limnol. oceanogr., 35(4), 1990, 7954308 q 1990, by the ...€¦ · depth intervals with a yellow...

Limnol. Oceanogr., 35(4), 1990, 7954308 Q 1990, by the American Society of Limnology and Oceanography, IIIC.

Planktonic community structure determines the fate of bacterial production in a temperate lake

Michael L. Pace, George B. McManus, 1 and Stuart E. G. Findlay Institute of Ecosystem Studies, New York Botanical Garden, Box AB, Millbrook 12545

Abstract

We examined the fate of planktonic bacterial production and the balance between bacterial growth and grazing mortality in the surface waters of Upton Lake, New York. Growth rates were measured by the incorporation of t3H]thymidine into DNA. Grazing rates on bacteria were determined with small cells produced by a mutant strain ofEscherichia coli and made either fluorescent or radioactive to monitor feeding. Bacterial community turnover times calculated from either growth or grazing rates ranged from 1.5 to 16 d. On the basis of these data and results from 29 other studies, most bacterial communities appear to have turnover times substantially > 1 d. Our measurements of feeding rates on bacteria frequently exceeded estimates of growth. Limitations of precision and doubts about the accuracy of methods make attempts to balance measurements of bacterial growth and grazing with current techniques unrealistic. The fate of bacterial production depends on planktonic community structure. Flagellates were the primary consumers of bacteria in winter and fall. At other times, Daphnia galeata consumed most of the bacterial production. Ciliates and rotifers were never important bacterial grazers. In Upton Lake large populations of Daphnia effectively “break” the microbial loop and funnel bacterial production to higher consumers.

In the plankton of most aquatic ecosystems, bacterial production is -30% of primary production on an areal basis (Cole et al. 1988). If we assume growth efficiencies of lo-50% (Pomeroy and Wiebe 1988), planktonic bacteria process more than half of the organic C produced in aquatic systems. Furthermore, these organisms either recycle or sequester substantial quantities of nutrients depending on nutrient conditions and trophic interactions (Caron et al. 1988).

The fate of bacterial production is uncer- tain. Bacteria are an important resource for the appendicularian Oikopleura dioica, the chrysophyte Dinobryon spp., and a variety of ciliates as determined by direct measurements of feeding on bacteria relative to

1 Current address: Chesapeake Biological Lab, Uni- versity of Maryland, Box 38, Solomons 20688.

Acknowledgments We thank B. Peierls, D. Lints, E. Funke, N. Under-

wood, and M. Holland for help in the lab and field. The help of A. Vasconcelos with aspects of the minicell methodology is acknowledged. Mr. and Mrs. C. Wood provided access to Upton Lake. S. Baines, H. Cyr, B. Riemann, D. Scavia, and an anonymous reviewer provided criticisms of the manuscript.

Our work was supported by the Mary Flagler Cary Charitable Trust and NSF grant BSR88-05893.

This paper is a contribution to the program of the Institute of Ecosystem Studies, the New York Botanical Garden.

estimates of consumer energetic demands (Kingetal. 1980;BirdandKalff1987,1989; Sherr and Sherr 1987). In contrast, crustacean zooplankton do not feed or grow efficiently on bacteria (see McManus and Fuhr- man 1988).

Early food-web models that included bacteria suggested that bacterial production was efficiently funneled to higher consumers (Pace et al. 1984; Fasham 1985). On the other hand, a direct experimental test of this idea by Ducklow et al. (1986) revealed that very little of the organic C incorporated by bacteria is passed up the food web. Recent theoretical analyses and reviews also suggest that bacteria are not a major source of C to higher consumers. Rather, the protozoans and microzooplankton which consume a combination of bacteria, cyanobac- teria, and eucaryotic algae represent a key linkage to higher trophic levels (Sherr et al. 1986; Vezina and Platt 1988; Sherr and Sherr 1988). Although the latter view is compelling, few direct simultaneous measurements of grazing on bacteria by the various potential consumers in the pelagic zone have actually been made.

Planktonic community structure may determine the pathway and degree of utilization of bacterial production by higher consumers. Bacteria are grazed by a wide variety

795

796 Pace et al.

of protozoans and metazoans, including mixotrophic algae, heterotrophic flagellates, ciliates, microzooplankton, and macrozooplankton (see Pace 1988; McManus and Fuhrman 1988). The relative abundance of these consumers varies dramatically among systems and seasonally within a system. If bacteria are grazed primarily by other microbes, then relatively little bacterial production will be transferred to higher consumers because of respiratory losses at each trophic step. On the other hand, direct pre- dation on bacteria by microzooplankton and macrozooplankton could funnel a significant fraction of bacterial production up the food web.

Here, we report measurements of bacterial growth and grazing on bacteria made in a series of experiments in a small, temperate lake. One purpose of our study was to assess the effect of community structure by measuring grazing on bacteria by both metazoans and protozoans under various community configurations. Determination of the major grazers allows us to assess the fate of bacterial production. In addition, simultaneous measurements of bacterial abundance and production allow us to assess the balance between bacterial growth and grazing to provide independent estimates of the turnover time of bacterial communities.

Methods Study site description and routine obser-

vations- Our study was carried out in Up- ton Lake, a small (18 ha) eutrophic system located in Dutchess County, New York. At a central station, weekly measurements of temperature and oxygen were made at l-m depth intervals with a Yellow Springs In- strument 57 meter. Upton Lake is covered with ice for -3 months. Thermal stratifi- cation developed rapidly in concert with ice- out and was maintained until late Novem- ber. Throughout the year, bottom waters were either anoxic or contained < 1 mg O2 liter- l.

Triplicate water samples were taken weekly from 2 m for estimates of chlorophyll and bacterial abundance. On the basis of previous profiles, these samples were rep- resentative of mixed-layer conditions. Sam- ples for chlorophyll were filtered through a

GF/F filter, frozen, and subsequently extracted in methanol. Chlorophyll was determined fluorometrically with corrections for pheopigments (Parsons et al. 1984). Bac- terial abundance was determined by staining 0.5-ml subsamples with DAPI (4,6-diamin- dino-2-phenylindole) at a concentration of 5 pg ml-l, otherwise following Porter and Feig (1980).

Experimental procedures-Five experiments were conducted at different times in 198 8 to represent different planktonic community configurations. An important feature of our study was the replication of all measures. A second important feature was that experiments were conducted during the day and at night on four of the five occa- sions. Crustacean zooplankton feeding in lake surface waters varies dramatically on a diel cycle because of vertical migration and increased feeding at night (Haney 198 5). It has also been suggested that bacterial growth or protozoan grazing may vary sys- tematically over 24 h (Hagstrom and Lars- son 1984; Fuhrman et al. 1985; Wikner et al. 1990). Our day and night measurements allowed us to assess this diel variation in growth and feeding rates.

