Taxon-specific and seasonal variations in flagellates grazing on heterotrophic bacteria in the oligotrophic Lake Annecy – importance of mixotrophy

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Taxon-speci¢c and seasonal variations in £agellates grazing on heterotrophic bacteria in the oligotrophic Lake Annecy ^ importance of mixotrophy Isabelle Domaizon  , Sylvie Viboud, Dominique Fontvieille Universite¤ de Savoie, CISM, UMR CARRTEL, 73376 Le Bourget du Lac, France Received 12 November 2002; received in revised form 22 September 2003; accepted 26 September 2003 First published online 23 October 2003

Abstract We investigated the taxonomic composition of flagellate assemblages and taxon-specific bacterial grazing rates of heterotrophic and mixotrophic flagellates in the oligotrophic Lake Annecy (France). The comparison of bacterial grazing rates to bacterial production demonstrated a high transfer efficiency from the bacterial compartment up to flagellates. Per capita grazing rates ranged from 1.2U103 to 5.1U106 bacteria l31 h31 for heterotrophic flagellates, and from 4.8U106 to 6.8U107 bacteria l31 h31 for mixotrophic flagellates. The main bacterial grazers were Katablepharis within heterotrophic flagellates and Dinobryon within mixotrophic flagellates. Our results show that bacterial ingestion by a given flagellate taxon changed seasonally and could vary up to 30-fold. We also provide evidence that mixotrophic flagellates represent an important link in the flux of materials through planktonic food webs in Lake Annecy, suggesting that the introduction of mixotrophs within functional groups could improve our understanding of carbon flux pathways. 9 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords : Heterotrophic nano£agellate ; Mixotrophic £agellate ; Grazing rate; Lake Annecy; Microbial food web

1. Introduction The recognition of the qualitative and quantitative importance of the microbial food web in freshwater ecosystems has greatly modi¢ed our approach to plankton ecology [1^3]. The ecological roles of heterotrophic £agellates and ciliates in freshwater pelagic environments were recently reviewed by Nakano [4], focusing especially on their roles as consumers of microbial plankton, food resources for zooplankton, and regenerators of inorganic nutrients. It is now well known that bacterivorous protists have the potentiality to regulate bacterial abundance and production and are therefore a keystone group in the transfer of picoplanktonic carbon to higher trophic levels [5^7]. However, although heterotrophic nano£agellates (HNF) are generally recognised as the primary consumers of bacterioplankton and picoplankton in both marine and fresh-

* Corresponding author. E-mail address : [email protected] (I. Domaizon).

water ecosystems [8^12], the number of records that show a lack of coupling between bacteria and HNF has increased in recent years [5,13,14]. Based on their investigation on bacterial grazing rates of heterotrophic £agellates, Cleven and Weisse [9] concluded that it seems obvious from the high variability of the data produced in the literature that we need to learn more about ingestion rates within a given £agellate population. Moreover, recent studies show the importance of mixotrophs as bacterial consumers [15^21], while their signi¢cance in regulating bacterial communities is still under discussion. Only a few studies have investigated seasonal succession and taxon-speci¢c grazing rates of £agellates [2,9,10,22^24]. None of these studies were under strict oligotrophic conditions and mixotrophic species were rarely taken into account. During this study, we investigated the taxonomic composition of £agellates and their bacterial consumption in the oligotrophic Lake Annecy. The goals were (i) to investigate the relative importance of di¡erent £agellate taxa (heterotrophic and mixotrophic £agellates) in bacterial regulation through grazing and (ii) to assess seasonal changes in taxon-speci¢c bacterial uptake rates. We as-

0168-6496 / 03 / $22.00 9 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/S0168-6496(03)00248-4

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sume that functional guilds could be more easily de¢ned from measured bacterivory impact of the di¡erent taxa and then the identi¢cation of relevant functional groups within protists would lead to a better understanding of the functioning and regulation of microbial food webs. The study is based on measurements of abundance and biomass of heterotrophic bacteria and £agellates, measurements of production (primary production and bacterial production), and evaluation of bacterial grazing rates of £agellates by the £uorescent microspheres method. This work is part of a larger study in which dynamic and seasonal bacterial grazing of ciliates and metazooplankton is investigated in addition to £agellate grazing in order to quantify carbon £ux from the microbial food web up to higher trophic levels and the relative importance of topdown and bottom-up forces in the regulation of bacterial populations.

2. Materials and methods 2.1. Study site and sampling Lake Annecy (24.5 km2 , altitude 446 m, latitude : 45.5‡N, longitude: 6.1‡E) is one of the largest alpine lakes in France [25]. This lake has a maximal depth of 65 m and is divided into two basins. Although nutrient concentrations are particularly low, phosphorus especially appearing as a limiting factor for algal production (high N/P), the oligotrophic Lake Annecy is characterised by a high ¢sh production according to the scienti¢c monitoring that has been conducted on the lake since the mid-1960s [26]. Our study was focussed on the epilimnion (0^10 m) at the routine sampling station located in the northern basin of the lake where water depth is at its maximum (65 m). This sampling station was de¢ned according to previous investigations conducted as part of the water quality monitoring [26]. The study period lasted from April to November 2001. Samples were taken in biweekly to monthly intervals (sampling dates : 3 and 18 April, 10 and 21 May, 5 and 19 June, 17 July, 14 August, 18 September, 15 October, 27 November). 2.2. Physicochemical variables Data on ambient parameters (temperature, dissolved oxygen, conductivity, chlorophyll a, nutrients) were collected by the CARRTEL-INRA team (Thonon-Les-Bains) as part of the scienti¢c monitoring of the lake and have been summarised in an annual report [26].

