the distribution of protozoa across a trophic gradient, factors

Journal of Plankton Research Vol.19 no.4 pp.491-518, 1997

The distribution of protozoa across a trophic gradient, factorscontrolling their abundance and importance in the plankton foodweb

Soon-Jin Hwang1 and R.T.HeathDepartment of Biological Sciences and Water Resources Research Institute, KentState University, Kent, OH 44242-0001, USA

'Present address: Ecosystem Restoration Department, South Florida WaterManagement District, West Palm Beach, FL 33416-4680, USA

Abstract The relative contribution of protozoan biomass to whole planktonic communities (phyto-plankton, picophytoplankton, bacterioplankton, protozoa and zooplankton) and factors important incontrolling protozoan abundance were investigated at two eutrophic coastal sites and two meso-oligo-trophic offshore sites in the central basin of Lake Erie, USA, from May through August in 1993 and1994. The abundance and biomass of heterotrophic nanoflagellates (HNAN) and ciliates (and alsoother plankton components) were significantly higher at the coastal sites than at the offshore sites.HNAN dominated numerically at all sites most of the time, but the biomass of phototrophic nano-flagellates (PNAN) was as high as that of HNAN, indicating that the average size of PNAN was larger.Percent protozoan carbon content was always higher at the offshore sites than the coastal sites, due torelatively lower phyto- and zooplankton biomass at the offshore sites. The percent contribution ofheterotrophic protozoans (both HNAN and ciliates) also showed the same trend. Correlationsbetween protozoan abundance and other parameters were stronger at the offshore sites than thecoastal sites. When correlating data over the coastal to offshore transect, both HNAN and ciliate abun-dances were significantly correlated with total phosphorus (TP) and the abundance of bacteria, naupliiand copepods. These results suggest that both bottom-up and top-down factors may be important incontrolling protozoan abundance, and suggest that protozoans are important as a carbon link in themicrobial food web of Lake Erie.

Introduction

Despite a long recognition of the presence of protozoa in pelagic systems, theirimportance in trophic dynamics has been recognized only recently (e.g. Mathesand Arndt, 1995). With the advent of epifluorescence microscopy (Hobbie et ai,1977; Caron, 1983), it became clear that protists are ubiquitous and comprise asubstantial fraction of the biomass of nano- and netplankton in aquatic systems(Pace and Orcutt, 1981; Porter et ai, 1985). Protozoans (mostly heterotrophicnanoflagellates) are also thought to be major consumers of bacterial andautotrophic picoplankton production (Bloem and Bar-Gilissen, 1989; Sherr et al.,1989; Weisse, 1990; Fahnenstiel and Carrick, 1991), and are themselves preyedupon by larger zooplankton (Carrick et al, 1991; Hartmann et ai, 1993; Pace andVaque, 1994; Hwang, 1995). Also, protozoan grazing of bacteria and picoplanktoncan play a pivotal role in the recycling of phosphorus and nitrogen (Jurgens andGude, 1990; Vadstein et ai, 1993; Karchman, 1994).

TYophic structure and interactions in aquatic food webs are recognized as beinghighly dynamic and much more complex than previously thought (Azam et al,1983; Porter et al, 1988; Arndt, 1993). It is, therefore, important to consider allcomponents of the planktonic food web when attempting to understand their rela-tive importance in the functioning of a partition or an entire aquatic food web.

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Unfortunately, the overall planktonic structure in aquatic systems is still poorlyunderstood. Only a few studies have examined the abundance and biomass of theoverall planktonic food web, including at least both protozoans and zooplankton(e.g. Pace and Orcutt, 1981; Mathes and Arndt, 1995).

The purpose of this study was to determine the abundance and carbon contentsof phototrophic and heterotrophic nanoflagellates and ciliates, and to identifyfactors that control the protozoan community. Also, we determined the total andrelative contribution of the protozoan biomass to the entire plankton biomass(bacterioplankton, picophytoplankton, phytoplankton, protozoa and zooplank-ton) at ecologically dissimilar study sites.

Method

Study sites

Measurements were conducted at four sites along a coastal to offshore transect inLake Erie: two coastal sites were located in the upper (site A) and lower (site B) partof Sandusky Bay, one of the most productive regions of Lake Erie, and two offshoresites (sites C and D) which were located in the central basin of Lake Erie (Figure 1).A few sites were not sampled because of weather problems (site A in June 1994, siteC in July 1993, site D in May 1994). Study sites were located during each trip by satel-lite detection using the Global Positioning System (GPS). Although the sites werelocated spatially separated, inter-site variability in terms of trophic condition, trans-parency, and phytoplankton and zooplankton assemblages was low within eachcoastal and offshore environment of Lake Erie (Herdendorf, 1975).

Sample collection and fixation

Our study focused on the growing season. Water samples were collectedmonthly between May and August during 1993 and 1994 from the middle of themixing zone at all sites (1 m at the coastal sites and 4.5 m at the offshore sites).The water depths were 2,3,15 and 18 m at sites A, B, C and D, respectively. Trip-licate water samples for water chemistry and chlorophyll a analysis were col-lected with a 4 1 Van Dorn sampler, stored in acid-rinsed 20 1 plastic bottles atambient temperature, and returned to the laboratory within 4 h. One hundredmilliliters of well-mixed aliquots were placed in separate Whirl-Pak bags forphytoplankton, picophytoplankton, bacterioplankton and protozoan enumer-ation. Triplicate zooplankton samples were collected with a Schindler-Patalastrap (61 um mesh size; 12 1 volume). Phytoplankton were preserved with 2 ml ofLugol's solution (Wetzel and Likens, 1991), and bacterioplankton were pre-served with 4% formalin (final concentration). Picophytoplankton and proto-zoan (nanoflagellates and ciliates) samples were preserved with 10 mlcacodylate-buffered glutaraldehyde (pH 7.4) to give a final concentration of2.5% in 10 mM cacodylate (Caron, 1983). Zooplankton (both rotifers and micro-crustaceans) were preserved with sucrose—formalin (4% final concentration)(Prepas, 1978), after adding 10% (v/v ratio) club soda solution (Gannon andGannon, 1975). Formalin fixation of zooplankton without adding club soda

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Protozoa in the plankton food web

•Study Location

OhioN

Locator Map

0 1000 2000 3000 4000 Kilometers

Sandusky Bay, Lake Erie

Pelee Isiandi

•D

Sandusky Bay

0 5 10 15 20 25 Kilometers

Fig. L Map of Lake Erie and Sandusky Bay showing the location of study sites. Sandusky is locatedin Huron, OH, USA.

caused a deformation of some soft-bodied rotifers (e.g. Synchaeta), which causedextreme difficulties in identification. Plankton samples were stored in therefrigerator (4-5°C) until analysis. All the filtration and staining procedures

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were conducted within 1-3 days after sampling, and filters were frozen untilenumeration.

