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Journal of Marine System

Variations of the abundance and nucleic acid content of heterotrophicbacteria in Beaufort Shelf waters during winter and spring

Claude Belzile a,⁎, Sonia Brugel a, Christian Nozais b, Yves Gratton c, Serge Demers a

a Institut des sciences de la mer de Rimouski, Université du Québec à Rimouski, 310 Allée des Ursulines, Rimouski, Québec, Canada G5L 3A1b Département de biologie et centre d'études nordiques, Université du Québec à Rimouski, 300 Allée des Ursulines, Rimouski,

Québec, Canada G5L 3A1c Institut national de la recherche scientifique, Centre Eau, Terre et Environnement, 490 de la Couronne, Québec, Québec, Canada G1K 9A9

Received 15 May 2007; received in revised form 11 December 2007; accepted 17 December 2007Available online 17 January 2008

Abstract

Depth profiles of heterotrophic bacteria abundance were measured weekly over a 6-month period from December to May inFranklin Bay, a 230 m-deep coastal Arctic Ocean site of the southeastern Beaufort Sea. Total bacteria, low nucleic acid (LNA) andhigh nucleic acid (HNA) bacteria abundances were measured using flow cytometry after SYBR Green I staining. The HNAbacteria abundance in surface waters started to increase 5–6 weeks after phytoplankton growth resumed in spring, increasing from1×105 to 3×105 cells mL−1 over an 8-week period, with a net growth rate of 0.018 d−1. LNA bacteria response was delayed bymore than two months relative to the beginning of the phytoplankton biomass accumulation and had a lower net growth rate of0.013 d−1. The marked increase in bacterial abundance occurred before any significant increase in organic matter input from riverdischarge (as indicated by the unchanged surface water salinity and DOC concentrations), and in the absence of water temperatureincrease. The abundance of bacteria below the halocline was relatively high until January (up to 5×105 cells mL−1) but thendecreased to values close to 2×105 cells mL−1. The three-fold bacterial abundance increase observed in surface waters in springwas mostly due to HNA bacteria, supporting the idea that these cells are the most active.© 2007 Elsevier B.V. All rights reserved.

Keywords: Heterotrophic bacteria; Flow cytometry; Beaufort Sea; Arctic shelves; Arctic Ocean.

1. Introduction

A very large amount of dissolved and particulateorganic matter enters the Arctic Ocean from thenumerous, large rivers discharging into this compara-tively small, strongly stratified ocean (Anderson 2002;Dittmar and Kattner 2003; Benner et al., 2005). The fate

⁎ Corresponding author. Tel.: +418 723 1986(poste)1332.E-mail address: claude_belzile@uqar.qc.ca (C. Belzile).

0924-7963/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.jmarsys.2007.12.010

of this organic material of terrestrial origin, whetherquickly re-mineralized or preserved for a long period oftime, has a significant impact on the global carbon cycle.Many biogeochemical studies have suggested a mini-mum extent of photochemical and biological reminer-alization of dissolved organic matter (DOM) in shelfwaters, resulting in the export of 25–50% of thismaterial to the North Atlantic via the East Greenlandcurrent (Benner et al., 2005 and references therein). Inthe Arctic Ocean, the low amount of incident photo-chemically active irradiance resulting from the high

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latitudes and low irradiance transmission through the icecover prevailing over most of the year makes photo-chemical reactions an unlikely candidate for a signifi-cant sink of organic material (see Bélanger et al., 2006).The reason why bacterial degradation of terrestrialorganic matter in the Arctic Ocean is low is subject todebate. The relatively weak photochemical alterationmentioned above may restrict the formation of biologi-cally labile DOM (Moran et al., 2000; Miller et al.,2002). The low temperature prevailing year-round in theArctic Ocean may also limit bacterial growth, althoughthe psychrophilic and psychrotolerant bacteria occurringin these waters (Yager et al., 2001; Connelly et al., 2006)can actively grow even at sub-zero water temperatures.Complex interactions between substrate limitation andtemperature effects are now viewed as the key to theunderstanding of the relatively low bacterial growth rateand growth efficiency often observed in polar waters(Pomeroy and Wiebe, 2001).