a sub-sample of the water used for microbe counting. Fifteen millilitres of the sample were incubated after adding [3 H]methylthymidine (9 nM ¢nal concentration, specific activity : 82 Ci mmol31 ) and non-labelled thymidine (9 nM ¢nal concentration) at in situ temperature in the dark. The samples were treated in duplicate and a formalin-¢xed control (2% ¢nal concentration) was systematically included. After 2 h incubation, 1 ml of formalin was added before macromolecules were extracted in 10% ice-cold trichloroacetic acid for 30 min. The samples were ¢ltered onto a polycarbonate membrane (0.22 Wm pore size) and rinsed with cold trichloroacetic acid before being radioassayed by liquid scintillation counting (Packard TriCarb Liquid Scintillation Analyser 1500). Cell concentration was computed from the production using a conversion factor (3.86U1018 cells mol31 [H3]Thymidine (TdR)) proposed by Strofek [28]. Photosynthetic assimilation was measured by the 14 C method according to Steeman Nielsen [29,30]. After 4 h incubation the samples (two light bottles and two dark bottles) were immediately ¢ltered onto 0.45 Wm pore size cellulose acetate membranes and were radioassayed by liquid scintillation counting. The assimilation rate (Wg C l31 h31 ) was computed according to the ¢ltered volume, the incubation time, the concentration of available mineral carbon and the amount of radiolabelled carbon. 2.4. Staining and enumeration of heterotrophic bacteria and £agellates Abundance of heterotrophic bacteria was determined from formaldehyde-¢xed samples (¢nal concentration 2%). Counting was carried out under an epi£uorescence microscope (Nikon Eclipse TE200) after staining with DAPI (1 Wg l31 ) and ¢ltration through black polycarbonate membranes (0.2 Wm pore size) according to the protocol described by Porter and Feig [31]. Bacterial biovolume was measured by a semi-automatic image analysis system (Lucia 4.6, Laboratory Imaging) and was converted to carbon units using the conversion factor (220 fg C Wm33 ) proposed by Simon and Azam [32]. Flagellates were ¢xed with glutaraldehyde (1% ¢nal concentration), stained with primulin [33] and collected onto a black polycarbonate membrane (0.8 Wm pore size). For £agellates, slides were prepared within 24 h after sampling and were stored at 325‡C to minimise losses of auto£uorescence [34]. Slides were observed at a 1250U magni¢cation using an epi£uorescence microscope (Nikon Eclipse TE200) under UV light for heterotrophic bacteria and HNF, and under blue light for pigmented £agellates. The C content of £agellates was then calculated using a conversion factor of 220 fg C Wm33 [35].

2.3. Bacterial production and primary production 2.5. Flagellate grazing Bacterial production was measured by incorporation of [3 H]thymidine according to Fuhrman and Azam [27] from

Grazing rates of £agellates on heterotrophic bacterial

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communities were estimated from the ingestion of £uorescent beads [2,36]. The protocol was similar to that used by Carrias et al. [10] and Thouvenot et al. [22]. Experiments were conducted in duplicate in 250-ml glass containers where tracer particles were added. A stock solution of tracer particles (0.5 Wm) was prepared from a concentrated solution of Fluoresbrite Plain Microspheres (Polysciences) and was treated with bovine serum albumin (0.5 mg ml31 ) to avoid clumping of particles [10,36]. The microsphere size was chosen according to the size of the bacteria in Lake Annecy (mean length of heterotrophic bacteria during the study was 0.7 M 0.28 Wm). The ¢nal concentration of microbeads in the experimental bottles was around 2U105 beads ml31 while bacterial abundance varied from 1.1 to 3.6 cells ml31 during the study period. Therefore, the concentration of microspheres in each experimental bottle was from 5 to 20% of the bacterial concentration in the lake. Bead concentration was estimated by epi£uorescence microscopy after ¢ltration onto polycarbonate ¢lters (0.2 Wm pore size). The plankton from 0 to 10 m were acclimatised for at least 5 min in 250-ml glass bottles before beads were injected. Based on preliminary measurements of predation kinetics in Lake Annecy, we chose an incubation time of 15 min. At all sampling dates, we conducted the grazing experiment in two experimental bottles in which we took and analysed samples at 0 min and 15 min. The incubation was stopped by adding ice-cold glutaraldehyde (2% ¢nal concentration). The microbeads ingested were enumerated after ¢ltering sub-samples from each bottle (30 ml) onto a 0.8-Wm polycarbonate membrane and staining with primulin as described for nano£agellate enumeration. Flagellates and microbeads were observed at a 1250U magni¢cation under UV light and blue light. Clearance rate (nl ind31 h31 ) was calculated for each taxon by dividing the number of ingested beads per hour by the bead concentration in the bottle. Ingestion rate of each taxon (bacteria £agellate31 h31 ) was calculated by multiplying the corresponding clearance rate by the heterotrophic bacterial concentration (abundance actually measured on the day of experiment). The grazing impact (bacteria h31 l31 ) of a taxon was estimated by multiplying its ingestion rate by its actual concentration. Bacterial grazing impact of di¡erent £agellate groups (heterotrophic, mixotrophic) was computed by summing grazing impacts of all taxa belonging to the same group. In order to compare loss of bacterial production due to £agellate grazing, bacterial ingestion rates were converted into carbon units using bacterial biovolumes and a conversion factor of 220 fg C Wm33 .