Measurement of limnological variables

Temperature (°C), dissolved oxygen (DO, mg I"1), conductivity (mS cm"2) andturbidity (NTU) were determined with a Hydrolab (Surveyor III) multiparameterwater quality monitor. Chlorophyll a concentrations (ug I"1) were determinedaccording to standard methods [American Public Health Association (APHA),1989], and total phosphorus concentrations (nM) were determined using theUnited States Environmental Protection Agency (USEPA) (1971) modificationof the Murphy and Riley (1962) method. Secchi depth (m) was measured with a20 cm (diameter) white disk.

Bacterioplankton enumeration and biovolume estimation

Bacterial abundances were determined using the fluorometric acriflavine stainingmethod (Bergstrom et al, 1986). At least 300 cells were counted at 1000X mag-nification using epifluoresence microscopy to determine sample bacterial abun-dance (cells ml"1).

Bacterial biovolume was determined at two representative coastal and offshoresites (A and D) in 1994 using the procedure of Scavia and Laird (1987). Acriflavine-stained bacterial cells were photographed on Ektachrome EES at ASA 1600, con-comitant with filtration of 0.66 um fluorescent beads (Polyscience) as an internalsize standard. Cell images were projected onto a white screen and cell volume wasdetermined by approximating shapes to regular geometric solids (Wetzel andlikens, 1991). Mean cell biovolume (um3 cell"1) was estimated from 100 cells.

The bacterial carbon was originally determined by a conversion factor in 1993[13.2 X 10"15 g C per bacterium; the value averaged from Laws et al (1984), Leesand Fuhrman (1987), Nagata (1988), Simon and Azam (1989) and Wylie and Currie(1991)]. However, we found that average bacterial volume at the coastal sites wassignificantly greater than that at the offshore site (P < 0.001, ANOVA, n = 100). Theuse of a cell-based conversion factor resulted in the significant underestimation (P <0.05) of bacterial carbon at the coastal site in 1994, when compared with a volume-based conversion factor. For this reason, we calculated the bacterial carbon of 1993with the cellular bacterial carbon derived from the biovolume in 1994 (19.62 ± 1.68fg C per bacterium at the coastal site and 13.77 ± 1.31 fg C per bacterium at the off-shore site). Because bacterial biovolumes between sites A and B and between sitesC and D were not different (ANOVA, P > 0.1, n = 100), we used the same cellularcarbon contents within each environment. Bacterial carbon content per cell wascalculated from the measured average bacterial biovolume per cell (um3) and vari-able Cbiovolume ratios depending on the cell size (Simon and Azam, 1989).

Picophytoplankton enumeration and biomass estimation

Picophytoplankton (<2 urn) were enumerated with unstained whole lake water(only in 1994). A few (2-5) milliliters of lake water samples (triplicate) werefiltered through 0.2 um black polycarbonate filters, and the filters were frozen

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until enumeration. At least 300 cells of fluorescing plankton were counted by epi-fluoresence microscopy (1000X). We did not distinguish picophytoplanktonaccording to their different color of pigment autofluorescences, instead wecounted and measured all the picophytoplankton fluorescing both orange andred. Picophytoplankton carbon content was determined from their measuredaverage volumes (n - 50). Hie bio volume estimate was converted to carbon usinga conversion factor of 121 fg C urn"3 (Watson et al.,\911).

Our procedure for preparing and counting picophytoplankton cells may under-estimate total picoplankton abundance due to fading of weak chlorophyll-fluorescing cells during the storage (Chisholm et ai, 1988) or destruction ofdelicate cells during the filtration (Murphy and Haugen, 1985). Although we didnot verify the amount of underestimation compared with the live sample, Fahnen-stiel and Carrick (1992) found that only slight differences (average 85%, range65-118%) in chlorophyll-fluorescing cells existed between live and fixed samplesin the Great Lakes waters. Thus, we consider our estimates of picophytoplanktonabundance to be reasonably accurate.

Phytoplankton enumeration and biomass estimation

At least 300 cells were counted using the procedure of Crumpton (1987) underhigh-power magnification (400 X) using differential interference-contrast optics.Phytoplankton identification was based on morphological characteristics, basedon descriptions in Taft and Taft (1971), Prescott (1978) and Streble and Krauter(1988).

Phytoplankton carbon content was determined from measured biovolumes. Foreach species, 10 cells in each sample were measured, and volumes ((am3 cell"1)were determined by approximating shapes to geometric solids (Wetzel andLikens, 1991). Cellular phytoplankton carbon content (ug C cell"1) was estimatedfor diatom and non-diatom cells after Strathmann (1967).

Protozoan enumeration and biovolume estimation

Nanoflagellates (2-20 um) and ciliates were enumerated using the fluorometric pro-cedure of Caron (1983). Primulin staining allowed heterotrophic nanoflagellates(HNAN) and phototrophic nanoflagellates (PNAN) to be differentiated by twofilter sets, which provided different UV excitations. HNAN abundance was thedifference between PNAN and total flagellate abundance. Triplicate aliquots (5-10ml for coastal samples and 20-25 ml for offshore samples) for flagellates and ciliateswere filtered through a 1.0 um black Nuclepore filter under low-pressure vacuum(it was verified that <0.75 kPa did not rupture protozoan cells) and stained withprimulin (2.5 ug mh1, final concentration). At least 300 cells were counted at lOOOxmagnification on the stained filter using the same epifluorescence microscopymethod described above. Nanoflagellate and ciliate abundances were determinedin the same manner as the bacterial abundances. Protozoans were identified usingthe morphological descriptions of Kudo (1960), Corliss (1979), Lee et al (1985),Streble and Krauter (1988), Pennak (1989) and Thorp and Covich (1991).

