Ice-brine and planktonic microheterotrophs from Saroma-ko Lagoon, Hokkaido (Japan): quantitative importance and trophodynamics

arine Systems 1II ( 1997) l49- 16 1

ers ’ a CNRS-URA 1944, BioL,-lr rmoar&e des Prutistes, hirersit Bluise Pascal ~Clemont-Ferrund II), les C&zeaux, F-631 77 Aubigre. Cedex. France b GEOTOP and D&par.ferneat des Scienc zs Biologiques, Universite du Quibec 0 Montr&al, CP 8588, Succ. ’‘Centre-Ville ’‘, Mont&al, f&t. H3C 3P8, Canada ’ INRS-Ockanologie, 310 all:e des Ursulines. Rimouski, (jut! G5E 3AI, Canada Received 11August 1994; accepted 12 Recember 1994

Biologists have rarely had the opportunity t ) investigate the community characteristics and dynamics of heterotrophic microorganisms in highly productive first-year sea ice. In this study, sterile seawater was used as a salinity buffer to extract the ice-brine microheterotroph communities (bacteria, flagellates and ciliates) from a coastal lagoon in Japan (Saroma-ko, Hokkaido; 44’N, 144”E) during the late winter (February-March) of 1992. This procedure reduced osmotic shock during the melting of ice cores and allowed the recovery of up to 323% more cells than the traditional melting method. Most of the organisms were concentrated in the bottom 3-4 cm of the ice, where abundances were up to 33 times higher than in the plankton. In ice and plankton samples, helterotrophic flagellates were dominated by small species (< 8 pm, mainly choanoflagellates) and cryothecomonad-type Icells while ciliates were dominated by a photosynthetic species, Mesodinium rubrum. in contrast to higher iatiiudes, iiic:tiseit snow cover appeared to fsor the development of protozoa beneath the relatively thin 30-40 cm ice cover of Saroma-ko Lagoon. Temporally, a successional sequence was observed between protozoa and the bacterial compartment. Bacteria decreased in abundance throughout the sampling period while protozoa increased or attained their maximum number in late winter, toward the end of the sampling period. These observations support previous suggestions of the existence of a functional microbial food web within the sea-ice community. Heterotrophic flagellate biomass greatly exceeded bacterial biomass in the sea ice (30-W X 1. Coupled with similar potential growth rates, this suggests the utilization of additional (non-bacterial) food items by ice-brine flagellates. Finally, the effects of salinity variations (ranging between 15 and 120 psu) on potential microheterotroph growth rates are discussed. Keywords: Arctic ice biota; salinity; bacteria; heterotrqphic

flagellates; ciliates; growth

uction

1.

First-year sea ice covers a maximum of ca. 14 X * Corresponding author. Phone: 33 73 40 78 36. Fax: 33 73 40 76 70. E-mail: [email protected].

IO6 km2 in the northern hemisphere (Comiso. 1986) and ca. 20 X lo6 km2 in the southern hemisphere

0924-7963/97/$17.00 Copyright 0 1997 Elsevier Scienu- B.V. All rights rese-ed. Pii s0924-7963(?6)0003~~-8

150

T. She-Ngando et al. / Jourr~d of Marine System 11 (1997) 149- 161

(Zwally et al., 1983). Aqueous interstices (brine) within sea ice constitute a habitat that differs from the water column by its smaller volume and elevated salinities, both of which influence biological activity and microorganism distribution (e.g., Grossi and Sullivan, 1985; Kottmeier and Sullivan, 1988; Garrison, 1991). The existence and diversity of microorganisms living in sea-ice brine and their hypothesized trophic interactions (Bradstreet and Cross, 1982; Garrison, 199 1) suggest that the ice biota develops a functional microbial food web. The interaction of the microbial food web with the larger polar marine ecosystem is not well known, although large consumers from the adjacent planktonic and benthic communities appear to feed on the ice biota (Conover et al., 1986). Homer (1985) and Legendre et al. ( 1992) have provided comprehensive reviews of recent studies in polar regions, most of which have dealt with algal production. With the exception of heterotrophic bacteria (Smith et al., 1989; Smith and Clement, 1990), biologists have rarely had the opportunity to investigate the quantitative dynamics of microheterotrophs in first-year ice in the Arctic. Data from the Antarctic are also very scarce (e.g., Garrison, 1991), but they clearly indicate that the ice contains a diverse heterotrophic protist assemblage (Garrison, 199 1; Garrison and Buck, 1991; Stoecker et al., 1993) that is as abundant and active in the brine inc!usions within the ice matrix as it is in the water column (Garrison et al,, 1986; Garrison and Buck, 1989). We present here findings related to the quantitative importance and trophodynamics of different microheterotrophs from both the ice and the water column of a seasonally ice-covered coastal lagoon in Japan @aroma-ko) during the late winter of 1992. In addition, we conducted experiments on osmotic shock and cell recovery as well as on the effects of salinity on potential community growth rates.

