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Journal of Plankton Research Vol.18 no.5 pp.683-714.19% Relative impacts of copepods, cladocerans and nutrients on the microbial food web of a mesotrophic lake Carolyn W.Burns and Marc Schallenberg Department of Zoology, University ofOtago, PO Box 56, Dunedin, New Zealand Abstract. Calaneid copepods, rather than cladocerans, frequently dominate the zooplankton of lakes in New Zealand. The potential consequences of this domination for the microbial community of mesotrophic Lake Mahinerangi, New Zealand, were determined by field experiments in which Boeckella and Daphnia were added to in situ enclosures in the presence and absence of added nutrients. Boeckella hamata at ambient densities (2 and 81~') rapidly and severely suppressed ciliate population growth over 4 days, even when microbial growth was enhanced by added nutrients, but effects of copepods on other components of the microbial community (bacteria, photosynthetic picoplankton, heterotrophic nanoflagellates, algae) were slight. In contrast, Daphnia carmata at the same densities (but 3-fold higher biomasses per litre) had a relatively weak effect on ciliates, suppressing ciliate abundance only after 4 days at 8 Daphnia I 1 (330 ^.g I 1 ); this daphniid density also depressed abundances of large bacterial rods, some photosynthetic picoplankton and the dominant alga, Cyclotella. These results highlight the relative importance of specific trophic linkages in a microbial food web; they also suggest that the dominance of Boeckella in many southern hemisphere lakes may account for relatively low ciliate abundances in these lakes. Introduction There have been numerous studies of the effects of nutrients (bottom-up) and predation (top-down) in regulating the biomass and productivity of phytoplankton and zooplankton in lakes (references in DeMelo et al., 1992); far less is known about how nutrients and consumers may interact to regulate planktonic microbial communities (ciliates, flagellates, picoplankton, bacteria). In nutrientlimited systems, consumers may influence microbial diversity and biomass directly by 'predation', and indirectly by the excretory release of nutrients. Several studies in northern hemisphere lakes have related decreases in biomass and growth rates of microbial organisms to increases in the biomass of zooplankton, particularly Daphnia (reviewed by Jtlrgens, 1994). These Daphnia-based studies form the basis of most current models of planktonic microbial food webs and grazer-mediated effects (Riemann and Christoffersen, 1993; Jiirgens, 1994). Calanoid copepods dominate the zooplankton biomass of the sea and are important components of the zooplankton of lakes, particularly in temperate latitudes. Comparatively little is known, however, about the effects of calanoid copepods on the microbial communities of lakes. Species of Diaptomus have the potential to suppress the growth of ciliates (Burns and Gilbert, 1993; Hartmann et al., 1993; Brett et al., 1994; Wiackowski et al., 1994) at least as effectively as a comparable biomass of Daphnia (Wiackowski etal., 1994). Thus, predation on ciliates by copepods is a potentially important link between the organisms that comprise the 'microbial loop' (Azam et al., 1983) and the classical food chain (nutrientsphytoplankton-zooplankton-fish) (Stoecker and Capuzzo, 1990; Pace and Funke, 1991; Burns and Gilbert, 1993; Wiackowski et al., 1994). Studies in Castle Lake, California, have compared the effectiveness of Daphnia, Holopedium, Diaptomus © Oxford University Press 683 C.W.Burns and M.Schallenberg and Diacyclops in altering algal and bacterial biomass, and in depressing ciliate growth rates, and hence in altering ciliate community structure (Brett et al., 1994; Wiackowski et al., 1994), but indirect effects of calanoid copepods on other heterotrophs have not been measured. Jiirgens et al. (1994a) described the effects of Daphnia spp. and mixed copepods (Eudiaptomus and three species of cyclopoids) on the largely heterotrophic, microbial community of a mesotrophic lake, but the relative effects of Daphnia and calanoids on net growth rates of autotrophic and heterotrophic components of microbial food webs have not been determined. Suspension-feeding calanoid copepods of the genus Boeckella dominate the zooplankton of many lakes in New Zealand throughout the year. Daphnia are absent or rare in many lakes, and common only briefly in a few mesotrophic and eutrophic lakes; predation pressure on mesozooplankton appears to be low as there are few invertebrate predators and no obligate planktivores (Chapman and Green, 1987). Thus, the crustacean zooplankton may be the 'top predators' in the pelagic zone of many New Zealand lakes for considerable periods of the year. One way of gaining insight into the relative importance of top-down and bottom-up regulation of populations and biomass in natural communities is by manipulating resources and consumers. Many studies of microbial communities in lakes have focused on only one or two components of the microbial loop; comparatively few have incorporated all of the major components, including nutrients and algae (exceptions include Christoffersen etal., 1990; Arndt and Nixdorf, 1991; Pace and Funke, 1991; Brett et al., 1994; Jurgens et al., 1994b). Pace and Funke (1991) used the manipulative approach in a study of the effects of nutrients and Daphnia on the heterotrophic microbes of two Michigan lakes, but effects of copepods were not examined; Jiirgens et al. (1994a) did not distinguish between calanoids and cyclopoids in their study of the effects of macrozooplankton on the bacterial community. The study by Brett et al. (1994) is the only one to compare the effects of Daphnia and a calanoid, Diaptomus, on natural microbial communities of a lake. In order to derive general predictions for the effects of cladocerans and copepods on the trophic dynamics of lakes, more studies on the relative impacts of cladocerans and copepods are needed. In our study, we use an experimental approach similar to that of Pace and Funke (1991) in which we manipulate nutrients and consumers in in situ enclosures to determine (i) the relative impacts of Daphnia and Boeckella on the microbial community of a mesotrophic lake, and (ii) the relative importance of top-down and bottom-up effects on algal biomass and the major components of the microbial community in summer. Study site and method Lake Mahinerangi (surface area 18.6 km2, mean depth of 6.2 m) is a mesotrophic, polymictic, reservoir. Daphnia carinata King and Boeckella hamata Brehm dominate the zooplankton biomass, reaching maximum densities of 6 and 9 1"' (adults and CV instars), respectively (Burns, 1992). This study was carried out from 6 to 10 December 1993 at a time when Lake Mahinerangi was isothermal at 13°C and well oxygenated, the chlorophyll a concentration was 3.1 p.g I"1 and the Secchi depth was 1.05 m. Suspended clay 684 Microbial food web of a mesotrophk lake [~2 mg I"1 ash-free dry weight (wt)] and colour (absorbance at 440 nm ~1.5 nr1) contribute to the consistently low transparency of the lake. Average zooplankton densities in the water column at the time were: adult Boeckella 2.25 I 1 (total copepodites 6.5 I 1 ), adult Daphnia 2 I"1 (total Daphnia 3.75 I"1) Bosmina 7.5 I"1, Ceriodaphnia dubia 12.2 I"1, Synchaeta (250 jim) 23 I"1 and Keratella 47 I"1. Water collected from 2 m depth with Van Dorn bottles was screened through 150 jjim mesh to remove large zooplankton and mixed in three large, covered barrels. Thirty, 4.25 1 polyethylene enclosures (Cubitainers) were filled with well-mixed water, after which nutrients and zooplankton were added in a combined factorial and gradient design (Table I). Nutrients (NH4C1 and KH2PO4), to give added concentrations of 120 ng nitrogen (N) I"1 and 10 jig phosphorus (P) I"1, were added to half of the enclosures. At the time of sampling, the inorganic nutrient concentrations in Lake Mahinerangi were 62 (xg I"1 N and 1.2 jo-g 1-' soluble reactive phosphorus (SRP) with a ratio of total nitrogen to total phosphorus (TN:TP) of 12:1. Zooplankton for addition to the enclosures were collected in the lake on the previous day by vertical hauls from 5 m, sorted to species and kept in filtered lakewater in the dark at 13°C. Egg-bearing D.carinata (body length 1.87 ± 0.012 mm SE) or B.hamata (prosomal length 1.06 ± 0.005 mm SE) were added at approximately ambient densities (2 I"1) and four times ambient; six enclosures contained no added crustaceans (Table I). Enclosures were assigned randomly to one of eight anchored buoys from which they were resuspended in the lake at a depth of 1 m below the surface for 4 days. Samples of the water that was used to fill the enclosures were collected for analysis of chlorophyll a, nutrients and microorganisms (see below). Enclosures were sampled after 1 day when grazing effects (top-down) of crustaceans were expected to dominate because 24 h is too short for most microorganisms