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Home , Dystrophic lake, Microbial food web, Microbial loop, Picoplankton, Planktivore

AQUATIC MICROBIAL Vol. 19: 67-80, 1999 1 Published September 6 Aquat Microb Ecol l

Effects of nutrients, planktivorous and depth on components of the microbial

'Ottawa-Carleton Institute of Biology, University of Ottawa, Ottawa, Ontario KIN 6N5, Canada 2~epartementde Sciences Biologiques, Universite de Montreal, Montreal. Quebec H3C 357, Canada 3National Water Research Insitute. Canada Center for Inland Waters, PO Box 5050, Burlington. Ontario L7R 4A6, Canada

ABSTRACT: An experimental study was conducted uslng large enclosures to examine the effects of nutrients, presence of planktivorous fish, and depth of the water colun~non the components of the . Enclosures (8 m in diameter) were installed, open to the sediments, at both 3.5 and 11 nl depths in Lac Croche, a small oligotrophic lake on the Canadian Shield The of proto- zoans, including obligate heterotrophic (HNAN) and potentially mutotrophic flagellates (MIXO),was examined in relatlon to the abundance of thelr prey, both heterotrophlc (HBAC) and picocyanobactena (PPICO). HBAC levels increased over the summer and were s~gnificantlyhigher in the nutrient enriched enclosures. While HBAC dld not respond in a consistent manner to the pres- ence of fish, the effect of nutrients was greater when fish were present. HBAC levels did not differ slg- nificantly between the shallcw and deep enclosures. In contrast, PPICO were signlf~cantlydffected by all 3 independent variables, nutrients had a strong overall negative effect on abundance and the pres- ence of fish tended to Increase their abundance particularly in the shallow enclosures Similarly, nutri- ent additions resulted in sigruficantly lower HNAN levels, but neither the presence of fish nor depth of the water column slgniflcantly affected HNAN. However, the lowest average HNAN concentrations were in the deep nutrient enriched enclosures without fish, where the highest populatlons of Daphnia middendorffiana developed. MIXO were most abundant early In the season (exceeding HNAN) but declined significantly by mid-summer and were generally not as abundant as HNAN. MIXO levels were significantly lower in the nutrient enriched enclosures. Depth of the water column had a signifi- cant effect on MIXO, but MIXO levels were not affected m a consistent manner by the presence of fish. Experimental results did not demonstrate that Increases in nutnent levels would lead to Increases in HNAN as current empirical models imply. There was no increase in HNAN abundance related to Increased bacterial abundance.

KEYWORDS: Microbial food web . . Nutrients . . Top-down control

INTRODUCTION sive research (Fenchel 1987, Porter et al. 1988). Het- erotrophic nanoflagellates (HNAN) are generally the During the past decade, the ecological role of proto- most abundant in the euphotic zone of zoa in aquatic systems has become the focus of exten- aquatic systems and are now considered an important component of pelagic food webs (e.g. Azam et al. 1983, Present addresses: Sherr & Sherr 1984). Several studies have recognized 'New York City Dept. of Environmental Protection, Bureau these protozoa as the principal consumers of bacteria of Water Quality, Supply and Protection, Corona, New and picoplankton. They control bacterial populations York 11368-5107, USA through and convert bacterial production into "'Ottawa-Carleton Institute of Biology, University of Ottawa, Ottawa, Ontario KIN 6N5, Canada larger particles, which can then be utilized as food by "Addressee for correspondence. larger protozoans and metazoans (e.g. Sherr & Sherr E-mail: [email protected] 1984, Andersen & Fenchel 1985, Sanders et al. 1989).