On each occasion we collected triplicate water samples from 2-m depth to measure chlorophyll, bacterial abundance, bacterial growth, grazing on bacteria, and the abundance of protozoans and metazoans. Water was collected by pooling several van Dorn samples into three separate 8-liter buckets. Buckets were returned to the lab and sub- sampled for chlorophyll and bacterial abundance as noted above. Abundances of heterotrophic and mixotrophic flagellates were determined in glutaraldehyde-preserved samples examined by epifluorescence microscopy (Haas 1982). A loo-ml sample from each bucket was preserved,by adding 1 ml of Lugol’s solution. Ciliates were later enumerated by settling a 1 O-25-ml subsample and scanning the entire subsample at 200 x with an inverted microscope. Micro- zooplankton were concentrated by passing 1 liter of water through a 3 5-pm sieve. These samples were preserved in a sucrose-For- malin solution and subsequently counted with an inverted microscope at 100 X magnification. Triplicate samples of macrozoo-

Fate of bacteria 797

plankton were taken with a pump described by Pace (1986). The outlet hose of the pump was passed through a 73-pm net. Samples were preserved in a sucrose-Formalin solution and enumerated with a stereomicro- scope at 25 x magnification.

Bacterial production -We measured bacterial production with the C3H]thymidine method following the protocol of Findlay et al. (1984). Preliminary evaluations indicat- ed that, based on three experiments conducted in 1987,25-50 nM of thymidine was sufficient to saturate incorporation and that uptake of [3H]thymidine into DNA was linear for at least 2 h. Isotopic dilution was estimated following the procedure of Find- lay et al. (1984) on a single occasion and was at most twofold.

During each experiment, we took six replicate lo-ml subsamples from each of three buckets (total, 18) and added 40 PCi of methyl [3H]thymidine (sp act-80 Ci mmol-l). Two of the six replicates were immediately killed by adding 2 ml of 5% For- malin to control for nonbiological absorp- tion of the radiolabel. Samples were incubated for 1 h. Incorporation of [3H]- thymidine was stopped by adding 2 ml of 5% Formalin. Samples were filtered on 47- mm, 0.2-pm polycarbonate filters and washed with ice-cold 5% trichloroacetic acid (TCA). Filters were frozen and subsequently extracted to isolate DNA following Findlay et al. (1984). Bacterial production was calculated from the radioactivity incorporated into DNA and a conversion factor of 2 x 1 018 cells produced per mole of thymidine incorporated (Fuhrman and Azam 1982; Moriarty 1986; Riemann et al. 1987). We checked our conversion factor (2 x 1 Ols) on one occasion by comparing bacterial growth in a dilution culture of lake water with thymidine incorporation into DNA. This dilution experiment was conducted in Feb- ruary 1988 over 46 h at lake water temperature (4°C). The factor 2 x lOI cells produced per mole thymidine incorporated gave good agreement between actual growth rates based on bacterial counts and thymidine incorporation converted to cell in- creases.

Minicell preparation-We used a modi- fication of the method of Wikner et al. (1986)

to measure grazing on bacteria. This technique exploits mutant strains of Escherichia coli, which divide to produce a small cell containing no DNA and a normal cell with a full complement of DNA. The small, agenomic cells are called minicells. They have the desirable properties of being both similar in size to most planktonic bacteria (0.6-0.7 pm) and incapable of cell division.

The minicell-producing strain X- 148 8 was obtained from the E. coli Genetic Stock Center, Yale University, and maintained on agar plates. In order to produce and isolate large numbers of minicells, we grew E. coli cultures overnight in Luria Bertani medium (Maniatis et al. 1982). Minicells were iso- lated from larger cells by sucrose-gradient centrifugation. Initially, the overnight culture was centrifuged in a Sorvall RCB-2 centrifuge with a GCA-fixed rotor at 2,000 rpm for 5 ‘min to remove debris and some large cells. The supernatant containing a mixture of minicells and larger cells was then sedi- mented by centrifugation at 6,000 rpm for 20 min. The pellet was resuspended in 2-3 ml of buffered gelatin saline (BSG-0.85% NaCl, 0.03% KH2P04, 0.06% Na,HPO,, and 100 pg of gelatin ml-l).

Cells were gently layered over 30 ml of sterile 5-20% (wt/vol) sucrose gradients and centrifuged at 4,000 rpm for 15 min in the centrifuge with an HS-4, swinging-bucket rotor. Bands of purified minicells were re- moved from the sucrose gradients, pelleted by centrifugation at 6,000 rpm for 20 min, and resuspended in BSG. The gradient centrifugation was repeated. Final concentrations of minicells were 1 Og-1 010 cells ml-l, and those of large cells were on the order of 1 O5 cells ml-‘. The minicells are diluted 4- 5 orders of magnitude in the feeding experiments (see below), so the residual large cells are present in the experiments at negligible concentrations.

Minicells were made either fluorescent to follow ingestion by protozoans or radioactive to follow ingestion by metazoans. Flu- orescent minicells were produced by labeling with 5-(4,6-dichlorotriazin-2-yl) aminoflu- orescein (DTAF), following the method of Sherr et al. (1987). Cells were stored in the refrigerator for 24-48 h before use. Imme- diately before using minicells in grazing ex-

798 Pace et al.

periments, suspensions were dispersed with a sonicator and subsamples counted with epifluorescence microscopy.

Radioactive minicells were produced by incubating cell suspensions in BSG with 250 ,uCi 13H]methionine (sp act - 80 Ci mmol-l) overnight at 37°C. To remove any large cells which may have grown in the overnight cultures, we layered minicells onto a sucrose gradient and separated them as described above. Radioactivity per cell was determined by filtering 0.1 ml of the final minicell suspension on 0.2~pm polycarbonate filters. Filters were washed twice with 2 ml of BSG to remove any unincorporated label, added to a scintillation vial, boiled for 1 h in 5% TCA, and radioassayed. Minicell concentrations were determined via acridine orange direct counts (Hobbie et al. 1977). We observed that the specific activity of the minicells (dpm cell-l) was constant between day and night experiments, implying that the minicells were not catabolizing the label between experiments while stored in cold BSG.

Grazing measurements -Three types of grazing measurements were conducted in each experiment. Total consumption of bacteria by all members of the community was measured by following the disappearance of fluorescent minicells in l-liter samples of lake water over 24 h. Grazing by heterotrophic flagellates, mixotrophs, and ciliates was measured by the ingestion of fluorescent minicells in 20-60-min experiments (depending on temperature). Grazing by macrozooplankton (>200 pm) and microzooplankton (3 5-200 pm) was measured by the incorporation of radioactive minicells in 30-min experiments.