2. Material and methods

2.1. Study site hmna-ko (4’N, 144”E) is a small and highly eutrophic lagoon (Fuji, 1979) (surface area 149.2 km*, maximum depth 19.5 m, mean depth 14.5 m,

SAROMABETSU RIVER

t

I

Fig. 1. Location of Saroma-ko Lagoon on the northeast coast of Hokkaido, Japan. The detailed map shows the topography of the lagoon (contour interval 2 m) and the sampling area (

circumference 92 km; Shirasawa, 1993) located on the northeast coast of Hokkaido, Japan (Fig. 1). The water mass characteristics of this lagoon reflect those of the Okhotsk Sea (Fujiyoshi et al., 1993) but are also influenced by freshwater inputs from two major rivers that reduce salinity to less than 32 psu (Shirasawa, 1993). From 1963 to 1978, the icecovered period lasted 110 f 10 days (Kikuchi, 1979) while it has been shorter (71 f 33 days) for the past decade (Taguchi and Takahashi, 1993). During our study, the ice cover started to form in the eastern part of the lagoon in early January (general surface currents divide the lagoon in two basins, east and west) and had melted by late March (Shirasawa et al., 1993). Ice algae develop during the period of ice coverage, forming an intensely colored layer at the bottom of the ice. This is probably the most southerly occurrence of ice algae in the northern hemisphere (Taguchi and Takahashi, 1993).

et al. / Jo~~r11~~ of

T. She-Ngmdo

conditions (30-40 cm).

cm internal diameter). Two adjacent ice sites were

33

an equal mixture of hand23

2.3. Experimertts or1 osmotic shock and cell meoven)

13 8 3 0

30

120

150

33 " UI

23 18

20

0

ne extraction proce ure was compared wit the traditional melting method to study the e melt-related osmotic shock on the abundance of microheterotrophs on six replicate ice cores. For each core, both ice-brine extracts and melted residual mash-ice (from which brine had been extracted) as well as subsamples of melted ice (with brine included) were fixed and counted (as indicated below) in duplicate. Ice cores and residual ice mash were both melted in a 20°C water bath. Samples were removed from the bath as soon as the ice had melted and were never above 0 t_ l”C, which was near the in situ temperature.

80

2.4. Analytic and counting methods 33 23

Ice and plankton organisms (subsampled in sterile

18

8

0

1

4 (x h

ceIlL3)

Abundance Fig. 2. Ice-brine microheterotrophnumbers vs. ice thickness in the Saroma-ko Lagoon.

containers) were immediately fixed by adding 0.5% (v/v) alkaline Lug01 solution followed by 3% (V/V) borate-buffered formalin and a few drops of 3% sodium thiosulphate to preserve the cells and decolo&e the Lug01 solution. All fixatives were filtersterilized (0.2 pm). This method of fixation reduces dissolution of naked ciliates (Sherr et al., 1989) and allows counting of ciliates (inverted microscopy) and bacteria and flagellates (epifhtorescent miCrOSCOpy) from the same sample. Epifluorescent microscopy offers the additional possibility of distinguishing het-

152

T.Sime-Ngando et nl. /Journal

ofhfurine

erotrophic cells (e.g., absence of pigment autofluorescence). Parallel aiiquots (concentrated for plankton samples, cf. Sime-Ngando et al., 1990) were immediately observed in vivo under stereoscopic and phase-contrast microscope (Dragesco and DragescoKern&s, 1986) to identify large-sized (> 10 cl,m) taxonomic groups. Processing of samples was generally completed within two hours of sampling. Samples for enumeration of bacteria and heterotrophic flagellates were filtered (after dilution of samples with high particle concentrations) onto 0.2 and 0.8 yrn_.npre size black polycarbonate filters, respectively, using 1.2 >)-Lmpore size cellulose acetate backing filters to obtain a uniform distribution of ceils. Subsamples for bacteria counts were treated with 4’6’-diamino-phenylindole (DAPI) (Porter and Feig, 1980) while subsamples for flagellate counts were treated with primuline (Caron, 1983) before filtration. The use of primuline allowed us to distinguish flagellates with no pigment autofluorescence, considered to be heterotrophic flagellates (Caron, 1983). Filters were mounted between a slide and glass cover slip with a nonfluorescent immersion oil prior to examination with an epifluorescent microscope equipped with a neofluar objective lens of 100/1.25 X . At least 500 bacteria and 200 flagellated cells were counted and sized with a micrometer in 20 to 50 fields (cf. Sime-Ngando et al., 199 1). A blank was routinely examined to control for contamination of equipment and reagents. Cell volumes were calculated from geomct& approximations and converted to carbon assuming 220 fg C pm-” for bacteria (Bratbak and Dundas, 1984) and heterotrophic flagellates (Bgrsheim and Bratbak, 1987). This conversion factor has previously been used by several (I_ authors for sea-ice microorganisms (Kottmeier and Sullivan, 1987; Smith et al., 1989). Cihates were counted by Uterrniihl’s inverted microscope method (Sime-Ngando et al., 1990) foilowing a 24-h sedimentation of 10 to 25 ml and 100 ml subsamples for ice and plankton samples, respectively. At least 100 cells were counted. Ciliate biomasses were calculated from the mean cell v01ume of each taxon, determined by measuring ceil dimension with a micrometer and approximating the cell shapes to geometrical figures. Carbon biomass was then estimated according to Putt and Stoecker (1989) assuming that 1 pm3 = 190 fg C.