O Inter-Research 1999 68 Aquat Microb Ecol 19: 67-80, 1999

Furthermore, HNAN play a key role in regulating piscivorous fish and large Daphnia species. However, nutrient flow through the of aquatic sys- in experimental studies HNAN are not always affected tems. Substantial amounts of organic carbon, nitrogen, by Daphnia additions (Pace & Funke 1991). There is and phosphorus contained in their prey (picoplankton) also evidence for efficient on HNAN by are released into the environment as regenerated min- other metazoans such as and calanoid cope- erals (e.g. Goldman & Caron 1985). pods (e.g. Sanders et al. 1994, Jiirgens et al. 1996) and Several factors potentially regulate the abundance of by (Arndt 1993). HNAN. The abundance of prey may be the most A third factor is thermal stratification, which may important factor regulating HNAN seasonal levels have profound effects on the relative importance of the (Fenchel 1982). A close coupling between HNAN and classical versus microbial food web (Cushing 1989). In bacteria has been observed in several systems (e.g. stratified water small cells tend to dominate Rassoulzadegan & Sheldon 1986) although not all (e.g. and and hence Pick & Hamilton 1994, Simek et al. 1997). In turn, bac- through the microbial loop is theoretically more signif- terial abundance or has been correlated icant (Porter et al. 1988, Kiarboe et al. 1990). In addi- with algal biomass or primary production over a wide tion, the relative importance of top-down versus bot- range of marine and freshwater systems (Bird & Kalff tom-up control may be quite different in shallow 1984, Cole et al. 1988). Berninger et al. (1991) de- versus deep lakes (Moss et al. 1994). scribed a significant correlation between bacterial In general, experimental tests of the role of nutrient abundance and HNAN abundance over a wide range supply versus predation in regulating HNAN have of freshwater systems, suggesting that system produc- been conducted in small containers or enclosures over tivity may be the most significant factor regulating short time periods (Riemann 1985, Pace & Funke 1991). HNAN. However, in temperate lakes of Eastern The effects of depth and thermal stratification on the Canada. HNAN abundance was not correlated with microbial loop in general have yet to be tested. In the bacterial abundance although total phosphorus ex- following study, large enclosures were used to investi- plained some of the variation observed between lakes gate the response of the microbial food web to altered (Tzaras & Pick 1994). A more extensive data analysis nutrients and overall food web structure. To address for a variety of aquatic systems by Gasol & Vaque the relative significance of nutrient loading (bottom- (1993) suggests that the relationship between HNAN up) or predatory (top-down) control on abun- and bacteria is not strong in any system. dance, we examined fertilized and unfertilized enclo- Secondly, the abundance of predators of HNAN may sures that either contained or excluded planktivorous be equally important. Zooplankton and ciliates are fish. These manipulations were examined in stratified both potential consumers of HNAN (Sanders & Porter (deep) versus continuously mixed (shallow) systems 1990). Seasonal declines in HNAN have been related within one lake. We examined the response of 4 main to increases in zooplankton (e.g. Jiirgens & Giide 1991, components of the microbial : the hetero- Jiirgens 1994, Sanders et al. 1994) or ciliates (Weisse et trophic bacteria (HBAC), the photosynthetic pico- al. 1990). Experimental additions of Daphnia partially (PPICO), which are also preyed on by proto- regulated HNAN abundance in lake container experi- zoan flagellates (e.g. Nagata 1988, Fahnenstiel et al. ments (Pace & Funke 1991). The correlation analyses 1991, Simek et al. 1997), the HNAN and potentially of Gasol & Vaque (1993) indicate that cladoceran mixotrophic flagellates (MIXO), which can also be abundance can explain a large fraction of the total important consumers of picoplankton (e.g. Porter 1988, variation in HNAN observed in lakes. Sanders & Porter 1988) as well as being frequently Because zooplankton community structure can be more abundant that HNAN in inland lakes (Pick & altered by fish planktivory (e.g. McQueen et al. 1986), Hamilton 1994). cascading effects on HNAN are possible. Rieman.n (1985) first showed experimentally that increases in planktivorous fish caused significant increases in MATERIALS AND METHODS HNAN. He attnbuted the increase to reduced preda- tion from 2 large cladoceran species (Daphnia cucul- Study site. The enclosures were installed in Lac lata and D. galeata). Subsequent enclosure experi- Croche (74" 00' N, 45'59' W), a small (4.82 ha), soft wa- men.ts confirmed these early results (Christoffersen et ter, slightly located at the Station de Bi- al. 1993). In comparing 2 lakes wlth contrasting food ologie des Laurentides of the Universite de Montreal, ap- web structure, Vaque & Pace (1992) found that the lake proximately 70 km north of Montreal on the Canadian dominated by planktivorous fish (and containing small Shield. The lake has a pH of 6.3 to 6.9 and dissolved or- cladoceran species) also contained the highest flagel- ganic carbon (DOC) levels range between 4 and 5 mg 1-' late abundance compared to the lake dominated by Spring total phosphorus levels average 10 pg I-'. Tzaras et al.: Nutrient, planktivorous fish and depth effects on mlcrobes

The lake is dimictic, with a maximuin depth of 11.5 m 1988). Epilimnetic total phosphorus levels in the and a mean depth of 8.5 m. The is at ap- enriched enclosures were about 4 times higher than proximately 3 m in early June and the lake remains in the non-enrich.ed ones (34 to 39 pg 1-' vs 8 pg I-'), strongly stratified until vertical mixing in early October with the shallow and deep enclosures maintaining The (depth of 1 % of surface radiation) is similar levels (Proulx et al. 1996). The resulting approximately 7 m during the summer months. Epil- and zooplankton biomass along with imnion summer temperatures range between 20 and estimates of primary production and heterotrophic 25°C while hypolimnetic water temperatures range bacterial production are summarized from various from 5 to 6°C. Additional information concerning mor- sources in Table l. phometric and physical characteristics of Lac Croche Fishless enclosures were seined to remove any fish. can be found in Lafond et al. (1990). Enclosures containing fish were stocked with a hybrid Enclosure and experimental design. Eight enclo- redbelly dace (Phoxinus eos X P. neogaeus), a plankti- sures were installed in a shallow area of the lake at a vore indigenous to Lac Croche. Approximately 160 to depth of 3 m, resulting in a total volume of 1.5 X 105 l 170 minnows, 6 to 8 cm in length and weighing 2.6 g enclosure-'. The second set of 8 enclosures was fish-', were added to give a final stocking of 5 g m-2. To installed in the deepest portion of the western basin of ensure that fish remained in the designated enclo- the lake at 11 m depth. The deep enclosures each had sures, fish fences measuring 0.4 m high were erected a total volume of 6.0 X 105 1. The volume of the epil- to prevent fish from jumping in and out of the enclo- imnion in the deep enclosures was approximately the sures. In addition, fish traps were installed in the fish- same as the total volume of the shallow enclosures. less enclosures to ensure that the fishless treatments The shallow enclosures were close to isothermal top to remained fishless for the duration of the experiment. bottom while the deep enclosures were thermally Only the deep fishless enclosures developed popula- stratified as of the middle of June, with a metalimnion tions of large cladocerans: Daphniamiddendorffiana extending from 3 to 5 m (Proulx et al. 1996). attained average biomass levels of 139 and 147 pg 1-' The enclosures were constructed out of nylon rein- in the deep C and N enclosures respectively (Table 1, forced polyethylene sheeting suspended in the water Proulx et al. 1996). column by an 8" (20.3 cm) polyurethane floating collar Sampling. Enclosures were sampled from June 19, supported by a 2" X 4" (5.1 X 10.2 cm) wood frame. A 1991 (Julian day 173), until September 14, 1991 metal chain anchored the bottom of each enclosure in (Julian day 254), resulting in a total of 7 sampling the sediment and prevented mixing of enclosure water with the surrounding lake water. Enclosures remained Table 1. Effects of nutrient additions and planktivorous fish on uncovered at both ends to allow for interaction with the measures of productivity and plankton biomass in the shallow underlying sediments and the atmosphere. A similar and deep enclosures. Values are summer averages (June 14 to enclosure design was used in the experiments con- August 29, n = 4 to 6) of integrated water column (shallow en- closures) or epilimnion (deep enclosures). PP: primary produc- ducted at Lake St. George by McQueen et al. (1986) tion as the average maximum photosynthetic rate, pg C 1-' h-' and Mazumder et al. (1988). (Perin et al. 1996). BP: bacterial production as the average The 3 treatment variables under investigation were thymidine assimilation rate, pM 1-' h-' (Howard 1994). Chl a: nutrients, presence of planktivorous fish and depth of chlorophyll a as an estimate of algal biomass, pg I-' ZB: zoo- plankton biomass, pgl-' (Proulx et al. 1996). DB: Daphn~abio- the water column for a 3 X 2 factorial design. Each vari- mass, pg 1-l (Proulx et al. 1996). CA: clliate abundance 1-' able was manipulated at 2 levels: presence versus (Marchessault & Mazumder 1997). Treatments: control, no absence and shallow versus deep accordingly. The nutrient addition and fishless; F, no nutrient ad&tion and fish resulting treatment combinations were: (1) nutrient present; N, nutrient addition and fishless; N + F, nutrlent and addition and fishless (N),(2) nutrient addition and fish fish present. na: not applicable present (N + F), (3) no nutrient addition and fishless (control) (C), and (4) no nutrient addition and fish pre- PP BP Chl a ZB DB CA sent (F). Each treatment combination had 2 replicates at each depth. Shallow Treatment combinations requiring nutrients re- Control 14 7 2.5 1966 0.4 na F 5 15 3.0 1004 1.0 na ceived nitrogen and phosphorus additions on a N 174 48 8.5 2701 0.4 na weekly basis at a N to P ratio of 13:l. Nitrogen was N+F 218 79 12.3 702 0.3 na added as sodium nitrate (NaN03) at a level of 47.97 Deep mg N m-2 d-l. Phosphorus was added in a liquid state Control 7 4 2.4 591 139 1100 as phosphoric acid (&PO4) at a level of 3.69 mg P F 7 7 2.6 204 9 2000 d-'. This fertilization rate represents a typical loading N 82 23 7.1 1032 147 180 N+F 254 15 9.1 391 33 2000 for a temperate eutrophic system (Mazumder et al. 70 Aquat Microb Ecol 19: 67-80, 1999

