AQUATIC MICROBIAL ECOLOGY Aquat Microb Ecol

Vol. 22: 301–313, 2000

Published October 26

Rates of growth and microbial grazing mortality of phytoplankton in a recent artificial lake Rémy D. Tadonléké, Télesphore Sime-Ngando* Laboratoire de Biologie des Protistes, UMR CNRS 6023, Université Blaise-Pascal (Clermont-Ferrand II), Les Cézeaux, 63177 Aubière Cedex, France

ABSTRACT: Dilution experiments were conducted from May to September 1998 in the epilimnion of a recently flooded reservoir (Sep Reservoir, Puy-de-Dôme, France: 46° 2’ N, 3° 1’ E), to estimate growth and microzooplankton (20 to 200 µm) grazing mortality of 2 size classes of the phytoplankton community. This community was dominated by < 25 µm cells, which averaged 77% (range 41 to 98%) of GF/F-collected chlorophyll a and 67% (27 to 99%) of total counts from inverted light microscopy. Total particulate DNA content was also significantly higher for the < 25 µm size class, compared to the > 25 to 200 µm size fraction. Micrograzers were largely dominated by ciliated protozoa (86 to 96% of total abundance), and also comprised rotifers and copepod nauplii. Experiments with and without added nutrients (N and P) indicated, together with changes in particulate protein, RNA and DNA, that phosphorus is a limiting element in the Sep Reservoir. Grazing activity of microzooplankton was significant on nanoalgae, averaging 0.38 ± 0.19 d–1 (range 0.16 to 0.66 d–1). The grazing activity balanced the daily production of the target algal community by 71 ± 11% (range 54 to 88%). Microzooplankton herbivory and the growth rates of nanoalgae were strongly correlated, suggesting the existence of an operating homeostatic interaction between the prey and the predators. The high mortality:growth ratio indicated that a substantial fraction of phytoplankton carbon is recycled in surface waters through microbial grazing. We conclude that microzooplankton herbivory provides an effective and substantial link to higher trophic levels in the Sep Reservoir, and might contribute to fueling planktonic communities with the limiting nutrient through regeneration. KEY WORDS: Phytoplankton · Microzooplankton · Growth · Grazing · Nutrient limitation · Dilution experiments · Reservoirs Resale or republication not permitted without written consent of the publisher

INTRODUCTION In the past few decades, developments in aquatic ecology have provided evidence that an important part of matter and energy can reach organisms of higher trophic levels through the functioning of the microbial loop (sensu Azam et al. 1983) or the microbial food web (sensu Rassoulzadegan 1993), which, at least, includes bacteria, autotrophic and heterotrophic flagellates and microzooplankton (i.e. protozoan ciliates and < 200 µm metazoa). Several studies in pelagic systems have shown that microzooplankton can (1) play an impor*Corresponding author. *E-mail: [email protected] © Inter-Research 2000

tant role in nutrient regeneration, especially in oligotrophic environments (e.g. Ferrier & Rassoulzadegan 1991), (2) control phytoplankton growth (e.g. Froneman & McQuaid 1997) and, at times, (3) provide a significant link to higher trophic levels (e.g. Stoecker & Capuzzo 1990, Sime-Ngando et al. 1995). Since aquatic ecology researchers have focused their attention on microbial food webs, numerous investigations have been conducted in order to quantify grazing activity and to understand the trophic role of micrograzers in planktonic systems. Field experiments have revealed that the proportion of the daily algal biomass production removed by microzooplankton typically ranges between 10 and 75% (Pierce & Turner 1992, Froneman & McQuaid 1997, Latasa et al. 1997). Most

302

Aquat Microb Ecol 22: 301–313, 2000

of these experiments have been conducted in coastal and oceanic environments and, in many cases, the dilution technique introduced by Landry & Hasset (1982) has been employed (e.g. Gifford 1988, Landry et al. 1995, 1997, 1998, Sime-Ngando et al. 1995, Froneman & McQuaid 1997, Latasa et al. 1997, Murrell & Hollibaugh 1998, Caron & Dennett 1999). This technique has rarely been used to investigate the grazing activity from microherbivores in freshwater ecosystems (e.g. Elser & Frees 1995). Furthermore, in these environments, studies concerning the functional role of micrograzers have mostly dealt with bacterivory, although some laboratory experiments showed that certain micrograzers grew better on algae than on bacteria (Walz 1983, Stemberger & Gilbert 1985, Ooms-Wilms 1997). In recent artificial lakes, the quantitative and functional importance of microheterotrophs have received very little attention (Paterson et al. 1997, Jugnia et al. 1999), or none at all as far as microzooplankton herbivory is concerned. The Sep Reservoir (Puy-de-Dôme, France) was formed and first flooded in 1995. Studies conducted in 1996 and 1997 in the reservoir showed that phytoplankton communities were largely dominated by nano-sized cells. In 1996, orthophosphate concentrations were low and sometimes undetectable, the phytoplankton biomass was characteristic of oligomesotrophic environments, and the primary production appeared to be strongly and negatively affected by the low P availability and by the water column instability. In 1997, the water of the reservoir exhibited a low chlorophyll a (chl a) (somewhat oligotrophic) high nutrient situation. During both 1996 and 1997, the N:P ratios (both total and inorganic) in the reservoir and its inflows were generally >16:1 (Redfield’s ratio) (Tadonléké 1999, Tadonléké et al. 2000). A study on metazooplankton feeding behavior in 1996 indicated that these

Fig. 1. Bathymetric map of the Sep Reservoir (Puy-deDôme, France), including the sampling area

organisms predate strongly on heterotrophic ciliates and flagellates in the Sep Reservoir (Thouvenot et al. 1999). From the above observations, we hypothesize that micrograzers play a significant trophic role in the food chain of this reservoir. Herein, we examine the phytoplankton growth rates and the herbivory activity from microzooplankton in the Sep Reservoir using a modified dilution technique. We also examine changes in the amounts of particulate protein, RNA and DNA, for evidence of the physiological responses of microbial communities to nutrient availability.