Laboratory experiments have greatly contributed toour understanding of bacterial growth in cold waters.However, the intricate relationships between bacterialgrowth and substrate availability at the molecular scale,grazing effects, nutrient regeneration and viral lysis aredifficult to reproduce in vitro. Time-series data ofbacterial abundance and activity offer an opportunity togain insight into the controls of bacterial growth in thenatural environment. However, spatial variabilitycoupled to water mass advection can complicate theinterpretation of time-series data acquired at a singlelocation. Only one published account of bacterialabundance over a multi-months scale exist for theArctic Ocean (Sherr et al., 2003). In the upper watercolumn of the central Arctic Ocean underneath a driftingice camp, these authors have shown a two-fold increasein bacteria abundance relative to winter values practi-cally synchronous to the phytoplankton spring bloomoccurring in June. This synchrony between hetero-trophic and autotrophic biomass increases suggested astrong dependence on autochthonous substrate forbacteria growth in the central Arctic Ocean.

Flow cytometric determination of bacteria abundancehave proven to be a very accurate (difference betweenduplicate water samples typically b7%), and fast (N20samples analyzed per hour) technique (Li et al., 1995;del Giorgio et al., 1996). When stained with afluorescent nucleic acid stain, two distinct bacterialpopulations can be identified: the low nucleic acid(LNA) and high nucleic acid (HNA) bacteria. Due totheir higher DNA and RNA content per cell, the HNAbacteria display higher fluorescence intensities. HNAcells have been shown to generally represent the more

active bacteria (Li et al., 1995; Gasol et al., 1999;Lebaron et al., 2001; Longnecker et al., 2006). The LNAbacteria were sometimes considered inactive or deadcells (Gasol et al., 1999), although there are nowevidences that they exhibit significant, although gen-erally low, metabolic activity (Zubkov et al., 2001; Maryet al., 2006; Sherr et al., 2006; Longnecker et al., 2006).In some situations, LNA per-cell activity can even behigher than HNA activity (Jochem et al., 2004; Zubkovet al., 2001). In addition to bacterial abundance, flowcytometry can therefore provide insights into relativebacterial activity.

This paper describes the depth distribution of totalbacteria and of LNA and HNA bacteria measuredweekly over a 6-month period from winter to spring inFranklin Bay, southeastern Beaufort Sea. Bacterialabundances are related to the hydrographic conditionsand phytoplankton and ice algae biomasses in orderto assess the seasonal variations and evaluate thepossible regulating mechanisms at this coastal ArcticOcean site.

2. Materials and methods

2.1. Study site and sampling

This study was conducted as part of the CanadianArctic Shelf Exchange Study (CASES), an internationaleffort under Canadian leadership to understand thebiogeochemical and ecological consequences of sea icevariability and change on the Mackenzie Shelf. Thestudy site is located in Franklin Bay, a 232 m deep,Beaufort Shelf site at 70° 02' N, 126° 18' W, 260 km tothe East of the Mackenzie River delta (for a map and amore detailed description of the site, see Riedel et al.,2006 or Seuthe et al., 2007). Water samples werecollected weekly through the moon-pool of the ice-breaker CCGS Amundsen from 10 December 2003 to28 May 2004 at 7–13 depths from just underneath theice to 10 m from the bottom of the water column. Duringthat period, Franklin Bay was ice-covered and icethickness increased gradually to reach 2.0 m at the endof the study (Riedel et al., 2006). Samples werecollected with a rosette fitted with 12 L Niskin bottlesand a SBE-9plus CTD. Because of possible contamina-tion or interference from the icebreaker, surface samples(3 and 10 m depth) were also collected at a site located500 m from the ship (sampling at this site was generallydone the day after sampling from the icebreaker).Comparison of the bacterial abundance measured at10 m in the moon-pool and at the distant site showedno significant differences (N=15, t-test P=0.783);

Fig. 1. Flow cytometric signature of the LNA and HNA bacteria.A) typical winter, near-surface populations (21 Feb. 2004, 10 m);B) typical springtime, near-surface populations (10 May 2006, 10 m);and C) typical deep-water populations (9 May 2006, 227 m). Thehorizontal black line indicates the limit between the two sub-populations. The warmer colors represent regions with a higherdensity of cells.

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bacterial abundances measured at the two sites aretherefore included in the analysis.

Samples for bacteria abundance determination werefixed with glutaraldehyde 0.1% final concentration,quickly frozen and stored at −80 °C until flow cytometricanalysis. Samples for chlorophyll a (CHLa) determina-tion were filtered onto Whatman GF/F filters, andanalyzed following 24 h extraction in 90% acetone at4 °C in the dark. CHLa concentrations corrected forpheopigments were measured with a 10-AU TurnerDesigns fluorometer (Parson et al., 1984).