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3. Results 3.1. Ambient parameters The study started on 4 April before strati¢cation began and water temperature varied from 7.6‡C (18 April) up to 21.4‡C (14 August). The lake was clearly strati¢ed from May to October. Transparency ranged from 2.5 to 9.6m (Secchi disc depth) (Fig. 1a). Phosphorus concentrations were low all through the study period and varied from 1 to 2 Wg l31 [26].

2.6. Analysis Statistical treatments mainly consisted of correlation analysis (Pearson correlation factor) to search for empirical relationships between variables.

Fig. 1. Seasonal variations of (a) temperature and transparency (Secchi disc readings), (b) heterotrophic bacterial concentrations and bacterial production, (c) mean biovolume and biomass of heterotrophic bacteria, and (d) primary production and chlorophyll a concentrations.

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3.2. Concentration, biomass and production of heterotrophic bacteria Abundance and biomass of heterotrophic bacteria respectively ranged from a minimum of 1.1U106 bacteria ml31 and 23.2 Wg C l31 (3 April) to a maximum of 3.6U106 bacteria ml31 and 78.9 Wg C l31 on 19 May (Fig. 1b). Filamentous forms were not observed during the study period. Heterotrophic bacteria were mainly represented by small-sized free cells (mean biovolume: 0.06 Wm3 ) (Fig. 1c). No correlation appeared between heterotrophic bacteria (abundance or biomass) and temperature, chlorophyll a concentration or primary production. Bacterial production reached its maximum (2.25 Wg C l31 h31 ) on 19 May and the annual mean was 0.77 Wg C l31 h31 (Fig. 1c). A second peak was observed in June when the maximal chlorophyll a concentration was observed. A signi¢cant positive correlation appeared between bacterial production and chlorophyll a concentration (r = 0.679; P = 5%).

identi¢ed were called respectively undetermined HNF 6Wm and undetermined HNF 3Wm. Heterotrophic £agellates varied between 290 and 1130 cells ml31 (annual mean : 750 cells ml31 ) (Fig. 2a). Their lowest abundance was observed in summer (minimum in September) and the highest during spring (maximum in April). The contribution of the di¡erent taxa to total HNF abundance and biomass is shown in Fig. 2b. The community was dominated by Katablepharis, Spumella, undetermined HNF 6Wm and undetermined HNF 3Wm. During the study period, the contribution of Katablepharis to total HNF abundance was rather high. Katablepharis (annual mean abundance: 5.4U102 cells ml31 ) represented up to 59% of total £agellate abundance on 5 June (Fig. 2b). In contrast, the proportion of chrysomonads (Spumella) was lower and represented from 3% (5 June) to 21% (3 April) of total £agellate abundance. In the epilimnion of Lake Annecy, the genus Bodo represented the main contributor within the Kinetoplastids. Their abundance and contribution to HNF were low

3.3. Phytoplankton primary production and chlorophyll a concentration Phytoplankton primary production varied between 0.9 (October) and 11.3 Wg C l31 h31 (June) in the 0^10-m surface layer (Fig. 1d). This low production is typical of this type of oligotrophic system. Similar values were measured during a previous study [37]. Chlorophyll a concentration ranged from 1.13 to 3.99 Wg l31 , the maximal concentration being recorded during June and July (Fig. 1d). For the last 4 years [38] similarly low values have usually been observed in the euphotic zone of Lake Annecy. 3.4. Taxonomic composition, abundance, biomass of £agellates Annual means for total £agellate abundance and biomass were respectively 2.8U103 cells ml31 and 16 Wg C l31 . HNF represented from 12 to 83% of the total £agellate abundance. This percentage reached a maximum in May. For all other sampling dates, pigmented £agellates outnumbered HNF. Due to a higher mean biovolume than heterotrophs, pigmented £agellates dominated £agellate biomass in the epilimnion throughout the whole study period. 3.4.1. Non-pigmented £agellates In most cases, £agellates were identi¢ed to the family or genus level. Within heterotrophic £agellates, major taxa such as Kinetoplastids, Choano£agellates and Chrysomonads (Heterokonts) could be identi¢ed under the epi£uorescence microscope. Some other £agellates, particularly colourless Cryptomonads (6 Wm long) and small uni£agellated cells (3 Wm long) which could not unequivocally be

Fig. 2. Seasonal variations of (a) heterotrophic £agellate concentration and biomass and (b) heterotrophic £agellate community composition in terms of abundance and biomass.