dates. Deep enclosures were not sampled during (Porter 1988), although they are rarely reported to Week 1. Thus in the statistical analysis, data col- consume bacteria in nature (e.g. Bennett et al. 1990). lected from Week 1 were not included. Between 60 and 100 nanoflagellate cells were counted The enclosures were fertilized on a weekly basis and from each sample or until 45 random whole field sampling was conducted every other week. The time views had been examined. interval between the sampling dates and the enrich- Statistical analyses. A repeated measures analysis of ment dates was constant throughout the sampling variance (RMANOVA) procedure was selected to eval- period. uate the treatment effects, any temporal effects, as Epilirnnetic water samples were collected using a well as their interaction effects on the seasonal abun- plexiglass integrated tube sampler (6.5 cm in diame- dance of bacteria and nanoflagellates throughout the ter) over a 0 to 3 m depth. All samples were collected 4 mo study period (June to September). from the midpoint of each enclosure between 08:OO The overall statistical model used was a Model I and 10:30 h. A total 16 1 of lake water were collected (fixed effects), 3-way RMANOVA. The data were ana- from each enclosure and stored in 20 1 opaque poly- lyzed using SAS software version 6.03 (SAS Institute ethylene containers which were pre-rinsed with sur- Inc. 1989). The model was constructed using 3 class face whole lake water prior to each use. Sub-samples variables (nutrients, fish, and depth) arranged in 2 lev- of whole lake water (90 ml) were preserved immedi- els (presence vs absence and shallow vs deep), and a ately with 10 m1 of 10% glutaraldehyde buffered time trial factor consisting of 7 sampling dates for with 0.1 M sodium cacodylate (pH 7). Samples were HBAC and 6 sampling dates for HNAN. The between- stored at 4°C in the dark for microbial slide prepara- subject effects are those due to the main treatment tions, which were made within 10 d of the collection effects, that is, the addition of nutrients, presence of date. fish, and depth of the water column. The response of Enumeration methods. Preserved lake water sam- the dependent variable to the treatment effects is aver- ples were filtered through 0.2 pm (for HBAC and aged over the entire sampling period. The error term is PPICO) and 1.0 pm (for nanoflagellate counts) Poretic calculated from the number of enclosures. filters prestained in a 0.2 % solution of Irgalan black in RMANOVA accounts for the between-subject 2% acetic acid. Direct counts of HBAC and HNAN effects, then focuses the analysis on the treatment were determined using DAPI (4'6 diamidino-2-phenyl- effects within each subject, that is, how the subject's indole) staining and epifluorescence rnicroscopy (Porter response varies with time. Therefore, the tested null &'Feig 1980). PPICO counts were performed using hypothesis is whether the response of the dependent green excitation (510 to 570 nm) of phycocyanin as variable to the different treatments is the same over described in Pick & Agbeti (1991). Non-phycoerythrin time. Specifically, the within-subject analysis tests de- dominated the picoplankton of Lac termine whether the slope of the best fit line through Croche and all the enclosures. enumerations were the obselvations plotted against time is similar for the conducted using a 100x oil immersion objective on a different treatment combinations (Gurevitch & Chester Zeiss Jenamed 2 epifluorescence microscope fitted 1986). The error terms associated with the within-sub- with a 50 W HBO mercury lamp for a final mag- ject analyses are derived from the number of repeated nification of 1200x. A minimum of 500 bacteria cells measures. was randomly counted per sample with the aid of an Microbial abundances were all log transformed to ocular grid. satisfy the assumptions of normality and homogeneity Nanoflagellate counts were conducted under both of the residuals. blue and UV light. HNAN could be distinguished from phototrophic flagellates based on the absence or presence of photosynthetic pigments. HNAN fluo- RESULTS resced weakly and appeared green under blue light and whitish blue under UV. Conversely, non-het- Heterotrophic bacteria erotrophic flagellates fluoresced red to orange under blue excitation owing to the presence of chlorophyll HBAC ranged from 3.1 X 10' to 5.7 X 106 cells ml-' a and other photosynthetic accessory pigments and in the shallow enclosures throughout the sampling yellowish under UV. We considered the MIX0 to period (Fig. 1A). There was a general increase in include the pigmented chrysophytes and prymnesio- HBAC throughout the summer months, with the phytes, which have been reported to consume bacte- highest cell abundance observed at the end of the ria (e.g. Porter 1988). In addition we included as sampling period (September 14, 1991). The nutrient potential pigmented cryptomonads, ennched enclosures (alone or in combination with which may consume bacteria in laboratory cultures fish) supported greater HBAC abundance than was Tzaras et al.: Nutrient, planktivorous fish and depth effects on microbes 71