Systems II (1997) 149- I61

2.5. Experiments concerning the qfeects of salinity on potential growth of the community To examine the effects of salinity changes on microheterotroph abundance, triplicate subsamples of an ice-brine extract taken on 1 March 1992 were prefiltered to remove (1) flagellates, ciliates and large predators (2 pm Nuclepore filters), (2) ciliates and large predators (20 p,rn Nuclepore filters) and (3) large predators only (200 pm Nitex netting). They were then incubated for 24 hours in the dark at N 0°C (ice bath). To prevent substrate limitation during incubation, the < 2 pm suspensions were enriched with 0.025% (wt/v, final concentration) yeast extract and inorganic nutrients (20 FM NH: and 2 @4 PO;) while a natural bacterial culture ( IO6 cells ml- I, final concentration) was added to the < 20 pm suspensions; the < 200 pm suspensions -were enriched with natural bacterial culture ( lo6 cellsml- ‘, final concentration) and microalgae ( lo4 cells ml- ‘). Natural cultures were from the lagoon. Prior to incubation, organisms in the prefiltered suspensions were diluted with distilled water or NaCl solution to obtain fourteen salinities ranging between 15 and 120 psu. Parallel aliquots were immediately fixed as described previously to serve as controls. Counts of bacteria (in < 2 pm suspensions), heterotrophic flagellates ( < 20 pm), and ciliates ( < 200 pm) were done in duplicate and compared by analysis of variance (ANOVA) with an a posteriori test, HIP Fisher least significanr iest (FLS; Sokal and Rohlf, 1981), for salinity-induced differences.

3. Results 3. I. Physical environment Based on physical consideratitins, our sampling period could be separated into two distinct parts. From the beginning of our field experiment until 1 March, air temperatures above freezing induced melting. Field observations showed an enlargement of brine channels, likely indicating melting of brine pocket walls (Gow and Tucker, 1990). Melt rates +)f 0.66, 0.42 and 0.72 cm day - * were measured on 28 and 29 February and on 1 March, respectively (Hudier and Ingram, 1993).

Table 1 Mean ratios ot Ice-brine extractecijice-meited iibtindaiiae estim&es for sea-ice mic~obeterotrophs fro Saroma-ko Lagoon Bacteria Nutural ice site 25 Feb

28 Feb 2Mar Open ice site 25 Feb

28 Feb 2 Mar Overall mean Overall std. dev.

agellates

Ciliates

0.51 0.67 0.84

1.33 1.40 2.49

3.23 2.07 1.27

0.77 0.85 0;70 0.72 0.13

1.43 1.52 1.30 1.58 0.45

1.90 1.38 3.00 2.14 0.81

smotic shock and cell recovery

nnelited-ice

During the night of I arch, 25 cm of snob accumulated on the ice, producing a hydrostatic prcssure head that induced upward flooding by underlying seawater (Hudier and Ingram, 1993). The development of a salty slush layer above the ice was observed on 2 March. The drop in air temperature that accompanied the snow storm induced new ice growth inside the brine channel network and a

ratios

overall extraction efGciency of 0.72 &.B3 (range 0.51 to 0.85) bas samples (Table 1). from which brine bacterial counts rep mean brine estim counts in residual mash ice were generally low (< 5%) compared to estimates from brine. ie!ded

a::

3.3. Community composition eterotrophic flagellate assemblages of the Saroma-ko ice-brine and plankton community con-

Table 2 Integrated (from 0 to 0.04 m and 0 to - 1 m for ice and plankton samples, respectively) abundance and biomass ( f standard error) of microheterotrophs in Saroma-ko. Values are averages for the entire study period (late February-mid-March 1992)

Free-living bacteria (10

Length

Natural ice site

(km)

Abundance

tocellxmg

Cm’ ‘! 2.54 (0.58)

Plankton

Open ice site Biomass 0.82 (0.2)

Abundance

Biomass

Abundance

Biomass

3.1 (0.99)

1.17 (0.43)

10.96 (1.56)

3.99 (0.70)

125.9 (27.1) 149.2 (561.4) 0.67 (0.32) 4.10 (1.72) 279.9 (80.9)

8.05 (2.50) 18.49 (5.58) 6.63 (2.64) 2.89 (0.48) 36.06 (8.56)

458.7 (85.2) 271.2 (36.7) 5.54 (4.9) 33.4 (13.0) 768.8 (109.0~

1.29(0.33) 3.56 (0.47) 3.54 (2.81) 1.28 (0.38) 9.67 (2.57)

Heterotrophicflagellates (IOn cellsm’ ‘, mg Cm- ‘)

Small unidentified Cryothecomonad-type cells Chrysomonad-type cells Dinoflagellates Total

<8 12 to 22 35 to 50 16 to 50

228.9 (91.1) 161.4 (75.5) 2.10 (1.76) 6.07 ( 1.62) 398.5 (165.3)

10.10 (2.52) 31.66 (13.02) 18.57 (14.95) 4.93 (1.11) 65.25 (30.42)

Phagotrophic ciliates (IO” cellsm- ‘, mg Cm- ‘)

Prostomatids Choreotrichs + oligotrichs Scuticociliates Other ciliates Total

17 to 68 19 to 190 13to39 31 to 133

(0.12) (0.01) (0.02) (0.01) (0.13)

0.63 (0.22) 0.55 (0.22) 0.10 (0.04) 1.04 (0.60) 2.32 (0.90)

0.10 (0.03) 0.02 (0.0 1) 0.01 (0.004) 0.04 (0.02) 0.17 (0.03)

0.20 (0.12) 0.22 (0.10) 0.03 (0.02) 0.8 1 (0.38) I .26 (0.40)

0.99 (0.48) 1.31 (0.24) 0.24 (0.07) 0.03 (0.01 J 2.57 (0.68)

0.99 (0.4 1) 6.94 (0.94) 0.60 (0.13) 0.16 (0.09) 8.69 (1.14)

Other “protozoa” (lo6 cellsm’ ‘, mg Cm- 2, Mesodinium rubrum a 15to35 0.83 (0.79)

1.74 (1.64)

1.60 0.06)

3.35 (2.21)

3.39 ( 1.00)

1.35 (0.40)

a Obligatory photosynthetic ciliate.