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throughout the study period (from 9 to 93 cells ml31 , i.e. from 3 to 10% of the total HNF abundance; Fig. 2b). Such a low concentration in oligo- to mesotrophic lakes is not surprising for this genus [9,10], Auer and Arndt [39] reported that the Bodonid Bodo saltans clearly increases in both frequency and abundance with increasing trophic status. 3.4.2. Pigmented £agellates High pigmented £agellate abundances were recorded from April to the end of May, while the maximal density was reached in July due to a high concentration of Dinobryon cells (Fig. 2b). Similar seasonal dynamics have been observed in other lakes [22]. The annual mean abundance and biomass for pigmented £agellates were respectively 1.9U103 cells ml31 and 12 Wg C l31 . The pigmented £agellate community was dominated by the Cryptomonads Cryptomonas, Rhodomonas and the Chrysophytes Dinobryon, Chrysidalis, Ochromonas. More occasionally we observed the Chrysophyte Mallomonas and the Euchlorophyte Chlamydomonas. Within these species, Dinobryon, Ochromonas and Cryptomonas were the main species that might develop a phagotrophic activity. Some species could even use more than two nutrition modes such as Ochromonas which is able to practise phagotrophy, photoautotrophy and osmotrophy and could also contain symbiotic bacteria [40]. Cryptomonads were characterised by a relatively high abundance during the whole study period. A ¢rst peak was observed in April for both Cryptomonas and Rhodomonas, and a second one in July or during late summer. Cryptomonas represented up to 87% of the pigmented £agellate abundance (19 June; Fig. 3b). The minimal concentration of Cryptomonads was recorded on 5 June, probably due to the high grazing pressure exercised by Cladocerans (mainly Daphnia) [41] that dominated the zooplankton community during this period. Chrysidalis abundance and biomass were rather low during the whole study period, maximal values being observed in October. Dinobryon represented up to 78% of pigmented £agellate abundance (29 May; Fig. 3b). Two peaks of abundance were observed, in May and July. Similar observations have been reported by Carrias et al. [10] in Lake Pavin, where Dinobryon grew preferentially during late spring and early summer at the time bacterial abundance and phytoplanktonic exudation were important. However, our results did not show any signi¢cant correlation between colonial Chrysomonad development and temperature as suggested by Pick et al. [42]. Mixotroph seasonal dynamics were closely related to those of phototrophic populations. A signi¢cant correlation was noted between pigmented £agellates and chlorophyll a concentration (r = 0.776; P = 5%). In Lake Annecy, the class of Chrysophycea was the second most numerous phytoplankton after the diatoms, whereas the class of Cryptophycea was the third most numerous [26].

Fig. 3. Seasonal variations of (a) pigmented £agellate concentration and biomass and (b) pigmented £agellate community composition in terms of abundance and biomass.

3.5. Bacterivory The grazing experiments were conducted using two replicates for each sampling date. Fig. 4 presents the mean value obtained for ingested rates and the values measured for each replicate. The range of variation between replicates was generally rather low (from 1.2-fold up to 2.6fold) compared to the range of seasonal variation. The greatest di¡erences between replicates were observed for Katablepharis, the ingestion rates measured for this taxon could vary up to 5.8-fold within the two replicates for one sampling (10 May). Per capita ingestion rates of £agellates (HNF+mixotrophs) ranged from 15 to 75 bacteria ind31 h31 and total grazing impact varied from 6.1U106 to 7.0U107 bacteria l31 h31 . We observed large taxon-speci¢c and seasonal di¡erences in the ingestion rates of major HNF and mixotrophic £agellates (Fig. 4). Bacterial losses due to total £agellate grazing varied from 23.7% to 180% of bacterial production (mean value during the study: 86.4% M 51.7%).

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Fig. 4. Seasonal variations of ingestion rates of (a) heterotrophic £agellates and (b) mixotrophic £agellates. Histograms represent mean values based on analysis of duplicates, and values for each replicate are also presented (horizontal lines) to visualise the range of variation between the two replicates.