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Flagellate and ciliate carbon contents were determined from their measuredvolumes. Cell biovolumes were measured in the same manner as phytoplankton.Biovolume estimates of PNAN and HNAN were converted to carbon using aconversion factor of 200 fg C um~3 (Strathmann, 1967) and 163 fg C um~3 [meanvalue of Fenchel (1982), Laws et al. (1984) and Borsheim and Bratbak (1987)],respectively. Ciliate biovolume was converted to carbon content using a factorof 110 fg C pm-3 (Weisse, 1991). Total biomass (ug C I"1) for each flagellate andciliate group was determined as population abundance times cell carboncontent.

Owing to protozoan cell shrinkage in fixed samples (Choi and Stoecker, 1989),our protozoan (especially ciliates) biovolumes and corresponding carbon con-tents may be underestimated. Choi and Stoecker (1989) demonstrated that thevolume of fixed protozoans was -20-55% lower than the cell volume of live cells.

Zooplankton enumeration and dry biomass estimation

Microzooplankton (40-200 um; rotifers and nauplii) samples were concentratedto a known volume in a plastic bottle having a 20 um mesh side window thatallowed water, but not zooplankton, to exit during rinsing of samples. Aliquotswere settled in a counting chamber, and at least 300 animals or the entire chamber(whichever came first) were counted at 80 X on an inverted microscope. Freshweights (ug) were determined from volumes (um3) of 20 individuals of eachrotifer and nauplius species (Peters, 1984). Dry weights (ug) were estimated as10% of fresh weight (Pace and Orcutt, 1981), and carbon content (ug C) was esti-mated as 48% of dry weight (Andersen and Hessen, 1991).

Macrozooplankton (>200 um; cladocerans and copepods) samples were con-centrated in the same manner as rotifer and nauplii samples, and enumerated at20-40X magnification. The entire sample was enumerated and abundances weredetermined as individuals per liter. Fresh weight was determined from an esti-mated length-weight relationship for the same species (Culver et al, 1985). Meandry weight (ug per animal) and carbon content (ug C per animal) were determinedusing the same method as for the microzooplankton.

Statistical analysis

Because picophytoplankton were estimated in 1 year (1994), their biomass wasnot included for yearly comparison of total plankton biomass. Two-way analysisof variance [Statistical Analysis Systems Institute (SAS), 1993] using dates andsites as the factors was used to compare plankton community abundance andbiomass. Correlations between protozoan abundance and other variables weredetermined with the SAS Stat PROC CORR program (SAS, 1993) for the com-bined coastal and combined offshore sites, as well as for the whole transect (allfour sites combined). The comparison-wise Type I error (a - 0.05) was amendedfor an experiment-wise error by Bonferroni correction (SAS, 1993). Thus, signifi-cant differences in correlations were defined as P < 0.004 and P £ 0.0007 insteadof P <, 0.05 and P <, 0.01, respectively.

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Results

Limnological conditions and dominant phytoplankton taxa

Filamentous cyanophytes (e.g. Oscillatoria spp. and Aphanizomenon flosaquae)dominated the coastal phytoplankton community; diatoms (e.g. Cyclotella oper-cidata, Flagilaria crotonensis and Asterionella formosa) dominated the offshorephytoplankton community (Table I). At the coastal sites, turbidity, specific con-ductivity and total phosphorus (TP) were higher than at the offshore sites(ANOVA, P < 0.05) (Table II). Secchi disk transparency and dissolved oxygenconcentrations were higher at the offshore sites (Table II). The higher turbidityand specific conductivity at the coastal sites were probably a function of sedimentloading from the Sandusky River.

Chlorophyll a, bacterial and picophytoplankton abundance, and bacterial carboncontent

Chlorophyll a concentrations were significantly greater at the coastal sites than atthe offshore sites (ANOVA, P < 0.01) (Table III). Coastal bacterial abundancewas always 2-3 times higher than offshore bacterial abundances (ANOVA, P <0.05). Chlorophyll a and bacterial abundance showed a decreasing gradient alongthe coastal to offshore transect at most times of both years. The greatest chloro-phyll a difference between coastal and offshore sites was observed in August 1994;differences in bacterial abundances along this transect were greatest in early 1993.Unlike chlorophyll a and bacterial abundance, there was no significant differencein picophytoplankton abundance between coastal and offshore sites during thesummer of 1994 (ANOVA, P > 0.05) (Table III). Picophytoplankton abundanceshowed a minimum in May, and it was similar for the rest of the months at all sites.Bacterial cellular carbon content (fg C cell"1) was always significantly higher atthe coastal sites compared to the offshore sites (ANOVA, P < 0.001). Bacterialcellular carbon varied significantly from month to month, and displayed the samevarying trends at the two sites (ANOVA, P < 0.001). The seasonally averagedcarbon content was found to be 19.62 fg C cell"1 and 13.77 fg C cell"1 at coastal andoffshore sites, respectively.

Table L Characteristics of study sites and dominant phytoplankton species. Dominant species listedwere >10% of total phytoplankton biomass. At the two coastal sites, one genus of Oscillatoriaaccounted for >50% of total biomass (range 46-90%)

Site

A

B

C

D

Description

Coastal (inner Sandusky Bay)

Coastal (outer Sandusky Bay)

Offshore (near central basinof Lake Erie)Offshore (central basin ofLake Erie)

Latitude

41°28'N

41°29'N

41°35'N

41°40'N

Longitude

82°53'W

82°45'W

82°30'W

82°10'W

Dominant phytoplankton

Oscillatoria, Aphanizomenon,VlothrixOscillatoria, Aphanizomenon,ChroococcusAsterionella, Cyclotella, Fragilaria,TabellariaAsterionella, Cyclotella, Fragilaria,Synedra

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Table IL Limnological variables at the coastal (A and B) and offshore sites (C and D) of Lake Eriein 1993 and 1994. Values of total phosphorus (TP) are means and standard errors are shown inparentheses (n = 3). ns indicates the site was not sampled

Temperature (°C)MayJuneJulyAugust

TUrbidity (NTU)MayJuneJulyAugust

Secchi depth (m)MayJuneJulyAugust

Dissolved oxygen(mgH)

MayJuneJulyAugust

Specific conductivity(mS cm-2)

MayJuneJulyAugust

TP(nM)May

June

July

August

1993

Coastal

Site A

16.422.926.226.0

187.027.024.026.0

0.520.500.450.43

9.165.306.896.10

0.4670.5130.4540.452

3448(877)3972(152)4114(341)4445(283)

SiteB

16.723.427.026.4

60.017.013.216.0

0.900.900.600.72

9.608.207.517.84

0.5130.4450.3730.359

4916(290)2711

(67)3171

(76)2325

(28)