Disappearance of fluorescent minicells was determined by adding minicells to 1 -liter subsamples in Whirlpak bags at 10% of natural bacterial concentrations. Controls were 0.2-pm-filtered lake water with added minicells (March, April, July experiments) or glutaraldehyde-poisoned lake water (July, September, October experiments). Experi- ments were run in the dark at lake water temperature either by placing the bags in constant temperature incubators (lake water ~20°C) or coolers (> 20°C). Minicell concentrations in initial, intermediate, and final

samples were determined by direct counts with epifluorescent microscopy. Initial tirne- course experiments revealed that declines in minicell concentration over 24 h were linear. In subsequent experiments we took only initial and final samples and, instead of using three replicates, duplicated each replicate for a total of six l-liter bags plus controls for each experiment. We did not observe significant changes in the concentration of minicells over 24 h in the controls. In the experimental containers, rates were determined either by linear regression in the time-course experiment or by difference between initial and final values in the end- point experiments.

Ingestion of fluorescent minicells by protozoans was determined following procedures developed by McManus and Fuhr- man (1986) and Pace and Bailiff (1987) for experiments with fluorescent particles. Sub- samples from the same bags used in the disappearance experiments were taken several times during the first hour of incubation. These samples were preserved with an equal volume of cold 4% glutaraldehyde, which minimizes particle losses from cells (San- ders et al. 1989; Bloem et al. 1989) and avoids the safety and precipitation prob- lems associated with the fixative containing acrolein, tannic acid, and glutaraldehyde recommended by Sieracki et al. (1987).

Ingestion of minicells by mixotrophic algae and heterotrophic flagellates was determined by scanning several random strips on filters prepared as by Pace and Bailiff (1987) with an Olympus 60 x 11.40 S-Plan Apo objective (total magnification 750 x). We observed that some species of heterotrophic flagellates (usually choanoflagel- lates) attached to algae and typically contained high numbers of minicells. These flagellates were often present at lower densities than unattached flagellates.

We counted the attached flagellates ac- cording to the type of algae to which they were attached (e.g. Fragilaria, Anabaena) by scanning the entire filter at lower magnification (300 x ) and shifting to a higher magnification to determine minicells per flagellate when these forms were encoun- tered. For each category of phagotroph (e.g. unattached heterotrophic flagellates, mixo-


trophs, Dinobryon, heterotrophic flagellates attached to Anabaena), clearance rates were calculated as

CR=I+Cx T (1)

where CR is the clearance rate in nl flagellate-l h-l, I the minicells flagellate-‘, C the concentration of minicells nl-’ in the experimental container, and T the incubation time (h). Community ingestion rates were calculated by multiplying CR for each category of phagotroph by the concentration of bacteria and summing across categories.

Ciliate ingestion of minicells was determined in a manner similar to the flagellates. Subsamples were taken after 20-38 min of feeding and fixed with ice-cold glutaraldehyde. Filters containing DAPI-stained ciliates (Pace and Bailiff 198 7) were scanned at low power (300 x ), and ciliates encoun- tered were checked for ingestion of minicells at high power (7 50 x ). Ciliates were cate- gorized by major taxa as determined in previous counts. Clearance rates for each taxon and for the entire community were calculated. Because rates of feeding by ciliates on minicells were relatively low, we determined clearance rates in only one. of the three replicates.

Experiments to measure feeding on radioactive minicells were conducted in clear polycarbonate bottles containing 2 liters of lake water. One replicate was killed by adding 100 ml of 37% Formalin to produce a 2% final solution. Radioactive minicells were added to the bottles at 10% of natural bacterial concentrations. Bottles were mixed thoroughly, and duplicate 5-ml subsamples taken from each to measure the radioactivity of the feeding suspension. These samples were filtered on 0.2~pm polycarbonate filters and rinsed with distilled water; the filters were then placed in a scintillation vial and frozen. At the end of a 30-min incubation, the entire 2-liter sample was filtered through a 200~pm sieve to collect macrozooplankton; subsequently l-2 liters of the filtrate were passed through a 35-pm sieve to collect microzooplankton. Plankton were washed off these sieves and immediately concentrated on 8 .O-pm polycarbonate filters. Each filter was rinsed thoroughly with

distilled water, placed in a scintillation vial, and frozen.

After completion of each paired day-night experiment, 1 ml of tissue solubilizer (NCS- Amersham) was added to the vials containing 8.0~pm filters, and the sample digested for 24 h to dissolve the animals and the filters. To the vials containing 0.2~pm filters, we added 1 ml of 5% TCA and boiled the samples for 30 min. All vials were subsequently radioassayed in a liquid scintillation counter. Community clearance rates were calculated for the > 200- and 35-200- pm size fractions with the equation

CCR=I+(SA x C) x V (2)

where CCR is the community clearance rate in liters h-l, I the radioactivity ingested by the size class, corrected for radioactivity absorbed by the control (dpm liter-’ h-l), SA the specific activity of the minicells (dpm minicell- l), C the concentration of minicells in the experimental bottles (minicells liter-l), and V the volume of the experimental container (liters). Community ingestion rates were calculated by multiplying clearance rates by the concentration of bacteria.

Rotifers dominated the 35-200-pm size class (Table 1)-primarily the genera Poly- arthra and Keratella. Incorporation of radioactivity by this size fraction was typically only two to three times, and sometimes less than or equal to, the radioactivity absorbed by the killed control. By contrast, radioactivity of the macrozooplankton (> 200 pm) was typically 10 times the radioactivity of the controls. The low radioactivity incorporated by microzooplankton indicates these animals were not feeding on bacteria at significant rates. Thus, we present results only for the macrozooplankton.

Results Annual cycle of chlorophyll and bacte-

ria - Chlorophyll was relatively high in winter under the ice and increased with the spring bloom that occurred in concert with ice-out (Fig. 1 a). Following the spring bloom, chlorophyll declined and oscillated between 2 and 10 pg liter-l for the remainder of the year. A bloom of Dinobryon divergens was observed in October which led to a modest increase in chlorophyll in fall. Chlorophyll


I= a. Apr

‘; loo- b. Jul

r ‘i- loo- 1

= ao-- ‘i-

L aJ k 60--

.e .k! - - ul m 40--

= =

s 8 20-- CD co

z 10 0

O- *IT

Growth Macro Flagellate Growth Macro Flagellate

-i 40 t

c. Sep

I

‘i- 15

L Jz

Growth Macro Flagellate

d. Ott

Growth Macro Flagellate Fig. 2. Measurements of bacterial growth and feeding on bacteria by macrozooplankton (> 200 pm) and

flagellates including mixotrophs in four experiments in 1988. Solid bars represent night measurements and open bars day measurements for experiments conducted on 13-l 4 April (note log scale), 13-l 4 July, 2 l-22 September, and 26-27 October. All values are means + 1 SD.