to multiply significantly in response to nutrients (e.g. Carrick et al., 1991), and after 4 days to allow the microbial community to respond to the combined effects of grazing and nutrient recycling (top-down and bottom-up effects) while minimizing enclosure effects (sedimentation, growth on enclosure walls) (e.g. Elser and Goldman, 1991; Pace and Funke, 1991). When the enclosures were sampled after 1 day, the water that was removed (8% of total volume) was replaced with freshly collected, 150 u,m-filtered lake water collected from 2 m and the nutrient-enriched enclosures received a second dose of nutrients at the same levels as the initial dose. The enclosures were resuspended at the incubation depth for a further 3 days. Table I. Treatments of enclosures and abbreviations for treatments. All treatments were replicated three times Low nutrients (no added nutrients) High nutrients (added nutrients) OL 2DL 8DL 2BL 8BL OH 2DH 8DH 2BH 8BH No Daphnia or Boeckella Daphnia 2 I"1 Daphnia 8 H Boeckella 2 1 1 Boeckella 8 H No Daphnia or Boeckella Daphnia 2 I"1 Daphnia 8 I 1 Boeckella 2 1' Boeckella 8 1' 685 C.W.Burns and M^diailenberg When the enclosures were retrieved on day 4, they were subsampled again for microorganisms, after which the entire contents of each enclosure were filtered through 150 n.m mesh to retrieve the zooplankton for biomass determinations; samples of the filtrate were taken for analysis of inorganic nutrients and chlorophyll a. Zooplankton were rinsed in distilled water, sorted under a dissecting microscope to remove any debris and decaying animals, dried at 50°C for 48 h and weighed on a Sartorius microbalance. All living zooplankton that were retrieved from an enclosure contributed to the total biomass for that enclosure. Chlorophyll a was measured spectrophotometrically (Shimadzu Model UV-120-01) on acetone-extracted samples (0.5 1) that had been collected on GF/F filters (Pridmore el al., 1983). P and N concentrations were determined spectrophotometrically using a Chemlab autoanalyser according to standard procedures (Wetzel and Likens, 1991); SRP was analysed using the antimony-ascorbate-molybdate method, NH4-N was measured by the phenol-hypochlorite method, NCyN was measured after reduction to NO2-N in a cadmium-copper column and TN and TP were measured after oxidation in the presence of boric acid and sodium hydroxide (Valderra, 1981). Samples of 300 ml for ciliates, small rotifers (<150 (xm) and algae were preserved with 1% Lugol's iodine, concentrated by sedimentation and counted at x300 magnification under an inverted light microscope. Samples for picophytoplankton (5 ml) andflagellates(15 ml) werefixedwith equal volumes of ice-cold 4% glutaraldehyde and collected on black polycarbonate 0.8 (im (flagellates) and 0.2 |im (picoplankton)filtersbefore staining with primulin (Bloem et al., 1986). Samples for bacteria were fixed with 0.5% formalin and frozen until analysis. Subsamples (1.5 ml) of the thawed samples were collected on black 0.2 \im filters, stained with DAPI (Porter and Feig, 1980) and counted immediately using fluorescence microscopy. Counts were made at xlOOO magnification under a Zeiss Axiophot microscope equipped with filter sets for UV excitation (BP365/11/FT395/LP397) for heterotrophic flagellates and bacteria, and green excitation (BP546/12/FT580/LP590) for picophytoplankton and photosynthetic flagellates. As so few picoplankton cellsfluorescedunder blue excitation (BP450490/FT510/LP520), we followed the procedure of Weisse and Kenter (1991) and used green excitation for enumerating all autotrophic picoplankton. This procedure may underestimate the abundance of eukaryotic picoplankton [see the Results, and Weisse and Kenter (1991)]. Net population growth rates (g, per day) of microorganisms in each enclosure were calculated as: and 8t = [in(c;g]/4 where Cn, C, and C, are the concentrations of a taxon or category initially, after 1 day and after 4 days, respectively; 1 and 4 are the incubation times in days. 686 Microbial food web of a mesotrophic lake The effects of zooplankton biomass on growth rates were examined by simple linear regression. Clearance rates, or the rates at which Daphnia and Boeckella removed microorganisms from the water [ml \ig (dry wt)~' day~'], were derived from the slopes of significant regressions (P < 0.05) that related microorganisms' growth rate to zooplankton biomass (p^g dry wt I 1 ). The effects of nutrients, zooplankton species and zooplankton density (excludes enclosures without added zooplankton) were tested by three-factor ANOVA; the effects of nutrients and zooplankton density on microbial abundance and clearance rates within zooplankton species, and including enclosures without added zooplankton, were tested by two-factor ANOVA. All data were log transformed before ANOVA to equalize variances. Results The concentrations of chlorophyll a, algae and microorganisms in Lake Mahinerangi at the start of the experiment are shown in Table II. Survival of the added zooplankton and their offspring in the enclosures at the end of 4 days was very high (97-100%, except for one enclosure with 75%). In addition to the added zooplankton, other small zooplankton at low densities were retrieved from most of the enclosures after 4 days: B.hamata nauplii (0.2-3.5 1"') and first instar copepodites (0.2^41"1), early copepodite instars of a small cyclopoid (0-1.4 I"1) and small saccate rotifers, probably Synchaeta (0-8.2 I"1). Densities of these 'other zooplankton' in the enclosures were not related to enclosure treatment (addition of nutrients, species and density of zooplankton). Table II. Initial concentrations of chlorophyll a and microorganisms in Lake Mahinerangi at the start of the experiment Initial concentration (1 SE) Algae Chlorophyll a (jig I 1 ) (cells 1 ') 3.45 380 000 930 (0.116) (70 000) (128) Picophytoplankton (cells ml 1 ) Red fluorescing (r-picoplankton) Orange fluorescing (o-picoplankton) 11 090 4300 (530) (230) 685 000 97 700 6900 782 700 (2470) (890) (860) (2040) Flagellates (cells ml 1 ) Heterotrophic nanoflagellates (HNF) Photosynthetic flagellates (PF) 2950 12.75 (930) (0.24) Ciliates (cells 1"') Large oligotrichs (>20 p.m) Small oligotrichs (£20 jun) Total ciliates 1258 400 2007 (190) (118) (322) Cyclotella (cells I 1 ) Staurastrum, Staurodcsmus Bacteria (cells m l ' ) Cocci (<1 p.m) Small rods (<1 (j.m) Large rods (~2 jim) Total small bacteria (<1 p.m) 687 C.W.Bums and M.Schallenberg 301 N : <O.0O1 N : <0.001 Dns B:n6 NxD ns NxB: ns OL DL BL OH DH =• 20- g 10 OL BH DL 300i BL OH DH BH DH BH TREATMENT TREATMENT 5001 N : p <0.001 N : p <0.001 N : p <0.001 D: ne B :ne NxD ns NxB: ns N : p <0 001 400 200 300 200 100 04 DL BL OH TREATMENT DH BH OL DL BL OH TREATMENT Fig. 1. Nutrient concentrations (u.g I', ± 1 SE) in enclosures after 4 days. OL, DL and BL refer to low nutrient (unenriched) enclosures without added zooplankton (OL), with added Daphnia (DL) and with added Boeckella (BL); OH, DH and BH are high nutrient (enriched) enclosures without added zooplankton (OH), with added Daphnia (DH) and with added Boeckella (BH). The results of twofactor ANOVA for treatment and interaction effects are shown in the top left of each graph, where N = nutrients, D = Daphnia and B = Boeckella. Nutrients The effects of nutrient enrichment on the levels of soluble and total N and P are shown in Figure 1. Nutrient enrichment significantly enhanced the concentrations of inorganic and total N and P in the enclosures (three-factor ANOVA: all F values for nutrient effects >24; all P = 0.0001), but there were no effects of zooplankton species or density on these concentrations, nor was there any evidence of interactions between zooplankton and nutrients (all P > 0.2). As the nutrient concentrations in enclosures containing 2 adult Daphnia I"1 and 2 adult Boeckella I"1 did not differ significantly from those containing 8 H of the same species in the same nutrient treatment, the zooplankton treatments were pooled within species and nutrient treatments for two-factor ANOVA (Figure 1). 