0.27 0.04 0.05 0.02 0.38

T. She-Nlpondo

154

et al. / Jtwnnl

of Marine Systems I I (1997) 149-161

sisted mainly of small ( < 8 Frn) unidentified flagellates (dominated by choanoflagellates) and cryothecomonad-type cells (Thornsen et al., 19911, which made up > 90% of total numbers (Table 2). cells and dinoflagellates Chrysomonad-like [dominated by a small ( < 20 Frn) Gymnodinium sp.1 werealso common. The phagotrophic ciliate community was dominated in the ice-brine by prostomatids, mostly small cells ( N 18 pm) of the genus Urotricha, which constituted 2 59% of phagotrophic ciliate abundance. The piankton assembiages of these organisms were typical of those known from other marine systems (cf. Sime-Ngando et al., 1992). They mainly consisted of ciliates of the orders Choreotrichida (dominated by Strobilidium spiralis, Strombidinopsis spp. and Tintinnopsis sp.) and Oligotrichida (dominated by Strombidium spp. and Laboea spp., including L. ov$ormis, L. strobila, and L. emergens), which together accounted for more than 50% of phagotrophic ciliate numbers. In both ice-brine and plankton, small bacterivorous scuticociliates (Dragesco and Dragesco-Kerneis, 1986) of the genus-type Uronema and Cyclidium were also common along with other phagotrophic ciliates, mainly hypotrichs (e.g., Euplotes sp.), the predatory Lacrymaria sp. and Didinium sp. Total ciliate numbers in both ice-brine and plankton were largely dominated by a photosynthetic species, Mesodinium rubrum (now known as Myrionecta rubrum), which were 1.3, 2.1 and 9 times higher in number than phagotrophic ciliates for the water column, natural ice site and open ice site, respectively (Table 2). Mesodinium rubrum is not included in our analysis as we focus here on heterotrophic microbes. 3.4. Standing stock

phagotrophic ciliates, respectively. These values were considerably higher than those recorded in the plankton: 0.05-0.20 X 10’” cells mm3 (bacteria), 0.211.00 X 10’ cellsm- 3 (flagellates), and 0.80-6.88 X lo6 cells m- 3 (ciliate& This may be related to light limitation of primary production in the water column (Kishino, 1993). Chlorophyll a concentration was also considerably higher in the bottom ice than in the water column (Hattori and Saito, 1993). Heterotrophic flagellates were the dominant mi. ---LA6 r.-., ,-c. .m ~~~~~~~~~~~~~~~~ 111,a &Jms o_f carbon biomass in the sea 6

---+-

a.

Abundance Blomcss

_

\

I

I

I

I

I

I

,

kb25 F&28 Mar2 Mar5 Mar9 Marl2 Marl6 10

Open ice site

8 h

or

FdQ5 Feb28 Mar2 Mar5 Mar9 Marl2 Marl6

40 35 8

30 25

Except for bacteria, microheterotroph

standing stocks were up to two times higher at the natural ice site (snow-covered site) than at the open ice site (snow-free site) (Table 2). A similar pattern was also found for the chlorophyll a concentration, which was about 3.2 times higher at the natural site than at the open site (Robineau and Legendre, 1993). At the natural ice site, cell numbers per m3 ranged from 0 24 to 1.21 X lo’* cells m- 3, from 4.55 to 33.50 X lb9 cells mV3 and from 1.50 to 28.20 x 10~ cells m- 3 for bacteria, heterotrophic flagellates and

6

6

20 4

15 ?Q I 5

2

1

o-o kb21 F&26 F&29 Mar2 Mar5 Mar9 Marl 2 Marl 6 Sampling

Date

Fig. 3. Integrated (from 0 to 0.04 m and 0 to - 1 m for ice and plankton samples, respectively) bacterial abundance and biomass vs. sampling date in the Saroma-ko Lagoon during late winter of 1992.

8

0

50

150 1 ci cn 100

200

FebtS

Fob28

Feb28

Mar2

Mart

War5

Mar5

its

Ma9

Mr9

site

Mar12

Marl2

Ma16

Mar16

Feb25

Feb28

Mar2

Mar5

Marl2

mMar16

Feb21

Feb26

Feb29

Mar2

abundance and biomass of the different heterotrophic

Par9

Open ice sita

Fig. 4. integrated (from 0 to 0.04 m and 0 to - 1 m for ice and plankton samples, respectively) sampling date in the Saroma-ko Lagoon during late winter of 1992.

vth

250

Feb25

Natural

flagellate

ar9

groups

VS.