2a). Strong day-night differences in the macrozooplankton grazing rates were due to diel vertical migrations by Daphnia (Table 1). Flagellate grazing rates were 10% (night) and 17% (day) of the macrozooplankton grazing rates. Overall, consumption of bacteria was far greater than growth during the April experiment. It is likely that zooplankton, in particular Daphnia, were driving the sharp declines of both algae and bacteria observed in April (Fig. 1).

In the July experiment, growth and grazing were approximately balanced (Fig. 2b). Total bacterial consumption by macrozooplankton plus flagellates was twice bacterial production at night and half of bacterial production during the day. Bacterial production was slightly higher in July relative to April, whereas grazing rates were lower, reflecting the decline in Daphnia abundance between the two experiments (Table 1). As in April, macrozooplankton grazing was substantially higher than flagellate grazing

both at night and during the day. Bacteria were relatively stable in summer with cell densities oscillating between 4 and 6 x log cells liter-l (Fig. 1). During this period, bacterial production and grazing were in balance with the primary fate of bacterial production being direct ingestion by Daphnia.

Two experiments were conducted in fall when water temperatures were declining (Fig. 2c,d). Bacterial production was lower during these experiments relative to spring and summer conditions. Total consumption of bacteria by macrozooplankton and flagellates exceeded growth estimates by about twofold to fourfold in September and October. Flagellate grazing was of increased significance, especially in October when mean flagellate feeding rates were higher than macrozooplankton rates (Fig. 2d). The shift in rates reflects changes in the community of bacterial consumers. In October, Daphnia densities were lower (Table 1) and D. divergens was abundant. Dinobryon is an

802 Pace et al.

Table 2. Abundance and grazing rates of heterotrophic and mixotrophic flagellates. All data are means f 1 SD except in some cases of low abundance where data were pooled from replicates. Data from consecutive dates represent day (D) and night (N) samples.

1988 D/N Flagellate type Abundance (No. ml-l)

Ingestion (mini. flag.? h-l)

Clearance (nl flag.3 h-l)

3 Mar D 13 Apr N 14 Apr D 13 Jul N

14 Jul D

21 Sep N

22 Sep D

26 Ott N

27 Ott D

Free heterotrophic Free heterotrophic Free heterotrophic Free heterotrophic Attached to diatoms Attached to blue-greens Free heterotrophic Attached to diatoms Attached to blue-greens Free heterotrophic Attached to diatoms Attached to blue-greens Attached to Dinobryon Dinobryon Free heterotrophic Attached to diatoms Attached to blue-greens Attached to Dinobryon Dinobryon Free heterotrophic Attached to diatoms Attached to blue-greens Attached to Dinobryon Dinobryon Free heterotrophic Attached to diatoms Attached to blue-greens Attached to Dinobryon Dinobryon

761f56 502k30 371k61 711a164 15.4k9.5

3.23 461+203 16.7+ 15.7

5.55 759+49 15.2k3.8 71.Ok45.6

3.31k2.4 42.3k22.9

1,335+31 14.0k6.2 88.8k30.6

5.6k3.6 39.7k7.8 789k 122 7.5k2.4 1.8k2.9

57.6k37.5 2,159&380

991+141 5.9k 1.5 1.4k2.0

59.1k2.1 2,121+441

0.37kO.09 2.46f0.42 2.8 1 kO.49 1.56kO.32 2.42+ 1.50

11.1 1.69kO.51 1.82kO.93

9.09 1.54kO.33 4.17 f2.55 9.48+ 1.08 5.49k0.88 7.20+ 1.60 2.33kO.14 2.79Zk1.18 8.59+ 1.25

3.81 4.72f0.94 0.84f0.39 2.901ko.53

3.17 1.24

1.12kO.15 1.13k0.38 2.47f0.31

2.05 kO.93 0.87kO.27

0.6220.12 5.32kO.71 5.3820.95 1.38kO.26 2.13k1.31

9.86 1.8 1 kO.68 1.81kO.78

9.59 1.85kO.49 5.ooIk3.03 11.4k1.81 6.67+ 1.44 8.62k1.81 2.65kO.08 3.14+ 1.23 9.74f0.87

4.13 5.36kO.94 1.18kO.65 4.03 f. 1.05

4.28 1.71

1.54f0.17 1.60k0.69 3.4320.68

2.90+ 1.44 1.23kO.50

effective consumer of bacteria (Bird and Kalff 198 7) and was the principal bacterial grazer in the October experiment (Table 2).

To test the hypothesis that grazing or growth rates vary significantly on a diel cycle, we used a two-factor ANOVA for the four experiments shown in Fig. 2. Factors were experiments and day vs. night. Growth and flagellate grazing did not vary significantly between day and night (growth: F = 0.29, P= 0.39, n = 22; flagellate grazing: F = 0.31, P = 0.58, n = 24). As expected, macrozooplankton grazing was significantly different between day and night (F = 26.27, P < 0.0001, n = 24), and we attribute this difference to vertical migration by Daphnia (Table 1).

Flagellate community composition and grazing- Free-living, nonpigmented flagellates (hereafter free-living heterotrophic flagellates) were typically the major protists consuming bacteria in Upton Lake. Den-

sities ranged from several hundred to > 1,000 cells ml-l, and clearance rates varied from 1 to 5 nl flagellate-’ h-l. The latter rates are somewhat lower than laboratory estimates of clearance rates for single species (Fenchel 1986a). These rates are, however, reasonable, particularly since the total community is likely to contain species which either do not consume bacteria or feed on bacteria at relatively low rates. A second group of heterotrophic flagellates was found attached to several species of large algae including the diatom Fragilaria, the blue-green Anabaena, and the chrysophyte Dinobryon. Attached flagellates ingested minicells at high rates (Table 2). Clearance rates up to 11 nl flagellate-l h-l were observed, but these flagellates were never abundant (< 100 cells ml-l). Consequently, attached flagellates were of little significance in the overall fate of bacterial production. Dinobryon was an important bacterial consumer when this


- 0.5

r; E 0.4

a,

4

g 0.3

E

3 0.2

= 8 0.1 .-

.- s

0.0 L ll!-NL ill-

243 f 41

150 J

r; -c 125

‘; 100

ki .e 75 -

22 50 x5 0

co 25 0 - 0

N DNDNDND

APr Jul SeP act

Fig. 4. Comparison of direct measurements of feeding by macrozooplankton and flagellates (hatched) to total community measurements of feeding based on minicell disappearance (black) for the night (N) and day (D) measurements conducted in 1988. All values are means -+_ 1 SD.