Offshore

SiteC

12.118.1ns24.7

1.50.9

ns\2

6.914.00

ns3.40

10.208.15

ns7.85

0.2720.266

ns0.274

181(54)

1094(178)ns

160(27)

SiteD

10.116.923.624.4

131.00.40.7

6314.206.854.40

12.148.326.327.15

0.2680.27002740.270

672(12)737

(159)493(46)

1005(38)

1994

Coastal

Site A

12.3ns24.7233

130.0ns65355.1

031ns0370.45

10.80ns9337.69

0.470ns0.4500.502

5912(1393)ns

1951(221)3070(144)

SiteB

11.824.524.824.4

54.017.044.018.7

0.601.170.470.75

12.809.106.888.46

0.42003700.4000.448

1142(165)1112(105)3701(407)1455(197)

Offshore

SiteC

8.817.624.422.7

20.00.1

18.50.1

3.0310.143.813.43

11.709.308.768.96

0.2800.2800.2600.266

484(78)704(26)885

(135)1558(101)

SiteD

ns18.123.822.1

ns5.31.00.1

ns5.955.136.28

ns9.808.889.63

ns0.2910.2600.258

ns

816(157)1187(59)513

(123)

Protozoan abundance and carbon content

Despite observed variation, total protozoan abundance was not statistically differ-ent between years or sites in each region (ANOVA, P > 0.05) (Figure 2).Protozoan abundance was significantly higher at the coastal sites than the off-shore sites for both years (ANOVA, P < 0.05) and the maximum total protozoanabundance at each site was usually observed in the middle of the season (July orAugust) for both years. HNAN dominated the total protozoan abundance at mostsites for both years. Average PNAN abundances were not significantly differentbetween coastal and offshore sites (ANOVA, P > 0.05). Ciliate abundance at

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Table III. Chlorophyll a, picophytoplankton and bacterioplankton abundances, and bacterial cellularcarbon. Picophytoplankton were measured in 1994 only. Bacterial cellular carbon was measured at tworepresentative sites (A and D) in 1994 only. Standard errors are shown in parentheses (n = 3). nsindicates the site was not sampled

Chlorophyll a (jtgH)May

June

July

August

Picophytoplankton(103 cells mr-')

May

June

July

August

Bacteria(10* cells ml"1)

May

June

July

August

Bacterial cellularcarbon (fgCcelh1)

May

June

July

August

1993

Coastal

Site A

84.8(2.0)19.4(0.3)

127.9(3.1)

133.3(4.8)

17.5(1.2)19.6(13)12.4(0.4)12.6(0.5)

SiteB

51.7(0.5)33.2(2.5)77.5(3.4)58.0(0.9)

10.9(0.3)17.6(1.2)10.8(0.3)11.3(0.4)

Offshore

SiteC

1.2(0.1)0.9

(0.1)ns

10.7(0.2)

3.5(03)6.1

(03)ns

6.7(0.2)

SiteD

1.5(0.01.0

(0.1)2.4

(0.1)4.5

(0.1)

3 3(0.1)5.5

(0.1)4.6

(0.4)5.8

(0.4)

1994

Coastal

Site A

24.1(3.0)ns

133.9(7^)

149.5(4.4)

4.5(0.7)ns

42.2(6.0)39.0(1.8)

11.5(0.7)ns

16.8(0.6)153(0-5)

22.60(233)17.02(2.13)22.43(1.60)6.42

(1-52)

SiteB

50.8(0.9)8.7

(0.4)45.1(2.4)41.1(3.0)

0 3(0.06)30.6(1.1)20.6(5.0)24.9(2.0)

8.1(0.2)8.6

(0.4)13.2(0.5)11.2(0.6)

Offshore

SiteC

1.2(0.3)0.4

(0.1)5.1

(0.2)2.7

(03)

12(0.02)38.0(0-5)32.8(3.0)383(1-5)

3.8(03)3.9

(0.1)7.4

(0.4)6.2

(0-4)

SiteD

ns

0.3(0.1)6.0

(1.5)1.1

(0.2)

ns

31.4(5.5)34.5(1.0)36.5(3.8)

ns

5.2(0.4)5.3

(0.2)6.3

(0.1)

15.05(1.75)12.00(1.60)16.82(1.60)11.20(1.05)

coastal sites (range 110-113 cells ml"1) was significantly higher by an order ofmagnitude compared to the offshore sites (range 1-22 cells ml"1) (ANOVA, P <0.01). The monthly abundance variation was greatest for PNAN.

Total protozoan carbon was significantly higher at the coastal sites than at theoffshore sites for both years (ANOVA, P < 0.05). The distribution of protozoancarbon was not consistent with abundance patterns because of different cell sizesin different taxa (Figure 3). In 1993, PNAN communities contained significantly

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HNAN abundance

site A slteB siteC site D site A slteB sKeC stteO

PNAN abundance

site A afteB sltaC slta D site A slteB slteC tile D

Ciliate abundance120

s»e A site B site C

Fig. 2. Mean heterotrophic nanoflagellate (HNAN), phototrophic nanoflagellate (PNAN) and ciliateabundances (cells ml"1) in 1993 and 1994 (n = 3). Note that the scales of y-axes differ.

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120

HNAN carbon

site A site B site C slteD site A slteB slteC slteD

PNAN carbon

150 H1993 1994

site A slteB slteC sNeD site A site B site C

Ciliate carbon

s*eA HOB s**C tNaD site A slteB srteC iNaO

Fig. 3. Mean heterotrophic nanoflageUate (HNAN), phototrophic nanoflageUate (PNAN) and ciliatebiomass expressed as carbon content (jig C I"1, n = 3). Note that the scales of y-axes differ.

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more carbon than those of HNAN (ANOVA, P < 0.05). PNAN usually containedthe highest amount of protozoan carbon at the offshore sites, while HNAN andPNAN both contained the highest amount of protozoan carbon at the coastalsites. Unlike abundance, protozoan carbon content was significantly greater in1993 than in 1994 (ANOVA, P < 0.01). Although similar taxa occurred in bothyears, large PNAN occurred in a higher proportion in 1993 than in 1994. Monthlyvariation in carbon content for all protozoa and the HNAN and ciliate group wassignificant only in 1994 (ANOVA, P < 0.05). The maximum HNAN and ciliatecarbon content was observed at the coastal sites during both years (ANOVA, P <0.05). The month-to-month variation in ciliate carbon was significant only in 1994(ANOVA, P< 0.05).