688 Microbial food web of a mesotrophic lake Table DL Results of three-factor ANOVA on log-transformed concentrations in enclosures on day 4 for chlorophyll a (jig 1'). and days 1 and 4 for Cyclotella (cells ml"1). The analyses exclude treatments without added zooplankton (OL and OH). Probabilities of 50.05 are shown in bold d.f. Nutrients (A) Species (B) AB Density (C) AC BC ABC Error 1 1 1 1 1 1 1 16 Chlorophyll a D 4 Cyclotella Dl Cyclotella D4 F P F P F P 78.61 25.45 2.52 7.44 2.47 0.70 0.01 0.000 0.000 0.132 0.015 0.136 0.415 0.941 0.93 5.02 0.06 0.32 0.16 2.31 1.36 0.351 0.039 0.816 0.580 0.690 0.148 0.260 40.76 4.37 0.04 14.90 0.89 8.46 0J3 0.000 0.053 0.840 0.001 0.360 0.010 0.571 14- N . p <0.001 N . p <0.001 D p <0.001 B : ns NxD p=0.04 NxB: ns 12" 10' 8 6 OL 8DL 8BL OH 8DH 8BH TREATMENT Fig. 2. Algal biomass, expressed as chlorophyll a (u,g I"1, ± 1 SE), in enclosures after 4 days. Abbreviations and the presentation of statistical results are as in Figure 1. Histograms for zooplankton treatments are for 8 animals I 1 . Algae Algal biomass, measured as chlorophyll a, increased after 4 days in response to nutrients with zooplankton species and density also having significant effects (Table III). After 1 day, a small species of Cyclotella (—10 jtm diameter), the most abundant alga, was significantly lower in enclosures containing Daphnia than in those with Boeckella (d.f. = 23, F= 5.23, P = 0.032). After 4 days, the concentrations of Cyclotella paralleled those of chlorophyll a, with significant effects of nutrients, zooplankton species and density, and a significant interaction term (Table III). In enclosures without added zooplankton, chlorophyll a responded to nutrient enrichment by increasing to 11 u,g I"1 which is >2.4 times that in the unenriched enclosures (Figure 2). When the responses in chlorophyll a are related to zooplankton biomass, algal biomass declined with increasing Daphnia biomass in the 689 C.W.Bums and M.Sctiallenberg presence and absence of added nutrients, but showed no significant responses to Boeckella biomass (Figure 3A). The growth rate of Cyclotella followed the same pattern, declining in the presence of Daphnia, but not Boeckella (Figure 3B). At the highest Daphnia concentration (~330 jig I"1), this decline corresponded to the daily removal of 37% of the Cyclotella cells. Picophytoplankton Two kinds of cells were distinguished based on their fluorescence with the green filter set: those that fluoresced red, and were generally more abundant, and those that fluoresced bright orange. Both are probably chroococcoid cyanobacteria that differ in the composition, or proportions, of their main phycobiliproteins (Reynolds, 1984; Maeda et ai, 1992; Weisse, 1993). We refer to the two types as r-picoplankton (red-fluorescing) and o-picoplankton (orange-fluorescing). The results of three-factor ANOVA for the effects of nutrients, zooplankton species and density on the abundance of eukaryotic picoplankton (r-picoplankton) and prokaryotic picoplankton (o-picoplankton) after 4 days are shown in Table IV. The abundance of r-picoplankton did not change in response to nutrients, species of grazers or grazer density after 1 day (three-factor ANOVA, all P > 0.5) or 4 days (Table IV); concentrations of r-picoplankton in enclosures that lacked zooplankton did not differ significantly from those in enclosures with added zooplankton (Figure 4). Abundances of o-picoplankton in enclosures after 1 day showed weak, but significant, effects of zooplankton species (three-factor ANOVA, F = 4.56, P = 0.048) and significant interaction terms (species x density; nutrients x species x density: 0.01 < P < 0.05). After 4 days, concentrations of o-picoplankton were lower in the presence of added nutrients, and lower in enclosures with Daphnia than Boeckella, with significant interaction effects (Table IV). When enclosures without added zooplankton are included in the analysis (two-factor ANOVA), the effects of nutrients on o-picoplankton concentrations disappear, but there are significant effects of Daphnia and of Boeckella-nutrient interactions (Figure 4). Bacteria Heterotrophic bacteria consisted primarily of small cocci and rods <1.0 (xm, but some large rods (~2 M-m), visible on primulin-stained filters with the UV filter set, were present initially at densities of 7 x 103 ml 1 (Table II). After 1 day, there were no significant changes in bacterial abundance related to nutrients, or to species or density of zooplankton (three-factor ANOVA). Densities of small bacteria in the enclosures increased significantly from 0.85 x 10* ml 1 (SE ± 0.03 x 106) on day 1 to 1.1 x 106 ml"1 (SE ± 0.06 x 10") on day 4 (P = 0.003). Concentrations of small bacteria, large rods and total bacteria were significantly higher in nutrientenriched enclosures on day 4, although small bacteria and total bacteria were less abundant in the presence of Daphnia than in the presence of Boeckella (Table V); interactions between nutrients, species and density affected the concentrations of small bacteria and total bacteria. When the enclosures without zooplankton were included in the analysis, there were no measurable effects of nutrients, Daphnia or Boeckella on the abundance 690 Microbial food web of a mesotrophic lake 100 200 300 400 ZOOPL BIOMASS (jig^) 1.01 B o u 100 200 300 400 ZOOPL BIOMASS (ugfl) Fig. 3. Relationship between algal indices and zooplankton biomass of Daphnia (circles) and Boeckella (squares) in nutrient-enriched (solid symbols) and unenriched (open symbols) enclosures. Linear regression lines were fitted to the data for each species and are shown for Daphnia, which is the only species for which the relationships were statistically significant (P<,0.05). (A) Regressions relating chlorophyll a (y, in p.g I -') to biomass of added Daphnia (x, in ng I 1 ) in enriched enclosures, when y = 10.194 - 0.0173J: (r2 = 0.642), and in unenriched enclosures, wheny = 3.8566 - 0.00397* (r2 = 0.524). (B) Regressions relating net growth rate (y, as g d a y ' ) of Cyclotella to biomass of added Daphnia (x, in jig I 1 ) in nutrient-enriched enclosures, when y = 0.77028 - 0.00113x (r2 = 0.758) and in unenriched enclosures, when y = 0.44398 - 0.00096* (r2 = 0.763). of small bacteria after 4 days (Figure 5), although net growth rates of cocci and small rods increased with increasing Boeckella biomass in the enriched enclosures (cocci: r2 = 0.408, P = 0.064; rods: r> = 0.636, P = 0.01). The large rods multiplied during the 4 day incubation period to attain densities, in the absence of Daphnia, of —10 x 103 ml 1 in the unenriched enclosures and 30 x 103 ml"1 in the enriched enclosures. In the unfertilized treatments, they comprised 0.8% of the total bacterial abundance on days 1 and 4; in the enriched enclosures, this percentage increased from 0.9% on day 1 to 2.4% on day 4. Boeckella had no effect on the abundance of these rods (Figure 6). Net growth 691 CW.Burns and M^challenberg Tabk IV. Results of three-factor ANOVA on log-transformed abundances (numbers ml 1 ) of orangefiuorescing photosynthetic picoplankton (o-pico), red-fluorescing picoplankton (r-pico), heterotrophic nanoflagellates (HNF) and photosynthetic flagellates (PF) in enclosures on day 4. The analyses exclude treatments without added zooplankton (OL and OH). Probabilities of £0.05 are shown in bold d.f. Nutrients (A) 1 1 Species (B) AB 1 Density (C) 1 AC 1 1 BC ABC 1 Error 16 o-pico PF HNF r-pico F P F P F P F P 7.15 13.6 1.69 1.91 1.50 0.01 10.2 0.017 0.002 0.213 0.186 0.238 0.922 0.006 1.46 0.27 0.10 0.17 0.% 3.64 0.89 0.245 0.614 0.761 0.681 0.343 0.075 0.358 1.53 0.14 0.27 1.77 1.06 0.00 0.501 0.235 0.709 0.611 0.202 0.318 0.972 0.489 9.78 0.13 0.69 1.90 0.064 2.84 0.178 0.006 0.725 0.419 0.187 0.804 0.111 0.679 rates of these rods declined with increasing Daphnia biomass in the presence and absence of added nutrients (Figure 6). Flagellates The abundance of heterotrophic nanoflagellates (HNF) in the enclosures ranged from 1300 to 5400 ml'; most were <5ujn. Variances within treatments were high and there were no measurable changes in HNF abundance in response to nutrients or zooplankton after 1 or 4 days and no significant nutrient-zooplankton interactions (Table IV, Figure 7). A large photosyntheticflagellate(PF) declined in abundance throughout the experiment, but was more abundant in nutrient-enriched enclosures after 4 days than in enclosures without added nutrients (Table IV, Figure 7). In nutrient-enriched enclosures, the growth rates of HNF after 4 days declined with Daphnia density and those of the photosynthetic flagellate declined with increasing Boeckella biomass (Figure 7). Ciliates Oligotrichs (Strombidium, Strobilidium, Halteria) comprised 83% of the total ciliates in Lake Mahinerangi (Table II). Approximately 76% of these oligotrichs were >20 u.m in length; the largest ciliate was Paradileptus. The effects of zooplankton and nutrients on ciliate abundances after 1 and 4 days are shown in Figure 8. When enclosures lacking added zooplankton are omitted from the analysis (three-factor ANOVA), there were no significant changes in total ciliate abundance in response to nutrient enrichment after 1 day (F = 0.054, P = 0.818) and 4 days (F = 3.44, P = 0.080). There were, however, significant effects of zooplankton species, density and species-density interactions on ciliate abundance after 1 day (three-factor ANOVA, all P < 0.02) and 4 days (all P ^ 0.0005). The growth rates of total ciliates after 1 day were unrelated to the presence of Daphnia, but decreased after 4 days with increasing Daphnia biomass in the presence and absence of added nutrients (Figure 9). Boeckella had highly significant 692 MicTobial food web of a mesotrophic lake OL DL BL OH DH TREATMENT Nns 100001 N r s D : p <0.01 B: ns NxD ns NxB p <0 05 8000" O 6000" 3 4000 2000 OL DL BL OH DH BH TREATMENT Fig. 4. Autotrophic picoplankton (ml"', ± 1 SE) in enclosures after 4 days, r-picoplankton = red-fluorescing cells; o-picoplankton = orange-fluorescing prokaryotic cells under a greenfilterset (see the text). Abbreviations and the presentation of statistical results are as in Figure 1. negative effects on total ciliate population growth after 1 and 4 days, in nutrientenriched and unenriched enclosures (all P < 0.001). To ascertain whether Boeckella depressed the population growth rates of large oligotnchs more strongly than those of small oligotnchs, which might be the case if Boeckella fed selectively on larger ciliates, the relationships between Boeckella biomass and oligotrich growth rates after 4 days were examined separately for oligotrichs that were £20 p.m (small) and >20 u.m (large) in the two nutrient treatments. The net growth rates of small ciliates were less than those of large ciliates (Figure 10). In the unenriched enclosures, the slope of the regression line for growth rate of large ciliates was significantly steeper than that of small ciliates (one-tailed r-test for differences between the slopes of the regression lines: t = 2, P < 0.05), but in the enriched enclosures the slopes of the regression lines did not differ (/= 1.472, P> 0.05). 