5'25 I June I July I August I September I 1 June I July I August l Ssptsmbsr 50 180 210 240 27 150 180 21 0 240 270 Sampling Date Sampling Date

Fig. 1. Log transformed mean heterotrophic bacterial abundance in (A) the shallow enclosures (3 m) and (B) the deep stratified enclosures (11 m) from June 19 to September 14, 1991. Error bars represent the standard deviation between replicate enclosures. Treatments were: C, control (no fish and no nutrient additions); F, planktivorous fish additions; N, nutrient additions; N + F, nutri- ent and fish additions. Numbers on the x-axis represent Julian days observed in the control and fish only enciosures (Fig. 2). HBAC trends in the deep enclosures were similar to those observed in the shallow enclosures (Fig. 1B). Mean cell abundance varied between 2.3 X 105 and 9.5 X 106 cells ml-' and maximum abundance was observed at the end of the sampling period (Septem- ber 14, 1991). There was also a slight drop in HBAC abundance during mid-summer around July 30, 1991. HBAC abundance in the nutrient enriched enclosures (with and without fish) was slightly higher than the control and fish only enclosures. Fish only enclosures had the lowest HBAC levels, but the lev- l- els were similar to those found in the control enclo- E0 W sures. l- The overall effects of the 3 treatment variables, i.e. IW nutrient additions (N), presence of planktivorous fish g 5.0 1 NUTRIENTS (F), and depth of the water column (D), as well as NUTRIENTS their interaction effects on HBAC, are summarized in ABSENT PRESENT Table 2. Mean seasonal HBAC levels were signifi- cantly higher in the enclosures with added nutrients m Flsh Shallow No Flsh Shallow (p = 0.001, Table 2, Fig. 2). While HBAC did not m Flsh Deep m No Flsh Deep respond in a consistent manner to the presence of fish (p = 0.632), a significant nutrient and fish inter- Fig. 2. Overall effects of the treatment variables on seasonal action effect was observed (N X F; p = 0.031). HBAC mean heterotrophic bacterial abundance. Treatments were: levels were higher in the enriched enclosures con- nutrient additions, presence of planktivorous fish and depth of the water column. Heterotrophic bacterial abundance has taining fish in comparison to those without fish been log transformed. Seven sampling dates were included in (Fig. 2). There was no significant difference in HBAC the analyses. Bars represent standard errors 72 Aquat Microb Ecol 19: 67-80, 1999

Table 2. Between-subject effects. Summary of the repeated Table 3 Within-subject effects. Summary of the 3-way measures 3-way RMANOVA procedure examining the effects RMANOVA repeated procedure exdmining the response of of nutrient additions (N), presence of planktivorous fish (F) log transformed ~cbacterial abundance to nutri- and depth of the water column (D) on the log transformed het- ent additions (N),presence of planktivorous fish (F) and depth erotrophic bacterial abundance ns: p > 0 07 of the water column (D) during the sampling period. W. sam- pling week. A total of 7 sampling dates were included in the analyses. ns: p > 0.14 Source SS df MS Fratio p value

N 11.22 I source d f F ratio p value I F 0.02 NxF 0.69 W D 0.43 WxN NxD 0.06 WxF FxD 0.05 WxNxF NxFxD 0.01 W X D Error 0.67 WxNxD WxFxD WxNxFxD abundance between the shallow and deep enclosures (p = 0.071) and no interactions with the other treat- ment variables (Table 2). Photosynihetic picoplankton Examination of the overall temporal effects (Table 3) indicated that HBAC abundance varied significantly In the shallow enclosures, PPICO levels were high- throughout the sampling period (p < 0.001). The sea- est in the nutrient enriched enclosures (with or with- sonal changes in HBAC abundance were influenced out fish) early in the sampling season (Fig. 3A). Cell by nutrients (p = 0.001) but were not influenced by abundance reached a maximum of 1.8 X 106 cells fish (p = 0.123). HBAC seasonal trends were signifi- rnl-' in the nutrient only enclosures during mid-sum- cantly different in the shallow versus deep enclo- mer (July 16), but dropped 3 orders of magnitude to sures (p = 0.006) and furthermore depended on 2.5 X lo3 cells ml-' on the following sampling date whether planktivorous fish were present (W X F X D; (July 30). PPICO levels in the nutrient only enclo- p < 0.001) or nutrients were added (W X N X D; p = sures remained considerably lower (<104 cells ml-') 0.010). in comparison to the other enclosures for the remain-