2.2. Flow cytometry

Frozen samples were quickly thawed in a 30 °Cwater bath and were stained with SYBR Green I(Invitrogen) following Lebaron et al. (1998). As a slightmodification to their protocol, 500 μL of Tris-EDTA10x buffer pH 8 (Laboratoire MAT) were added to500 μL of sample in order to insure a constant pH atwhich the fluorescence quantum yield of SYBR Green Iis optimum. Dilution with the buffer also avoidedcoincidence despite the relatively high flow rate of60 μL min−1 used during the flow cytometric analysis.Two μL of a solution of 1 μm Fluoresbrite beads(Polysciences) were added to each tube as an internalstandard and allowed to verify there was no degradationof the side scatter signal despite the relatively high flowrate used. Bacteria were counted with an Epics Altraflow cytometer (Beckman Coulter) fitted with a 488 nmlaser operated at 15 mW. The green fluorescence ofnucleic acid-bound SYBR Green I was measured at525±5 nm. The analyzed sample volume was deter-mined from the change in mass corrected for a deadvolume of 50 μL (the water volume taken from thesample tube but not yet counted when data acquisitionis stopped). The cytograms obtained were analyzedusing Expo32 v1.2b software (Beckman Coulter). Thesame regions of the side scatter vs. green fluorescenceplots were ascribed to LNA and HNA bacteria for thewhole sampling period (Fig. 1). Archaea could not bediscriminated from Bacteria using this protocol; there-fore in the remaining text, bacterial abundance includesboth Archaea and Bacteria. SYBR Green I-stainedpicocyanobacteria that could be confused with hetero-trophic bacteria when using the Epics Altra defaultconfiguration were present in very low numbers duringthe whole study period (at most 50 cells mL−1, i.e. lessthan 0.05% of the bacterial abundance; S. Brugel,unpublished data).

In order to test the accuracy of the flow cytometricbacterial count method, 40 samples were stained and

counted in duplicates. The results showed this techniqueto be accurate, with a root mean squared difference of4.5% between duplicates.

Variations of the measured variables with depth andtime were plotted using Ocean Data View v.3.1(Schlitzer 2006).

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3. Results

3.1. Hydrography

The water column in Franklin Bay remained stronglystratified during the whole study period (Fig. 2A and B).

Fig. 2. Variation with depth and time of A) water temperature; B) salinity; C)in Franklin Bay between 10 December 2003 and 28 May 2004. The near-icebreaker are those appearing on the day following the full profiles.

The shallow (b30 m) Polar Mixed Layer (PML) of cold(b−1.1 °C), relatively fresh (b31) water overlaid highersalinity, Pacific-derived water. The transition betweenPML and the underlying water masses was character-ized by a sharp temperature maximum in the halocline at30–40 m depth, with the Halocline Arctic Layer (HAL)

chlorophyll a concentration; and D) total bacteria abundance measuredsurface data points corresponding to the site located 500-m from the

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grading into the Deep Atlantic Layer (DAL) of saline(34.5) and warmer (N0 °C) water at 200 m. There wereimportant differences between the depths usuallyassociated with these layers (see Carmack and Kulikov,1998) and the corresponding depths in Franklin Bay; thePacific layer was much thinner at the overwinteringstation, and the 0 °C isotherm was at 200 m instead of∼300 m as observed in the Beaufort Sea. In December

Fig. 3. Temporal variation of ice algae chlorophyll a concentration (CHLa) atvariations of C) phytoplanktonic CHLa concentration; D) total bacteria abundC–F, only data from the top 11 m of the water column are included. Black dpresented at Table 1.

2003, a hydrodynamic event pushed DAL higher up thewater column and resulted in temporarily warmer, muchmore saline water at the surface (Fig. 2A and B). Thisevent accounted for most of the seasonal variability inthe water column, especially in the first 30–40 m. Frommid-February and until the end of the sampling seasonon 28 May, very little changes in temperature andsalinity were observed. Temperature in the PML

(A) a high snow site and (B) a low snow site, and near-surface temporalance; E) LNA bacteria abundance; and F) HNA bacteria abundance. Inots represent the data included in the exponential increase calculations

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remained colder than −1.1 °C over the full study period.Salinity near the ice-water interface steadily increasedfrom 28.1 to 30.9, with most of the change occurringbefore March. Snow cover melting started only in earlyJune (Riedel et al., 2006). The stamukhi retaining thefreshwater from the Mackenzie River (see Carmack andMacdonald 2002) broke up in early June only. Thesalinity data indicate little change in the influence of theMackenzie River in Franklin Bay until the end of thesampling season.