SeP act

Fig. 3. Total community grazing rates measured by following the disappearance of minicells in four experiments in 19 8 8. Black bars- night measurements; white bars-day measurements. All values are means *l SD.

species reached high densities during a fall bloom (Table 2). Other mixotrophic Aagel- lates were rare and never significant grazers.

Ciliate community composition and grazing-Total ciliate grazing varied from 1.2 to 96 x lo4 bacteria liter-’ h-l with lowest rates in the October experiment and highest rates in July. These rates were typically < 10% of the total flagellate grazing (Fig. 2). The ciliate fauna of the surface waters con- sisted primarily of choreotrichs (Strombid- ium spp. and Halteria sp.) and haptorids (Mesodinium sp. and Askenesia sp.). Some species of choreotrichs ingested minicells, but clearance rates were low (range l-20 nl ciliate-’ h-l) relative to measurements made on similar species feeding on larger particles (Fenchel 1986b). Haptorids did not ingest minicells.

Total community grazing rate - Esti- mates of grazing by the entire planktonic community on bacteria were made by following the disappearance of minicells over 24 h. No change was observed during the March experiment (data not shown) reflecting the relatively low grazing rate under the ice. Losses of minicells were observed at other times of the year, and specific daily loss rates were calculated from these changes (Fig. 3). Loss rates were typically in the range of 0.1-0.2 d-l except in July, when higher rates of 0.4 and 0.3 d-l were observed.

Total community grazing estimates should agree with the direct measures of feeding on bacteria by macrozooplankton and flagellates, since these two groups ac-

count for most of the grazing. There was good agreement between the two estimates only in the October experiments where t-tests revealed no significant differences (P > 0.1) between the estimates (Fig. 4). Poor- est agreement between the two methods was observed in April, when total grazing estimates were threefold to fourfold lower than the sum of zooplankton and flagellate grazing (Fig. 4). Differences between the estimates were highly significant for the night measurements (P = 0.004), but because of high variability in replicate total grazing estimates (Fig. 4), differences were of lower significance for the day measurements (P = 0.07). In July, total community estimates were greater than the direct measures of feeding in the day (P = O-03), but there were no significant differences between night measurements (Fig. 4). Finally, direct grazing estimates were significantly greater than total community measurements in Septem- ber (night: P = 0.06; day: P = 0.04).

Comparison of turnover-Our estimates of bacterial growth, direct feeding on bacteria by flagellates and macrozooplankton, and total community grazing rates can be compared in terms of turnover times by di- viding bacterial abundance by each rate estimate. Turnover times as measured by three different techniques are all > 1 d (Table 3). Exceptionally slow turnover times of 50- 100 d were observed under the ice in March.

804 Pace et al.

Table 3. Turnover times (in days) of bacteria in Upton Lake based on estimates of growth, grazing, and total mortality. -.

TOtal

1988 Growth Grazing mortality

3 Mar 55.9 90.9 - 13 Apr 11.4 1.5 5.9 14 Apr 9.2 3.0 9.1 13 Jul 6.8 3.4 2.5 14 Jul 6.1 12.7 3.7 21 Sep 14.7 3.8 7.0 22 Sep 15.9 4.0 7.0 26 Ott 16.3 4.4 6.1 27 Ott 13.3 6.2 5.5

More rapid turnover was observed in spring, summer, and fall experiments with most estimates falling in the range of 3-15 d. In general, shorter turnover times were calculated based on grazing measurements than on measurements of growth.

Discussion Some of the discrepancy between the total

mortality and grazing estimates illustrated in Fig. 4 could be attributed to measurement error. In the minicell disappearance experiments we usually observed only a 1 O- 15% decline in minicells over 24 h. Such small declines are difficult to detect reliably because of the inherent variability associated with direct microscopic counts. Mea- surements of minicell disappearance may not be sufficiently sensitive to accurately detect community rates of grazing on bacteria in systems where bacteria do not turn over rapidly. In addition, direct measurements of feeding were based on short-term estimates which minimize containment effects on the organisms. It may be problematic to compare these short-term estimates to those made over 24 h, given possible containment effects in these longer experiments and the diel rhythms of feeding by organisms like Daphnia (Haney 1985).

The fate of ingested minicells in the long- term experiments is unknown. Minicells which are ingested may be assimilated or pass through consumers and be egested. In the latter case, they will likely be compacted in fecal material as observed for fluorescent particles (Pace and Bailiff 1987), but some could redisperse and be counted, thus caus-

ing an underestimate of community grazing rate.

Previous studies have considered the relationship between bacterial growth and losses due to grazing and have inferred that growth and grazing are in close balance (McManus and Fuhrman 1988; Sanders et al. 1989; Bloem et al. 1989). On the other hand, Sherr et al. (1989) made simultaneous measurements of growth and protozoan grazing and found that growth estimates consistently exceeded grazing estimates in a Georgia estuary. Our study is among the first to attempt to measure feeding on bacteria by all the major consumers including heterotrophic flagellates, mixotrophs, ciliates, microzooplankton, and macrozooplankton. We found that growth rates were considerably lower than feeding rates when Daphnia was abundant. If we had measured feeding by microbial phagotrophs alone, we would have concluded that growth was approximately balanced by flagellate grazing. In Upton Lake, however, feeding rates of macrozooplankton and flagellates com- bined were significantly greater (t-tests, P ( 0.1) than growth except in March and July experiments, when the two estimates were similar.

Differences between growth and grazing estimates were probably due to the uncer- tain accuracy of our methods, which makes comparisons among them difficult. The thymidine method we used measures the incorporation of radioactivity into DNA iso- lated by chemical extraction procedures. Previous evaluations of our chemical extraction suggest that our method of isolating DNA is > 90% efficient (Findlay et al. 1984; unpubl. data). There is uncertainty about converting estimates of radioactivity in DNA into measures of cells liter-’ h-l (Mor- iarty 1986). We attempted to verify our conversion with a dilution growth approach, which gave a value that agrees well with many previous estimates (Fuhrman and Azam 1982; Moriarty 1986), an extensive empirical study (Riemann et al. 1987), and ecosystem C budget constraints (Cole et al. 1989).

We also checked our method to be sure that we were using saturating quantities of the isotope and to examine the possibility


of isotope dilution by external pools of nu- cleic acid precursors. We saturated uptake, and isotope dilution appeared to be low. Nevertheless, some isotope dilution by competing external pools, internal de novo synthesis, and community variability in the ability to incorporate and utilize thymidine for DNA synthesis could easily change the thymidine-based estimates by a factor of two or more. Furthermore, recent studies suggest that the conversion factor varies in freshwater environments (Smits and Rie- mann 1988; Coveney and Wetzel 1988).