Taxonomic composition

Despite large differences in protozoan abundances and biomass, most taxa wereobserved at both coastal and offshore sites. Several taxa dominated throughout thestudy period at most times. HNAN community composition was similar at bothcoastal and offshore sites during the summer. Chrysomonads {Chromulina sp. andSpurmella sp.), choanoflagellates, and a small unidentified zoofiagellate dominatedHNAN biomass at all sites during the summer (Figure 4). Chromulina sp. was thesmallest HNAN (2-3 X 3-4 um) and most numerically dominant HNAN taxon atall sites over the whole study period. Choanoflagellates such as Codosiga sp. andMonosiga sp. accounted for a similar proportion of biomass at most sites.

At both the coastal and offshore sites, PNAN biomass was dominated by crypto-monads (i.e. Cryptomonas erosa and Chroomonas minuta) and chlamydomonads(i.e. Chlamydomonas spp.) throughout most of the summer, with a prymnesiophyte{Chrysochromulina sp.) becoming more important at the offshore sites (Figure 4).Dinobryon divergens (chrysophyte) was only observed at the offshore sites and attimes accounted for a substantial fraction of PNAN biomass.

Ciliate biomass was dominated by oligotrichids and a scuticociliate both at thecoastal and offshore sites, with similar proportions of total protozoan biomass atmost sites during the summer (Figure 4). However, total abundance and speciesrichness at each date were always much higher at the coastal sites (Table IV). Also,the most abundant ciliate was different among different sites: Cyclidium at sites Aand C, Tintinnopsis at site B and three ciliates at site D. Some ciliates (Stentor,Urotricha and Vorticella) were observed only at coastal sites. Strobilidium,Tintinnopsis and Coleps were rare at the offshore site and observed only in 1 month.

Carbon allocation in planktonic communities

Total plankton carbon was significantly greater in 1993 than in 1994 (ANOVA, P <0.05), and was significantly greater at coastal sites than at offshore sites (ANOVA,P < 0.01) during both years (Figure 5). A significant monthly variation in totalplankton carbon was observed only at the offshore sites (ANOVA, P < 0.05) forboth years; the maximal carbon content was measured in July and August. Unlikeother plankton components, picophytoplankton carbon was not different betweencoastal and offshore sites (ANOVA, P > 0.05).

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The percent carbon allocation in planktonic communities differed betweencoastal and offshore sites. Percent protozoan, picophytoplankton and bacterialcarbon was much greater at the offshore sites (Figure 6). The highest percentageof protozoan carbon was always found at offshore site C, except during August1994. Both percent phytoplankton and zooplankton carbon were always higher atthe coastal sites (Figure 6).

Percent heterotrophic protozoan (both HNAN and ciliate) carbon, out ofheterotrophic consumer carbon including HNAN, ciliates and metazoans(rotifers, nauplii, cladocerans, copepods), was usually greater at offshore sites thanat coastal sites (Figure 7). Zooplankton carbon was usually greater than theheterotrophic protozoan carbon, except during May 1994, when the reverse wasobserved at all sites. HNAN accounted for most of heterotrophic protozoancarbon, yet ciliates were more important at the coastal sites.

Correlation between protozoan abundance and other variables

Correlations between protozoan abundance and other variables were generallystronger at the offshore sites than at the coastal sites (Table V). At the offshoresites, HNAN were negatively correlated with picophytoplankton, and PNAN andciliates were positively correlated, but none of the variables were significantly cor-related with protozoan abundances at the coastal sites. However, when all of thedata were pooled from both coastal and offshore sites, TP, chlorophyll a, and bac-teria, nauplii and copepod abundances were positively correlated with bothHNAN and ciliate abundances. Ciliates and rotifers were positively correlatedwith HNAN abundances.

Most of the ciliate taxa and bacterial abundance were not significantly correlatedat coastal and offshore sites; only Cyclidium had a strong negative correlation withbacteria (Table VI). However, over the whole transect, Cyclidium, Strombidiumand Strobilidium were significantly correlated (positive) with bacteria.

Discussion

Protozoans comprise a substantial fraction of the plankton food web at both theeutrophic coastal and meso-oligotrophic offshore sites in Lake Erie during thesummer. The relative contribution of protozoans, both to total plankton biomassand to total consumer biomass, was consistently greater at the offshore sites.Resource availability may be more evident than predation in explaining the vari-ation of protozoan abundance, but direct correlations also suggest that protozoansare a potentially important food source for zooplankton in the Lake Erie eco-system.

Protozoan abundance

As has been reported elsewhere (Porter, 1984; Beaver and Crisman, 1989; Car-lough and Meyer, 1989), our data showed that the highest total protozoan abun-dances and biomass levels were found at the eutrophic coastal sites. Oligotrophic

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S.-J.Hwang and HTJieath

Chromulina SputrmOa

Chrysochromulina

Chlamydomonas

Dinobryon

m •

r10

Cryptomonas erosa

ins

i \

1M4

JOHgotrichkJ ciliates Scuticociliatid ciliates

lakes are typically characterized by low ciliate densities (<10 cells ml"1), whereasmore productive lakes exhibit greater abundance (Beaver and Crisman, 1989).Porter (1984) reported the range of ciliate abundances in lakes of differing trophic

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m

m

I•• I

ChoanoflagelNda

IMS 1«(

iJ-.Uli, , B | l l .liL, Ik

HNANsp.

1-0

4'

a

Chroomonas minuta

I N

Jli J I

1t»4

ft

m

1-

Tintinnid ciliates

rJ,rJ,JI , . , , 1 I...Fig. 4. Percent biomass of dominant protozoan species. HNAN sp. is a colorless nanoflagellate (8-9x 4-5 um, width x length, with two equal length flagellae). Choanoflagellida includes both Monosigasp. and Codosiga sp. Dinobryon was observed only at offshore sites. Note that the scales of y-axesdiffer.

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Table IV. Ciliate taxa and range of abundances (cells ml"1) found during the summer of 1993 and 1994.Average abundances of two summers are shown in parentheses, nf indicates the species was not found;a single number indicates that it was found on only one date

Order

ChoreotrichidaScuticociliatidaOligotrichida

Prorodontida

HeterotrichidaSessilida

Taxa

Strobilidium sp.Cyclidium sp.Haltariasp.Strombidium virideTintinnopsis sp.Coleps sp.Urotricha sp.Stentor sp.Vorticella sp.