3.2. Ice algae and phytoplankton biomass

The ice algae CHLa in the bottom 4 cm of the ice at ahigh snow and a low snow cover sites had alreadystarted to increase when sampling started in lateFebruary (Fig. 3A and B; see also Riedel et al., 2006).At all time, CHLa concentration in the bottom ice wasabout 3 orders of magnitude higher than in surface water(see below) and the concentrations at the low snowcover site were in average 5 times higher than at the sitewith high snow cover. CHLa concentration in thebottom ice increased exponentially at the relatively highrates of 0.079 d−1 and 0.088 d−1 at the low snow andhigh snow sites, respectively (Table 1).

Near-surface phytoplanktonic CHLa concentrationdecreased steadily in Franklin Bay during Decemberand January from 0.08 to 0.03 μg L−1, but started toincrease exponentially in mid-February (Figs. 2C and3C). CHLa concentration increased at an exponentialrate of 0.033 d−1 (Table 1) until it reached 0.30 μg L−1

by mid-April. CHLa concentration then decreasedduring four weeks to values near 0.10 μg L−1 beforeit increased again to reach maximum concentrations of0.41 μg L−1 in mid-May. CHLa then decreased quicklyto reach 0.14 μg L−1 on the last sampling day (28 May).The low light availability resulting from the increasing

Table 1Parameters of exponential rate of increase of ice algae chlorophyll a conheterotrophic bacteria abundance

Exponential rate ofincrease (d−1)

Correlationcoefficient

Ice algae CHLa (low snow site) 0.0793 0.968Ice algae CHLa (high snow site) 0.0882 0.978Phytoplanktonic CHLa 0.0328 0.975Total bacteria 0.0143 0.879LNA bacteria 0.0135 0.856HNA bacteria 0.0179 0.934

Exponential rates were calculated as the slope of Ln CHLa or Ln abundancsignificant, with Pb0.001). Doubling time was calculated as Ln(2) divided bof the sampling season.

ice thickness (Riedel et al., 2006) and the continuousgrowth of ice algae probably contributed to the declinein phytoplankton biomass. Photosynthetically availableradiation (PAR) measured under the ice indeed remainedlow during the full sampling period (see Riedel et al.,2008-this issue), implying that the decreases in CHLaconcentrations were not the result of decreasingintracellular concentrations of CHLa of phytoplanktoncells becoming photo-adapted to higher irradiance.Below the PML, CHLa concentration remainedb0.07 μg L−1 at all time, except on 4 May where0.12 μg CHLa L−1 was measured at 50 m.

3.3. Bacterial abundance

Total bacteria abundance varied widely with depth andtime, from 1.4×105 to 7.3×105 cells mL−1. Bacterialabundance was higher in December than during theremaining of winter, and sometimes reached relativelyhigh concentrations below the PML (as high as5×105 cells mL−1; Fig. 2D), a phenomenon not observedduring the remaining of winter and during springtime.From the end of January until the end of March, totalbacteria abundance at all depths varied within therelatively narrow range of 1.4×105 to 2.8×105 cellsmL−1. Bacterial abundance in the surface layer (0–11 m)started to increase exponentially in late March at a rate of0.014 d−1 (Fig. 3D and Table 1) to reach 4.7×105 cellsmL−1 in May. During the whole sampling period,minimum abundance generally occurred at 150 m, theapproximated location of the oxygen minimum (notshown), with higher abundances above and below thisdepth. In surface waters, bacterial abundance measuredover the whole 6-months period was positively correlatedto phytoplanktonic CHLa concentration (log bac-teria=5.633+(0.202 log CHLa); r=0.62, Pb0.001;N=72). Bacterial abundance in surface water was also

centration (CHLa), and of near-surface phytoplanktonic CHLa and

Beginning ofexponentialgrowth

End ofexponentialgrowth

Doublingtime (d)

Time lag relativeto phytoplanktonicCHLa (d)

– 8 May 2004 9 –– 28 May 2004 8 –09 Feb 2004 11 Apr 2004 21 –27 Mar 2004 16 May 2004 48 4728 Apr 2004 28 May 2004 51 7921 Mar 2004 22 May 2004 39 41

e vs. days, using the data points illustrated in Fig. 3 (all slopes werey the exponential rate of increase. 28 May 2004 corresponds to the end

952 C. Belzile et al. / Journal of Marine Systems 74 (2008) 946–956

positively correlated to ice algae biomass (low snow site:log bacteria = 5.289+ (0.104 log CHLa); r=0.76,Pb0.001; N=35; high snow site: log bacteria=5.369+(0.087 log CHLa); r=0.81, Pb0.001; N=33).