Similar concerns could be raised about the minicell method. We have verified in independent studies that flagellates, ciliates, and Daphnia consume minicells at expected rates. For mixed communities of consumers, we assume that the spherical minicells, which are 0.6-0.7 pm in diameter, ad- equately represent the diversity of bacteria in terms of size, shape, and taxonomy for all grazers. These assumptions are not en- tirely valid. For example, Glide (1988) observed that the size composition of bacteria in Lake Constance shifted, which presum- ably resulted from Daphnia grazing most intensely on larger bacteria and bacterial ag- gregates. In this context, our method may represent a conservative estimate of grazing.

Both the thymidine and minicell methods, however, are among the best techniques now available to measure bacterial processes. Given current uncertainties about accuracy, it is probably unrealistic to ask whether bacterial production and grazing mortality are in balance. Rather, diverse methods help constrain the possible range of bacterial activity and identify the major pathways of bacterial consumption.

Several studies in both marine and freshwater systems have presented evidence for rapid turnover times (0.2-2 d) of bacterial communities based on thymidine incorporation into macromolecular pools, cali- brated with dilution growth experiments (Kirchman et al. 1982; Ducklow and Hill 1985; Scavia and Laird 1987). Bacteria in Upton Lake do not turn over so rapidly. Our conclusion of slower turnover rates is based on three different approaches, including growth measurements, direct estimates

of grazing by several types of consumers, and disappearance rates of tracer bacteria.

While the studies cited above have argued that turnover is rapid, others support the view of slower turnover times. For example, Cole et al. (1988) summarized data from studies where both primary production and bacterial production were measured in units of pg C liter-l d- l. Using these data, we calculated bacterial turnover times for 29 cases where the thymidine method was used and bacterial abundance was available. We converted abundances into C with a factor of 20 fg C cell-l (Lee and Fuhrman 1987). Average turnover time was 4.5 d with a me- dian of 3.7 and a range of 0.5-14. Upton Lake turnover times fall within this range, except in winter. The literature data and the Upton Lake data are based on epilimnion communities only. Recent field studies in lakes also provide evidence for slow bacterial turnover times in the epilimnion, metalimnion, and hypolimnion (Sanders et al. 1989; Bloem et al. 1989). We conclude that community turnover times of planktonic bacteria in Upton Lake and in most systems are considerably > 1 d.

A clear result of our study concerns the relative importance of various microbial phagotrophs as bacterial consumers. In Up- ton Lake free-living heterotrophic flagellates and mixotrophs such as Dinobryon ac- count for most Protist grazing. Ciliates were not important grazers nor were attached heterotrophic flagellates, although for the latter, per-cell grazing rates were high. The surface-water ciliates of the lake are typical of those found in the epilimnion of other lakes (Pace 1982), and we infer that epilimnetic ciliates are, in general, not important grazers of bacteria. Our results are very similar to those found by Sanders et al. (1989) for Lake Oglethorpe, Georgia, where grazing was measured with fluorescent micro- spheres. In that lake heterotrophic and niix- atrophic flagellates accounted for 68-90% of the bacterial ingestion, whereas ciliates accounted for a relatively low percentage.

Daphnia consumes most of the bacteria1 production in Upton Lake. Earlier studies have concluded that Daphnia, and metazoans in general, are relatively unimportant as bacterial consumers (see Pace 1988;

806 Pace et al.

McManus and Fuhrman 1988; Sanders et al. 1989). Daphnia feeds on bacteria with a lower efficiency than it does on algae (Porter et al. 1983), and bacteria alone are a poor food for Daphnia (Pace et al. 1983). In systems, however, where large populations of Daphnia develop, these cladocerans can consume a substantial portion of the bacterial production as noted during seasonal Daphnia maxima in several lakes (Pedros- Alio and Brock 1983; Geertz-Hansen et al. 1987; Giide 1988) and in enclosure experiments when dense populations of Daphnia develop (Riemann 1985; Geertz-Hansen et al. 1987). Kankaala (1988) studied a Daph- nia Zongispina population that maintained high densities (5-326 animals liter-l) and found that these animals could graze 3-48% of bacterial biomass daily.

In Upton Lake, if we assume Daphnia accounts for all the feeding on bacteria by macrozooplankton, we obtain estimates of clearance rates ranging from 0.1 to 0.4 ml animal-l h-l, in good agreement with expected rates based on earlier studies of daphnids (Peterson et al. 1978; Porter et al. 1983; B@sheim and Andersen 1987; Bern 1987; Kankaala 1988). The estimates of bacterial consumption by Daphnia are equivalent to or greater than our estimates of bacterial production for the April through October experiments.

Grazing by Daphnia is likely to be the predominant fate of bacterial production in lakes where daphnids are consistently abundant. In Upton Lake, Daphnia densities varied from - 10 to > 100 animals liter-’ excluding the sample taken when the lake was covered with ice. Densities in this range are not unusual, as demonstrated by McCauley et al. (1988) who summarized data on algal biomass and the abundance of Daphnia in 35 lakes worldwide. In 11 of those 35 lakes the average Daphnia density exceeded 10 animals liter-l. This average was based only on observations following the spring algal-zooplankton bloom and preceding winter decline. The averages, therefore, did not include high-density pe- riods such as we observed during the April bloom and which occur commonly in many lakes. In contrast, bacteria appear to be consumed primarily by flagellates in lakes with

low Daphnia densities (Riemann 1985). For example, Sanders et al. (1989) found heterotrophic and mixotrophic flagellates to be the primary bacterial grazers in Lake Ogle- thorpe where cladocerans including Daph- nia are abundant only seasonally, with densities > 10 animals liter-l only briefly during spring or fall blooms.

Microbial food webs have recently been viewed as primarily regenerative in the sense that nutrients contained in picoplankton are rapidly turned over by the microbial phagotrophs that consume picoplankters (Pom- eroy and Wiebe 198 8). A correlate of this idea is that processes mediated by heterotrophic bacteria are “loops” wherein matter cycles rapidly and is only passed up the food web by protozoan intermediaries. These concepts have been developed primarily in marine systems where the dominant zooplankton are copepods. Copepods cannot graze as effectively as Daphnia on small particles-including in some cases the smallest heterotrophic flagellates. Daphnia, because of their broad feeding abilities, are able to “break” the microbial loop and directly consume both algae and a broad spectrum of heterotrophic microbes (Porter et al. 1979). Microbial food webs may differ between systems with and without abundant populations of Daphnia (Stockner and Por- ter 1988).