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Per capita ingestion rates of HNF were from 1 to 29.8 bacteria ind31 h31 . The maximal value was recorded in June and the lowest in August (Fig. 4a). Nakano et al. [43] similarly reported a low ingestion rate of HNF during August in the mesotrophic Lake Biwa. Spumella, Katablepharis and undetermined HNF 6Wm were the main bacterial grazers within colourless £agellates. Bacterial ingestion by Kinetoplastida was quanti¢ed for two dates only (3 and 18 April) when ingestion rates were low, 1.1 and 1.2 bacteria ind31 h31 , respectively. For several other sampling dates, Kinetoplastida occurred in too low concentrations to allow a correct estimation of their ingestion rates. A similar problem appeared for Choano£agellida and undetermined HNF 3Wm which sometimes were in rather low concentration. However, we were able to measure the uptake of beads by Choano£agellida and to verify that undetermined HNF 6Wm never ingested microspheres. Choano£agellida ingestion rates were low and were positively correlated with bacterial production (r = 0.880; P = 1%) and chlorophyll a concentration (r = 0.630; P = 5%). The ingestion rates measured for Spumella varied from 0 to 15.6 bacteria ind31 h31 , the highest value being observed on 19 June. A positive correlation between Spumella ingestion rates and primary production was noted (r = 0.930; P = 1%). However, the impact of Spumella predation on bacteria was found to be rather low during this study due to the relatively low abundance of this taxon. Spumella consumed up to 7.9U105 bacteria l31 h31 (maximum observed in July), which represented less than 5% of the bacterial production (Fig. 5a). The highest loss of bacterial production via Spumella grazing was observed in November (7.1% of the bacterial production), at a time when the predation rate was low. While some authors previously concluded that Katablepharis did not ingest microbeads [10,22], during this study, similarly to Cleven and Weisse [9], we identi¢ed Katablepharis as a bacterivore, as several peaks in ingestion rate appeared for this taxon during the study period (3 April, 10 May, 15 October ; Fig. 4a). No correlation appeared between ingestion rates of Katablepharis and bacterial abundance or production. It seems that bacterial abundance did not in£uence the bacterial activity of Katablepharis. Due to their high abundance and relatively large ingestion rates, Katablepharis consumed up to 4.2U106 bacteria l31 h31 (15 October) which represented 62.3% of the bacterial production (Fig. 5a). The impact of undetermined HNF 6Wm on bacterial communities was lower (maximal grazing rates in July: 2.32U106 bacteria l31 h31 = 13.5% of the bacterial production). Finally, total HNF consumed up to 6.1U106 bacteria l31 h31 (15 October; Fig. 5a). The comparison of predation rates to bacterial production showed that HNF consumed from 0.1 to 62.4% of the bacterial production. The minimum value was recorded in August and the maximum in October due to the bacterivory of Katablepharis. Among HNF the most important

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taxa as bacterial grazers were Katablepharis, undetermined HNF 6Wm and Spumella, which could consume respectively 0^62.3%, 0^13.5% and 0^7.1% of the bacterial production. Periods of highest HNF predation impact were in April (due to undetermined HNF 6Wm and Bodo) and in October (due to Katablepharis). Ingestion rates of mixotrophic £agellates were generally higher than those measured for HNF. Per capita they varied from 10.7 to 54.8 bacteria ind31 h31 (Fig. 4b). The minimal and maximal value were recorded during May (respectively on 29 and 10 May). Bacterial grazing within mixotrophs was mainly dominated by Dinobryon and Cryptomonas (Fig. 5b). However, Chrysidalis and Ochromonas were also occasionally responsible for a quite considerable grazing pressure. Dinobryon ingestion rates £uctuated between 6.1 and 19.1 bacteria ind31 h31 , except on 10 May when an ingestion rate of 43.2 bacteria ind31 h31 was measured (Fig. 5b). Such a high ingestion rate had already been reported for Dinobryon by some authors but it was generally described as occasional, so that, over an annual period, this mixotroph was generally considered an unimportant bacterivore [9,10]. The particular feature in our study was that Dinobryon occurred at most of the sampling dates (except in June) and always had bacterivorous activity. Our results showed that the ingestion rate of Dinobryon was highly correlated with bacterial abundance (r = 0.905; P = 1%) and bacterial production (r = 0.840; P = 1%). We did not ¢nd a similar correlation reported in the literature, Dinobryon is more generally considered an occasional bacterivore [44]. Similarly, we observed rather high ingestion rates for Cryptomonas,

Fig. 5. Seasonal variations of grazing impact from (a) heterotrophic £agellates and (b) mixotrophic £agellates.

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varying from 3 to 24 bacteria ind31 h31 , whereas this genus is usually considered a negligible bacterivore [24] During our study, due to their high abundance and ingestion rates, mixotrophic £agellates, and particularly Dinobryon, appeared as major bacterial grazers (Fig. 5a,b). Mixotrophs consumed up to 6.8U107 bacteria l31 h31 (maximum observed in April). A quite similar result was observed in July when mixotrophic £agellates were responsible for the loss of 49.1% of the bacterial production. The mean loss of bacterial production due to grazing by mixotrophic £agellates was 152% ( M 98.2). On particular dates during spring, late summer and autumn (May, August, September, October) grazing measurements showed that mixotrophic £agellate grazing was higher than bacterial production, mainly due to the bacterivory of Dinobryon; mixotrophic £agellates removed 193% of the bacterial production on 29 May. Of course, the rather large uncertainty associated with such comparisons of bacterial production and grazing loss rates derived from microsphere or £uorescently labelled bacteria (FLB) experiments must be considered. Any extrapolation of the data to discuss the regulation impact of £agellates on bacteria should be considered in view of this uncertainty. However, our results clearly demonstrated that mixotrophs were important bacterial grazers in Lake Annecy.

4. Discussion Our study is the ¢rst to provide information on the composition, dynamics, and grazing impact of £agellates in the oligotrophic Lake Annecy. A few studies have investigated bacterial grazing by HNF-speci¢c taxa, and extremely rarely studies have dealt with taxon-speci¢c bacterial grazing for both HNF and mixotrophic £agellates, particularly under oligotrophic conditions. Concentrations of HNF measured in Lake Annecy were close to those measured in other lakes [9,10,21]. The main di¡erence concerning this group was the large dominance of Cryptomonad forms (Katablepharis, undetermined HNF 6Wm) compared to Chrysomonad cells (Spumella) which were usually reported as the dominant species within HNF [2,9,10,22,45]. As regards pigmented £agellates the speci¢c feature in Lake Annecy was the numerical importance of Dinobryon which was observed on nine out of the 11 sampling dates, while in several lakes a high occurrence of mixotrophic species and particularly Dinobryon was restricted to a relatively short period during summer [9,46]. 4.1. Potential shortcomings of the £uorescent microbeads method Bene¢ts and advantages of microspheres and FLB techniques have been extensively discussed in the literature [9,22,47^50]. In this study we used the tracer approach