Sampling Date Sampling Date Fig. 3. Log transformed mean picocyanobacteria abundance in (A) the shallow enclosures (3 m)and (B)the deep stratified enclo- sures (11 m) from June 19 to September 14, 1991 Error bars represent the standard deviation between replicate enclosures. Treatments were: C, control (no fish and no nutrient additions); F, plankt~vorousfish additions; N, nutrient additions: N + F, nutn- ent and fish additions. Numbers on the x-axis represent Julian days Tzaras et al.: Nutrient, planktivorous fish and depth effects on microbes 73

Table 4. Between-subject effects. Summary of the 3-~vay 6 RMANOVA examining effects of nutrient additions (N),pres- n ence of fish (F), and depth of the water column (D) on log transformed picocyanobacteria abundance. ns: p > 0.33 E

Source SS df MS Fratio p value

N 63.05 1 63.05 78.58 <0.001 F 22.12 1 22.12 27.57 <0.001 NxF 12.27 1 12.27 15.29 0.005 D 62.54 1 62.54 77.96 <0.001 NxD 34.16 1 34-16 42.58 <0.001 FxD 0.37 1 0.37 0.46 ns NxFxD 0.86 1 0.86 1.07 ns Error 6.42 8 0.80

der of the sampling period. Enriched with fish (N + F) enclosures exhibited high PPICO levels in early NUTRIENTS NUTRIENTS July, but PPICO levels declined rapidly from 1.3 X ABSENT PRESENT 106 to 7.0 X 104 cells ml-' during Week 3 and remained relatively constant around 2.0 X 105 cells ml-' for the remainder of the summer months. PPICO Fish Shallow m No Fish Shallow levels in the control and fish only enclosures Flsh Deep m No FishDeep remained at 105 cells n~l-'throughout the entire sam- pling period. Fig. 4. Overall effects of the treatment variables on seasonal A greater range in PPICO abundance was observed mean picocyanobacteria abundance. Treatments were: nutri- in the deep enclosures in comparison to the shallow ent additions, presence of planktivorous fish and depth of the enclosures (Fig. 3B). Maximum cell abundance was water column. Heterotrophic bacterial abundance has been log transformed. Seven sampling dates were included in the observed in the N + F enclosures during Week 2 of analyses. Bars represent standard errors sampling (July 4). However, PPICO levels declined steadily and from the fourth sampling week PPICO abundance in the N + F enclosures varied between 102 enclosure (N X D; p < 0.001). PPICO levels were sig- to 103 cells ml-' for the remainder of the summer. Sim- nificantly lower in deep enclosures with added nutri- ilarly, PPICO abundance in the nutrient only enclo- ents in comparison to the shallow ones with added sures was initially higher than the control and fish only nutrients (Fig. 4). enclosures during early summer. PPICO abundance RMANOVA within-subject tests (Table 5) revealed declined 3 orders of magnitude following the third that seasonal variation in PPICO levels depended on sampling date. PPICO levels remained relatively sta- nutrient additions (p = 0.003) and the depth of the ble at 103 cells ml-l until the end of the sampling period, when densities reached a minimum of 102 cells ml-l. Control and fish only enclosures exhibited virtu- Table 5. Within-subject effects. Summary of the 3-way ally identical trends throughout the sampling period RMANOVA procedure examining the response of log trans- (Fig. 3). In both cases, PPICO abundance did not devi- formed picocyanobacteria abundance to the addition of nutri- ate greatly from 10' cells ml-l. ents (N), presence of fish (F), and depth of the water column (D) during the sampling period. W: sampling week. A total of The RMANOVA between-subject analysis indi- 5 sampling dates were included in the analyses cated that PPICO abundance was significantly af- fected by all 3 treatment variables (Table 4). Mean PPICO levels decreased with nutrient additions (p < Source df F ratio p value 0.001); increased in the presence of fish (p < 0.001); W and were more abundant in the shallow enclosures WxN compared to the deep (p < 0.001) (Fig. 4).The effect WxF of nutrients was influenced by the presence of fish WxNxF (N X F; p = 0.005). Mean PPICO levels were lower in WxD WxNxD the nutrient enriched enclosures with no fish in com- WxFxD parison to those with fish (Fig. 4). Similarly, the effect WxNxFxD of nutrients was influenced by the depth of the 74 Aquat Microb Ecol 19: 67-80. 1999

210 240 Sampling Date Sampling Date

Fig. 5. Mean heterotrophic flagellate abundance in (A) the shallow enclosures (3 m) and (B) the deep stratified enclosures (I l m) during the sampling period June 19 to September 14, 1991. Error bars represent the standard deviation between replicate enclosures. Treatments were: C, control (no fish and no nutrient additions); F, planktivorous fish ad&tions; N, nutrient additions; NF, nutrient and fish additions. Numbers on the x-axis represent Julian days

water column (p = 0.002) but did not depend consis- closures and lowest in the nutrient e~chedenclosures. tently on the presence of fish (p = 0.371). In the nutrient enriched enclosures (with or without fish) HNAN levels generally remained well below 2.0 X 103 cells ml-l. A sharp increase in HNAN abundance Heterotrophic flagellates was observed in the nutrient enriched enclosures on the last sampling date, but HNAN levels still remained HNAN abundance ranged from 0.09 to 7.6 X 103 cells lower than what was found in the control and fish only ml-' in the shallow enclosures (Fig. 5A). Maximum enclosures. HNAN was found in the control enclosures at the end of Seasonal HNAN data for the deep enclosures indi- the sampling period on September 14. For most of the cated similar trends to what was observed in the shal- summer, excluding the second sampling week, HNAN low enclosures (Fig. 5B). HNAN abundance ranged abundance was highest in the control and fish only en-