We suggest that nutrient cycling and respiration by heterotrophic microbes will be slower and the flow of C to higher trophic levels will be greater in systems with Daph- nia. In marine systems dominated by copepods and freshwater systems without Daphnia, heterotrophic microbes will dis- sipate more of the primary production in respiration, and the flow of C to higher trophic levels will be lower. This hypothesis requires comparative study of systems with and without Daphnia to fully quantify the interactions and assess the postulated significance of these organisms in controlling microbial heterotrophy. If correct, the hypothesis identifies a major difference between the nature of pelagic food webs in freshwater and marine systems, although larvaceans, when abundant, may be the marine analog of Daphnia.

In conclusion, the fate of bacterial pro-


duction in Upton Lake is largely a function of planktonic community structure. The three primary bacterial consumers in the surface mixed layer were Daphnia, Dinobry- on, and heterotrophic flagellates. Variations in the relative abundances of these consumers over the season influence how bacterial production enters the food web. In winter and fall, flagellates and Dinobryon are the primary consumers of bacteria; in spring and summer, Daphnia are the primary consumers. Factors determining the relative abundances of varying bacterial predators among lake ecosystems seem to be crucial in determining the pathways of bacterial utilization in pelagic food webs.

References

BERN, L. 1987. Zooplankton grazing on [methyl- 3H]thymidine-labelled natural particle assem- blages: Determination of filtering rates and food selectivity. Freshwater Biol. 17: 15 l-l 59.

BIRD, D. F., AND J. KALFF. 1987. Algal phagotrophy: Regulating factors and importance relative to phy- tosynthesis in Dinobryon (Chrysophyseae). Lim- nol. Oceanogr. 32: 277-284.

-, AND -. 1989. Phagotrophic sustenance of a metalimnetic phytoplankton peak. Limnol. Oceanogr. 34: 155-l 62.

BLOEM, J., F. M. ELLENBROEK, M.-J. B. BAR-GILISSEN, AND T. E. CAPPENBERG. 1989. Protozoan grazing and bacterial production in stratified Lake Vetch- en estimated with fluorescently labeled bacteria and by thymidine incorporation. Appl. Environ. Microbial. 55: 1787-1795.

B~RSHEIM, K. Y., AND S. ANDERSEN. 1987. Grazing and food size selection by crustacean zooplankton compared to production of bacteria and phytoplankton in a shallow Norwegian mountain lake. J. Plankton Res. 9: 367-379.

CARON, D. A., J. G. GOLDMAN, AND M. R. DENNETT. 1988. Experimental demonstration of the roles of bacteria and bacterivorous protozoa in plankton nutrient cycles. Hydrobiologia 159: 27-40.

COLE, J. J., N. F. CARACO, D. L. STRAYER, C. OCHS, AND S. NOLAN. 1989. A detailed carbon budget as an ecosystem-level calibration of bacterial respiration in an oligotrophic lake during midsum- mer. Limnol. Oceanogr. 34: 286-296.

-, S. FINDLAY, AND M. L. PACE. 1988. Bacterial production in fresh and saltwater: A cross system overview. Mar. Ecol. Prog. Ser. 43: l-10.

COVENEY, M. F., AND R. G. WETZEL. 1988. Experi- mental evaluation of conversion factors for the [3H]thymidine incorporation assay of bacterial secondary productivity. Appl. Environ. Micro- biol. 54: 20 18-2026.

DUCKLOW, H. W., AND S. M. HILL. 1985. Tritiated thymidine incorporation and the growth of het-

erotrophic bacteria in warm-core rings. Limnol. Oceanogr. 30: 260-272.

-, D. A. P~RDIE, P. J. LEB. WILLIAMS, AND J. M. DAVIES. 1986. Bacterioplankton: A sink for carbon in a coastal marine plankton community. Sci- ence 232: 865-867.

FASHAM, M. J. R. 1985. Flow analysis of materials in the marine euphotic zone. Can. Bull. Fish. Aquat. Sci. 213, p. 139-162.

FENCHEL, T. 1986~. The ecology of heterotrophic microflagellates. Adv. Microb. Ecol. 9: 57-97.

- 1986b. Protozoan filter feeding. Prog. Pro- tistol. 1: 65-l 13.

FINDLAY, S. E. G., J. L. MEYER, AND R. T. EDWARDS. 1984. Measuring bacterial production via rate of incorporation of [3H]thymidine into DNA. J. Mi- crobiol. Methodol. 2: 57-72.

FUHRMAN, J. A., AND F. AZAM. 1982. Thymidine incorporation as a measure of heterotrophic bacterioplankton production in marine surface waters: Evaluation and field results. Mar. Biol. 66: 109- 120.

-, R. W. EPPLEY, A. HAGSTR~M, AND F. AZAM. 1985. Diel variations in bacterioplankton, phytoplankton, and related parameters in the South- em California Bight. Mar. Ecol. Prog. Ser. 27: 9- 20.

GEERTZ-HANSEN, O., M. OLESEN, P. K. BJBRNSEN, J. B. LARSEN, AND B. RIEMANN. 1987. Zooplankton consumption of bacteria in a eutrophic lake and in experimental enclosures. Arch. Hydrobiol. 110: 553-563.

GLADE, H. 1988. Direct and indirect influences of crustacean zooplankton on bacterioplankton of Lake Constance. Hydrobiologia 159: 63-74.

HAAS, L. 1982. Improved epifluorescence microscopy for observing planktonic micro-organisms. Ann. Inst. Oceanogr. Paris 58: 261-266.

HAGSTR~M, A., AND U. LARSSON. 1984. Diel and seasonal variation in growth rates of pelagic bacteria, p. 249-262. Zn J. E. Hobbie and P. J. 1eB. Williams [eds.], Heterotrophic activity in the sea. Plenum.

HANEY, J. F. 1985. Regulation of cladoceran filtering rates in nature by body size, food concentration, and diel feeding patterns. Limnol. Oceanogr. 30: 397-411.

HOBBIE, J. E., R. J. DALEY, AND S. JASPER. 1977. Use of Nuclepore filters for counting bacteria by flu- orescence microscopy. Appl. Environ. Microbial. 33: 1225-1228.

KANKAALA, P. 1988. The relative importance of algae and bacteria as food for Daphnia longispina (Cladocera) in a polyhumic lake. Freshwater Biol. 19: 285-296.

KING, K. R., J. T. HOLLIBAUGH, AND F. AZAM. 1980. Predator-prey interactions between the larvacean Oikopleura dioica and bacterioplankton in en- closed water columns. Mar. Biol. 56: 49-57.

KIRCHMAN, D. L., H. W. DUCKLOW, AND R. MITCHELL. 1982. Estimates of bacterial growth from changes in uptake rates and biomass. Appl. Environ. Mi- crobiol. 44: 1296-l 307.