as a ‘near standard’ method but of course, we have to consider possible problems inherent in the technique. First, some species may have had some selective grazing behaviour against microspheres as arti¢cial preys, and the extrapolation of the data should be considered accordingly. However, Boegnik et al. [51] recently reported that the types of particles (inert or living) do not induce selectivity for interception-feeding bacterivorous nano£agellates. The authors showed that the introduction of beads into natural food (mixture of bacteria) did not modify the ingestion rates of three di¡erent £agellates (Spumella, Ochromonas, Cafeteria) and that no signi¢cant di¡erence appeared between beads and bacteria in terms of capture e⁄ciency. This study [51] concluded that selective food uptake rather depended on food concentration for Spumella and Ochromonas. A signi¢cant selection of beads (beads rejected or non-ingested) appeared only for relatively high food concentrations (from 2.5U107 cells ml31 to 3.3U107 cells ml31 ). During our experiment we did not observe such high concentrations of bacteria and we assumed that there was no strong selection between beads and bacteria. Secondly, a problem inherent in the use of microspheres is the possible quick egestion of ingested beads and FLB by some Chrysomonads as demonstrated by Boegnik et al. [52]. We relied on the conclusions of several authors who previously demonstrated that the number of microspheres ingested by £agellates increases with time up to a maximal value [47,50,53]. They considered that during the linear increase of particle numbers in vacuoles, there is no egestion of particles. After the plateau is reached, the particle number in £agellates is relatively constant through time and represents a balance between ingestion and egestion. Based on this predation kinetics, the ingestion rates are assessed by using data in the linear part of the curve [47,50,53]. We used this kind of predation kinetics to choose the time of incubation used during our experiment. Predation kinetics were performed on three dates (April, June, September) for the microbial communities in Lake Annecy. The incubation times tested were 0, 5, 10, 15, 20, 25 and 30 min. We observed a linear increase of the ingested particle numbers during a period which ranged from 15 to 30 min according to taxa. During this linear increase we considered that there was no egestion. Egestion appeared after 20 min for Dinobryon, while for Choano£agellates we observed a linear increase in ingested particles for up to 30 min. For Spumella and Katablepharis, egestion appeared after 15 min and we noticed that no particles were ingested by Spumella for a 5-min incubation. As a result of these observations, for the seasonal study of grazing we chose to minimise handling e¡ect and counting, and we carried out the enumeration of ingested microspheres for two incubation times only: 0 min and 15 min. Thirdly, the size selectivity of bacterivorous £agellates is an often neglected problem in FLB or microspheres tech-

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niques. We cannot exclude the possibility that larger bacterivores such as Katablepharis prefer larger feed sizes and do not e⁄ciently graze on small particles such as the microbeads. However, the use of microspheres still appeared to us to be the best compromise for the simultaneous estimation of bacterial consumption by £agellates (results presented in this paper), ciliates and metazooplankton (Domaizon et al., in preparation), because beads resist digestion and are easy to see in guts or vacuoles. Associated with a taxonomic identi¢cation of grazers, this method has the primary advantage of taking into account the variability of ingestion rate within each taxon and is especially useful for considering the still poorly studied bacterivorous activity of mixotrophic species. 4.2. Interest of taxon-speci¢c ingestion rates We observed large di¡erences between taxon-speci¢c ingestion rates of major £agellates (both heterotrophs and mixotrophs) and high seasonal variations. Per capita in-

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gestion rates of HNF ranged from 0 to 30 bacteria HNF31 h31 ; Cleven and Weisse [9], who considered HNF grazing in Lake Constance by the FLB method, reported very similar values of ingestion rates (from 0 to 31 bacteria ind31 h31 ). As regards mixotrophs, we measured per capita ingestion rates varying from 10 to 55 bacteria ind31 h31 which demonstrated that uptake rates by mixotrophs may also greatly vary seasonally. In order to compare taxon ingestion rates of £agellates we included this study in literature data (Table 1), assuming that the microspheres technique and the alternative methods such as FLB were internally consistent, and ignoring problems originating from di¡erent experimental protocols. As concerns HNF and more particularly Spumella, Choano£agellida, Bodonids, our data fall generally within the range but toward the lower end of previously published data (Table 1) [2,9,10,22,24,45,54^56]. In contrast, Katablepharis, which is generally considered a facultative bacterivore [10,22], appeared to have quite considerable bacterivorous activity ; however, its bacterial uptake rates varied seasonally by a factor of up to 8. The larger range for seasonal

Table 1 Ingestion rates of heterotrophic and mixotrophic £agellates obtained by various methods in lakes di¡ering in their trophic status

Heterotrophic £agellates Spumella-Monas-like cells

(Spumella 2^6 Wm) (Spumella s 6 Wm)