T. Sime-Ngando et al. /Journal of Marine Systems I I (1997) 149-161

T. She-Ngando

et al. /kxwrtnl

of

wine Systems I I

t 1907) d49--16/

lS7

Table 3 One-way ANOVA results showing effects of salinity on the abundance of sea-ice microheterotrophs after a 24-h incubation (cf. Fig. 6) Group

Source of variation

df

MS

Bacteria (IO’” cellsm-‘1 Heterotrophic flagellates (lo3 cellsm-“) Phagotrophic ciliates (lo6 cellsm-‘)

between salinities within sahnities between salinities within salinities between salinities within salinities

13 14 13 14 13 14

0.04 0.01 0.02 0.003 0.01 0.00 1

f=-ratio 5.49. /I = 0.00 16 6.20, p = 0.0009

ice (Table 2).When numbers were integrated froiii G to 0.04 m of bottom-ice thickness, heterotrtiphic flagellate biomass greatly exceeded (30-60 x ). 3.5. TroplzndWarnics

5 -

2.7

2.3 0.5

-t-=---d 0

20

40

60

Heterotrophic

0.011 0

80

100

120

flagellates

20

40

60

,

I

I

40

60

80

140

1

100

120

I

I

100

120

140

0.4

(u

0.39

k vi = 8 0.2% * F )( O.l-

acterial abundance and biomass generally decreased with time at the natural ice site and in the plankton (Fi,.0 3). In contrast, heterotrophic flagellate abundance and biomass generally increased with he at the natural and open ice sites (Fig. 4). The peak in flagellate number and biomass coincided with the minimum in bacterial abundance and biomass at the natural ice site (Figs. 3 and 4). Phagotrophic ciliate abundance and biomass peaked in March at the natural ice site and in the plankton (Fig. 5). Bacterial biomass at the naturaf ice site was significantly ,vrre!ated with the biomass of the small negatively co unidentified flagellate group ( i’= - 0.75, p < 0.05, df = 5) and with that of the prostomatid ciliates dominated by small Urotricha ( r = =*+ 0.79,p < 0.05, df= it!, both of which were observed with ingested bacteria in their food vacuoles (Eime-Ngando, in prep.). Large variations in ciliate abundance at both ice sites were apparent from late February to early March (Fig. 5), probably related to the melting ol’ brine pocket walls followed by the drainage and complete flushing of the brine channel networks that occurred during this period. 3.6. Salinity e$jkcts and potential growth

0.0

! 0

20

1

80

i

140

Salinity (oAo) Fig. 6. Effects of salinity on the abundances of ice-brine bacteria. heterotrophic flagellates, and phagotrophic ciliates after 24-h incubations in non-limiting substrate conditions. Horizontal dotted lines show the initial cell numbers (i.e.. before incubation). Error bars represent standard de\*iations for triplicate incubations.

Salinity effects on microheterotroph density and 24-h potential growth are shown in Fig. 6. Variations due to counting among replicates were generally low while variations among different samples were highly significant ( p < C.002, Table 3), indicating salinity effects. These effects were more pronounced for

158

T. She-Ngcmdo et al. / Joumal of Mclrine Sytettts 1 I f 1997) 149-161

heterotrophic protists than for bacteria. In all salinity solutions, bacteria increased by t.28- to l-48-fold. However, cell numbers in high salinity suspensions (> 70 psu) were significantly lower ( p < 0.05, FLS test) compared to other suspensions (cf. Fig. 6). The salinity range tolerance of heterotrophic protists was narrow compared to that of bacteria (Fig. 6). In both low (< 30 psu) and high (> 60 psu) salinity suspensions, flagellate and ciliate abundance decreased significantly ( p < 0.05, F’LS test), by 467% and 2-75%, respectively. In contrast, cell numbers of these organisms increased significantly ( p < 0.05, ANOVA) in intemrediate salinity suspensions (30-60 psu), which included natural ice-brine salinity ranges, up to 1.47-fold (45 psu) and 2.25-fold (50 psu) for flagellates and ciliates, respectively (Fig. 6). J. Discussion This study provides the first observations of standing stocks and trophodynamics of different microheterotroph compartments in both plankton and ice-brine biota at the southern limit of sea ice in the northern hemisphere. Our ice-brine extracting procedure allowed recovery of up to 323% more protozoan cells than the traditional melting method (Table 1). Garrison and Buck ( 1986) found that over 70% of cell losses can be prevented for both flagellates (including dinoflagellates) and ciliates by melting ice cores in a large volume of sterile seawater. In this study, the considerable variability between melting and extraction results for all microheterotrophs, and the brine to melted-ice ratios that yielded an overall extraction efficiency of < 100% for bacteria (Table l), precludes any simple use of correction factors. Bacteria present in relatively high abundances in the residual mash-ice melt (i.e., from which brine had previously been extracted) may come from the residual brine and/or from the recovery of cells frozen into the ice at the time of formation. The absence of protozoa in the mash ice may be due either to their ability to avoid being frozen into the ice or to osmotic shock during the melting of the mash ice. The importance of osmotic shock may have been clearer if we had also melted mash ice in sterile seawater. We believe that buffering salinity and OSmotic changes is important when sampling sea-ice microorganisms and that further testing of extraction