Site A

4-13 (6)15-77 (31)2-13 (5)4-47 (24)5-44(15)3-5(4)1-3 (2)

nf3-5(4)

SiteB

1-5 (3)2-17(11)3-30 (10)1-17 (10)2-53 (23)1-3 (2)21-3 (2)1

SiteC

11-16 (6)1-4(3)1-9(3)2nfnfnfnf

SiteD

21-7 (3)1-6(3)l-*(3)11nfnfnf

Zooplankton carbon

* « M l AC AD AA A « * C AD

• C AD

Bacterial carbon

* « AS AC AD

* « A l AC AD AA A l AC m,0

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Phytoplankton carbon

•b> tub —C * 0

Picophytoplankton carbon

A rtat 4bC HlD

Fig. 5. Mean total phytoplankton, picophytoplankton, bacterioplankton, protozoa and zooplanktonbiomass (fig C I"1, n = 3). Protozoa includes HNAN, PNAN and ciliates. Zooplankton includes rotifers,nauplii, copepods and cladocerans. Note that the scales of y-axes differ.

status as 18-71 cells ml"1 in mesotrophic lakes, 55-145 cells ml"1 in eutrophiclakes and 90-215 cells ml"1 in hypereutrophic lakes. The ciliate abundances weobserved at the coastal and offshore sites in Lake Erie are in the range ofeutrophic and oligotrophic systems, respectively (Porter, 1984).

The flagellate abundances observed during this study are similar to thosereported in other freshwater systems (cf. Carlough and Meyer, 1989). Protozoanabundances reported at our study sites are also similar to those reported in otherGreat Lakes. Typically, a range of 1-10 X 104 cells ml"1 of nanoflagellates wasreported among Lakes Huron, Michigan and Ontario, depending on their trophicstatus (Pick and Caron, 1987; Carrick and Fahnenstiel, 1989). Ciliate abundancesamong those Great Lakes were reported within the range of 1-10 cells mH(Taylor and Heynen, 1987; Carrick and Fahnenstiel, 1990).

Factors affecting protozoan abundance and distribution

The correlation analysis provides information on direct relationships among vari-ables. However, with a large set of data matrix some significant correlations(-5%) could arise by chance alone (SAS, 1993). In order to eliminate thisproblem, we corrected the significance level of correlation, which omitted some

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S--J.Hwang and RXHeath

00

ao

w

40

20

0

rJjj|

May 1993

:

'! ii; - l - i j .

n n r

1 • L: i 1J• I -J -Jm-Jm

ere

May 1994

JUM1993 June 1994

(— — 1 _ —t—

ii

§1111

July 1993 July 1994

C * D

August 1993 August 1994

Fig. 6. Percent carbon allocation (yg C I"1, n = 3) among plankton communities.

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May 1993


May 1994

Fig. 7. Percent carbon allocation (pg C I"1, n = 3) of heterotrophic consumer plankton (HNAN, dil-ates and zooplankton).

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Table V. Pearson's correlation coefficients between protozoan densities and measured variables.Correlations at the coastal and offshore sites were performed on the data of two sites in each region(n = 14; n = 7 for picophytoplankton), and also on data from all four sites (n = 29; n = 14 forpicophytoplanlcton). HNAN and PNAN represent heterotrophic nanoflagellates and phototrophicnanoflagellates, respectively. Significant correlations were defined as *P < 0.05 and **P < 0.01

Coastal sitesTemperature (°C)DO(mgH)pHTP(nM)Chlorophyll a (ugh1)Bacteria (cells ml"1)Picophytoplankton (cells mh1)HNAN (cells mH)PNAN (cells mh1)Ciliates (cells mh1)Rotifers (ind. h1)Nauplii (ind. H)Qadocerans (ind. h1)Copepods (ind. H)

Offshore sitesTemperature (°C)DO(mgh' )PHTP(nM)Chlorophyll a (fig I"1)Bacteria (cell ml"1)Picophytoplankton (cells mh1)HNAN (cell mh1)PNAN (cell ml"1)Ciliates (cells mh1)Rotifers (ind. h1)Nauplii (ind. h1)Cladocerans (ind. h1)Copepods (ind. H)

WholeTemperature (°C)DO (mg h1)PHTP(nM)Chlorophyll a (fig h1)Bacteria (cell mh1)Picophytoplankton (cells mh1)HNAN (cell mh1)PNAN (cell ml"1)Ciliates (cells mh1)Rotifers (ind. h1)Nauplii (indh1)Cladocerans (ind. h1)Copepods (ind. h1)

HNAN

-0.0040.0830.4460.5540.4380.2620.256-

-0.1950.213

-0.0230.178

-0.2860.401

-0.0760.394

-0.4960.256

-0.2720345

-0.993**-0.131

-0.2180.222

-0.052-0.2300.037

0.164-0.0090.3310.745* •0.678**0.679**

-0.051—0.1240.573*0.4060.591**0.1670.643**

PNAN

0.0480.101

-0.061-0.224-0.156-0.1950.071

-0.195_0.021

-0.397-0.081-0.498-0.378

0.706-0.4310.1140.64203990.5230.6370.131-0.801**0.5940.4220.2860378

0331-0.1210.0910.1690.1410.2470.1810.124-0.289

-0.0010335

-0.191-0.001

Ciliates

-0.4840.085

-0.0350.443

-0.1760.428

-0.5540.0210213-

-0.2010.077

-0.2820.084

0.485-0.3690.2790.3880.5710.2910.3620.801

-0.218-0.2180.4800.2380.361

0.028-0.1430.2420.742**0.456* •0.761**

-0.4260.573*0289-03360.629**0.2020.541*

significant correlations at the offshore sites. Results of the correlation analysisshould be interpreted with caution because significant correlations do not necess-arily mean direct causal relationships, and because a variable can co-vary withothers Also, an expected predation effect (negative correlation) could turn out to

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Protozoa In the plankton food web

Table VL Correlation coefficients between ciliate groups and bacterial abundance. Significantrelationships were defined as *P < 0.05, **P < 0.01 and ***P < 0.001. Numbers in parentheses aresample numbers. Strombidium and Tintinnopsis at offshore sites were not analyzed due to smallsample numbers (n = 3)

CyclidiumHaltariaStrombidiumStrobilidiumTintinnopsis

Coastal

-0.829* (9)-0.086 (13)0366 (15)0345 (6)

-0.258 (15)

Offshore

0.441 (11)0.489 (10)0.408 (12)--

Whole

0.812***0.1170.618***0.762*0.069

(20)(18)(27)(7)

(19)

be a positive correlation (resource control), presumably because of higher growthrates of prey than predation rates, or because of an indirect effect as a result ofincreases in abundances of prey with an increase in resource availability. This mayexplain some positive correlations between prey and predator relationships in thisstudy and others (e.g. James and Hall, 1995; Mathes and Arndt, 1995). Forexample, although often the entire bacterial production is consumed by HNAN(Bloem and Bar-Gilissen, 1989; Sanders et al, 1989; Hwang and Heath, 1997), thegeneral relationship between HNAN and bacterial abundances in a variety ofaquatic systems is described to be positive (e.g. Berninger et al, 1991), and thetemporal change in bacterial abundance in a system is usually in a very narrowrange (e.g. Hobbie et al, 1977).