There was a clear distinction between the cytometricsignature of LNA and HNA bacteria and it remained atthe same green fluorescence intensity for the whole6 months (Fig. 1). The mean green fluorescenceintensity of the LNA bacteria (0.10–0.13 fluorescenceunits normalized to 1 μm Fluoresbrite beads) and HNAbacteria (0.35–0.45 fluorescence units) varied littleduring the whole observation period. The mean greenfluorescence intensity of HNA cells was 3.4±0.2 timeshigher than mean LNA green fluorescence, a value closeto that found off the Oregon coast (Longnecker et al.,2006) but slightly lower than measured in a wide rangeof aquatic ecosystems by Bouvier et al. (2007).

Fig. 4. Variation with depth and time of A) LNA bacteria abundance; B) HNAHNA bacteria (%HNA).

LNA and HNA bacteria abundance respondeddifferently to the autotrophic biomass increase. HNAbacteria abundance in surface waters started to increase5–6 weeks after phytoplankton growth resumed inspring, increasing from 1×105 to 3×105 cells mL−1

over a 8-week period, with a net growth rate of0.018 d−1 (Figs. 3F and 4B; Table 1).

HNA bacteria abundance in surface waters waspositively correlated to phytoplanktonic CHLa concen-tration (log HNA bacteria=5.510+(0.335 log CHLa);r=0.72, Pb0.001; N=72). LNA bacteria response wasdelayed by more than two months relative to thebeginning of the phytoplankton biomass accumulationand had a lower rate of increase of 0.013 d−1 (Figs. 3Eand 4A; Table 1). LNA bacteria abundance in surfacewaters was not correlated to phytoplanktonic CHLaconcentration (r=0.169; P=0.16; N=72).

bacteria abundance; and C) percentage of total bacteria represented by

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An interesting feature was the near-bottom increase inHNA bacteria abundance observed from early April untilmid-May (Fig. 4B). These HNA bacteria had a cytometricsignature not markedly different from those observed atthe same time near the surface, indicating of similaramount of nucleic acid per cell, and similar light scatteringcharacteristics suggesting a similar size (Fig. 1).

The percentage of total bacteria represented by HNAbacteria (%HNA) in the surface waters increased from aminimum of 41% in January to reach 71% in mid-May(Fig. 4C). In near-surface waters, %HNA was stronglycorrelated to phytoplanktonic CHLa, %HNA increasingwith increasing CHLa concentration (%HNA=72.91+(16.92 log CHLa); r=0.802, Pb0.001; N=72). Thiscorrelation was much stronger than the correlationbetween total bacteria abundance and CHLa (seeabove). The highest %HNA values were observedbelow the PSM in December and early January. Nearthe bottom, %HNA remained high for the whole studyperiod, with values in the range 60–74%.

4. Discussion

This report of bacterial abundance variations through-out the water column over winter and spring at a coastalArctic Ocean site contrasts with results obtained in thecentral Arctic (Sherr et al., 2003). While the increase inbacterial abundance was simultaneous to the phytoplank-ton biomass increase in the central Arctic (Sherr et al.,2003), in Franklin Bay itwas delayed by 41 and 79 days inthe case of HNA and LNA bacteria, respectively. The rateof increase of bacteria abundance in Franklin Bay duringtheir exponential growth in spring (0.014 d−1; a doublingtime of 48 days) was much lower than that measured bySherr et al. (2003) in the Central Arctic (0.025 d−1; adoubling time of 28 days). Surface water bacterialabundances in Franklin Bay were slightly higher thanthose measured in the central Arctic; considering the 0–40m depth zone (as in Sherr et al., 2003) average bacteriaabundance during December–May in Franklin Baywas 2.61 ± 0.81× 105 mL− 1, compared to 1.76 ±0.28×105 mL−1 in the central Arctic during Novem-ber–May. However, surface water bacterial abundances inFranklin Baywere lower than thatmeasured in the CentralArctic during summer i.e., 3.23±0.70×105 mL−1 inJune–September 1998 (Sherr et al., 2003) or 3.8±0.8×105 to 6.4±0.4×105 mL−1 during July–September1994 (Sherr et al., 1997).