METHODS Study site and sampling. The Sep Reservoir (Fig. 1) was formed and first flooded in 1995 by damming the Sep Stream for the summer irrigation of an agricultural region known as ‘Haute-Morge’, located in the French Massif Central (ca 46° 2’ N, 3° 1’ E). In addition to the Sep Stream, water from the ‘Les Riaux’ stream also flows into the reservoir. The water leaving the reservoir flows into the ‘La Morge’ river, which had a very low summer discharge (< 0.5 m3 s–1) before the dam construction. At its full supply level, the reservoir contains about 5 million m3 of water, has an area of 33 ha, a mean depth of 14 m and a maximum depth of 37 m. During the present study, the sluices of the reservoir were opened in May for irrigation and the depth of the deepest point (sampling station) varied between 30 m (last sampling date) and 37 m (first sampling date). Additional details on the site description can be found in Tadonléké et al. (2000). Samples were collected every 2 wk at 1 m depth with a Van-Dorn bottle, from May to September 1998, at the deepest point of the reservoir situated about 150 m from the dam (Fig. 1). Previous studies in 1996 and

Tadonléké & Sime-Ngando: Phytoplankton growth and mortality in reservoirs

1997 showed that the 0 to 4 m zone of the reservoir water column is permanently well mixed and represents the epilimnion during the thermal stratification period (Tadonléké 1999). Initial physico-chemical, biochemical and biological analyses. Physical (temperature, light), chemical (nutrients), biochemical (chl a, protein, DNA, RNA), and biological (phytoplankton and microzooplankton composition and counts) parameters were obtained at the time of collection of the reservoir water used in the dilution experiments. Temperature was measured in situ using a digital display multiparameter apparatus YSI GRANT/3800 probe. Light penetration (photosynthetically available radiation, PAR, 400 to 700 nm) in the water column was measured with a Li-Cor probe equipped with a submersible photoelectric cell (Model Li 189). The euphotic depth (Z eu) corresponded to the depth at which 1% of the subsurface irradiance penetrated. Nutrient concentrations (nitrate and orthophosphate) were determined spectrophotometrically on 0.2 µm (acetate cellulose membranes were used) filtered subsamples, using AFNOR (1990) standard methods. Nitrate (N-NO3) was assayed by reduction with cadmium followed by reaction with sulfanilic acid, while orthophosphate (P-PO4) was determined by the molybdate-ascorbic acid method. At the beginning of the experiments, samples for chl a concentrations and for protein, DNA and RNA particulate contents were passed through nitex nylon screening to obtain 4 µm cells) and microzooplankton (20 to 200 µm protists and micrometazoa) were determined from microscopical analyses of samples used in the dilution experiments. Samples for phytoplankton, ciliates and micrometazoa were preserved with Lugol’s iodine (final concentration 1% v/v) and prepared for inverted light microscopy, i.e. sedimentation of 40 to 500 ml subsamples in settling chambers for 24 h. Samples for microheterotrophic dinoflagellates were fixed with glutaraldehyde (final concentration 1% v/v), stained with the fluorochrome DAPI and prepared for inverted epifluorescence microscopy (sedimentation of 100 to 300 ml subsamples in settling chambers for 24 h) equipped with the appropriate exciter/barrier filter set for UV (330 to 380 nm) excitation. At least 400 phytoplankton cells were counted for each sample (10% accuracy; Lund et al. 1958). The whole settling chamber was scanned for ciliate (magnification = ×400) and < 200 µm metazoan (magnification = ×100) counts. At least 200 ciliates were counted per sample. The numbers of apoplastidic dinoflagellates (> 20 µm) were low and < 50 cells were generally counted per sample. Several book guides were used for phytoplankton (Bourrelly 1966, Germain 1981) and microzooplankton (Corliss 1979, Pourriot & Francez 1986, Finlay et al. 1988, Foissner & Berger 1996) morphological identification. Dilution experiments. Phytoplankton growth and microzooplankton herbivory were examined using a modified dilution technique (Landry & Hasset 1982, Landry 1993, Landry et al. 1995, 1997, 1998, Latasa et al. 1997, Caron & Dennett 1999). Diluent for all experiments was obtained by gently (< 50 mm Hg) filtering lake water through Whatman GF/F glass fiber filters and, subsequently, through 0.2 µm acetate cellulose filters, in order to minimize cell bursting and the enrichment of filtrates. Measured volumes of diluent were added first to the incubation bottles for all diluted samples, and then raw lake water was gently added without bubbling to a prescribed level. Raw samples were prescreened through a 200 µm nitex netting to eliminate large (> 200 µm) zooplankton. All carboys, experimental flasks and nitex screening were soaked in 10% HCl and rinsed in distilled-deionized water prior to each experiment. Experimental flasks were 2 l clear polycarbonate bottles filled to the neck to reduce agitation during incubation. All dilution bottles were prepared and incubated in triplicate. Dilution series consisted of bottles containing 100, 50, 75 and 10% raw reservoir water with nutrient

304

Aquat Microb Ecol 22: 301–313, 2000

enrichment (N as NaNO3 at 20.3 µM l–1, and P as K2HPO4 at 1.3 µM l–1). Three additional replicates of raw experimental samples were also prepared without added nutrients and incubated, as well as diluent controls in which chlorophyll concentrations were always undetectable before and after incubations. Incubations were performed in situ for approximately 24 h, and then all bottles were resampled and analyzed for changes in chlorophyll concentrations. In addition, all bottles containing raw samples (i.e. with and without nutrient enrichment) were also resampled and analyzed for particulate protein, DNA and RNA concentrations. As for the initial analysis, incubated samples for chlorophyll, protein, DNA and RNA concentrations were passed through nitex screening before analysis to obtain 0.7 to 25 and 25 to 200 µm size fractions. Growth and mortality rates of phytoplankton were calculated from changes in chlorophyll concentrations over the length of the dilution experiments using linear regression analysis. Our terminology follows that of Landry et al. (1995,1997, 1998), Latasa et al. (1997), and Caron & Dennett (1999). Growth rates of the phytoplankton assemblages in the enriched bottles were determined from the y-intercepts of the regressions of apparent growth rate in the bottles versus dilution. Phytoplankton mortality rates due to grazing (g) were determined from the slopes of the regressions. Phytoplankton growth rates in the unenriched bottles (µ 0) were determined from net (apparent) growth rates of the phytoplankton in the unenriched, undiluted bottles (q 0) and g, as follows: µ 0 = q 0 + g. According to Paranjape (1987) and to Gifford (1988), µ 0, g and the initial concentration of chlorophyll (C i) were used to calculate pertinent functional parameters: (1) the apparent (or net) chlorophyll production, P a = (C i × e(µ 0 –g)) – C i, (2) the potential (i.e. apparent plus grazed) chlorophyll production, P t = (C i × eµ 0) – C i, and (3) the grazed fraction of the potential chlorophyll production, GP t = (C i × eµ 0) – [C i × e(µ 0 –g)]. Statistical treatments. Statistical treatments mainly consisted of correlation analysis to establish the empirical relationships between variables. The 2 size fractions of chlorophyll, protein, DNA and RNA were compared by a 1-way non-parametric analysis of variance (Mann-Whitney U-test).