It is clear that resources (TP and chlorophyll a) most likely control protozoanabundances over the whole transect. This is the general pattern that has beenobserved in many freshwater systems, presumably because more nutrients andphytoplankton increase protozoan abundances through direct and/or indirectcausal relationships (Porter et al, 1988; Beaver and Crisman, 1989; Laybourn-Parry and Rogerson, 1993; James and Hall, 1995).

A strong negative correlation suggests that picophytoplankton are probablycontrolled by HNAN at the offshore sites. It has been demonstrated that HNANgrazing on picophytoplankton is a major source of loss of production in LakesMichigan and Huron (Fahnenstiel and Carrick, 1991), and Lake Constance(Weisse, 1988).

Small HNAN were the dominant protozoans at all sites and their abundanceswere significantly correlated with the bacterial abundance in this study, suggest-ing the importance of bacteria in the diet of HNAN. Protozoans (mainly HNAN)are major bacterial grazers in various pelagic systems (Bloem and Bar-Gilissen,1989; Weisse, 1990; Berninger et al, 1991; Chrzanowski and Simek, 1993). With theaverage estimated grazing rate of 20 bacteria per HNAN h"1 and 650 bacteria perciliate h"1 (Hwang and Heath, 1997), both HNAN and ciliates consumed as muchas 16-30% day"1 and 12-22% day"1 of bacterioplankton at the coastal and at theoffshore sites, respectively. These estimated protozoan grazing rates on bacteriaare similar to those reported in other studies (Fenchel, 1982; Bloem and Bar-Gilis-sen, 1989; Epstein and Shiaris, 1992).

HNAN and ciliate abundances were directly correlated with zooplankton(copepods including nauplii) abundances. Zooplankton predation effect on

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protozoa was not evident with correlation analysis because of the positiverelationship. Presumably, bottom-up effects (e.g. TP, chlorophyll a and bacteria)may be stronger than top-down effects in controlling protozoan abundance.However, the significant correlations suggest the potential importance of proto-zoa as a food source for copepods. Gasol et al. (1995) found, from correlationanalysis, that when the effects of resource availability were removed, the clado-cerans became an important determinant of HNAN abundance. A significantgrazing relationship between protozoa and zooplankton at coastal and offshoresites of Lake Erie was demonstrated experimentally in another study during thesame time period (Hwang, 1995). A unit dry weight (ug) of zooplankton (bothcladocerans and copepods combined) cleared nanoflagellates at the rates of 1.20(range 0.43-4.03) and 9.36 (range 2.12-20.99) ml day"1 in a coastal (site A) andoffshore (site D) regions. These are similar to predation rates found in LakeMichigan (Carrick era/., 1991). Burns and Schallenberg (1996) demonstrated thatin a mesotrophic New Zealand lake, a calanoid (Boeckella) had significant grazingeffects on the ciliate community with a biomass specific clearance rate from 0.4 to5.2 ml ug"1 dw day"1. Protozoans are a potential food source for macrozoo-plankton because they are ubiquitous, are within the grazing size spectrum forzooplankton and are more nutritious than algae (Fenchel, 1987; Stoecker andCapuzzo, 1990). This may be the case in the Lake Erie ecosystem, although thisstudy did not show a direct negative relationship between the two. A directgrazing relationship between zooplankton (rotifers, cladocerans and copepods)and PNAN, however, was suggested at coastal sites (Table V).

The significant correlations between ciliates and both PNAN (offshore sites)and HNAN (both coastal and offshore combined) suggest that high numbers ofnanoflagellates may have supported high ciliate abundances. Many ciliates areknown to feed on a variety of food sources, including bacteria, nanophytoplank-ton and small nanoflagellates (Sheldon et al., 1986; Fenchel, 1987; Capriulo, 1990).

Ciliate abundance and composition may depend on food size and abundance aswell as habitat characteristics (e.g. Capriulo, 1990). We observed that ciliatesassociated with benthic habitats, such as Stentor and Coleps, were only observedat shallow coastal sites. Vorticella were also observed only at coastal sites, whichwas not surprising because Vorticella is a highly efficient suspension feeder (Simeket aL, 1996), and their occurrences were associated with colonies of a cyanobac-terium, Anabaena. Fenchel (1980) suggested that ~5 X 106 bacteria ml"1 was athreshold concentration to support small bacterivorous ciliates (e.g. Cyclidium).Although bacterial abundance was significantly lower at offshore than coastalsites, their abundances still often met the threshold for ciliate predation definedby Fenchel (1980). However, Cyclidium abundance was significantly related tobacterial abundance only at the coastal sites. We also found that the planktonicciliate taxonomic composition at all sites was similar. This suggests that not onlythe range of bacterial abundances at both the coastal and offshore sites may notsignificantly affect the ciliate composition, but also that mixotrophy of ciliates maybe important.

Markedly high bacterial abundance at the coastal sites, however, might affectbacterivorous ciliate abundances. The size spectrum preferred by each ciliate

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species is a function of mouth diameter, body size and morphology (Fenchel,1980). Among the coastal ciliates, small Cydidium sp. were most dominant. Giventhe resource partitioning by ciliates on the basis of body size, it was not unex-pected to find that small ciliates dominated at the coastal sites. Beaver andCrisman (1989) found that smaller ciliates (20-30 um; mostly scuticociliates) weredominant in eutrophic Florida lakes, while larger oligotrichid ciliates (40-50 |im)dominated oligotrophic systems. Because many ciliates also feed on a variety ofdifferent plankton (cf. Capriulo, 1990), the much higher ciliate abundance at thecoastal sites may also be related to significantly higher phytoplankton andnanoflagellates at the coastal sites.