It is noteworthy that over most of the Arctic winter anduntil late March, bacterial growth in surface waters wasbalanced by the loss terms, resulting in nearly constantbacterial abundances. Bacteria are known to show protein

synthesis and cell growth duringwinter even in the centralArctic Ocean (Sherr and Sherr 2003), although at rates 5–10 times lower than during summer. The marked increasein bacterial abundance measured in Franklin Bay in Aprilwas apparently not the consequence of an increase inallochthonous organic matter input from the MackenzieRiver. This lack of increase in allochthonous organicmatter is suggested by the unchanged salinity of thesurface waters. Moreover, the few reliable DOC con-centrations measurements available from the surfacewaters (14 dates between the end of February and theend of May; Lisa Miller, pers. comm.) show no increaseduring that period. The increase in bacterial abundancealso occurred before any water temperature increase sincesurface water temperature was nearly constant with valuesvarying only −1.66 to −1.69 °C from 26 February untilthe end of the study period on 28 May, ruling outtemperature as the factor preventing a bacterial abundanceincrease earlier in spring.

The delayed response of bacterial abundance relativeto autotrophic biomass increase may be explained by atight top-down control on bacteria or by a limitation ofbacterial growth by substrate availability. The relativelylow nutrient concentrations prevailing over winter andspring in the surface waters of the Beaufort Sea Shelf(Carmack and Macdonald 2002; Carmack et al., 2004)make inorganic nutrients concentration a likely regulat-ing factor in Franklin Bay. Estimations of bacterialproduction (BP) from 3H-leucine and 3H-thymidineincorporation assays showed BP to be very low in thesurface waters during March (b20 μg C m−3 d−1) but toincrease abruptly to ∼60 μg C m−3 d−1 on 11 April(Garneau et al., submitted for publication). BP duringthe remaining of April and during May was highlyvariable (∼20–160 μg C m−3 d−1) but generally higherthan during winter (Garneau et al., submitted forpublication). The BP rates estimated for the month ofMarch correspond roughly to the production of 2×103

bacteria mL−1 d−1 (assuming 10 fg C per cell), which isequivalent to 1% of the bacterial abundance at that time.From the 3H-leucine and 3H-thymidine incorporationdata, it therefore appears that BP was so low until mid-April that no significant increase in bacterial abundancecould be detected. The increase in BP on 11 Apriloccurred within two weeks of the beginning of LNA andHNA bacteria exponential increase in abundance. Thedelayed response of the bacteria in terms of abundanceand production might therefore be due to a delaybetween the autotrophic biomass increase and therelease of labile DOM by ice algae and phytoplankton.

It has been established that bacterial abundance canbe a poor indicator of bacterial activity in polar

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environments (Yager et al., 2001). In this highlyseasonal environment, balanced growth cannot beassumed and cell division may not be the first responseto new resources availability. If the main bacterialresponse in Franklin Bay was an increase in individualcell biomass, it is very likely that the fluorescence ofnucleic acid-bound SYBR Green would increase due tothe higher amount of RNA per cell often accompanyingan increase in cell size (e.g., Kemp et al., 1993).However, there was very little change in meanfluorescence intensity of the LNA bacteria (0.10–0.13fluorescence units) and HNA bacteria (0.35–0.45fluorescence units), and the variations were not relatedto the ice algae or phytoplankton biomass. An increasein %HNA was not observed before April either. Theseresults suggest that cell division and individual cellbiomass increase were both delayed relative to theinitiation of autotrophic growth. In agreement with thisview, Garneau et al. (submitted for publication) havefound a significant correlation between 3H-leucine and3H-thymidine incorporation indicating continuous bac-terial protein production coupled to cell divisionthroughout the year in Franklin Bay.