Katablepharis Choano£agellida

Bodonids Mixotrophic £agellates Cryptomonas Dinobryon

D. bavaricum D. cylindricum Ochromonas

a

Ingestion rates (bacteria ind31 h31 )

Methoda

Site

Trophy degree

Reference

0^15.6 1.6^27 0^14.1 10^14 10^15 0^5 6^31 14^38 3^23 21 0^8.1 0^5 0^3.1 1.7^33.6 0^11 13^73 13^37 8^42 53 0^10 36

Microspheres Microspheres Microspheres FLB FLB FLB FLB Live observation Microspheres FLB Microspheres FLB Microspheres Microspheres FLB Microspheres FLB Microspheres FLB Microspheres FLB

Lake Annecy Lake Pavin Reservoir Sep Lake Erie Lake Constance Lake Constance Lake Constance

Oligotrophic Oligo-mesotrophic Oligo-mesotrophic Mesotrophic Meso-eutrophic Meso-eutrophic Meso-eutrophic

Lake Oglethorpe Reservoir Rimov Lake Annecy Lake Constance Lake Annecy Lake Pavin Lake Constance

Eutrophic Eutrophic Oligotrophic Meso-eutrophic Oligotrophic Oligo-mesotrophic Meso-eutrophic

Lake Erie Lake Oglethorpe Reservoir Rimov Lake Annecy Reservoir Rimov

Mesotrophic Eutrophic Eutrophic Oligotrophic Eutrophic

this [10] [22] [45] [24] [9] [9] [54] [2] [55] this [9] this [10] [9] [56] [45] [2] [55] this [55]

3^24 0^14 6^43.2 2.4^35.3 63.2^137.6 8^38 6^12 0^5.4 0^3 6.4^87 2^53 0^38.3

Microspheres Microspheres Microspheres Microspheres Microspheres Microspheres Microspheres FLB Microspheres Microspheres Microspheres FLB

Lake Annecy Reservoir Sep Lake Annecy Lake Pavin Reservoir Sep Lake Oglethorpe Lake Oglethorpe Arti¢cial pond Lake Annecy Lake Pavin Lake Oglethorpe Arti¢cial pond

Oligotrophic Oligo-mesotrophic Oligotrophic Oligo-mesotrophic Oligo-mesotrophic Eutrophic Eutrophic Eutrophic Oligotrophic Oligo-mesotrophic Eutrophic Eutrophic

this study [22] this study [10] [22] [2] [2] [16] this study [10] [2] [16]

FLB: £uorescent labelled bacteria

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study

study study

study

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variations was observed for Spumella within heterotrophic £agellates (from 0 to 15 bacteria ind31 h31 ) and for Dinobryon within mixotrophs (from 6 to 43 bacteria ind31 h31 ). The large di¡erences between taxon-speci¢c ingestion rates of major £agellates and high seasonal variations show that all HNF did not act similarly as primary bacterivores. These results suggest that considering the taxonomic composition of £agellates may provide a better understanding of trophic structures and thus energy £ow within the microbial loop. When assessments of overall HNF grazing are made, they are generally based on the bacterial abundance/HNF abundance ratio. We have derived this type of overall evaluation from our data with the aim of making a comparison with our taxon-speci¢c assessment of bacterial grazing. It is generally considered that a minimal proportion of 1000 bacteria per heterotrophic £agellate characterises a microbial food web where £agellates preferentially consume bacteria [57]. During our study this ratio was generally higher than 1000 and varied from 981 to 8275 (mean value: 3239) showing that bacteria in the euphotic zone of Lake Annecy were abundant enough to support the development of the heterotrophic £agellates that were present. Jugnia et al. [57] proposed using the formula established by Starink et al. [58] to evaluate the fraction of the bacterial production consumed by heterotrophic £agellates. When applied to our data, this formula suggested that the fraction of bacterial production consumed by HNF would vary from 20% to 109% (annual mean 55%). The results we obtained from the microspheres method showed that HNF consumed from 0.1 to 62.4% of the bacterial production (annual mean: 17.9%). For most of the sampling dates, the impact of HNF predation measured by the microspheres method was then lower than that estimated from the ratio bacteria/HNF according to the Starink formula. The estimated HNF predation based on the microsphere method was higher for one sampling date only, in October, when both the ingestion rates and the abundance of Katablepharis were particularly high. The signi¢cantly di¡erent results given by these two approaches highlight the di⁄culty in comparing studies where impact of predation is assessed by di¡erent methods. 4.3. Mixotrophy in the microbial loop of Lake Annecy A few studies concluded that mixotrophic £agellates are unimportant bacterial grazers in freshwater pelagic food webs [9,10,46]. On the contrary, we observed that mixotrophic £agellates could play an important role in the regulation of bacterial communities in Lake Annecy. Although per capita ingestion rates of HNF could be higher than ingestion rates of mixotrophs, the total grazing impact of mixotrophs, particularly due to Dinobryon, and to a lesser extent to Cryptomonas, was most of the