RESULTS Initial physico-chemical, biochemical and biological parameters Physico-chemical environment Temperatures at the depth sampled for the phytoplankton growth and mortality experiments fluctuated around 21°C (Table 1). Values increased by few degrees (3 to 6°C) from the beginning of the experiments (May 20) to August 13, and then decreased similarly until the end of sampling (September 10). The Z eu was always > 4 m. N-NO3 concentrations decreased conspicuously from ~1.7 to 0.3 mg N l–1 during the experimental period. P-PO4 concentrations were generally under 9 µg P l–1 and exhibited no consistent temporal pattern. Undetectable P-PO4 concentrations appeared on July 16 and August 13 (Table 1).

Particulate biochemical compounds Simple statistics for the particulate concentrations of protein, RNA, DNA and chl a are given in Table 2. For the 2 latter variables, concentrations from the nanoplankton communities (0.7 to 25 µm size fraction) were significantly higher (Mann-Whitney U-test, p < 0.01) than those from the microplankton assemblages (25 to 200 µm size fraction, Table 2). For both size classes, the concentrations of protein, RNA and DNA were generally higher between July 16 and September 10 than at the beginning of the experimental period. Due to the 25 to 200 µm size fraction, the total concentrations of protein and chlorophyll reached their maximum on July 16, coinciding with undetectable P-PO4 concentrations (Table 1, Fig. 2A to D).

Phytoplankton Phytoplankton communities analyzed under inverted light microscope (i.e. > 4 µm in size) were dominated by chlorophytes over the experimental period,

Table 1. Temporal changes and mean (SD) values for the physico-chemical parameters at the depth sampled for the phytoplankton growth and mortality experiments. 0* = undetectable Parameter Temperature at 1 m (°C) Euphotic depth (m) N-NO3 (mg l–1) P-PO4 (µg l–1)

May 20

Jun 13

Jul 2

Jul 16

Jul 30

Aug 13

Aug 27

Sep 10

Mean (SD)

20.7 6 1.66 17.4

18.6 6 0.98 5

22.1 6 0.897 8.1

21.4 5 0.684 *0*

23.2 5.5 0.69 5.4

24 4.5 0.781 *0*

21.3 6 0.458 8.2

19.6 5.5 0.278 5.4

21.3 (1.8)1 5.56 (0.56) 0.8 (0.4) 7.3 (4.7)

305

Tadonléké & Sime-Ngando: Phytoplankton growth and mortality in reservoirs

Table 2. Simple statistics for the initial particulate biochemical and phytoplankton variables under study and comparison (MannWhitney U-test) of the 2 size classes. For each variable, mean value is followed by the standard deviation in brackets and the related range of values are given on the second line Variables Protein RNA DNA Chl a Phytoplankton abundance (4–200 µm)

Total

< 25 µm fraction (%)

> 25 µm fraction (%)

U

p

608.1 (148.1) µg l–1 354.4–812.5 9.9 (5.8) µg l–1 3.7–18.6 2.6 (1.0) µg l–1 1.4–3.8 9 (4.4) µg l–1 3.6–15.8 5.44 (3.2) × 106 cells l–1 0.99–10.7

56 (15) 37–85 59 (18) 36–87 72 (15) 52–97 77 (19) 41–99 67 (26) 27–99

44 (15) 15–63 41 (18) 13–64 28 (15) 3.0–48 23 (19) 1.0–59 33 (26) 1.0–73

45.0

0.172

40.0

0.401

60.0

0.003

61.0

0.002

52.0

0.036

except on July 16 and 30 when the large diatom Strobilidium sp. and Halteria sp. Other algivores Asterionella formosa was the most abundant species included prostomatids Urotricha furcata and Balanion (Table 3). Other large (> 25 µm) algal cells (not listed in planctonicum, hypotrichs Euplotes sp., and an hymenTable 3 because of their low numbers: 4 µm) species with their cell numbers and length numbers and particulate biochemical of the largest axis of the cell or the colony (*). For each date, species are ranked from most to least abundant compounds were noted: (1) phytoplankton total abundances (range: 1 to 11 × 106 cells l–1) were dominated by Date Species Length of No. of cells largest axis (µm) (×106 cells l–1) nanocells (4 to 25 µm size fraction). The densities of these small cells avera May 20 Sphaerocystis schroeteri* 30 2.82a aged (± SD) 67 ± 26% of total algal Elakatothrix sp. 21 0.84 abundance, and were significantly Jun 13 Volvox aureus* 192–420 0.57 Rhodomonas minuta 9 0.54 (Mann-Whitney U-test, p < 0.05) Jul 2 Chlorocloster sp. 6 0.27 higher than those of microalgae (25 to Cyclotella meneghiniana 13 0.21 200 µm) (Table 2). (2) Algal cell densiAsterionella formosa* 70 0.11 ties were higher in the last 5 samples Scenedesmus ecornis v. alternans* 9 0.10 Jul 16 Asterionella formosa* 70 6.8 than in the first 3, and (3) the highest Sphaerocystis schroeteri* 18 1.93 abundance of microalgae and of total Jul 30 Asterionella formosa 70 2.18 cells were recorded on July 16 when Sphaerocystis schroeteri* 18 1.81 the P-PO4 concentration was undeBotryococcus braunii* 147 0.86 tectable (Table 1, Fig. 2A,D,E). Chlorella minima 5 0.57 Aug 13

Micrograzers Microzooplankton communities (20 to 200 µm, protists and micrometazoa) were largely dominated by ciliated protozoa which represented 86 to 96% (mean = 91 ± 5%) of total counts. Classified by feeding preference (cf. SimeNgando & Hartmann 1991), algivorous ciliates were largely dominated by naked oligotrichs: Strombidium viride,

Aug 27

Sep 10

a

Chlorella minima Scenedesmus acutus* Cyclotella meneghiniana Scenedesmus apiculata* Sphaerocystis schroeteri* Indeterminated chlorophytes Scenedesmus acutus* Scenedesmus apiculata* Cyclotella meneghiniana Chlorella minima Scenedesmus denticulatus* Indeterminated chlorophytes Scenedesmus acutus*

5 8 13 5 18 5 8 5 13 5 9 5 8

17% of the cells belong to the < 25 µm size class

2.42 1.01 0.89 0.70 0.67 1.91 1.74 1.47 1.47 1.78 1.78 1.58 0.80

306

Aquat Microb Ecol 22: 301–313, 2000

Tintinnopsis sp.). Algivorous species were the most abundant (> 80% of the total abundance) within the ciliate community, except for the last 2 sampling dates when bacterial feeders (mainly the small scuticociliates Cyclidium sp. and Uronema nigricans) dominated (> 50% of the total abundance) this community (Fig. 3A). Other bacterivorous ciliates were represented by prostomatids Urotricha sp., gymnostomatids Askenasia sp. and peritrichs Vorticella sp. Heterotrophic dinoflagellates (> 20 µm) mainly included apoplastidic Gymnodinium sp., while unpigmented microflagellates (excluded from comparisons because of their marked scarcity during the study) were represented by a few euglenid-like cells. The number of heterotrophic dinoflagellates generally represented < 3% of the total abundance of protozoa. Micrometazoan community was dominated by rotifers, except on the first sampling date when nauplii accounted for 75% of the total abundance (Fig. 3B). Quantitatively, microprotist abundances fluctuated from 2.4 to 6.3 × 10– 3 cells l–1 (mean ± SD = 4.1 ± 1.6 × 10– 3 cells l–1), while micrometazoa numbers varied from 94 to 428 ind. l–1 (mean = 272 ± 123 ind. l–1). Microprotist abundances were highest from August 13 to September 10, which followed the period of highest micrometazoan densities. The algivorous ciliate Strobilidium sp. progressively disappeared in the experimental samples from the beginning of the study to August 13 (Fig. 3).