693 C.W.Buras and M-Schallenberg Table V. Results of three-factor ANOVA on log-transformed concentrations (numbers ml ') of small bacteria (< I u.m). large rods and total bacteria in enclosures on day 4. The analyses exclude treatments without added zooplankton (OL and OH). Probabilities of £0.05 are shown in bold df Nutrients (A) Species (B) AB Density (C) AC BC ABC Error Small bacteria Large rods Total bacteria F P F P F P 5.133 4.37 2.80 1.08 1.72 0.02 8.23 0.038 0.053 0.113 0.314 0 209 0.877 0.011 51.99 2.75 0.00 3.66 0.87 1.89 1.09 0.000 0117 0.994 0.074 0.365 0.188 0.313 6.16 4.60 2.84 0.91 1.54 0.04 8.52 0.025 0.048 0.111 0.354 0.232 0.854 0.010 1( 2.01 DAY 4 N . ns Dns NxD: ns DAY1 OL 8DL 8BL OH N: ns B:ns NxB: ns 8DH 8BH TREATMENT Fig. 5. Bacteria (x l0*ml ', ± I SE) in enclosures after 1 day (open columns) and 4 days (solid columns). Abbreviations and the presentation of statistical results are as in Figure I. Histograms for zooplankton treatments are for 8 animals I '. The weight-specific rates at which Daphnia and Boeckella cleared microorganisms from the water are shown in Table VI. Clearance rates for small particles <10 [Lm (bacteria, flagellates, Cyclotella) are lower than those for ciliates. Boeckella cleared ciliates from the water at rates of 3.18-5.85 ml \ig (dry wt)-' day 1 , which are an order of magnitude higher than those at which Daphnia removed ciliates. Keratella was the only small rotifer present in the enclosures (Figure 11). Abundances ranged from 0 to 56 rotifers I 1 , but were unrelated to enrichment, species or concentration of crustacean zooplankton (three-way ANOVA, all P > 0.3). Discussion In studies of complex food web interactions in lakes, it is tempting to impose perturbations that are unrealistically large in order to ensure measurable outcomes. 694 Microbial food web of a mesotrophic lake 401 N:p<0.001 D:p<0.01 NxD ns N:p<0001 Bns NxB: ns » A 30' I 20- O 10- OL 8DL 8BL OH 8DH 8BH TREATMENT B 100 200 300 400 ZOOPL BIOMASS (ug/1) Fig. 6. (A) Large rod-like bacteria (x 10-' ml"1, ± 1 SE) in enclosures after 4 days. Abbreviations and the presentation of statistical results are as in Figure 1. Histograms for zooplankton treatments are for 8 animals I 1 . (B) Relationship between net daily growth rates of large rods (y, as g day-') in enclosures on day 4, and Daphnia biomass (x, in (ig I'). In enriched enclosures (black circles), y = 0.4643 - 0.00076* (H = 0.387, P = 0.0734); in unenriched enclosures (open circles), y = 0.06773 - 0.00063* (r2 = 0.450, P = 0.0478). Whereas the results of extreme 'presses' may shed light on processes and pathways involved in ecosystem functioning, they cannot readily be extrapolated to describe the relative importance of the various interactions in the lake itself. In our study, the concentrations of TN, TP and zooplankton in the enclosures at the end of the incubation period fall within the range of concentrations that have been recorded in Lake Mahinerangi (Malthus, 1986; Burns, 1992). For this reason, the scope and scale of the responses of the microbial community in the enclosures are likely to be realistic reflections of those that occur in the lake. Responses after 1 day—top-down effects Our objective in sampling the enclosures after 1 day was to identify and separate the dominant top-down effects of Daphnia and Boeckella on algae and the microbial community from the combined effects of consumption (top-down) and 695 C.W.Bums and M-Schallenbcrg N : p <0.01 N : p <0 06 Dns Bns MxDire NxBrs 8OOO 7000 • B 6000 500040003000 2000 1000 OL OH a OH BH DH DL TREATVBfT BH TREATNBCT c 0.3 4 BM- I", z 0.0 I m -01 \ m 100 200 300 ZOOPL BIOUASS (|igfl) 400 100 200 300 400 ZOOPL BIOMASS Ctg/I) Fig. 7. (A) and (B) Flagellates (ml 1 , ± 1 SE) in enclosures after 4 days. HNF= heterotrophic nanoflagellates (A); PF = photosynthetic flagellate (B). Abbreviations and the presentation of statistical results are as in Figure 1. (C) and (D) Relationship between net growth rates of flagellates (g day-') in enriched enclosures on day 4, and zooplankton biomass (jig 1 ') of Daphnia (circles) and Boeckella (squares). (C) Regression line relating net growth rate of heterotrophic nanoflagellates (y) to Daphnia biomass (*), where y = 0.1586 - 0.000665* (^ = 0.430, P = 0.055). (D) Regression line relating net growth rate of a photosynthetic flagellate (y) to Boeckella biomass (x), where y = 0.154 - 0.002607* (r2 = 0.854, P< 0.001). nutrient enhancement (bottom-up) after 4 days. Through their feeding activities, Daphnia and Boeckella exerted two different, and strong, top-down effects on the microorganisms in Lake Mahinerangi. Adult D.carinata at moderate densities (2-81"1) lowered the concentration of the dominant alga, Cyclotella, significantly, but had no measurable impact on any components of the microbial food web. In contrast, Boeckella reduced the concentration of ciliates severely and had no measurable direct effects on other microorganisms, or algae. These responses are consistent with the well-documented prowess of large Daphnia to clear algae from water in short-term grazing experiments (reviewed by Lampert, 1987) and recently documented abilities of freshwater calanoids (Diaptomus spp., Epischura) to cull ciliates (Burns and Gilbert, 1993; Hartmann etal., 1993; Wiackowski et al., 1994). 6% Microbial food web of a mesotrophic lake 50001 DAY1 N rts B: p < 0 001 NxB:ns 4000- DAY 4 N : p < 0.05 D : p < 0 006 NxDns N:ns B:p<0.001 NxBns 3000 2000 1000 OL 8DL 8BL 8DH 8BH TREATMENT Fig. 8. Total ciliates (number 1', ± 1 SE) in enclosures after 1 day (open columns) and 4 days (solid columns). Abbreviations and the presentation of statistical results are as in Figure 1. Histograms for zooplankton treatments are for 8 animals I 1 . Responses after 4 days—bottom-up effects The strong positive effects of inorganic nutrient additions on the levels of soluble inorganic N and P and total N and TP in the enriched enclosures (Figure 1) confirm that the enrichment procedures were successful. The presence of crustaceans in the enclosures had no effects on concentrations of NH4-N and PO4-P after 4 days, probably because the highest biomass of zooplankton used (—330 (ig 1"') was too small for rates of nutrient remineralization to be detected, and because inorganic nutrients released by excretion and egestion of the zooplankton are taken up rapidly by algae. In a study of nutrient recycling by crustacean zooplankton in Lake Washington, Lehman (1980) found that enclosures enriched with >600 ng (dry wt) I"1 of Daphnia and copepods had higher concentrations of NH4-N, PO4-P and dissolved P after 2 days than those containing 40-160 (xg 1~' zooplankton, but that these differences disappeared after 3 days. In mesotrophic Castle Lake, Elser and Goldman (1991) detected no increase in SRP after 4 days in enclosures containing 425 n-g \~l Daphnia, although NH4-N increased. As the highest biomass of added zooplankton in our study was —330 u.g I"1, and the period of in situ enclosure was 4 days, we did not expect to find differences in nutrient concentration that could be related to differences in zooplankton biomass, although increases in soluble inorganic N, associated with the presence of Daphnia, and increases in SRP, associated with the presence of Diaptomus, have been reported in enclosures after 8 days (Brett et ai, 1994). The addition of nutrients had positive effects on algal biomass and the growth rates of a small diatom, Cyclotella, a large photosynthetic flagellate, and bacteria, but no measurable effects on the abundance of r-picoplankton, HNF and ciliates (Table VII). The strong stimulatory effect of nutrient additions on the growth of Cyclotella indicates that it was nutrient limited and is consistent with the low concentrations of soluble inorganic nutrients in Lake Mahinerangi at the time of the study. In