Table 7. Within-subject effects. Summary of the 3-way Table 6. Between-subject effects. Summary of the 3-way RMANOVA procedures examining the response of log trans- RMANOVA procedures examining the treatment effects of formed heterotrophic flagellate abundance to the addition of nutrients additions (N), presence of fish (F), and depth of the nutrients (N),presence of fish (F), and depth of the water col- water column (D) on the log transformed dependent variable umn (D) during the sampling period. W: sampling week. A heterotrophic flagellate abundance. ns: p > 0 130 total of 6 sampling dates were included in the analyses

Source SS df F ratio p value Source d f F ratio p value

N 16.50 1 154.39 <0.001 W 5 92.19 <0.001 F 0.30 1 2.84 ns WxN 5 16.71 0.009 NxF 0.21 1 1.96 ns WxF 5 11.05 0.019 D 0.004 1 0.04 ns WxNxF 5 17.17 0.008 NxD 0.91 1 8.55 0.019 WxD 5 41.58 0.002 FxD 0.04 1 0.38 ns WxNxD 5 8.18 0.032 NxFxD 0.002 1 0.02 ns WxFxD 5 36.18 0.002 Error 0.11 8 WxNxFxD 5 45.44 0.001 Tzaras et al.: Nutrient, planktivorous fish and depth effects on microbes 7

from 0.1 to 4.5 X 103 cells ml-'. Fish only and control enclosures followed very similar patterns, with HNAN increasing after the second sampling week (July 16) for the duration of the summer. The nutrient enriched enclosures, alone or in combination with fish, exhib- ited much lower HNAN than in the control and fish only enclosures. Fertilized enclosures maintained HNAN levels generally less than 1.5 X 103 cells ml-' except in the N only enclosures, where there was a slight increase to 1.8 X 103 cells ml-' in September. Between-subject RMANOVA revealed that nutrient additions had a highly significant effect on HNAN (p < 0.001) (Table 6). HNAN levels were reduced when nutrients were added (Fig. 6). HNAN were neither affected by the presence of fish (p > 0.130) nor by the depth of the water column (p = 0.851). There was, however, a significant N X D interaction (p = m 0.019). The nutrient effect observed was greater in o NUTRIENTS NUTRIENTS the deep enclosures than in the shallow enclosures ABSENT PRESENT (Fig. 6). HNAN levels in the deep fertilized enclo- sures were considerably lower in comparison to the Flsh Shallow No Fish Shallow fertilized shallow. Flsh Deep m7] NO Flsh Deep Within-subject RMANOVA indicated that seasonal variation in HNAN abundance was due to all 3 treatment Fig. 6. Overall effects of the treatment variables on seasonal variables (Table 7). The HNAN response to the addition mean heterotrophlc flagellate abundance. Treatments were: nutrient additions, presence of planktivorous fish and depth of nutrients (p = 0.009), presence of fish (p = 0.019), and of the water column. Heterotrophic flagellate abundance has depth of the water column (p = 0.002), including all the been log transformed. Six sampling dates were included in interaction effects of these treatment combiiations, var- the analyses. Bars represent standard errors ied considerably during the 4 mo sampling period.

150 180 210 240 270 Sampling Date Sampling Date

Fig. 7. Mean mixotrophic flagellate abundance in (A) the shallow enclosures (3 m) and (B) the deep stratified enclosures (11 m) during the sampling period June 19 to September 14, 1991. Error bars represent the standard deviation between replicate enclo- sures. Treatments were: C, control (no fish and no nutrient additions); F, planktivorous fish additions; N, nutrient additions; N + F, nutrient and fish additions. Numbers on the x-axis represent Julian days 76 Aquat Microb Ecol 19: 67-80, 1999

Table 8. Between-subject effects. Summary of the 3-way Table 9. W~thin-subject effects. Summary of the 3-way RMANOVA procedure examining the treatment effects of RMANOVA examining the response of log transformed nutrient additions (N),presen.ce of fish (F),and depth of the mixotrophic flagellate abundance to nutrient additions (N), water column (D) on the log transformed dependent variable presence of fish (F), and depth of the water column (D) dunng rnixotrophic flagellate abundance. ns: p > 0.071 the sampling period. W: sampling week. A total of 6 sampling dates were included in the analyses Source SS F ratio p value Source Fratio p value N 33.95 F 0.009 W 5 NxF 5.58 W X N 5 D 14.45 WxF 5 NxD 1.65 WxNxF 5 FxD 1.77 WxD 5 NxFxD 11.91 WxNxD 5 Error 3.27 WxFxD 5 WxNxFxD 5