Dilution experiments Phytoplankton growth rates and grazing mortality Growth rates of nanophytoplankton (µ 0n) and microphytoplankton (µ 0m) in unenriched bottles of the dilution series fluctuated moderately, from 0.23 to 1.15 and 0.18

Fig. 2. Temporal changes in the concentrations of particulate biochemical compounds (caught on Whatmann GF/F filters, nominal porosity = 0.7 µm) and phytoplankton abundance (from inverted light microscopy counts, i.e. > 4 µm) in the epilimnion of the Sep Reservoir (1 m, 1998), for 2 size classes

Tadonléké & Sime-Ngando: Phytoplankton growth and mortality in reservoirs

307

Fig. 3. Temporal changes in the abundances of different populations of microzooplankton (20 to 200 µm) in the epilimnion of the Sep Reservoir (1 m, 1998)

to 0.5 d–1, respectively (Table 4). The mean (± SD) value for µ 0n (0.57 ± 0.29 d–1) was significantly higher (Mann-Whitney U = 52.5, p = 0.031) than that of µ 0m (0.32 ± 0.12 d–1). In contrast to µ 0m, µ 0n increased during the first 3 sampling dates. Thereafter, both variables exhibited similar temporal variations, characterized by a general decreasing trend (Table 4). For all dilution experiments, the regression coefficients (i.e. g) of chlorophyll-based apparent growth rates in nutrient-enriched bottles versus dilution were significant (p < 0.05) for nanophytoplankton, but always non-significant (p > 0.05) for microphytoplankton (Table 4). Accordingly, we conclude that larger

cells of the latter size class were not significantly fed by micrograzers during our experiments. The significant nanophytoplankton mortality rates due to grazing (g n) fluctuated from 0.16 to 0.66 d–1 (mean ± SD = 0.38 ± 0.19 d–1) and exhibited a temporal pattern similar to that of µ 0n. Microzooplankton grazing coefficient thus represented a significant fraction of µ 0n (ca 50 to 90%) during our experiments. Net nanophytoplankton growth rates (q 0n) in the unenriched bottles (q 0n = µ 0n – g n) fluctuated from 0.09 to 0.49 d–1, and decreased from July 2 until the end of the experimental period (Table 4). µ 0n exceeded g n for all experiments, but q 0n were 19 to 50% of µ 0n. For the

308

Aquat Microb Ecol 22: 301–313, 2000

Table 4. Nano- and microphytoplankton growth rates (µ 0, without nutrient addition) and grazing coefficients (g). q 0: net growth rates in the undiluted samples without nutrient addition; r2: coefficient of determination for the regression of apparent growth against the dilution; (P): positive slope. *p < 0.05; **p < 0.01 Date µ 0 (d–1) May 20 Jun 13 Jul 2 Jul 16 Jul 30 Aug 13 Aug 27 Sep 10

0.38 0.73 1.15 0.69 0.43 0.37 0.52 0.23

Nanophytoplankton (< 25 µm) q 0 (d–1) g (d–1) 0.11 0.11 0.49 0.27 0.22 0.09 0.10 0.07

0.27 0.62 0.66 0.42 0.21 0.28 0.42 0.16

nanoplankton size fraction (0.7 to 25 µm), the potential (i.e. net plus grazed) chlorophyll production (P tn) and the fraction of this potential production that was grazed (GP tn) peaked together on July 2. Lowest values of these variables were noted on July 30 for GP tn and on August 13 for P tn, when P-PO4 concentration was undetectable (Table 1, Fig. 4). Both variables were strongly correlated (r = 0.973, p < 0.001, n = 8). For all experiments, GP tn represented 54 to 88% (mean ± SD = 71 ± 11%) of P tn.

Effects of nutrient addition Effects of nutrient addition on chlorophyll-derived phytoplankton growth rates and on the concentration of particulate protein, RNA and DNA are shown in Table 5. On the first sampling date, nutrient additions were unfavorable for all these variables. Negative effects of nutrient addition were also observed for protein, RNA and DNA of the 25 to 200 µm size class on the second sampling date. For the rest of the experi-

Fig. 4. Rates of the potential production (i.e. net + grazed) and the microbial grazing mortality of phytoplankton biomass (chlorophyll a) in the epilimnion of the Sep Reservoir (1 m, 1998), determined using dilution experiments

r2 0.69* *0.88** *0.82** 0.53* 0.59* 0.55* *0.79** *0.77**

Microphytoplankton (> 25–200 µm) µ 0 (d–1) g (d–1) r2 0.50 0.34 0.19 0.30 0.25 0.18 0.44 0.23

0.04 0.06 0.02 0.04 00000.059 (P) 0.07 0.04 0.03

0.20 0.04 0.04 0.10 0.07 0.22 0.02 0.04

ments, nutrient addition enhanced algal growth rates and the amount of protein and nucleic acids. For these experiments, increased percentages of these variables as a result of nutrient amendments were generally higher for the < 25 than for 25 to 200 µm size class, except for RNA. For both size classes, the stimulating effect of nutrient addition was higher for RNA, followed by DNA, algal growth and protein. In both size classes, the highest percentages of increase in phytoplankton growth rates (August 13) and in particulate protein contents (July 16 and/or August 13) were obtained when P-PO4 concentrations were undetectable (Tables 1 & 5).