contrast, r-picoplankton did not respond to nutrient enrichment, which 697 C.W.Bums and M^challenberg 02- OAY1 LOWNUTTBQfTS 0.0- ° A -021 •oV -06 -08 •10 •12 100 200 300 400 100 ZOOPL BtOWASS (p»fl) 200 300 ZOOPL BIOMASS (Mflfl) DAY1 HCHNUmBENTS c L KB 3 » CO -04 3 -08 0 • \ 0 \ • °\ , • N—, 200 100 300 ZOOPL BtOIIASS (MB/0 100 200 300 ZOOPL BtOUASS (Jltfi) Fig. 9. Relationship between net growth rates (y, as g day 1 ) of ciliates on days 1 and 4 and zooplankton biomass (x, in jig 1') in unennched (low nutrients) and enriched (high nutrients) enclosures. Linear regressions were fitted to the data for Daphnia (open circles) and Boeckella (solid squares); regression lines are shown only when the relationships are statistically significant (Pi 0.05). After 1 day, ciliate growth rate decreased with increasing Boeckella biomass in unenriched enclosures (A) asy = -0.242 0.0058&r (H = 0.884), and in enriched enclosures (C) as y = -0.1793 - 0.005341* (r2 = 0.841). After 4 days, ciliate growth rate decreased in unenriched enclosures (B) with increasing Boeckella biomass as y = 0.07297 - 0.00451 \x (r2 = 0.871), and with increasing Daphnia biomass as y = 0.3353 - 0.00051* (r* = 0.575); in enriched enclosures (D), ciliate growth rate decreased with increasing Boeckella biomass as y = 0.1352 - 0.0041 ]6x (r2 = 0.911), and with increasing Daphnia biomass as y- 0.1132 0.00043001(^ = suggests that they were not nutrient limited, or that any nutrient-stimulated population growth was balanced by losses of cells to grazers. The negative response of o-picoplankton to nutrients in the presence of zooplankton implies that enrichment affected them adversely in addition to a depression in their population growth caused by Daphnia. The hypothesis that high nutrient levels may depress, rather than stimulate, population growth of picoplankton is consistent with observations that picoalgal abundance tends to decrease with increasing lake trophy (Stockner and Antia, 1986; Burns and Stockner, 1991), and competitive superiority of eukaryotes over prokaryotes in the ability to take up nutrients from enriched waters (Weisse, 1993), although changes in abundance and species of grazers accompanying eutrophication cannot be ruled out. Some evidence suggests that the relatively low N:P ratio in the fertilized enclosures (12:1) may selectively favour eukaryotic algae rather than prokaryotic picoplankton. Chemostat 698 Microbial food web of a mesotropbic lake 0.4 LOW NUTRIENTS A 0.2J I X 5 OOtl -02-0.4 • -0.6 D -0.8 100 200 BOECKELLA BIOMASS (jig/1) HIGH NUTRIENTS B 200 BOECKELLA BIOMASS (ng/1) Fig. 10. Relationship between net growth rates (g day 1 ) of oligotrich ciliates on day 4 and Boeckella biomass (u.g I ') in unenriched (low nutrients) and enriched (high nutrients) enclosures. Linear regressions were fitted to the data for small oligotrichs (£20 u.m, open squares) and large oligotrichs (>20 u.m, solid squares). (A) Low nutrients. Net growth rates (y) of small oligotrichs decreased with Boeckella biomass (x) as y = -0.12201 - 0.00362.V (r' = 0.672. P = 0.007); net growth rates of large oligotrichs decreased as y = 0.13701 - 0.00541* (r2 = 0.842, P< 0.001). (B) High nutrients. Net growth rates (y) of small oligotrichs decreased with Boeckella biomass (jr) as y = -0.2694- 0.003 \&x(r> = 0.397, P = 0.069); net growth rates of large oligotrichs decreased as y = 0.22598 - 0.0O478J (r* = 0.922, P< 0.001). studies with natural plankton showed that Synechococcus dominated at a N:P supply ratio of 20, whereas diatoms and Scenedesmus dominated at ratios of 2 and 7 (Pick and Lean, 1987). Bacteria have high affinity for inorganic N and P [e.g. Caron et al. (1988) and references therein]. It is not surprising, therefore, that nutrient additions stimulated bacterial population growth rates after 4 days. Bacterial growth was enhanced by nutrients only in the presence of zooplankton (Table V) and not when zooplankton were absent (Figure 5), probably because protistan grazers, primarily 699 C.W.Bums and M.SchaUenberg Table VI. Clearance rates [ml jig (dry wt)~' day 1 ] derived from the slopes of significant regressions (P <, 0.05) that relate growth rate negatively to biomass of Daphnia and Boeckella. L and H refer to enclosures without (L) and with (H) added nutrients Daphnia Algae Chlorophyll a Cyclotella Picophytoplankton r-picoplankton o-picoplankton Bacteria Large rods Flagellates HNF PF Ciliates Total, day 1 Total, day 4 Boeckella L H L H 0.29 0.96 0.6 1.13 - - - 0.68 0.63 0.76 _ - 0.66 - _ 2.6 0.51 0.43 Oligotrichs 220 JJUTI Oligotrichs >20 u.m 0.42 5.85 4.51 3.61 5.4 5.34 4.12 3.18 4.77 heterotrophic flagellates and ciliates, were more abundant in enclosures that lacked zooplankton and were suppressing the bacteria (see below). Responses after 4 days—top-down effects When present at a density of 8 adults I"1 for 4 days, Daphnia strongly depressed algal biomass and the growth rate of Cyclotella. Daphnia cleared Cyclotella from the water at rates of 0.96-1.13 ml u-g (dry wt)-' day 1 (Table VI) or ~2 ml Daphnia-* rr1. The adult D.carinata in Lake Mahinerangi were relatively large (1.87 mm) and this rate is similar to maximum rates recorded for other large-bodied Daphnia (Lampert, 1987). In contrast, Boeckella at the same density had no impact on algal biomass or the growth rate of Cyclotella after 4 days. Several factors may have contributed to this difference between Daphnia and Boeckella in their impact on phytoplankton. (i) Calanoid copepods feed selectively and prefer large to small algae so that their weight-specific clearance rates for small algae are lower than those of less selective daphniids. When exposed to monocultures of algae at low concentration, female B.hamata clear Cyclotella and Cryptomonas at maximum rates of 0.2-0.7 ml animal"1 h"1 (Burns and Xu, 1990a; Burns and Hegarty, 1994) compared to 3-10 times this rate for large D.carinata (this study; Ganf and Shiel, 1985). Even at these maximum rates, and not allowing for algal growth, Boeckella at a density of 8 females I"1 would clear Cyclotella from <14% of an enclosure in a day. (ii) Adult Boeckella are smaller and weigh less than adult D.carinata so that 8 Boeckella I"1 had approximately the same biomass as 2 Daphnia I-1; this latter daphniid density had no measurable impact on algal biomass or Cyclotella growth rate. 700 Microbial food web of a mesolrophic lake ^ BL OH BM TREATMENT 1 Fig. 11. Keratella (number I- , ± 1 SE) in enclosures after 1 day (open columns) and 4 days (shaded columns). Abbreviations are as in Figure 1. Therefore, we cannot discount the possibility that higher densities of Boeckella (~330 u.g I"1 or 22 adult females 1"') may also have had detectable top-down effects on Cyclotella. The absence of any detectable response of r-picoplankton to the presence of crustaceans is consistent with low rates of clearance of autotrophic picoplankton by Daphnia and Diaptomus (Bogdan and Gilbert, 1984; Lampert and Taylor, 1985; Fahnenstiel et ai, 1991). However, grazing by Daphnia probably accounts for the negative effect of this cladoceran on the growth rate of o-picoplankton in the enriched enclosures. Although the dominant consumers of coccoid cyanobacteria are likely to be smallflagellatesand ciliates (e.g. Fahnenstiel etal., 1991), Daphnia removed Synechococcus in Schdhsee (Lampert and Taylor, 1985) and o-picoplankton have been observed in enormous numbers in the guts of cladocerans (Burns and Stockner, 1991). As Daphnia have poor ability to feed selectively (Lampert, 1987), and hence are unlikely to be able to discriminate between r- and o-picoplankton, we attribute their significant negative effect on o-picoplankton and non-significant effect on r-picoplankton to high variances of the r-picoplankton data that increase the probability of a Type II error. Other explanations are possible, however. Picoplankton are at the lower end of the size range of particles that most large Daphnia can remove efficiently (B0rsheim and Andersen, 1987; Lampert, 1987). The o-picoplankton cells may have been slightly larger than those of the r-picoplankton and hence have been retained more efficiently by D.carinata. The average net growth rates of ciliates in enclosures over the first 24 h were negative, even in the absence of added crustaceans (Figure 9). These net negative rates may reflect the sensitivity of some ciliates to containment (Taylor and Johannsson, 1991). For example, Strombidium disappeared at rates of -0.77 to -0.2 day-' from enclosures in Lake Michigan (Carrick et al., 1992); it was also present in our enclosures. The multiplication of more robust species during the 4 day incubation in Lake Mahinerangi resulted in positive mean growth rates for ciliates >20 u.m in enclosures without added zooplankton of 0.03 day 1 (no added nutrients) to 0.14 day 1 (enriched). Although low relative to the potential rates of increase of oligotrichs in the presence of abundant food (Taylor and Johannsson, 1991), these rates fall within the range of 0-1.4 day 