Mixotrophic flagellates lowest number of MIXO, usually less than 1.0 X 103 MIX0 abundance in the shallow enclosures cells ml-l. reached a maximum early in the sampling season but In the deep enclosures, MIXO levels also peaked declined steadily throughout the remainder of the in early July (6.4 X 103 cells ml-l) in the N + F treat- summer (Fig. ?A). The highest MIXO levels were ment (Fig. ?B).However, MIXO numbers sharply observed in the control enclosures during early July declined to less than 0.01 X 103 cells ml-' by early (5.1 X 103 cells ml-l). MIXO levels in the fish only August and close to zero afterwards until the com- enclosures were generally lower and followed a simi- pletion of the study. The MIXO levels in the control lar pattern to what was observed in the control and nutrient enclosures also peaked in early July enclosures. The fertilized enclosures exhibited the (between 1.5 and 2.5 X 103 cells ml-') and remained below 0.4 X 103 cells ml-' for the remainder of the season. The fish only enclosures exhibited a more gradual decline and supported higher MIXO levels at the end of the season in comparison to the other treatments. MIXO levels were significantly lower in the fertilized enclosures (p < 0.001, Table 8, Fig. 8). There was also a significant depth effect (p < 0.001). MIXO levels were generally lower in the deep enclosures in comparison to the shallow. However, this depth effect was not clear in the MIXO seasonal trends. MIXO levels were not affected in a consistent manner by the presence of planktivorous fish. However, there was a significant N X F interaction effect (p = 0.006). Seasonal mean MIXO data indicate that the effect of nutrients was greater in the enclosures without fish (Fig. 8). Nutrient enclosures with no fish exhibited lower MIXO levels than enriched J enclosures with fish. Seasonal variation of MIXO abun- dance depended on the addition of nutrients, presence of SHALLOW DEEP fish, and depth of the water column (Table 9). Seasonal m Fish with Nutrients m No Fish with Nutrients changes were d~fferentin each treatment. rish N~ ~~t~i~~t~[III] NO Flsh No Nutrients Fig. 8. Overall effects of the treatment variables on seasonal DISCUSSION mean mi.xotrophic flaqellate abundance. Treatments were- nutrient additions, pesence of plankt~vorousflsh and depth of the water column. Mixotrophic flagellate abundance has Of the independent been ]oq transformed. samplinq- - dates were included in clearly had the most significant effecton the abundance the analyses. Bars represent standard errors of all 4 components of the microbial food web. Although Tzaras et al.: Nutrient, planktivorous fish and depth effects on microbes 77

there were significant temporal changes in abundance in that the N + F enclosures (particularly the shallow all the components, there was a strong overriding effect ones) maintained high PPICO may help to explain of the nutrient additions on the patterns observed. While why some eutrophic lakes have high PPICO and nutrients significantly increased the abundance of others have insignificant populations (Stockner 1991). HBAC, the other 3 components (PPICO, HNAN and Christoffersen et al. (1993) also observed a summer MIXO) were, on average, negatively affected by nutri- increase in picoalgae (mainly cyanobacteria) in fish ents. In the case of PPICO, this effect was predictable enclosures relative to fishless ones deployed in a 'based on empirical studies across lake systems (Pick shallow eutrophic lake. 1991).Similarly, because most of the MIXO were chrys- Fish alone had no significant effect on HBAC, HNAN ophytes, the decline in their abundance with nutrient or MIXO abundance. This is in contrast to other enclo- additions was also a predictable response based on sure studies, where the presence of planktivorous fish empirical studies of chrysophyte distributions and ex- (and the associated removal of large daphnids) led to perimental fertilization of lakes (see review by Sandgren higher densities of HBAC and HNAN (e.g. Christof- 1988). fersen et al. 1993, MarkoSova & Jezek 1993).However, However, the significant decline in HNAN with the these studies have generally been conducted in eu- addition of nutrients was a surprising result given the trophic lakes. positive correlations between HNAN and HBAC In this study, although there was no main fish effect, reported in the literature (Berninger et al. 1991) and there were some significant nutrient-fish interactions. the positive (albeit weak) correlation between HNAN HBAC abundance was highest under nutrient condi- and total phosphorus (Tzaras & Pick 1994). In the fer- tions in the presence of fish. There were no significant tilized enclosures, HNAN concentrations were always interactions with fish for HNAN but the decline in significantly lower than in the non-fertilized enclo- abundance with enrichment was most pronounced in sures; the decline cannot be explained by a lack of the absence of fish in the deep enclosures. It would prey because there was a significant increase in the have been interesting to determine whether size shifts fertilized enclosures of HBAC abundance as well as occurred in the HNAN community in addition to abun- productivity (Table 1, Howard 1994). The increase in dance. Since fish treatments generally tend to increase HBAC was likely triggered by the increases in algal the importance of small cells (Vanni 1987, Mazumder biomass and primary production in the enriched enclo- et al. 1988, Proulx et al. 1996), a negative effect of fish sures (Table 1, Perin et al. 1996, Proulx et al. 1996). It on HNAN biomass might have been detected if there would have been interesting to examine whether there was an overall decrease in average size of HNAN. were also size shifts in the bacterial community with The lack of a significant fish effect on HNAN abun- the various treatments as observed for the algal com- dance was not due to a lack of change at the level of the munity (Proulx et al. 1996). zooplankton community. In the enclosures containing In contrast to this study, Riemann (1985)found that nu- planktivorous fish, the biomass and size distribution of trient additions had no significant effect on HNAN zooplankton were significantly altered (Table 1, Proulx within smaller experimental enclosures for 2 dates in et al. 1996). This occurred principally by mid- to late early summer for which data were provided. However, it sunlnler since it took about 4 wk following the removal of should be noted that the lake he studied was highly eu- fish in the fishless enclosures for the large bodied clado- trophc and nutrients may not have been limiting the sys- ceran Daphnia middendorffianato develop (Mazumder tem since HBAC biomass was not stimulated by nutrient & Lean 1994). Interestingly enough, such populations did additions. In the short-term (4 d) bag experiments of not develop at all in the shallow fishless enclosures. In Pace & Funke (1991) nutrient additions also led to no the enclosures containing fish, the zooplankton commu- change in HNAN in one lake (Peter),but a significant in- nity was dominated by rotifers, small microzooplankton crease was observed in the other lake studed (Paul).The measuring between 41 and 200 pm. Increased 2 lakes in this study were not very different in their abundance with the addition of planktivorous fish is con- trophic status and the authors suggested that the differ- sistent with other experimental studies (e.g. Mazumder ences were due to differences in predation pressure. et al. 1990, Lazzaro et al. 1992). The presence of planktivorous fish alone (+F) only Many planktonic rotifers are relatively unselective, had a significant effect on the abundance of PPICO. feeding on particles in the size range of 0.5 to 20 pm On average PPICO abundance was higher in the (Rothhaupt 1990). Although this size range includes presence of fish. The exact mechanism behind this HNAN and HBAC as well as PPICO, the impact of result cannot be determined but the presence of rotifers is generally considered to be much less signifi- planktivorous fish may reduce loss rates via a reduc- cant than that of large cladocerans (Arndt 1993). The tion in grazing or nutrient supply rates increase in rotifers in the fish enclosures did not have a may have been altered. In any case, the observation negative impact on HNAN. 78 Aquat Microb Ecol19: 67-80, 1999