methods is warranted (e.g., effects of brine volume and salinity on extraction efficiency, brine extraction vs. melting in sterile seawater). The general domin,rnce of small flagellates (mainly choanoflagellates), cryothecomonad flageland spirotrich (e.g., lates, and prostomatio choreotrichs and oligotrichs) ciliates as well as the photosynthetic ciliate Mesodinium rubrum (Table 2) closely resembles results from sea-ice and plankton communities in the southern hemisphere (Fenchel and Lee. 1972; Corhss and Snyder, 1986; G?*rrison and Buck, 1989; reviewed in Garrison, 1991; Thomsen et al., 1991; Stoecker et al., 1993). Mesodznium rubrum, which was found at densities up to about 2 X lo5 cells l- ’ in the bottom ice of Saroma-ko Lagoon is also very common in the Antarctic, where maximum densities of 1O5 cells 1- ’ have been reported (Garrison and Buck, 1989). Higher microheterotroph standing stocks and chlorophyll a concentrations (Robineau and Legendre, 1993) at the natural ice site (snow-covered site) compared to the open ice site (snow-free site) (Table 2) suggest that reflectance, scattering, and absorption of solar irradiance by snow cover were favorable to ice biota productivity. This contrasts with observations at higher latitudes, where annual sea ice is thicker and snow cover generally inhibits development of ice biota (Gossclin et al., 1986; Palmisano et al., 1987). Sunny weather and thin ice cover (30-40 cm) prevailed during our sampling period at SW mako Lagoon. Since differences between the twf:: ice sites were on the order of 2-3 times fol most measured variables, it is important to consider “the influence of the natural spatial variability of the milieu on our interpretation of the effects J snow cover. Robineau et al. (1997-this issue), in an analvsis of spatial heterogeneity at Saroma-ko, found that snow depth and ice thickness (and thus light f.e~ls below the ice) were quite uniform throughout the lagoon. Natural variability in algal biomass, which could bias between-site comparisons, became significant at spatial scales in excess of 70 m, apparently in relation to differences in bottom-ice salinity (Robineau et al., 1997-this issue). On the basis of this analysis, Robineau et al. concluded that comparisons between the natural and open ice sites, which were < 15 m apart, were not confounded by smallscale heterogeneity.

F&25

F&28

Mar2

Mar5

Mar9

Marl2

Marl6

eier et al.,

In

0.06 0.04

16

12 10 8 6 4 2 0 Fekl

scb?6 F&29 Mar2 Mar5 Mar9 Ma 12 Mxl6

Fig. 7. Integrated (from 0 to 034 m and 0 to - 1 m for ice and plankton samples, respectiy4y) abundance and biomass of tl@ photosynthetic ciliate Mewdiniumrubrum vs. sampling date in the Saroma-ko Lagoon during late winter of 1992.

Although the photosynthetic ciliate M. rubrum was excluded from comparisons, its dynamics (Fig. 7) suggest that the photosynthetic compartment was in a decreasing phase during this study, similar to the trend observed for bacteria at the natural ice site and in the plankton (Fig. 3) as well as for chlorol?hyll a concentrations (Robineau and Legendre, 1993). Decreases in abundance and biomass of M. rubrum were evident and rapid from 25 February to 5 March at both ice sites and from 29 February to 9 March in the plankton (Fig. 7). The general increase in heterotrophic protists (such as flagellates) to a maximum

brine channel networks dtuing the beginning of our sampling period probably affected the dynamics of organisms such as phagotrophic ciliates, whit ied greatly during the early sampling period at both ice sites (Fig. 5). Our salinity-growth experiments indicated that protozoan communities were more affected by changes in brine salinity than bacteria since they grew faster in the suspensions with salinities near that of the natural brine (Fig. 6). The observed successional sequence suggests a strong trophic coupling between heterotrophic protists and bacteria in Saroma-ko Lagoon during late winter. This may have involved predator-prey interactions, as the biomass of bacterivorous protists was significantly negatively correlated with bacterial biomass at the natural ice site. However, the average heterotrophic flagellate biomass in the ice was much higher (30-60 X ) than the bacterial biomass (Table 2), even though the growth rate experiments showed both groups to have similar potential growth rates at the natural brine salinity range (Fig. 6). This suggests that bacteria cannot meet the energy demands of the bacterivorous flagellates and that the latter require additional (non-bacterial) food items for growth and survival. Other possible flagellate food sources include viruses (Gonz’alez and Suttle, 1993) and high molecular weight dissolved organic matter (Marchant and Scott, 1993: Tranvik et al., 1993) as