1 which have been recorded for 701 C.W.Bums and M.Scfiallenberg Table VII. Summary of responses of microbial food web organisms in Lake Mahinerangi to nutrients and crustacean grazers after 4 days Signs indicate statistically significant (P<0.05) increases (+). decreases (-) or no changes (NS) in abundance or net growth rate in nutrient-enriched enclosures relative to unenriched enclosures and in nutrient-enriched enclosures with grazers relative to nutrientenriched ones without grazers (ANOVA, or linear regression*) Response to Nutrients Grazers Daphnia Boeckella Algae Chlorophyll a Cyclotella + + - NS NS Picophytoplankton Red-fiuorescing Orange-fluorescing NS NS NS NS NS Bacteria Cocci Small rods Large rods Total small bacteria NS NS + NS NS NS +* +* NS +* Flagellates HNF PF NS + _* NS NS Ciliates Large oligotnchs Small oligotrichs Total ciliates NS NS NS NS _* NS ciliates in Lakes Michigan and Ontario (Taylor and Johannsson, 1991; Carrick et al., 1992). Factors that may have contributed to the relatively low net growth rates in our experiments are: (i) differential sensitivity of species to containment; (ii) differential growth rates of ciliates (Carrick etal., 1992) and the possible domination by slow-growing species; (iii) the temperature at the time of the study (13°C); (iv) resource limitation (see below); (v) predation. Potential metazoan predators of ciliates (small saccate rotifers, nauplii and early copepodites) were relatively sparse in our enclosures (<91 '), but in view of their ability to clear ciliates at rates of up to -20 ml animal-1 day-' (Burns and Gilbert, 1993; Gilbert and Jack, 1993), they cannot be dismissed as potential contributors to the low net rates of ciliate growth. After 4 days in the presence of Daphnia, densities and growth rate of ciliates were depressed (Table VII). Negative effects of Daphnia on ciliates have been reported in several studies (Porter et al., 1979; Carrick etal., 1991; Pace and Funk, 1991; Wickham and Gilbert, 1991,1993; Jack and Gilbert, 1993,1994; Brett etal., 1994; Wiackowski etal., 1994). In our study, D.carinata did not suppress small and large ciliates differentially according to size, as has been noted in other studies (Wickham and Gilbert, 1993; Jack and Gilbert, 1994; Wiackowski et al., 1994). In studies in Star Lake, populations of small ciliates were depressed by D.pulex more than those of large ciliates (Wickham and Gilbert, 1993; Jack and Gilbert, 1994), whereas large ciliates were affected more than small ones by the Daphnia in Castle 702 Microbial food web of a mesotrophk lake Lake (Wiackowski et al., 1994). Several factors may account for a lack of differential response between small and large ciliates in the presence of Daphnia in our study, and for the difference in effects of Daphnia in the studies cited above: (i) different authors use different size thresholds between 'large' and 'small' ciliate categories (e.g. —30 ^m for Wiackowski etal., 1994; 20 \im in our study); (ii) different intrinsic rates of increase between large and small ciliates; (iii) species-specific differences among ciliates in the ability to avoid competition (exploitative or interference), or consumption, by Daphnia that are unrelated to size (Jack and Gilbert, 1993; Wickham and Gilbert, 1993). Daphnia can lower the growth rates and abundance of ciliates directly by predation, interference, or both, and indirectly by exploitative competition for the same food resource [Jack and Gilbert (1994) and references therein]. The rates at which D.carinata removed ciliates from the water in enriched and unenriched enclosures [0.43-0.51 ml (xg (dry wt)~' day 1 ] are slightly less than those at which they cleared Cydotella (Table IV), which is consistent with ciliates being an 'incidental catch' in the inhalant current of Daphnia during their routine collection of algae and with avoidance behaviours of some ciliates (Jack and Gilbert, 1993; Wickham and Gilbert, 1993). This interpretation accounts for a daphniid effect on ciliates being detected after 4 days at the highest daphniid density, but not after 1 day. The clearance rates for ciliates that we measured would have allowed daphniid densities of 8 I"1 to remove ciliates from only 15% of the volume of an enclosure after 1 day, and 2 Daphnia V to remove ciliates from the same volume after 4 days. In contrast, Boeckella at 2 and 8 animals I 1 had strong negative effects on ciliate abundance and growth after 1 and 4 days. This difference is reflected in weightspecific clearance rates that are an order of magnitude higher than those of Daphnia (Table VI) and per capita rates that are 3-4 times higher than those of Daphnia. Boeckella is a suspension feeder that grows and reproduces well on purely algal diets (Burns and Xu, 1990b; Xu and Burns, 1991). Our finding that Boeckella has a much stronger negative effect on ciliates than Daphnia differs from that of Brett et al. (1994) and Wiackowski et al. (1994), who found that Daphnia rosea was as effective as the calanoid, Diaptomus novamexicanus, in Castle Lake in depressing ciliate growth. Daphnia were also more effective than copepods in depressing ciliates in enclosures in mesotrophic Schohsee (JUrgens et al., 1994a), although the effects of a calanoid (Eudiaptomus gracilis) cannot be separated from those of equally numerous cyclopoids (Thermocyclops oithonoides, Cyclops kolensis, Cyclops abyssorum). Our finding that Boeckella removed large ciliates (>20 jun) faster than small ones in enriched enclosures (Figure 10) is consistent with those of others who have reported that large ciliates are affected by crustacean zooplankton more adversely than small ones (Arndt and Nixdorf, 1991; Wiackowski et al., 1994). Flagellates are generally considered to be the major food resource of oligotrich ciliates, although bacteria and autotrophic picoplankton are also consumed, sometimes at high rates (reviewed by Laybourn-Parry, 1992). If the ciliate populations in Lake Mahinerangi were regulating flagellate abundance through predation (top-down control), we might expect tofindan inverse relationship between flagel703 C.W.Burns and M-Schallenberg late abundance and ciliate abundance. There was no correlation (P > 0.5). Instead, ciliate numbers increased with flagellate abundance in the nutrient-enriched enclosures (log-transformed data, n = 15, r2 = 0.2914, P = 0.0378), which implies that ciliate abundance may have been determined, in part, by HNF availability. There was no significant relationship between ciliate and flagellate populations in the unenriched enclosures, presumably because HNF were resource limited themselves (see below). The mean net growth rate of HNF in Lake Mahinerangi in the absence of crustaceans, 0.15 day 1 (Figure 7), falls within a range of 0.04-0.29 day 1 for HNF in Lake Michigan during the spring isothermal period (Carrick etal., 1992) and is at the lower end of a range of rates reported for HNF in mesotrophic Lake Constance in early summer (Weisse, 1991). Growth of HNF did not change in the presence of Boeckella, probably because copepods feed inefficiently on particles in the size range of the small (2-5 n,m) HNF which predominated (Sherr et al., 1986; Bogdan and Gilbert, 1987). In the presence of Daphnia, the growth rate of HNF decreased in the enriched enclosures (Table VII). As HNF growth rates were not depressed by Daphnia in the unenriched enclosures, which contained fewer bacteria for them to feed on, it is unlikely that suppressed rates in the presence of Daphnia were an indirect consequence of exploitative competition between Daphnia and flagellates for bacterial food resources; rather, the low HNF growth rates are likely to be a direct consequence of larger volumes of water cleared by Daphnia in the enriched enclosures (Table VI) and hence greater rates of consumption of HNF by Daphnia (top-down control). Christoffersen et al. (1993) report cladoceran control of HNF in eutrophic conditions. As an indirect indication of whether bacterial abundance might be limiting flagellate population growth in Lake Mahinerangi, correlations were carried out between HNF density and bacterial abundance in the presence and absence of added nutrients. HNF density was positively correlated with bacterial abundance in enclosures without added nutrients (n = 15, r = 0.580, P = 0.0235), but not in those with added nutrients. These results imply a strong coupling between bacteria and HNF that is broken by increased nutrient enrichment. A positive correlation exists between nanoplankton and bacteria over a wide variety of freshwater habitats and latitudes, and is consistent with a predator-prey interaction between HNF and bacteria (Berninger et al., 1991). Heterotrophic flagellates have been identified as the major consumer of bacteria in lakes, particularly when Daphnia are scarce (e.g. Sanders et al., 1989; Pace et al., 1990; Fukami et al., 1991). Strong coupling between HNF and bacteria at certain times of the year has also been inferred from studies in a variety of lakes (e.g. Nagata, 1988; Bloem and BSr-Gilissen, 1989; Weisse, 1991), although not in others (e.g. Pace and Funke, 1991; Carrick et al., 1992). Based on mean densities of bacteria and HNF in the unenriched enclosures of 1.13 x 106 bacteria ml 1 and 3000 HNF ml 1 , respectively (Figure 5), and a mean grazing rate of 15 bacteria HNF-1 Ir1 (Jurgens and Stolpe, 1995), the HNF in Lake Mahinerangi could remove 95% of the bacterial standing stock per day. Even if the flagellate grazing rates were considerably lower (2 nl flagellate-1 h~'; Jurgens, 1994), HNF still had approximately twice the potential of Daphnia to control bacterial abundance in Lake Mahinerangi (top-down control). 