DISCUSSION Initial phytoplankton standing stocks and empirical observations The phytoplankton biomass and the nutrient environment during our dilution experiments were typical of oligomesotrophic lakes, similar to the conditions observed in 1996 when algal production was severely affected by low P-PO4 concentrations. In 1996, available P-PO4 in the surface waters of the reservoir explained only about 50% of the primary production, indicating the importance of nutrient sources such as regenerated P for algal activity in this milieu (Tadonléké 1999, Tadonléké et al. 2000). We speculate that, during this study, the conditions for phytoplankton development in the surface waters of the Sep Reservoir in 1998 were similar to those previously recorded in 1996, with a prevalence of P deficiency. This is apparently in agreement with the low and at times undetectable P-PO4 concentrations during both years. At the depth sampled for phytoplankton growth and grazing mortality experiments, the total abundance and biomass of phytoplankton communities as well as the total concentration of particulate DNA were signifi-

–19 (0.2; 0.13) (0.16; 0.1) –31 (0.75; 0.6) (0.51; 0.13 19 (0.69; 0.3) (0.82; 0.3) 36 (1.39; 1) (1.89; 1.4) 17 (0.92; 0.2) (1.08; 0.24) 9 (1.45; 0.73) (1.58; 1.1) 30 (0.57; 0.1) (0.74; 0.2) 38 (0.96; 0.15) (1.32; 0.79) 24.8 (11.6) –11 (2.4; 1.1) (2.08;1.2) 62 (1.92; 0.9) (3.1; 0.8) 33 (1.99; 1.01) (2.65; 0.99) 12 (2.35; 1.3) (2.64; 0.84) 46 (1.82; 1.1) (2.66; 0.9) 5 (2.77; 0.8) (2.9; 0.63) 47 (3.57; 0.78) (5.26; 1.2) 37 (2.98; 0.61) (4.09; 1.2) 34.6 (20.1) –20 (3.7; 0, 9) (2.9; 1.1) –45 (4.57; 1.6) (2.48; 0.87) 57 (4.86; 0.64) (7.62; 0.73) 78 (6.34; 0.95) (11.27; 1.5) 80 (4.07; 1.9) (7.35; 2.2) 39 (6.32; 2.1) (8.8; 1.4) 20 (5.78; 0.92) (6.91; 1.21) 17 (4.65; 1.23) (5.45; 0.88) 48.5 (27.7)

DNA > 25–200 µm < 25 µm > 25–200 µm

10.3 (8.2)

4

17

24

0

11

5

11

–14

–9 (466.1; 18.5) (425.6; 12.3) 14 (205; 21.4) (234; 38.5) 7 (251.8; 31.2) (269; 18.6) 30 (371.5; 9.2) (485.5; 17.1) 13 (361.8; 18.3) (408.2; 10.6) 15 (455.5; 24.1) (523.3; 16.4) 0 (409; 31.6) (409.2; 23.8) 8 (308.7; 33) (333.8; 20.2) 12.4 (9.3)

–37 (140; 20.6) (87.9; 7.2) –15 (236.7; 10.7) (200.9; 14.9) 8 (246.8; 6.1) (267.6; 12.5) 3 (501.3; 14.9) (515; 21.4) 4 (401.9; 12.4) (418.3; 29.2) 20 (274.1;13.9) (328.2; 21.4) 2 (174.8; 15.3) (178; 9.7) 3 (354; 12.8) (364.2; 15.1) 6.7 (6.9)

–19 (9.05; 1.4) (7.28; 1.5) 35 (4.79; 1.9) (6.48; 1.6) 24 (4.15; 1.05) (5.14; 1.9) 62 (6.39; 1.3) (10.35; 1.1) 75 (3.04; 0.35) (5.32; 1.3) 55 (11.4; 1.7) (17.6; 1.1) 26 (16.2; 2.3) (20.5; 1.6) 11 (16.98; 3.1) (18.83; 2.42) 41(23.2)

RNA < 25 µm Proteins > 25–200 µm < 25 µm > 25–200 µm

6

11

24

31

46

16

19

Jun 13

Jul 2

Jul 16

Jul 30

Aug 13

Aug 27

Sep 10

Overall mean (SD) 21.9 (13.4)

–6 May 20

GR < 25 µm Date

Table 5. Percent changes in phytoplankton growth rates (GR) and the amount of biochemical components following nutrient addition for the 2 plankton size classes, calculated as (V/V0–1) × 100, where V is the value with nutrients added and V0 the value without nutrient addition. For biochemical variables, V0 and V are mean values (µg l–1) for 3 replicates and are given in brackets for each sampling date on the first and the second line, respectively. Each of these values is followed by the corresponding standard deviation

Tadonléké & Sime-Ngando: Phytoplankton growth and mortality in reservoirs

309

cantly (Mann-Whitney U-test, p < 0.05) dominated by small cells (< 25 µm), similar to previous observations made in 1996 and 1997 (Tadonléké 1999, Tadonléké et al. 2000). This supports our working hypothesis on the significant trophic role of microbial grazing in the food chain of the Sep Reservoir. Indeed, dominance of small cells in the plankton and phytoplankton community is generally indicative of a situation where the microbial loop (Azam et al. 1983) and the microbial food web (Rassoulzadegan 1993) play a much larger role than the ‘classic’ web in the overall metabolism of pelagic systems (e.g. Legendre & Le Fèvre 1995).

The in situ dilution method To experimentally test the potential role of microherbivory in the Sep Reservoir, we conducted experiments using the dilution approach introduced by Landry & Hasset (1982). This approach implies setting up experimental samples with and without nutrient enrichments (Landry 1993, 1994, Landry et al. 1995) and this was interesting in our case study, given the possibility of inorganic P limitation in the Sep Reservoir. The method also provides reliable estimates of phytoplankton growth and grazing mortality with a minimal manipulation of live plankton samples (Landry & Hasset 1982, Gifford 1988), and when incubations are performed in situ, such as in this study, the dilution method makes it possible to avoid potential bias and artifacts due to photoadaptation known from ‘out situ’ incubators (McManus 1995, Caron & Dennett 1999). The depths of the 1% incident light level in the present study were always > 4 m, and chlorophyll concentrations and algal densities were not unusually high relative to highly productive lakes, or as low as those representative of highly oligotrophic freshwaters (Wetzel 1983). This suggests that the functional parameters derived from our dilution experiments were not significantly affected by the prey density constraint (Landry & Hasset 1982, Elser & Frees 1995), nor by differences in algal cell exposure to light which may occur in turbid waters when experimental samples are diluted (Murrell & Hollibaugh 1998). Based on preliminary tests during this study (data not shown) and on a previous study (Sime-Ngando et al. 1995), no significant changes in nutrient concentrations occurred in our experimental diluent as a result of the filtration process, compared to the raw lake samples. However, these raw samples were prescreened through a 200 µm nitex netting to eliminate mesozooplankton, mainly because measuring the grazing activity of these organisms in our 2 l experimental flasks was judged inaccurate. Our phytoplankton growth and microzooplankton grazing mortality rates might thus have been some-

310

Aquat Microb Ecol 22: 301–313, 2000

what biased compared to the natural rates, which include interference from mesozooplankton. Other potential bias of the dilution method is the confinement and the absence of cell migration in the experimental bottle (Landry 1994). A recent study in a eutrophic marine station also indicated that the growth rates of tintinnid and oligotrich ciliates were negatively affected by dilution, while no consistent dilution effect was shown by predacious ciliates and rotifers (Dolan et al. 2000). The fitted algal growth and grazing mortality rates observed in this study should thus be extrapolated to nature with care.