160

T. Sittte-Ngcurdo et cd./Jowtrtrl of Muritw S_vstcttt.s I I (1997) 149-161

well as microalgae or other protists for larger-sized flagellates. Further evidence for the use of alternative food sources by bacterivorous flagellates in Saromako comes from a study by Marchant (1990), who found that Diaphauoecn grundis, a loricate choanoflagellate that was abundant in the bottom-ice community of Saroma-ko Lagoon in March 1988, was not able to ingest microspheres larger than 1 p,m in size. Bottom-ice bacteria in our study were generally larger (l-3 p,m), while approximately l/6 of the total flagellate biomass (at the natural ice site) was composed of very small choanoflagellates ( < 8 pm) (Table 2). The apparent shortage of bacterial prey and their inaccessibility to a significant fraction of the flagellate biomass both suggest the existence of important energy flow pathways to the flagellate compartment other than that provided by bacterivory. We are presently completing analyses of bacterivory in Saroma-ko Lagoon that should permit us to quantify the energy flow from bacteria to bacterivorous protists. Acknowledgements We thank R. Gagnon for technical assistance and the advisors and participants of the SARES project for collaboration during time spent on the Saroma-ko Lagoon. Thanks are also extended to the staff of the Saroma Tohayo-Kumial Laboratory (Hokkaido, Japan) for logistic support, to Drs. D. Stoecker and K. Buck for helpful comments, and to Dr. J.-C. Therriault and L. Devine for correction of the English text. Dr. E. Hudier provided physical data and Dr. B. Robineau supplied the map of Saroma-ko LagoonThis work was supported by the Japan Science and Technology Fund (JSTF, External Affairs and International Trade Canada) and the National Sciences and Engineering Research Council of CanadaThis is a contribution to the SARES @aroma-Resolute Studies) joint Canada-Japan project and to the CNRS-URA 1994 (Biologie ComparGe des Protistes) Research Programmes. References Borsheim, K.Y. and Bra&k. G.. 1987. Cell volume to cell carbon conversion fxtor for a bacterivorous Moms sp. enriched from seawater. Mar. Ecol. Prog. Ser., 36: I71 - 175.

Bradstreet, M.S.W. and Cross, W.E., 1982. Trophic relationships

at high arctic ice edges. Arctic, 35: l-12. Bratbak, G. and Dundas, I., 1984. Bacterial dry matter content and biomass estimations. Appl. Environ. Microbial., 48: 755-757. Caron, D.A., 1983. Technique for enum_.ation of heterotrophic and phototrophic nanoplankton, using epifluorescence microscopy, and comparison with other procedures. Appl, Environ. Microbial., 46: 49 l-498. Comiso, J.C., 1986. Characteristics of Arctic winter sea ice from satellite multispectral microwave observations. J. Geophys. Res.. 91: 975-994. Conover, R.J., Herman, A.W.. Prinsenberg. S.J. and Harris, L.. 1986. Distribution of and feeding by the copepod Pseudocu1utut.sunder fast ice during the arctic spring. Science, 232: 1245- 1247. Corliss, J.O. and Snyder, R.A., 1986. A preliminary description of sevecxl new ciliates from the Antarctic, including Cohtziimbus grusrei n. sp. Protistologica, 22: 39-46. Dragesco, J. and Dragesco-Kerneis, A.. 1986. CiliCs libre!: de I’Afrique intertropicale. Introduction B la connaissance 1’6ude des Cilih. Orstom, Paris. 560 pp.

et 3

Fenchel, T. and Lee, CC., 1972. Studies on ciliates associated with sea ice from Antarctica. I. The nature of the fauna. Arch. Protistenkd., 114: 23 I-236. Fuji, A., 1979. Enviromnental capacity of coastal water determined by the aquaculture of scallop. Bull. Coastal Oceanogr., 17: 44-49 (in Japanese). Fujiyoshi, Y.. Shibanuma, S. and Kajiwara, M., 1993. Variability in water mass characteristics in Lake Saroma. Bull. Plankton Sot. Jpn., 39: 158- 159. Garrison, D.L.. 1991. Antarctic sea ice biota. Am. Zool., 31: 17-33. Garrison, D.L. and Buck, K.R.. 1986. Organism losses during ice melting: a serious bias in sea isc cot,imunity studieh. Polar Biol., 6: 237-239. Garrison, D.L. and Buck, K.R., 1989. The biota of Antarctic pack ice in the Weddell Sea and Ann&c peninsula regions, Polar Biol., 10: 211-219. Garrison, D.L. and Buck, K.R., 1991. Surface-layer sea ice assemblages in Antarctic pack ice during the austral spring: eavironmental conditions, primary production and community structure. Mar. Ecol. Prog. Ser., 75: 161- 172. Garrison, D.L., Sullivan, C.W. and Ackley, SF., 1986. Sea ice microbial communities in Antarctica. Bioscience, 36: 243250. Gonzhlez, J.M. and Suttle, C.A., 1993. Grazing by marine nanoflagellates on viruses and virus-sized particles: ingestion and digestion. Mar. Ecol. Prog. Ser., 94: l-10. Gosselin, M.. Legendre, L., Therriault, J.-C., Demers, S. and Rochet, M., 1986. Physical control of the horizontal patchiness of sea ice microalgae. Mar. Ecol. Prog. Ser., 29: 289-298. Gow, A.J. and Tucker III, W.B., 1990. Sea ice in the polar region. In: W.O. Smith (Editor), Polar Oceanography. Academic Press, San Diego, CA, pp. 47- 122. Grossi, SM. and Sullivan C.W., 1985. Sea ice microbial communities V. The vertical zonation of diatoms in an Antarctic fxt ice community. J. Phycol., 21: 401-409.