704 Microbial food web of a mesotrophic lake Daphnia at 8 I"1 (the highest biomass in our study) would only have removed ~8-27% of the standing stock of bacteria, based on rates of bacterial clearance by 1.87 mm Daphnia of 0.4 ml animal1 h"1 (Jtirgens and Stolpe, 1995) or 1.4 ml animal 1 h 1 (Porter et al., 1983). Our finding that the correlation between HNF and bacterial abundance disappeared in the enriched enclosures is consistent with the hypothesis that the HNF in Lake Mahinerangi are resource (bacteria) limited (bottom-up control). Berninger et al. (1991) concluded that the abundance of heterotrophic nanoplankton in nutrient-poor systems is largely determined and controlled by the abundance of bacteria; Andersen and S0rensen (1986) drew similar conclusions from their study of a eutrophic, coastal marine environment. In Lake Constance, the production of HNF appears to be limited in spring by the availability of bacteria (Weisse, 1991). The growth of HNF in our study appears to have been controlled from below when levels of inorganic nutrients were low, and from above at high nutrient levels. The biomass-specific clearance rates of Daphnia and Boeckella for flagellates and ciliates in Lake Mahinerangi ranged from 0.42 to 5.85 ml u-g (dry wt)-' day-' (Table VI). This range is similar to that of 0.4-5.2 ml jxg (dry wt)"1 day-' recorded for the clearance of flagellates and ciliates by macrozooplankton, primarily Diaptomus and Daphnia, in Lake Michigan (Carrick et al., 1991). The biomassspecific clearance rate of D.carinata for the smallflagellatesin Lake Mahinerangi, 0.66 ml (ig (dry wt)-' day 1 , which we calculated from the slope of the regression relating daphniid biomass to net growth rate of HNF (Table VI), lies within a range of biomass-specific rates of 0.46-0.77 ml fxg (dry wt) 1 day 1 for adult D.magna and D.hyalina feeding on the colourless chrysomonad Spumella (JUrgens and Stolpe, 1995). The biomass-specific clearance rates (Table VI) can be used to calculate the proportions offlagellateand ciliate standing stocks consumed by macrozooplankton per day. When present at densities of 8 individuals I"1 D.carinata consumed 19.8% of the HNF standing stock and 13-15% of the standing stock of ciliates per day. This estimate for ciliate consumption falls near the lower end of the range for Daphnia grazing on Strobilidium sp. in Storrs Pond, NH (Wickham and Gilbert, 1993) and is considerably higher than the 2.8% of ciliate standing stock consumed by macrozooplankton in Lake Constance (Weisse, 1990). In contrast to Daphnia, Boeckella at a density of 81"1 consumed between 49 and 70% of ciliate standing stock per day. If the ciliates in Lake Mahinerangi are primarily algivorous, as the dominance of oligotrichs implies (Fenchel, 1980), our finding that they are consumed so effectively by Boeckella suggests the existence of a strong nanoplankton-ciliate-calanoid coupling in this lake. Comparable couplings may also occur in lakes containing diaptomids. Equations that relate the rates at which copepods clear ciliates to calanoid body size (prosomal length) have been derived for species of Diaptomus and Epischura (Burns and Gilbert, 1993). The rates at which adult female B.hamata cleared ciliates from Lake Mahinerangi (Table VI) are equivalent to per capita rates of 48-88 ml animal"1 day*1 and bracket the rate of 68 ml animal"1 day-1 that is predicted for a diaptomid of the same mean prosomal length of the B.hamata in our study (1.06 mm). Thus, Boeckella and Diaptomus appear to be functionally equivalent in their potential ability to control ciliates. 705 C.W.Burns and M.Scfaallenberg Keratella populations were unaffected by the presence of Daphnia and Boeckella in our study. Although Daphnia, particularly large Daphnia, can suppress rotifers, including several species of Keratella (Gilbert, 1988; Wickham and Gilbert, 1991), suppression is not always evident, either because some species of rotifers have morphologies or behaviours that make them less susceptible to interference (e.g. Gilbert, 1988), or possibly because rotifer data from field experiments can be highly variable (e.g. Wickham and Gilbert, 1993). As Keratella abundances varied greatly within treatments in our study, we cannot exclude the possibility of a Type II error; it is worth noting, however, that the rotifer populations in Castle Lake, California, were not suppressed by Daphnia or Diaptomus at densities of 15 I"1 (Brett et at, 1994). The average turnover time of bacterial biomass in the enclosures in our study, calculated from net growth rates, was ~8 days which is within the reported range of several days to weeks for temperate lakes (Jlirgens and Glide, 1991, 1994). Bacterial abundance was positively correlated with chlorophyll a in enclosures without added nutrients (n = 15, r = 0.644, P = 0.0095), which is consistent with the hypothesis that prod ucts of algal exudation and lysis provide the main source of energy for bacterial production (Azam etai, 1983); small bacteria increased only in the nutrient-enriched enclosures in which algal biomass had increased markedly. These observations support the hypothesis that bacterial growth in Lake Mahinerangi was nutrient limited (bottom-up control). The increase in populations of large rods over 4 days in the fertilized enclosures without added macrozooplankton is probably a result of selective grazing by ciliates and flagellates favouring an increase in bacterial morphologies that are more resistant to protozoan grazing, and a poorer ability of larger rods to compete with small single bacteria for substrates in the unenriched enclosures (Glide, 1989), although substrate enhancement by the grazing activities of rotifers and small crustaceans (<150 |im) may also have stimulated the growth of these rods (Arndt et ai, 1992; Peduzzi and Herndl, 1992). Daphnia had strong negative effects on the density of large bacterial rods (~2 n.m), but no measurable effects on the abundance of smaller rods and cocci (<1 urn). Several authors report negative effects of Daphnia on bacterial populations in lakes (e.g. Riemann, 1985; Christoffersen et ai, 1990; Pace et al., 1990; Jiirgens et al., 1994a; MarkoSovi and Jezek, 1993), but these are at higher daphniid densities than in our experiments. Others report no effects of Daphnia on bacterial abundance (Pace and Funke, 1991; Wickham and Gilbert, 1993; Brett etal., 1994). Shifts in the morphometry of bacteria from large rods andfilamentsin the absence of Daphnia to small cocci in the presence of Daphnia have been reported (e.g. Glide, 1988; MarkoSova" and Jezek, 1993; Jurgens et al., 1994a). These shifts are attributed to the less efficient retention by Daphnia of small bacteria compared to larger cells (Brendelberger, 1985; DeMott, 1985; Glide, 1988; reviewed by Jurgens, 1994), and size-selective grazing by protozoans in the absence of Daphnia that increase the proportion of grazer-resistant larger forms (Glide, 1988). Boeckella had no effect on populations of large rods, probably because they were too small for the copepod to collect. Thisfindingis consistent with well-documented inabilities, and inefficiencies, of calanoid copepods to collect small algae and bacteria (Bogdan and Gilbert, 1984, 1987; Sanders et al., 1989; Pace et al., 706 Microbial food web of a mesotropbic lake 1990), and their preferences for larger food particles (Vanderploeg, 1990). The presence of Diaptomus had no effect on bacterial abundance in enclosures in Castle Lake (Brett etal., 1994). When copepods dominated the macrozooplankton in mesotrophic Schohsee, they caused an increase in bacterivorous protozoa which grazed on small bacteria so that the bacterial assemblage consisted primarily of large filamentous forms (Jlirgens et al., 1994a). The dramatic increase in small bacteria that occurred in the enriched enclosures in the presence of Boeckella was associated with the decline in ciliate abundance in these enclosures due to boeckellid predation. The net growth rates of cocci and small rods in these enclosures were strongly related inversely to ciliate densities (regression analysis on log-transformed data, n = 6; cocci: r2 = 0.877, P = 0.0058; small rods: r2 = 0.838, P = 0.0105). These results are consistent with the regulation of small bacteria in these enclosures through