Large riverine inputs to the Beaufort Self makeFranklin Bay a site with high concentration of terrestrialDOM (Anderson 2002; Dittmar and Kattner 2003). Thefact that a bacterial production of∼2×103 bacteria mL−1

d−1 corresponding to only ∼1% of the bacteria standingstock was balanced by the bacterial loss terms over mostof the winter and spring season suggests a low transfer ofterrestrial DOM to the bacteria grazers. The highest BPmeasured in spring by Garneau et al. (submitted forpublication) i.e. 160 μg C m−3 d−1 on 15 May,corresponds roughly to 16×103 bacteria mL−1 d−1, or5% of the bacterial abundance at that time (bacterialproduction was then lower than the loss terms sincebacterial abundance was declining slightly). The transferof allochthonous carbon to the higher trophic levelstherefore represents a relatively low transfer of energyduring both seasons. Estimates of bacterivory in surfacewaters of the central Arctic Ocean were in the same rangeas those inferred in Franklin Bay from BP and bacteriaabundance dynamics (i.e. 1.2×103 to 46×103 bacteriamL−1 d−1; Sherr et al., 1997). Payet and Suttle (2008-thisissue) have found relatively large variations in viralabundance in Franklin Bay and suggested viruses areimportant agents of bacterial mortality, implying an evenlower transfer of bacterial carbon to grazers.

Most of the bacterial abundance increase was due toHNA bacteria. This is in agreement with many previousreports that these cells are the most active (Gasol et al.,1999; Longnecker et al., 2006 and refs therein).

Nevertheless, LNA bacteria abundance doubled inspring, suggesting these cells are capable of significantmetabolic activity and growth, as was often observed(Gasol et al., 1999; Lebaron et al., 2001; Sherr et al.,2006 and refs therein). Grazing can also affectdifferently HNA and LNA cells, the HNA cells beinggrazed preferentially (Gasol et al., 1999; Corzo et al.,2005). The very low %HNA observed in surface watersduring winter (i.e. b50%) might therefore be related to atight top-down control of these cells.

Although the highest bacterial abundance wasgenerally observed in the surface mixed layer, thehighest %HNAwas generally observed near the bottomof the water column. Wells et al. (2006) have foundArchaea in relatively high numbers in bottom waters ofthe Beaufort Sea, specially in nepheloid layers wherethey represented ∼20% of the Bacteria+Archaeaabundance. Similarly, in the Chukchi Sea and theCanada Basin of the western Arctic Ocean, Kirchman etal. (2007) have found Archaea relative abundance toincrease from about 10% of prokaryotes in surfacewaters to as much as 40% in samples from 100 to 200 m.In Franklin Bay, the cytometric signatures of the nearbottom cells and those near the surface were not verydifferent (Fig. 1). Because Archaea in the Arctic Oceantend to be larger than Bacteria by 1.5 to 2-fold(Kirchman et al., 2007), they likely had more nucleicacid per cell and would be HNA cells. Wells andDeming (2006) have recently found grazing rates inbottom waters of Franklin Bay during winter to be lowor undetectable; this low grazing may also explain theaccumulation of HNA cells in bottom waters.

5. Conclusions

In conclusion, bacterial abundance in Franklin Baywas tightly controlled by a combination of bottom-upand top-down factors that prevented its increase beforeApril, nearly two months after phytoplankton and icealgae growth resumed. One interesting avenue of futureresearch is to establish links between bacteria phyloge-netic composition (e.g., Garneau et al., 2006) and theirflow cytometric signatures (see Zubkov et al., 2001;Mary et al., 2006). Together with flow sorting oftargeted cells, this will improve our understanding of therelationships between bacteria phylogenetic diversityand function. The marked differences between bacterialabundance dynamics in Franklin Bay and that in thecentral Arctic Ocean (Sherr et al., 1997) also highlightthe importance of gaining a better understanding of theheterotrophic processes in the large Arctic shelvesduring winter and spring.

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Acknowledgments

We appreciated the constant support from the officersand crew of the CCGS Amundsen and the help providedby colleagues during the 6-months long experiment inthe Arctic. We also thank Julien Le Coq and NoémieDenieul for technical assistance. We thank AndreaRiedel, Christine Michel and Michel Gosselin forproviding the ice algae biomass data and Lisa Millerfor sharing unpublished DOC data. We acknowledgetwo anonymous reviewers for their constructive criti-cisms. This work was supported by grants from NSERCof Canada to S.D. and Y.G. The flow cytometry facilityat ISMER was funded by the Canada EconomicDevelopment agency. This is a contribution to theCanadian Arctic Shelf Exchange Study (CASES)research program.

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