatively stained for one minute with 2% uranyl acetate and then rinsed with deionized distilled water. Two grids per cryoconite were examined for visibly infected bacterial cells at 80 kV and 20 000–40 000× magnification. A cell was considered infected when two or more viruses were visualized inside a cell based on a dark staining and a clearly recognizable round or hexagonal capsid structure. Normally, counting cells with such low numbers of virus particles inside increases the risk of counting cells that are simply resting on top of viruses as infected bacteria. This is because ultrafugation is the usual method to pellet both virus and host on to TEM grids (Bettarel et al 2006). In this study, the low speed centrifugation used to pellet bacteria, but not viruses, greatly reduced the free viruses. This allowed use of a lower detection limit of two phages per cell in a similar fashion to Säwström et al (2007). The frequency of infected cells (FVIC) and burst size (B_z) were calculated from the mean and standard deviation of the six cryoconite holes.

Virus production (VP) was determined in cryoconite sediment samples using the dilution method (Mei and Danovaro 2004). A 1:10 sediment: virus-free water dilution was achieved for each of three sub-replicates per cryoconite hole in three holes from Svalbard and five from Greenland. Virus-free water (13.5 ml) was added to 1.5 ml cryoconite sediment to significantly reduce encounter rates and new virus infections. Each incubation was conducted for 24 h in the dark at in situ temperatures. To determine the virus increase, subsamples of 100 μl were removed at approximately 4 h intervals and immediately fixed with 0.02 μm-filtered glutaraldehyde (2% final conc.) before slide preparation and viral counts by epifluorescence microscopy as above. VLP were counted at each time point. VP was calculated by plotting a first order regression of VLP against time during the first increase in VLP numbers in each of the three sub-replicates. To confirm viruses were not simply detaching from the sediment and influencing counts, a killed control incubation was performed by adding glutaraldehyde (2% final conc.) to an extra replicate from each sample location and treated as above. All counts were normalized to dry weight by desiccation at 105 ° C for 24 h.

To determine the significance of lysogeny in cryoconite holes, the induction agent mitomycin C was added to triplicate sediment dilutions as above to a final concentration of 1 μg ml⁻¹. Treatments were incubated for 24 h before a 100 μl subsample was removed, fixed and made into a slide, as described above. Triplicate incubations in which mitomycin C was not added, served as controls. The percentage of lysogenic bacteria was calculated according to Weinbauer and Suttle (1996).

Bacterial carbon production (BCP) was measured alongside VP in each cryoconite hole (n = 8) by ³H-leucine incorporation, modified from the method of Smith and Azam (1992) (see supplementary information 1, available at stacks.iop.org/ERL/8/045021/mmedia). Briefly nine 1.5 ml subsamples from each cryoconite hole were removed and transferred to sterile 1.7 ml microcentrifuge tubes. Incorporation was started by addition of ³H-leucine (100 nM final conc.). Three of the tubes were incubated as killed controls by the immediate addition of 100 μl glutaraldehyde. The tubes (six live, three killed) were incubated at in situ temperature for 3 h and incorporation was stopped by addition of 100 μl glutaraldehyde (2% final conc.). BCP was converted to new bacterial cell production (NCP) by dividing BCP by the conversion factors calculated from the biovolume (10.68–30.53 fg C cell⁻¹) to produce a range of values.

Viral turnover (VT) was calculated by dividing VP rates by initial viral abundances. The virus mediated mortality (VMM) of bacterial cells (cells lysed g⁻¹ h⁻¹) was calculated by dividing VP values by the viral burst size (B_z). VMM was further divided by the NCP rate (cells g⁻¹ h⁻¹), calculated from ³H-leucine incorporation, to give a range of value for the virus induced bacterial mortality (VIBM) in each incubation expressed as a percentage of bacterial production.

All data were tested for normality prior to statistical analysis using a Kolmogorov–Smirnov test conducted in SPSS (16.0) or SigmaPlot (12.0).

Virus abundances in independent cryoconite holes in both Greenland and Svalbard ranged from 5.62 × 10⁸ to 24.5 × 10⁸ VLP g⁻¹ dry weight sediment (ML and GrIS 11 km respectively), with a mean across all sites of 14.8 ± 7.3 × 10⁸ VLP g⁻¹ (table 1). Bacterial abundances ranged from 2.68 × 10⁸ to 19.6 × 10⁸ cells g⁻¹ (RG2 and GrIS 5.4 km respectively), with mean abundances of 8.62 ± 6.8 × 10⁸ cells g⁻¹. We can report that the coefficient of variation of VLP and BDC within cryoconite holes was generally less than 0.3, which was similar to the variation between cryoconite holes from the same site.