bacterivory by ciliates (top-down control) (Sherr and Sherr, 1987; Sanders et al., 1989; Christoffersen et al., 1990), although direct predatory control of bacteria by ciliates in our study is unlikely because ciliate densities in Lake Mahinerangi at the time of our study were too low (<5 ml"1) to have a significant grazer impact. Even if they consumed bacteria at a rate of 420 bacteria day 1 ciliate"1, the estimated rate for ciliate bacterivory in Lake Vechten (Bloem and Ba'rGilissen, 1989), or at a rate of 15 120 bacteria day"1 ciliate-1, which is the maximum rate measured by Sherr et al. (1989) for large marine ciliates, the ciliates in Lake Mahinerangi could have removed only <1% to 7% of the bacterial biomass per day. If the enhanced bacterial biomass in enclosures containing copepods were an outcome of reduced bacterivory by HNF, we might expect to find an inverse relationship between the abundances of bacteria and HNF. Bacterial abundance in the nutrient-enriched enclosures containing Boeckella was weakly correlated, inversely, with the abundance of HNF (Spearman rank correlation, n = 6, rs = -0.714, P = 0.110), which is consistent with predatory control of bacterial populations by heterotrophic flagellates. Increased bacterial production in the presence of macrozooplankton (cladocerans and copepods) has been reported and attributed to stimulatory effects of nutrients released by the grazing activities (sloppy feeding, excretion, defaecation) of the macrozooplankton (Crisman etal., 1981; Peduzzi and Herndl, 1992). Monomeric carbohydrates released by marine copepods enhanced the growth of bacteria, particularly in oligotrophic waters (Peduzzi and Herndl, 1992). Although inorganic nutrients are unlikely to have been limiting bacterial growth in the enriched enclosures, soluble substrates released by Boeckella during feeding and excretion may also have stimulated bacterial growth in our experiments (bottom-up regulation). The fact that the grazing effects of Daphnia on components of the microbial food web were evident after 4 days and in the presence of added nutrients indicates that top-down effects were stronger than nutrient effects in determining microorganism abundances in the presence of this zooplankter. The strong predatory impact of Boeckella on ciliates occurred in the presence and absence of added nutrients, indicating that ciliate populations were regulated more strongly by topdown control than by bottom-up effects; this conclusion is supported indirectly by the lack of any positive correlations between the population growth rate, or 707 CW.Burns and M.Schallenberg abundance, of ciliates and their potential food resources of bacteria and autotrophic picoplankton at both nutrient levels (Spearman rank correlations between ciliate net growth rate and microorganism concentrations, all P > 0.2). Two factors may have contributed to a lack of detectable changes in abundance or growth rates of bacteria, picophytoplankton and flagellates in response to these predatorinduced depressions in ciliate biomass: (i) the potentially weak grazing impact of a relatively low total ciliate abundance in Lake Mahinerangi and (ii) the incubation period. Although 4 day incubations have been used in studies that are comparable to ours (e.g. Elser and Goldman, 1991; Pace and Funke, 1991), a longer incubation period may have resulted in detectable responses to changes in ciliate abundance. The bacterial populations in Lake Mahinerangi, particularly those of large bacteria, are controlled from the bottom up by nutrients; large bacteria are also strongly controlled from the top down by Daphnia, whereas small bacteria are weakly controlled from the top down by HNF. Model The relative effects of Daphnia, Boeckella and inorganic nutrients on the microbial community of Lake Mahinerangi are summarized in Figure 12. Inorganic nutrients had strong stimulatory effects on the growth of phytoplankton, a large photosyntheticflagellateand large bacterial rods; weaker bottom-up effects were evident in the growth of small bacteria, HNF and ciliates in response to increased resources. In the presence of Boeckella, the top-down effects virtually ceased at the level of ciliates. In contrast to Boeckella, Daphnia 'broke open' the microbial loop by feeding directly on ciliates,flagellatesand large bacteria, as well as on phytoplankton, including some picoplankton. These contrasting effects of a cladoceran and a Fig. 12. Effects of Daphnia, BoeckeUa and nutrients on the pelagic microbial food web of Lake Mahinerangi. Solid lines indicate top-down effects; broken lines indicate bottom-up effects. Increasing strengths of the impacts are indicated by increasing thickness of arrows. Trophic and resource interactions that were not detected in this study have been omitted. 708 0.32 0.90 3.45 South Island Lake Wakatipu Lake Manapouri Lake Mahinerangi 2007 50 640 5100 4020 980 Actual ciliates (numbers I"1) 1920 3330 6860 2600 16 790 17 970 Predicted ciliates (numbers \-') 19.2 29.3 2.6 37.7 30.4 22.4 (%) Actual/predicted C.W.Burns (unpublished) C.W.Burns (unpublished) This study James el al. (1995) Morrow (1995) James et al. (1995) Reference • * Q- | 2 I veb of a mesotrophic s 0.57 18.21 20.65 North Island Lake Taupo Lake Rotorua Lake Okaro Chlorophyll a (M-gl1) Table VIII. Concentrations of chlorophyll a and ciliates in the open waters of New Zealand lakes. Empirically determined ciliate densities are expressed as a percentage of those predicted from chlorophyll a concentration according to the equation of Pace (1986): log Y = 3.547 + 0.538 log X, where Y is ciliate density (numbers I"1) and X is chlorophyll a (p.g I"1). Data for North Island lakes are annual means based on 4-12 samples per lake llcrobi C.W.Burns and M.Sctiallenberg copepod on the microbial food web of Lake Mahinerangi are consistent with predictions concerning the structures of microbial food webs in the presence and absence of abundant Daphnia (Porter et al., 1988; Stocknerand Porter, 1988; Pace et al., 1990; Wylie and Currie, 1991; Riemann and Christoffersen, 1993; Jurgens, 1994; Jurgens and Giide, 1994). If also consistent with hypotheses and models of energy flow differences in £>ap/i«/a-dominated and copepod-dominated systems (Scavia and Fahnenstiel, 1988; Wylie and Currie, 1991; Riemann and Christoffersen, 1993), they imply that in New Zealand's copepod-dominated lakes, more of the carbon that is fixed in photosynthesis will be lost to heterotrophic respiration (bacteria, flagellates, ciliates) and less will be available to higher trophic levels than in the Z)fl/?/m/a-dominated lakes of the northern hemisphere. By highlighting the relative importance of specific trophic linkages in the microbial food web of a mestrophic lake, Figure 12 identifies some of the difficulties and limitations inherent in adopting too simplistic a view of energy flow in microbial food webs as being controlled either by resources or by consumption (top-down versus bottom-up). Figure 12 also illustrates the concurrent impacts of both resource availability (bottom-up) and consumption (top-down) on the dominant alga and main components of the microbial food web. For example, the net growth rate of Cyclotella responded strongly to nutrients (bottom-up), but was equally strongly depressed by the grazing activities of Daphnia (top-down); the total ciliate growth rate increased with increasing flagellate resources, and decreased in response to predation by Daphnia and Boeckella, whereas bacterial growth rates responded positively to increases in substrate concentration and negatively to the grazing impacts of flagellates and Daphnia. The results of our study provide empirical support for the finding of Gasol et al. (1995), based on statistical analyses of the abundance of HNF in 16 Quebec lakes in relation to food (bottom-up) and predation (top-down) variables, that both food (bacteria) and predation (Daphnia) predicted HNF abundance reasonably well. The distinctly different effects of a cladoceran and a calanoid on the microbial community of Lake Mahinerangi prompt us to predict that in ecosystems that are dominated by suspension-feeding calanoid copepods, either permanently, as in some lakes and marine systems, or seasonally, as in many temperate lakes that do not freeze, ciliate densities will be low relative to those that occur when calanoids are less abundant. Calanoid copepods (Boeckella, Calamoecia), rather than Daphnia, tend to dominate the mesozooplankton of New Zealand lakes throughout the year (Chapman and Green, 1987). Estimates of ciliate density are available for only a few lakes in New Zealand, but in all of them ciliate densities are low relative to those in temperate lakes of similar trophic state elsewhere (Table VIII). In light of the strong top-down control of ciliates by Boeckella in our study, it is tempting to speculate that calanoid dominance may contribute to low ciliate abundance in New Zealand lakes. 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