LEE, S., AND J. A. FUHRMAN. 1987. Relationships

808 Pace t

between biovolume and biomass of naturally de- rived marine bacterioplankton. Appl. Environ. Microbial. 53: 1298-l 303.

MCCAULEY, E., W. W. M URDOCH, AND S. WATSON. 1988. Simple models and variation in plankton densities among lakes. Am. Nat. 132: 383-403.

MCMANUS, G. B., AND J. A. FUHRMAN. 1986. Bac- terivory in seawater studied with the use of inert fluorescent particles. Limnol. Oceanogr. 31: 420- 426.

-,AND-. 1988. Control of marine bacterioplankton populations: Measurement and significance of grazing. Hydrobiologia 159: 5 l-62.

MANIATIS, T.,E.F. FRITSCH,AND J. SAMBROOK. 1982. Molecular cloning: A laboratory manual. Cold Spring Harbor Lab.

MORIARTY, D. J. W. 1986. Measurement of bacterial growth rates in aquatic systems from rates of nu- cleic acid synthesis. Adv. Microb. Ecol. 9: 24% 292.

dation and zooplankton grazing on natural populations of freshwater bacteria. Appl. Environ. Mi- crobiol. 52:101-107.

-, P. K. BJBRNSEN, S.NEWELL, AND R. FALLON. 1987. Calculation of cell production of coastal marine bacteria based on measured incorporation of [3H]thymidine. Limnol. Oceanogr. 32: 47 l-476.

SANDERS, R. W., K. G. PORTER, S. J. BENNETT, AND A. E. DEBIASE. 1989. Seasonal patterns of bacterivory by flagellates, ciliates, rotifers, and cladocerans in a freshwater planktonic community. Limnol. Oceanogr. 34: 673-687.

SCAVIA, D., AND G. A. LAIRD. 1987. Bacterioplankton in Lake Michigan: Dynamics, controls, and significance to carbon flux. Limnol. Oceanogr. 32: 1017-1033.

PACE, M. L. 1982. Planktonic ciliates: Their distri- bution, abundance, and relationship to microbial resources in a monomictic lake. Can. J. Fish. Aquat. Sci. 39: 1106-l 116.

--. 1986. An empirical analysis of zooplankton community size structure across lake trophic gradients. Limnol. Oceanogr. 31: 45-55.

- 1988. Bacterial mortality and the fate of bacterial production. Hydrobiologia 159: 4 l-50.

-, AND M. D. BAILIFF. 1987. Evaluation of a fluorescent microsphere technique for measuring grazing rates of phagotrophic microorganisms. Mar. Ecol. Prog. Ser. 40: 185-193.

--, J.E. GLASSER,ANDL. R. POMEROY. 1984. A simulation analysis of continental shelf food webs. Mar. Biol. 82: 47-63.

--, K. G. PORTER, AND Y. S. FEIG. 1983. Species- and age-specific differences in bacterial resource utilization by two co-occurring cladocerans. Ecol- ogy 64: 1145-l 156.

PARSONS, T. R., Y. MAITA, AND C. M. LALLI. 1984. A manual of chemical and biological methods of seawater analysis. Pergamon.

PEDR~S-ALI~,C.,AND T.D. BROCK. 1983. Theim- pact of zooplankton feeding on the epilimnetic bacteria of a eutrophic lake. Freshwater Biol. 13: 227-239.

SIERACKI, M. E., L. W. HAAS, D. A. CARON, AND E. J. LESSARD. 1987. Effect of fixation on particle re- tention by microflagellates: Underestimation of grazing rates. Mar. Ecol. Prog. Ser. 38: 251-258.

SHERR, B. F., E. B. SHERR, AND R. D. FALLON. 1987. Use of monodispersed, fluorescently labeled bacteria to estimate in situ protozoan bacterivory. Appl. Environ. Microbial. 53: 958-965.

- - AND C. PEDR~S-ALI& 1989. Simul- taneous measurement of bacterioplankton production and protozoan bacterivory in estuarine water. Mar. Ecol. Prog. Ser. 54: 209-2 19.

SHERR, E. B., AND B. F. SHERR. 1987. High rates of consumption of bacteria by pelagic ciliates. Nature 325: 710-711.

-,AND- . 1988. Roles of microbes in pelagic food webs: A revised concept. Limnol. Oceanogr. 33: 1225-l 227.

-- AND G.-A. PAFFENH~FER. 1986. Phagotrophic protozoa as food for metazoans. A “missing” trophic link in marine pelagic food webs? Mar. Microb. Food Webs 1: 61-80.

SMITS, J. D., AND B. RIEMANN. 1988. Calculation of cell production from [3H]thymidine incorporation with freshwater bacteria. Appl. Environ. Micro- biol. 54: 22 13-22 19.

PETERSON, B.J., J.E. HOBBIE, AND J.F. HANEY. 1978. Daphnia grazing on natural bacteria. Limnol. Oceanogr. 23: 1039-1044.

STOCKNER, J. G., AND K. G. PORTER. 1988. Microbial food webs in freshwater planktonic ecosystems, p. 69-83. In S. R. Carpenter [ed.], Complex interactions in lake communities. Springer.

VEZ;INA, A. F., AND T. PLAY. 1988. Food web dynamics in the ocean. 1. Best-estimates of flow net- works using inverse methods. Mar. Ecol. Prog. Ser. 42: 269-287.

POMEROY, L. R., AND W. J. WIEBE. 1988. Energetics WIKNER, J., A. ANDERSSON, S. NORMARK, AND A. of microbial food webs. Hydrobiologia 159: 7-18. HAGSTR~M. 1986. Use of genetically marked

PORTER, K. G., AND Y. S. FEIG. 1980. The use of minicells as a probe in measurements of preda tion DAPI for identifying and counting aquatic micro- on bacteria in aquatic environments. Appl. En- flora. Limnol. Oceanogr. 25: 943-948. viron. Microbial. 49: 19-23.

-- - AND E. F. VETTER. 1983. Morphol- og;, flow iegimes and filtering rates of Daphnia,

F. RA~SOUUADEGAN, AND A. HAGSTR~M. 1990. Periodic bacterivore activity balances bac-

Ceriodaphnia, and Bosmina fed natural bacteria. terial growth in the marine environment. Limnol. Oecologia 58: 156-l 63. Oceanogr. 35: 3 13-324.

--, M. L. PACE, AND J. F. BATTEY. 1979. Ciliate protozoans as links in freshwater planktonic food chains. Nature 277: 563-565.

RIEMANN, B. 1985. Potential importance of fish pre-

Submitted: 24 August 1989 Accepted: 9 January 1990

Revised: 15 March 1990

2 al.

limnol. oceanogr., 35(4), 1990, 7954308 q 1990, by the ...€¦ · depth intervals with a yellow...

Documents