1993. Vertical distribution of copepods and die1 change of their feeding un er ice in Lake Saroma. ull. Plankton Sot. Jpn., 39: 167-169. Ho R.A., 1985. Ecolog gae. . er (Editor), Sea Ice oca , pp. 83- 103. Hudier, E.J.-J. and Ingram, R.G., 1993. Effect of sea water percolation through first year ice. Proc. 8th Intl Symp. Okhotsk Sea and Sea Ice, pp. 3.51-354 (extended abbtract). Kikuchi, K., 1979. Water quality of Lake Saroma during the period from 1967 to 19’*1. J. Hokkaido Fish. Exp. Sm., 36: 233-254 (in Japanese). Kishino, M., 1993. Spectral light environment in/under sea ice in Lake Saroma. Bull. Plankton Sot. Jpn., 39: 161-163. Kottmeier, S.T. and Sullivan, C.W.. 1987. LLte winter primary production and bacteria: production in sea ice and seawater west of the Antarctic Peninsuia. Mar. Ecol. Prog. Ser., 36: 287-298. Kottmeier, S.T. and Sullivan, C.W., 1988. Sea ice microbial communities (SIMCO). 9. Effects of temperature alld salinity on rates of metabolism and growth of autotroph*j and heterotrophs. Polar Biol., 8: 293-304. Kottmeier, S.T., Grossi, S.M. and Sullivan, C.W., 1987. Sea ice microbial communities VIII. Bacterial production in annual sea ice of McMurdo Sound, Antarctica. Mar. Ecol. Prog. Ser., 35: 57%i86. Legendre, L., Ackley, S.F., Dieckmann, G.S., Guliiksen, B., Homer, R,, Hoshiai, T., Melnikov, I.A., Reeburgh, W.S., Splindler, M. and Sullivan, C.W., 1992. Ecology of sea ice biota. 2. Global significance. Polar Biol., 12: 429-444. Marchant, H.J., 1990. Grazing rate and particle size selection by the choanoflagellate Diaphanneca grartdis from the sea-ice of lagoon Saroma ko, Hokkaido. Proc. NIPR Symp. Polar Biol., 3: l-7. Marchant, H.J. and Scott, F.J., 1993. Uptake of sub-micrometer particles and dissolved organic material by Antarctic choanoflagellates, Mar. Ecol. Prog. Ser., 92: 59-64. Palmisano, AC., SooHoo, J.B., Moe, R.L. and Sullivan, C.W., 1987. Sea ice microbial communities. VII. Changes in underice spectral irradiance during the development of Antarctic sea ice microalgal communities. Mar. Ecol. Prog. Ser., 35: 165173. Porter, K.G. and Feig, Y.S., 1980. The use of DAPI for identifying and counting aquatic microflora. Limnol. Oceanogr., 25: 943-948. Putt, M. and Stoecker, D.K., 1989. An experimentally determined carbon:volume ratio for marine “oligorrichous” ciliates from estuarirte and coastal waters. Limnol. Oceanogr., 34: 10971103. Robineau, B. and Legendre, L., 1993. The core program at Saroma-ko: biomass of algae. Proc. 8th Intl. Symp. Okhotsk Sea and Sea Ice, pp. 373-374 (extended abstract). Robineau, B., Legendre, L., Fortier, L., Kishino, M. and Kudoh, S., 1997. Horizontal heterogeneity of microbial biomass in the

1. Plankton Sot. Jpn., 39:

Ingram, G., Belanger, C., Peltola, P., Takahashi, S., suyama, M., udier, E., Fujiyoshi, Y., Kodama, Y. and Ishikawa, N.. l993.‘Atmospheric port for Saroma-ko Lagoon of the Studiesj Project, 1992. Lqw Temp. 69-167. Sime-Ngando, T., Hartmann, .J. and Groli&re, CA., 1990. Rap quantification of planktonic ciiiates: comparison of im live counting with other methods, Appl. Environ. Microbial., 56: 2234-2242. Sime-Ngando, T., Bourdier, G., Amblard, C. and Pinel-Alloul, B., 1991. Short-term variations in specific biovolumes of different bacterial terms in aquatic ecosystems. Microb. Ecol., 21: 2 11-226. Sime-Ngandn, T., Jttniper, K. and V&zina, A., 1992. Ciliated protozoan communities over Cobb Seamount: &crease in biomass and spatial patchiness. Mar. Ecol. Prog. Ser., 89: 37-51. Smith, R.E.H. and Clement, P., 1990. Heterotrophic activity and bacterial productivity in assemblages of microbes from sea ice in the high Arctic. Polar Biul., 10: 351--357. Smith, R.E.H., Clement, P. and Cota, G.F., 1989, Population dynamics of bacteria in arctic sea ice. Kicrob. Ecol., 17: 63-76. Sokal, R.R. and Rohlf, F.J., 1981. Biometry. Freeman, New York, NY, 859 pp. Stoecker, D.K., Buck, K.R. and Putt, AI., 1993. Changes in the sea-ice brine community during the spring-summer transition, McMurdo Sound, Antarctica. II. Phagotrophic protists. Mar. Ecol. Prog. Ser., 95: 103-113, Takuchi, S. and Takahashi, M., 1993. Summary of the Plankton Colloquium on the ecosysrtem of Lake Saroma under sea Ice covered condition in winter. Bull. Plankton Sot. Jpn., 39: 152-156. Thornsen, H.A., Buck, K.R., Bolt, P.A. and Garrison, D.L., 1991. Fine structure and biology of Crpthecomonas gen. nav. (Protista incertae sedis) from the ice biota. Can. J. Zool., 69: 1048- 1070. Tranvik, L.J., Sherr, E.B. and Sherr, B.F., 1993. Uptake and BJtilizationof “colloidal DOM” by heterotrophic flagellates in seawater. Mar. &ol. Prog. Ser., 92: 301-309. Zwally, H.J., Parkinson, CL. and Comi,io, J.C., 1983. Variability of Antarctic sea ice and clunges in. carbon dioxide. Science, 220: 1005-1012.