Phytoplankton growth in unenriched experimental bottles Chlorophyll-derived phytoplankton growth rates in unenriched bottles of the dilution series observed in this study (Table 4) are in good agreement with the observations of temperate marine and estuarine studies using the dilution technique (e.g. Landry & Hasset 1982, Sime-Ngando at al. 1995, Latasa et al. 1997, Catano et al. 1998, Landry et al. 1998, Murrell & Hollibaugh 1998). Our rates are also in agreement with those (0.21 to 0.98) observed in the Subalpine Castle Lake (northern California, USA) using a dilution approach (Elser & Frees 1995). Using a mean C:chlorophyll ratio of 40 that is typical of the Sep Reservoir (Tadonléké 1999), the apparent algal production rates calculated for 1998 during this study (mean = 71.8 ± 40.1 mg C m– 3 d–1) were similar to those determined in 1996 (mean = 72.6 ± 29.1 mg C m– 3 d–1) and in 1997 (mean = 64.9 ± 45.4 mg C m– 3 d–1) using 14C incorporation, supporting the idea of an overall annual equity of algal production in the Sep Reservoir, despite substantial differences in standing stocks and species composition (Tadonléké et al. 2000). This also indicates that the phytoplankton growth rates determined using the dilution method in this study were minimally affected due to methodological artifacts. As expected, nanophytoplankton growth rates (µ 0n, < 25 µm) were higher than those of microphytoplankton (µ 0n, 25 to 200 µm), except for the first samples (Table 4), in which the large chlorophyte Sphaerocystis schroeteri, known as an r-strategist (Arauzo & Alvarez Cobelas 1994), was the numerically dominant species (Table 3). Relatively high microphytoplankton growth rates (µ 0m) were also noted on June 13 when Volvox aureus was the dominant species, and on July 16 and 30 when Asterionella formosa dominated the algal assemblages, a P shortage being apparent on July 16 (Tables 1 & 3). V. aureus is known to be a rapid growing species (Reynolds 1998) and A. formosa generally has a competitive advantage in P-deficient envi-

ronments because of its low half-saturation constant for P uptake (Reynolds 1998). The results discussed above suggest that changes in species composition in relation to P concentration might account for most of the temporal variations in this study.

Effects of nutrient addition on phytoplankton growth Nutrient enrichment enhanced phytoplankton growth rates and the concentrations of particulate protein, RNA and DNA in almost all of the samples used for the dilution experiments. The only negative effects of nutrient addition, i.e. for all these variables and for the 2 size fractions, occurred during the first sampling date when ambient N-NO3 and P-PO4 were at their highest concentrations (1660 and 17.4 µg l–1, respectively; Table 5). From this sampling date, N-NO3 concentration in the reservoir decreased progressively during the study, while P-PO4 concentration decreased drastically in the second samples and remained low (around 5 µg l–1) or undetectable till the end of the study (Table 1). Given this difference in the temporal fluctuations of the 2 elements and the general low concentrations of P in the Sep Reservoir compared to those of N, we suspect that P rather than N was the main limiting element in our experimental flasks. This did not contradict empirical observations from annual standing stocks of nutrients and algal assemblages in the Sep Reservoir, and was apparently also in agreement with some of the experimental observations. For example, the highest percentages of the increase in algal growth rates and in particulate protein contents as a result of N and P addition were obtained, for the 2 size fractions, on July 16 and/or on August 13 when P-PO4 was undetectable in the reservoir (Tables 1 & 5). For the 2 particulate size fractions, the stimulating effects of nutrient addition were more pronounced for nucleic acids than for protein (Table 5), indicating a prevalence of P shortage rather than of N deficiency. It has been experimentally shown that nucleic acids are indeed more sensitive to P than to N deficiency while, in contrast, proteins are more sensitive to N than to P depletion (Ganf et al. 1986, Berdalet et al. 1994, 1996). It has also been shown that in Pdepleted algal cultures, P supply is accompanied by a rapid synthesis of ATP, followed by an increase in RNA concentrations (Sakshaug & Holm-Hansen 1977, Laws & Bannister 1980).

Microbial herbivory and its ecological implications Microbial grazing coefficients (i.e. g) for our dilution experiments were significant only for nanophyto-

Tadonléké & Sime-Ngando: Phytoplankton growth and mortality in reservoirs

plankton prey (g n), while larger microphytoplankton cells which were represented mainly by diatoms or large colonial species apparently escaped from micrograzer pressure (Tables 3 & 4). Similar results from dilution experiments are currently reported in coastal and oceanic waters where larger algae might be less susceptible to microbial grazing (e.g. Strom & Welschmeyer 1991, Landry et al. 1998, Caron & Dennett 1999). Our values of g n (range: 0.16 to 0.66 d–1, mean ± SD = 0.38 ± 0.19 d–1) fell within the range of those (0.06 to 1.19 d–1) reported for various coastal and open ocean waters using the dilution method (e.g. Landry & Hasset 1982, Sime-Ngando at al. 1995, Latasa et al. 1997, Catano et al. 1998, Landry et al. 1998, Murrell & Hollibaugh 1998). In the Subalpine Castle Lake, microbial grazing rates (0.05 to 0.22 d–1, mean = 0.14 d–1) estimated using a dilution approach (Elser & Frees 1995) were lower, likely because of the saturation of micrograzer feeding that did not occur in the experiments cited above. Using the C:chl a ratio of 40 (Tadonléké 1999), the maximum food concentrations (MFC) in our undiluted experimental samples (145.6 to 630.4 µg C l–1, mean = 358.9 ± 175.7 µg C l–1) were lower than those reported in the literature. For the dominant grazers present during our study, MFC are generally > 500 µg C l–1 for herbivorous ciliates (Müller & Schlegel 1999 and references therein) and comprise between 500 and 2500 µg C l–1 for rotifers (Rothhaupt 1990). The degree to which algal production is consumed by micrograzers has significant ecological implications for nutrient cycling and energy flows in pelagic systems. A low mortality:growth ratio implies a situation in which the potential for carbon export from the surface waters (sinking) is high. In contrast, a high mortality:growth ratio implies that the phytoplankton carbon is largely recycled in surface waters through microbial grazing and does not contribute significantly to sinking particle flux (Caron & Dennett 1999). It is likely that this was the case in the present study where algal community was dominated by nanocells, for which the ratios of g n to µ 0n were substantially high, with 54 to 88% (mean = 71 ± 11%) of the potential production being removed by micrograzers (Table 4, Fig. 4). In addition, algal production and microbial herbivory were strongly correlated (r = 0.97, p < 0.001, df = 6), which generally suggests that grazers are implicated in the maintenance of their rapidly growing prey at approximately steady state, indicative of the existence of an homeostatic regulation between predators and prey (Burkill et al. 1987, Ruiz et al. 1998). In such a dynamic feedback system, the microbial grazing is well known to fuel the system with regenerated nutrients (Ferrier & Rassoulzadegan 1991, Landry et al. 1997).