time higher than the impact of heterotrophs. On average, only 17.9% ( M 19.2) of the bacterial production was removed by HNF during the study period, while the mean loss of bacterial production due to mixotrophs grazing was 152% ( M 98.2). Cleven and Weisse [9] reported similar results for the HNF grazing impact in Lake Constance. The authors observed that only 9.7% ( M 12.4%) of the bacterial production was removed by HNF on an annual average. They suggested that grazers other than small HNF signi¢cantly contributed to the total bacterial mortality. We assume that the high ingestion rates of mixotrophic £agellates in the epilimnion of Lake Annecy could be a consequence of the low soluble reactive phosphorus (SRP) concentrations. Olrik [44] demonstrated that it is possible for mixotrophic Chrysophytes to solve their demand for phosphorus by ingestion of bacteria. The results of Hitchman and Jones [16] con¢rmed this observation showing that, in Lily Pond (England), the Dinobryon population ingested FLB to supplement their own phosphate requirements when SRP was low. Similarly, our results pointed out that when Dinobryon was present, its bacterial ingestion rate was always important under the P-limited conditions that are quite permanent in the oligotrophic Lake Annecy (SRP concentrations varied from 1 to 2 Wg l31 during the study [26]). Bird and Kal¡ [15] showed that Dinobryon were also able to assimilate organic carbon from the ingestion of prey, in addition to other nutrients such as phosphorus. Such results suggested that the importance of mixotrophic protists as bacterial grazers could be higher in oligotrophic systems than in systems with higher trophic status. However, Sanders et al. [2] demonstrated that mixotrophic £agellates could play a signi¢cant role as bacterivores under eutrophic conditions. In the eutrophic lake they studied, mixotrophs displayed a strong seasonal impact, the highest values being recorded in winter and spring. In Lake Annecy, the maximal impact of mixotroph bacterivory was recorded in August. Our results cannot be compared to other oligotrophic systems, due to the absence of available data on taxonspeci¢c grazing in oligotrophic freshwater systems. However, some comparisons can be made with marine systems where several recent studies and reviews deal with the importance of mixotrophs [17,20,21,58^61]. As in freshwater systems, in marine environments mixotrophy is considered an adaptation to low food environments in order to supplement nutrients [21,58,60,61]. For example, Havskum and Riemann [21] demonstrated that bacterivorous mixotrophic £agellates were responsible for 86% of the entire £agellate bacterivory in the upper layer of the Bay of Aarhus (Denmark) which is characterised by low salinity and low inorganic nutrients. However, Dolan and Perez [59], discussing the costs, bene¢ts and possible uses of mixotrophy in marine oligotrichs, suggested that it could not be closely linked to the exploitation of low food environments, but probably serves a variety of purposes. Sim-

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ilarly, Havskum and Hansen [20] concluded that the additional phototrophic metabolism of mixotrophs could not be only related to the availability of dissolved inorganic nutrients but probably gives the mixotrophs a better ability to compete with strictly heterotrophic protists in environments where prey concentration or production is low. This point was investigated by Rothhaupt [62] who demonstrated that both £agellates (obligate bacterivores and mixotrophs) e¡ectively competed for food bacteria, showing that Spumella built up lower biomass compared to the larger Ochromonas due to the supplementary photosynthetic capabilities of the latter. The apparent contradictions in the literature as regards the role of mixotrophs are probably due to the variety of nutritional strategies that exist within mixotrophic protists themselves [17,63,64]. Di¡erent types of mixotrophs have been described, based on the extent to which phototrophy and phagotrophy are used [18,63,64]. Some mixotrophs are primarily autotrophic while others mainly use heterotrophy to ful¢l their energy requirements. Some authors have de¢ned di¡erent physiological types within mixotrophs based on their nutritional behaviour [18,63,64]. Mixotrophic £agellates identi¢ed in Lake Annecy essentially belong to type II of the Stickney model [18] and more exactly to subgroups IIA and IIB which, according to Stoecker, primarily concern phototrophic species that can have a bacterivorous activity when dissolved inorganic nitrogen is limiting (e.g.Dinobryon) or when a trace organic growth factor is limiting (e.g. Cryptomonas). Jones [63] categorised Cryptomonas as a primarily phototrophic species that ingests preys only at very low rates to meet requirements for cell maintenance. In contrast, during our study, rather high ingestion rates were measured for Cryptomonas compared to those measured for HNF taxa. Most of the studies dealing with the identi¢cation and the role of mixotrophic groups were conducted in marine systems [17,18,20,59,60,65], and references on lakes are still lacking; however, it is clear that, in all systems, mixotrophs increase the complexity of nutrients and energy £ows by functioning as both producers and consumers. Our study demonstrated the important role that mixotrophs could play in the regulation of bacterial communities stressing the importance in developing a functional partitioning of all microbial compartments where mixotrophs especially would be included, and which could be incorporated into general models of food webs to better distinguish possible carbon pathways within the pelagic food webs.

Acknowledgements We thank Ian Jones (CEH Windermere, UK) for correcting the English text. We also wish to thank Dr J.F. Carrias for his advice concerning methodology on grazing experiments, J.C. Hustache and J.P. Moille from the Hy-

327

drobiology Laboratory of INRA Thonon for their valuable help during ¢eld samplings. This work was supported by the Re¤gion Rho“ne-Alpes as part of the Contrat Plan Etat Re¤gion (CPER) ‘Durable management of Lake Annecy ^ Productivity and Functioning’ coordinated by Dr D. Gerdeaux, CARRTEL-INRA Thonon (74) France.

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