The lowest abundance of HNAN occurred in the Interestingly, the response of HNAN to the various deep nutrient enriched enclosures without fish. Al- treatments tracked more closely the response of PPICO though these enclosures contained Daphnia popula- than that of HBAC. PPICO can be an important food tions, cladoceran biomass was not much higher than in source for HNAN (Sirnek et al. 1997).Therefore it is pos- the control enclosures where HNAN populations were sible that the HNAN growth rates were reduced in treat- the highest (Table 1). This result is in contrast to sev- ments where PPICO abundance was significantly lower, eral descriptive and experimental studies, which have leading to lower HNAN abundance. HNAN abundance indicated that populations of large Daphnia have neg- was most depressed in the enriched, fishless, deep en- ative impacts on HNAN. Jiirgens (1992) found that the closures where PPICO were also at their lowest concen- lowest HNAN numbers were found in German lakes trations. This hypothesis would require testing with di- where Daphnia species dominated the zooplankton. In rect grazing experiments that would estimate the the experiments of Riemann (1985), enclosures con- relative importance of PPICO and HBAC to the growth taining fish had elevated levels of HNAN presumably of HNAN. because of reduced predation from large Daphnia. In An alternative explanation for the decline in his fishless enclosures, zooplankton (particularly cla- HNAN is that with phytoplankton for docerans) increased in abundance whereas HNAN dissolved nutrients leads to declines in HNAN in the abundance decreased and could not fully account for nutrient enclosures. Although HNAN are generally the bacterial turnover. Furthermore, Riemann (1985) considered to obtain their mineral nutrients by feed- concluded that in enclosures without fish, the clado- ing on bacteria, there is no evidence to suggesl that cerans were effective grazers on bacteria. In the bag they cannot also obtain dissolved inorganic nutrients experiments of Pace & Funke (1991), the cascading across the cell membrane as do phytoplankton. It is effect of Daphnia was usually stronger than the nutri- possible that under the conditions of continuous ent effect in determining final densities of HNAN. nutrient additions larger simply outcompeted Interestingly, more recently, Pace et al. (1998) also the HNAN for nutrients. Larger algae were stimu- found that HNAN did not increase in response to lated by nutrient additions at least in the deep enclo- whole lake fertilization and that mortality from preda- sures (Proulx et al. 1996). tion (but not necessarily due to Daphnia) accounted for There was also no effect of depth on HBAC and the patterns observed. HNAN abundance while there was a depth effect on In the present study, the underlying cause for the PPICO and MIXO. This depth effect was probably not strong negative impact of nutrients on HNAN is not due to any significant differences in nutrient concen- known. The results would support the general concept trations since both the shallow and deep enclosures that the microbial loop is weaker in eutrophic condi- maintained similar nutrient levels in the epilimnion tions than in oligotrophic systems (Porter et al. 1988). during the summer (Proulx et al. 1996). Originally, the Predation by Daphnia is not the explanation, but it is enclosures were installed at 2 different depths to still possible that predation by other grazers not mimic stratified (deep) and continuously mixing (shal- impacted by Daphnia was occurring. For example, cil- low) systems. When a deep lake stratifies, nutrient lev- iates are common in eutrophic water where bacterial els tend to decline in the surface waters and accumu- densities are high and some species may graze on late below the thermocline. In contrast, in shallow small HNAN and pico- and , thus lakes, which continuously mix from top to bottom, providing another link to higher trophic levels. In nutrients get recirculated. An increase in HBAC and mesotrophic to eutrophic lakes where bacterial densi- HNAN levels in the shallow enclosures relative to the ties are high, ciliates have been shown to play a key deep enclosures was anticipated, presuming that the role in the energy transfer to higher trophic levels (e.g. shallow enclosures would accumulate higher nutrient Pace 1982, Weiss et al. 1990). Beaver & Crisman (1982) levels. However, Lac Croche has extremely nutrient- found that concentrations varied from less than poor sediments which appeared to adsorb nutrients 10 to around 200 cells ml-' in Florida lakes and that and the shallow enclosures did not accumulate more there was a significant correlation between ciliate num- nutrients when compared to the deep enclosures. bers and chlorophyll a. If ciliates were responsible for In this study, heterotrophic flagellates and their the decline of HNAN in the enclosures, then one would principal food , heterotrophic bacteria, were expect ciliate abundance to increase with nutrient not tightly coupled. Enclosures with increased nutri- additions and not be affected by the presence of plank- ent levels supported higher bacterial levels but did tivorous fish. However, for the deep enclosures, abun- not show a corresponding increase in heterotrophic dance was highest in both the fish enclosures, followed flagellates. Heterotrophic nanoflagellate levels were by the control and was lowest in the enriched fishless significantly reduced with the addition of nutrients. treatment (Table 1, Marchessault & Mazumder 1997). The presence of planktivorous fish altered zooplank- Tzaras et al.. Nutrient, planktivorous fish and depth effects on microbes 79

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Editorial responsibiljty: Fereidoun Rassoubadegan, Subm~tted:May 29, 1998; Accepted. February 23, 1999 Villefranche-sur-Mer, France Proofs received from author(s): August 20. 1999