311

Conclusions In conclusion, the results from this study demonstrate that phytoplankton growth and microbial herbivory in the Sep Reservoir are characteristic of oligotrophic waters and that phosphorus is potentially a limiting element in this reservoir. Microzooplankton significantly grazed on nanoalgae, in contrast to larger algae which apparently escape this grazing pressure. Microbial grazing seemed to maintain the small algal cells at approximate steady state and likely fuel the system with the limiting nutrient through regeneration. Our findings support the idea that, following the flooding of a reservoir, matter and energy might flow mainly through microorganisms. This may be of great importance for lake and reservoir studies, since existing models that describe modifications of food webs in lakes through biomanipulation (e.g. Carpenter et al. 1985, McQueen et al. 1986) do not actually take into account microzooplankton herbivory. Acknowledgements. We gratefully acknowledge financial support from the European Community and from the following French national and regional organizations: ‘Ministère de l’Environnement’, ‘Agence de l’eau Loire-Bretagne’, Conseil Régional d’Auvergne’, ‘Conseil Général du Puy-de-Dôme’, and ‘Syndicat des Agriculteurs Irrigants de la Haute Morge’. We also thank the numerous people who were conscripted as field assistants during this study, namely D. Gilbert, L.-B. Jugnia and D. Sargos. We also thank Dr E. Berdalet for constructive comments on an earlier version of the manuscript. This work is a contribution to the SEP Project and to the UMR CNRS 6023 (Biologie des Protistes) research programmes, and is part of the fulfillment of the requirements for a PhD (Doctorat d’Université) degree from the Université Blaise Pascal by R.D.T.

LITERATURE CITED AFNOR (Association Française de Normalisation) (1990) Eaux méthodes d’essais. 4ème édn. Mason, Paris Arauzo M, Alvarez Cobelas M (1994) Phytoplankton strategies and time scales in a eutrophic reservoir. Hydrobiologia 291:1–9 Azam F, Frenchel T, Field JG, Gray JS, Meyer-Reil LA, Thingstad F (1983) The ecological role of watercolumn microbes in the sea. Mar Ecol Prog Ser 10:257–263 Berdalet E, Latasa M, Estrada M (1994) Effects of nitrogen and phosphorus starvation on nucleic acid and protein content of Heterocapsa. J Plankton Res 16:303–316 Berdalet E, Marrasé C, Estrada M, Arin L, MacLean ML (1996) Microbial community responses to nitrogen- and phosphorus-deficient nutrient inputs: microplankton dynamics and biochemical characterization. J Plankton Res 18:1627–1641 Bourrelly P (1966) Les algues d’eau douce. I. Algues vertes. N. Boubée ed, Paris Burkill PH, Mantoura RFC, Llewellyn CA, Owen NJP (1987) Microzooplankton grazing and selectivity of phytoplankton in coastal waters. Mar Biol 93:581–590

312

Aquat Microb Ecol 22: 301–313, 2000

Caron DA, Dennett MR (1999) Phytoplankton growth and mortality during the 1995 northeast monsoon and spring intermonsoon in the Arabian sea. Deep-Sea Res II 46: 1665–1690 Carpenter SR, Kitchell JF, Hodson JR (1985) Cascading trophic interactions and lake productivity. BioScience 35: 634–639 Catano U, Uriarte I, Villate F (1998) Herbivory of nanozooplankton in polyhaline and euhaline zones of a small temperate estuarine system (Estuary of Mundaka): seasonal variations. J Exp Mar Biol Ecol 227:265–279 Corliss JO (1979) The ciliate protozoa: characterization, classification and guide to the literature, 2nd edn. Pergamon Press, New York De Madariaga I, Joint I (1992) A comparative study of phytoplankton physiological indicators. J Exp Mar Biol Ecol 158: 149–165 Dolan JR, Gallegos CL, Moigis A (2000) Dilution effects on microzooplankton in dilution grazing experiments. Mar Ecol Prog Ser 200:127–139 Dortch Q, Roberts TL, Clayton JR Jr, Ahmed SI (1983) RNA/DNA ratios and DNA concentrations as indicators of growth rate and biomass in planktonic organisms. Mar Ecol Prog Ser 13:61–71 Elser JJ, Frees DL (1995) Microconsumer grazing and sources of limiting nutrients for phytoplankton growth: application and complications of nutrient-deletion/dilution-gradient technique. Limnol Oceanogr 40:1–16 Fara A, Berdalet E, Arin L (1996) Determination of RNA and DNA concentrations in natural plankton samples using Thiazole orange in combination with DNAse and RNAse digestions. J Phycol 32:1074–1083 Ferrier C, Rassoulzadegan F (1991) Density-dependent effects of protozoans on specific growth rates in pico- and nanoplankton assemblages. Limnol Oceanogr 36:657–669 Finlay BJ, Rogerson A, Cowling AJ (1988) Collection, isolation, cultivation and identification of freshwater protozoa. Freshwater Biological Association, Ambleside Foissner W, Berger H (1996) A user-friendly guide to the ciliates (Protozoa, Ciliophora) commonly used by hydrobiologist as bioindicators in rivers, lakes and waste waters, with notes on their ecology. Freshw Biol 35:375–482 Froneman PW, McQuaid C D (1997) Preliminary investigation of the ecological role of microzooplankton in the Kariega Estuary, South Africa. Estuar Coast Shelf Sci 45:689–695 Ganf GG, Stone SJL, Oliver RL (1986) Use of protein to carbohydrate ratios to analyse for nutrient deficiency in phytoplankton. Aust J Mar Freshw Res 37:183–197 Germain H (1981) Flore des Diatomées des eaux douces et saumâtres. Boubée ed, Paris Gifford DJ (1988) Impact of grazing by microzooplankton in the Northwestern Arm of Halifax Harbour, Nova Scotia. Mar Ecol Prog Ser 47:249–258 Jugnia LB, Tadonléké RD, Sime-Ngando T, Devaux J, Andrivon C (1999) Bacterial population dynamics, production and heterotrophic activity in a recently formed reservoir. Can J Microbiol 45:747–753 Landry MR (1993) Estimating rates of growth ang grazing mortality of phytoplankton by the dilution method. In: Kemp PF, Sherr BF, Sherr EN (eds) Hanbook of methods in aquatic microbial ecology. Lewis Publishers, Boca Raton, p 715–722 Landry MR (1994) Methods and controls for measuring the grazing impact of planktonic protisis. Mar Microb Food Webs 8:37–57 Landry MR, Hasset RP (1982) Estimating the grazing impact of marine micro-zooplankton. Mar Biol 67:283–288