VLP and bacterial abundances were positively and significantly related across all sample sites (regression equation y = 0.988x + 6.121 × 10⁸,n = 12, R² = 0.76,P < 0.001) with a virus to bacteria ratio (VBR) between 1.2 and 4.4 (mean 2.1 ± 1). Over the 42 km transect of the ablation zone of the GrIS (figure 2), VLP abundances and BDC varied significantly (ANOVA, P < 0.001). Moving from the ice sheet margins to the interior, VLP abundances increased by a factor of three (ANOVA, P < 0.001 followed by post hoc Scheffé, P < 0.001) to a maximum at 11 km of 24.5 × 10⁸ VLP g⁻¹. BDC also showed a seven fold increase between site RG (1 km) and their maximum at 5.4 km of 19.6 × 10⁸ cells g⁻¹ (ANOVA, P < 0.001, followed by post hoc Scheffé, P < 0.001). A planned 65 km site was also investigated but found to be above the snowline and insufficient cryoconite debris was obtained for counting.

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For the purposes of comparing abundance variability, samples from Greenland were divided into outlet glacial locations and the ice sheet itself. Stibal et al (2012b) previously demonstrated that the marginal sites of the GrIS (<3.6 km from the margin) are physically and chemically different from the ice sheet interior because of their slope and proximity to deglaciated areas. Hence all cryoconite holes (n = 5) from sites RG1 and 2 (Greenland) (∼1 km from the ice sheet margin) were considered part of the Russell Glacier (figure 1). Abundance data from 5.4 to 42 km on the transect across the GrIS (n = 12) were considered together as representative of the main ablation zone of the ice sheet and are subsequently referred to as GrIS sites. Cryoconite holes from ML and AB represented Svalbard cryoconite holes (n = 3). Using the above criteria, there were significant differences in VLP abundances between sites (ANOVA P = 0.001). GrIS VLP abundances (sites 5.4–42 km) were approximately 2–4 times greater than the glacial sites in Svalbard and Greenland (post hoc Scheffé, P < 0.01). However, no significant differences were detected in VLP abundance between Svalbard and Greenland glacial sites (post hoc Scheffé, P > 0.05). Mean bacterial abundances were approximately 4 times greater on the main GrIS (Sites 5.4–42 km) than the Svalbard or Greenland glacial sites (ANOVA P < 0.001 followed by post hoc Scheffé, P < 0.05), but again no significant differences were found between Svalbard and Greenland glacial bacterial counts (post hoc Scheffé, P < 0.05).

Visible virus infection in bacterial cells was clearly resolved using TEM (figure 3) by visualizing over 140 bacteria. Bacterial cells exhibited very low numbers of visible virus particles with a range of between 2 and 4 viruses per cell (mean 2.4 ± 0.5). No cells appeared replete with viruses. All viruses infecting bacteria were round or hexagonal in shape and were approximately 30–90 nm across. The frequency of visibly infected cells (FVIC) was calculated as 21 ± 6%.

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Rates of virus production (VP) in independent cryoconite holes ranged from 4.41 × 10⁷ VLP g⁻¹ h⁻¹ (RG2) to 8.98 × 10⁷ VLP g⁻¹ h⁻¹ (ML) with a mean of 7.06 ± 1.8 × 10⁷ VLP g⁻¹ h⁻¹ across all sites (table 1). No increase in VLP abundances was detected in the killed controls and lysogeny was also not detected in any of the mitomycin C incubations during the VP experiments. Viral turnover (VT) was calculated to range from 0.04 h⁻¹ (RG2) to 0.16 h⁻¹ (ML). Mean bacterial biovolume from cryoconite hole sediment was calculated to be 0.102 μm³, thus the mean bacterial carbon content (CC) for cryoconite heterotrophic bacteria was calculated to range from 10.68 to 30.53 fg C cell⁻¹. Bacterial carbon production (BCP) in cryoconite holes ranged from 35.4 to 73.3 ng C g⁻¹ h⁻¹ (ML and RG2 respectively), (mean 57.8 ± 12.9 ng C g⁻¹ h⁻¹). Using the CC conversion values BCP was equivalent to a mean new cell production (NCP) rate of 1.89–5.41 × 10⁶ cells g⁻¹ h⁻¹. Mean bacterial turnover, expressed as the ratio of NCP to bacterial abundance, ranged from 0.137 to 0.393 d⁻¹. VP was significantly related to bacterial abundance and could be predicted by the regression equation y = 0.298x − 3.325 × 10⁷ (n = 8,R = 0.943, P < 0.005). There was no significant correlation between bacterial production and virus production (n = 8,R = 0.56, P > 0.05), nor any correlation between bacterial production and bacterial abundance (n = 8,R = 0.46, P > 0.05). By using the burst size derived from TEM measurements, viruses were calculated to account for the mean abatement of 598%–1710% of the heterotrophic bacterial production across all sites (table 1).