Temperature is thought to be an important factor affecting the temporal andspatial distribution of protozoans (Carrick and Fahnenstiel, 1989), and their activi-ties (Sorokin and Paveljeva, 1978). A strong (but not significant) correlation wasobserved between temperature and PNAN abundance at offshore sites. Interest-ingly, the correlation between temperature and ciliate abundance at the coastalsites was negative, indicating that other factors were involved in explaining ciliateabundances. Temperature may not significantly affect protozoan abundanceunder warm-water conditions, when there is little thermal variation between thesesites.

There were no significant relationships between protozoan abundances andother parameters at coastal sites, suggesting that biological interactions might beobscured by more complicated interactions among biological and physicochemi-cal factors. The very turbid and eutrophic coastal area of Lake Erie is subject tofluctuating allochthonous carbon and nutrient inputs, which probably controlmicrobial productivity (Tranvik, 1989; Karner et ai, 1992). This fluctuation innutrient loading may make it difficult to predict temporal variation in protozoanabundance and shifts in assemblage composition.

When allochthonous carbon loading is high, usually during the summer due tomore frequent storm events, an imbalance may occur between dissolved organiccarbon (DOC) release by phytoplankton and bacterial productivity (i.e. lowerDOC release than bacterial production). Hwang (1995) found that algal DOCrelease was lower than bacterial production at the coastal site during most of thesummer, indicating the importance of allochthonous carbon as a bacterial sub-strate during this study. It may be that although the coastal region supports highprimary production, autochthonous DOC released by algae may not be enough tosupport high bacterial production. Scavia and Laird (1987) reported in LakeMichigan that the discrepancy between phytoplankton supply and bacterialrequirement for DOC during the summer was substantial. They found that thedecrease in the winter-spring carbon source during the summer was coincidentwith the decrease in epilimnetic bacterial productivity.

The role of protozoa in food web dynamics

The high proportion of protozoa to total planktonic biomass at both the coastaland offshore sites suggests the potential importance of their role as a carbon linkto higher trophic levels. Additionally, the relative percentage of heterotrophic

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protozoans to total heterotrophic carbon (consumer carbon) also emphasizesprotozoan importance. Only a few comparative studies have considered both pro-tozoans and zooplankton in freshwater systems. However, protozoan biomassaccounted for a considerable range of zooplankton biomass among a variety offreshwater systems: 3-78% (Pace and Orcutt, 1981; Mathes and Arndt, 1994);annual average of 40-48% (Carrick and Fahnenstiel, 1990; Salbrechter and Arndt,1994; Schmidt-Halewicz, 1994). The relative importance of protozoan biomass tozooplankton found in this study is higher than that found in other studies.

Functioning of the food web and carbon flux through the microbial food webmay vary with trophic conditions and the plankton community structure (Paceet al, 1990; Weisse et al., 1990; Wylie and Currie, 1991). Our findings also show thatthe relative importance of protozoan biomass to both total plankton and hetero-trophic consumer plankton biomass is greater at mesotrophic offshore sites com-pared to the eutrophic coastal sites, suggesting that the protozoans may be a moreimportant carbon link at the offshore sites. Bacterial-HNAN coupling has beenfound to be greater in systems with less bacterial abundance and weaker insystems with high bacterial abundances (Gasol and Vaque, 1993; Hwang andHeath, 1997). However, different seasons in a range of trophic systems may resultin different microbial interaction patterns The contribution of protozoans to zoo-plankton biomass has been reported to increase from 20% in mesoeutrophic lakesto -50-60% in hypereutrophic lakes during springtime (Mathes and Arndt, 1994).

Although the highly eutrophic coastal sites supported high primary production,the composition of phytoplankton may also have affected the functioning of themicrobial food web. The coastal phytoplankton community was dominated byfilamentous cyanophytes, which may not be readily grazed by zooplankton (Porterand McDonaugh, 1984; Sorrick, 1995). Instead,protozoans may have been favoredas a food source by zooplankton at the coastal sites (Hwang, 1995).

We found the same trend of picophytoplankton relative importance across thetrophic gradient as for protozoans and bacteria. The relative importance of pico-phytoplankton to total plankton carbon was much greater at offshore sites. Alsopicophytoplankton carbon was 1.1, 2.3, 9.9 and 15.6% of total phototrophiccarbon at sites A, B, C and D in 1994, respectively. This result agrees with otherfindings, in which the contribution of picophytoplankton to primary productionand phototrophic biomass increases in oligotrophic environments (Li et al., 1983;Stockner and Antia, 1986). Picophytoplankton were demonstrated to have asimilar growth rate to bacterioplankton (Weisse, 1988), and grazing mostly byHNAN and ciliates is the major cause of the loss of their production (Weisse, 1988;Fahnenstiel and Carrick, 1991). Thus, the greater proportion of picophytoplank-ton biomass at the offshore sites indicates their potential importance as a carbonsource for higher trophic levels.

Conclusions

The results of this study show that (i) protozoans are an important food web com-ponent, in terms of biomass, at sites of differing trophic status, yet their relativeimportance was more evident at meso-oligotrophic offshore sites than at the

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highly eutrophic coastal sites in Lake Erie, perhaps due to more complicatedinteractions at coastal sites; (ii) protozoan abundance is more strongly relatedwith both biotic and abiotic variables at mesotrophic offshore sites than ateutrophic coastal sites. This study suggests that protozoans may be an importantcarbon link to the higher trophic levels. The importance of protozoans may begreater in less productive systems.

Acknowledgements

The authors greatly acknowledge the suggestion of Dr Peter Larventyev on ciliateidentification and the assistance of Matt Sorrick in the field work. The commentsof Dr Karl E.Havens, Dr Alan D.Steinman, Andy Rodusky,Therese East and twoanonymous reviewers improved this manuscript. We thank Dr R. Tom James forassistance in the statistical analysis of protozoan data and Mark Brady in con-struction of the map. This study was supported by Ohio Sea Grant CollegeProgram, grant nos NA90AA-D-SG496: OSG R/ES-3 andR/ES-5, and by a doc-toral dissertation award to S.-J.H. from the International Association for GreatLakes Research.

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Received on July 31,1996; accepted on December 3,1996

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the distribution of protozoa across a trophic gradient, factors

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