Landry MR, Kirshtein J, Constantinou J (1995) A refined dilution technique for measuring the community grazing impact of microzooplankton, with experimental tests in the central equatorial Pacific. Mar Ecol Prog Ser 120: 53–63 Landry MR, Barber RT, Bidigare RR, Chai F, Coale KH, Dam HG, Lewis MR, Lindley ST, McCarthy JJ, Roman MR, Stoecker DK, Verity PG, White JR (1997) Iron and grazing contraints on primary production in the central equatorial Pacific: an EqPac synthesis. Limnol Oceanogr 42:405–418 Landry MR, Brown SL, Campbell L, Constantinou J, Liu H (1998) Spatial patterns in phytoplankton growth and microzooplankton grazing in the Arabian Sea during moonsoon forcing. Deep-Sea Res II 45:2353–2368 Latasa M, Landry MR, Schlüter L, Bidigare RR (1997) Pigment-specific growth and grazing rates of phytoplankton in the Central Equatorial Pacific. Limnol Oceanogr 42: 289–298 Laws EA, Bannister TT (1980) Nutrient- and light-limited growth of Thalassiosira fluviatilis in continous culture, with implications for phytoplankton growth in ocean. Limnol Oceanogr 25:457–473 Legendre L, Le Fèvre J (1995) Microbial food webs and the export of biogenic carbon in oceans. Aquat Microb Ecol 9: 69–77 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275 Lund JWG, Kipling C, Le Cren D (1958) The inverted microscope method of estimating algal numbers and the statistical basis of estimations by counting. Hyrobiologia 11: 143–170 McManus GB (1995) Phytoplankton abundance and pigment changes during simulated in situ dilution experiments in estuarine waters: possible artifacts caused by algal light adaptation. J Plankton Res 17:1705–1716 McQueen DJ, Post JR, Mills EL (1986) Trophics relations in freshwater pelagic ecosystems. Can J Fish Aquat Sci 43: 1571–1581 Müller H, Schlegel A (1999) Responses of three freshwater planctonic ciliates with different feeding modes to cryptophyte and diatom prey. Aquat Microb Ecol 17:49–60 Murrell MC, Hollibaugh JT (1998) Microzooplankton grazing in the northern San Francisco Bay measured by the dilution method. Aquat Microb Ecol 15:53–63 Nelson DL, Cox MM (2000) Lehninger principles of biochemistry. Worth Publishers, Inc, New York Ooms-Wilms AL (1997) Are bacteria an important food source for rotifers in eutrophic lakes? J Plankton Res 19:1125–1141 Paranjape MA (1987) Grazing by microzooplankton in the eastern Canadian arctic in summer 1983. Mar Ecol Prog Ser 40:239–246 Paterson MJ, Findlay D, Beaty K, Findlay W, Schinler EU, Stainton M, McCullough G (1997) Changes in the planktonic food web of a new experimental reservoir. Can J Fish Aquat Sci 54:1088–1102 Pierce RW, Turner JT (1992) Ecology of planktonic ciliates in marine food webs. Rev Aquat Sci 6:139–181 Pourriot R, Francez AJ (1986) Introduction pratique a la systematique des organismes des eaux continentales françaises. 8. Rotifères. Extrait du Bulletin mensuel de la Société Linnéenne de Lyon No. 5. Association Française de Limnologie ed, Lyon Rassoulzadegan F (1993) Protozoa patterns in the Azam-Ammerman’s bacteria-phytoplankton mutualism. In: Guerrero R, Pedròs-Aliò C (eds) Trends in microbial ecology. Spanish Society for Microbiology, Barcelona, p 435–439

Tadonléké & Sime-Ngando: Phytoplankton growth and mortality in reservoirs

313

Reynolds CS (1998) What factors influence the species composition of phytoplankton in lakes of different trophic status? Hydrobiologia 369/370:11–26 Rothhaupt KO (1990) Changes in the functional responses of the rotifers Brachionus ruben and Brachionus calyciflorus with particle size. Limnol Oceanogr 35:24–32 Ruiz A, Franco J, Villate F (1998) Microzooplankton grazing in the Estuary of Mundaka, Spain, and its impact on phytoplankton distribution along the salinity gradient. Aquat Microb Ecol 14:281–288 Sakshaug E, Holm-Hansen O (1977) Chemical composition of Skeletonema costatum (Grev.) Cleve and Pavlova (Monochrysis) lutheri (Droop) Green as function of nitrate-phosphate-and iron-limited growth. J Exp Mar Biol Ecol 29: 1–34 SCOR-UNESCO (1966) Determination pf photosynthetic pigments in seawater. UNESCO Monogr Oceanogr Method 1:11–18 Sime-Ngando T, Hartmann HJ (1991) Short-term variations of the abundance and biomass of planktonic ciliates in a eutrophic lake. Eur J Protistol 27:249–263 Sime-Ngando T, Gosselin M, Roy S, Chanut JP (1995) Significance of planktonic ciliated protozoa in the lower St. Lawrence Estuary: comparison with bacterial, phytoplankton and particulate organic carbon. Aquat Microb

Ecol 9:243–258 Stemberger RS, Gilbert JJ (1985) Body size, food concentration and population growth in planktonic rotifers. Ecology 66:1151–1159 Stoecker DK, Capuzzo JM (1990) Predation on protozoa: its importance to zooplankton. J Plankton Res 12:891–908 Strom SL, Welschmeyer (1991) Pigment-specific rates of phytoplankton growth and microzooplankton grazing in the open subartic Pacific Ocean. Limnol Oceanog 36: 50–53 Tadonléké DR (1999) Structure et fonctionnement des peuplements phytoplanctoniques dans un réservoir récemment mis en eau. PhD thesis, Université Blaise Pascal/Clermont-Ferrand II, Aubière Tadonléké DR, Sime-Ngando T, Amblard C, Sargos D, Devaux J (2000) Primary productivity in a recently flooded resevervoir. J Plankton Res 22:1355–1375 Thouvenot A, Debroas D, Richardot M, Devaux J (1999) Impact of natural metazooplankton assemblage on planktonic microbial communities in a newly flooded reservoir. J Plankton Res 21:179–199 Walz N (1983) Individual culture and experimental population dynamics of Keratella cochlearis (Rotatoria). Hydrobiologia 147:209–213 Wetzel RG (1983) Limnology, 2nd edn. Saunders, London

Editorial responsibility: Fereidoun Rassoulzadegan, Villefranche-sur-Mer, France

Submitted: February 7, 2000; Accepted: August 29, 2000 Proofs received from author(s): October 20, 2000

314

Aquat Microb Ecol 22: 301–313, 2000