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Grazing of two toxic Planktothrix species by Daphnia pulicaria: potential for bloom control and transfer of microcystins LAURA OBERHAUS1,2, MALORIE GE´LINAS3, BERNADETTE PINEL-ALLOUL3*, ANAS GHADOUANI4 AND JEAN-FRAN ¸ OIS HUMBERT1,5 1´

EQUIPE DE MICROBIOLOGIE AQUATIQUE, INRA-UMR CARRTEL,

75 AV. DE CORZENT,

BP

511, 74203 THONON-LES-BAINS

2 CEDEX, FRANCE, CENTRE

D’ENSEIGNEMENT ET DE RECHERCHE EAU, VILLE, ENVIRONNEMENT (CEREVE), ENPC, 6–8 AV. BLAISE PASCAL, 77455 MARNE-LA-VALLE´E CEDEX 2, FRANCE, 3 GRIL, DE´PARTEMENT DE SCIENCES BIOLOGIQUES, UNIVERSITE´ DE MONTRE´AL, C.P. 6128 SUCC. CENTRE VILLE, MONTRE´AL, QUE´BEC, CANADA H3C 3J7, 4 AQUATIC ECOLOGY AND ECOSYSTEM STUDIES, SCHOOL OF ENVIRONMENTAL SYSTEMS ENGINEERING, THE UNIVERSITY OF WESTERN AUSTRALIA, 35 STIRLING 5 HIGHWAY, M015, CRAWLEY, WESTERN AUSTRALIA 6009, AUSTRALIA AND INSTITUT PASTEUR-CNRS URA2172, UNITE´ DES CYANOBACTE´RIES, 25– 28 RUE DU DR ROUX,

75724 PARIS CEDEX 15,

FRANCE

*CORRESPONDING AUTHOR: [email protected] Received February 26, 2007; accepted in principle April 12, 2007; accepted for publication July 30, 2007; published online August 7, 2007 Communicating editor: K.J. Flynn

The role of zooplankton in the control of cyanobacterial blooms and the transfer of cyanotoxins to higher trophic levels are of great importance to the management of water resources. Many studies have focused on the cyanobacterium Microcystis, but few have examined the interactions between zooplankton and filamentous cyanobacteria. In this study, we provide experimental evidence for the potential grazing of two toxic strains of filamentous cyanobacteria, Planktothrix rubescens and P. agardhii, by Daphnia pulicaria, and for transfer of toxins in the planktonic food chain. We determined clearance rates (CRs) by adult and juvenile D. pulicaria of the two Planktothrix strains, Scenedesmus acutus and a mixture of S. acutus cells with P. rubescens culture filtrate. Filament lengths were analyzed, and microcystin (MCY) presence in Daphnia was assessed using the Protein Phosphatase-2A (PP-2A) Inhibition Assay. The two Planktothrix strains were equally grazed by D. pulicaria, but at lower CRs than S. acutus. Potential anti-grazer toxins in P. rubescens filtrate did not inhibit Daphnia grazing. Small P. rubescens (,100 mm) filaments were preferentially grazed by adult D. pulicaria, suggesting their limited ability to control a Planktothrix population during a bloom. Large quantities of MCYs were found in unstarved Daphnia previously exposed to Planktothrix, whereas quantities were significantly smaller in individuals starved for 24 h before preservation. This indicated a potential for transfer of toxins in the food chain by Daphnia, especially immediately after ingestion of toxic cyanobacteria.

I N T RO D U C T I O N Planktothrix agardhii and P. rubescens are potentially toxic, filamentous cyanobacteria that proliferate in many temperate lakes throughout the world (Feuillade, 1994; Walsby et al., 1998; Kurmayer and Ju¨ttner, 1999; Ernst et al., 2001; Humbert et al., 2001; Nu¨rnberg and Lazerte, 2003; Oberhaus et al., 2003; Poulickova et al., 2004; Jacquet et al., 2005). Their widespread proliferation has caused much concern, due to the limitations put on water uses during blooms and the potential toxicity of these organisms. Top-down control of blooms

through predation by herbivores can be considered among the different ecological processes involved in decreasing cyanobacterial proliferation. However, contrasting results have been observed concerning the ingestion of cyanobacteria by zooplankton, making it difficult to conclude on the efficiency of zooplankton grazing in controlling cyanobacterial growth. Some studies have provided evidence for ingestion of cyanobacteria by zooplankton (Fulton, 1988; DeMott and Moxter, 1991; Epp, 1996), suggesting that grazing can provide a control mechanism for cyanobacterial blooms (Boon et al., 1994), or that cyanobacteria can be a

doi:10.1093/plankt/fbm062, available online at www.plankt.oxfordjournals.org # The Author 2007. Published by Oxford University Press. All rights reserved. For permissions, please email: [email protected]

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complementary resource for zooplankton (Kurmayer, 2001). But most studies show evidence for decreased grazing of zooplankton on cyanobacteria when compared with other species of phytoplankton, suggesting this mechanism to be insufficient to control cyanobacterial proliferation (De Bernardi and Giussani, 1990; DeMott et al., 1991; Rohrlack et al., 1999a; Lu¨rling, 2003; Ghadouani et al., 2004). Previous studies have suggested decreased grazing of cyanobacteria to be due to mechanical interference, as related to food or herbivore size (Webster and Peters, 1978; Hawkins and Lampert, 1989; Gliwicz and Lampert, 1990; DeMott et al., 2001; Ghadouani et al., 2004), as well as to chemical modes of feeding inhibition in zooplankton (DeMott and Moxter, 1991; Jungmann and Benndorf, 1994; Lu¨rling, 2003) and mussel larvae (Dionisio Pires et al., 2003). Many examples of the toxic effects of cyanobacteria on zooplankton exist (Infante and Abella, 1985; Nizan et al., 1986; Weithoff and Walz, 1995; Thostrup and Christoffersen, 1999; Rohrlack et al., 1999a; Rohrlack et al., 2005), suggesting that the dominance of phytoplankton communities by proliferating cyanobacteria may negatively affect crustacean zooplankton communities (Ghadouani et al., 2003; Chen et al., 2005). Several authors have shown that zooplankton responses to cyanobacteria vary among species, or even strains or clones that are tested (DeMott et al., 1991; Kirk and Gilbert, 1992; Hietala et al., 1995; Epp, 1996; Repka, 1996; Kurmayer and Ju¨ttner, 1999). In addition to better understanding the potential impact of zooplankton on the growth of cyanobacteria populations, the potential transfer of cyanotoxins in the pelagic food web through the grazing of toxic cyanobacteria by cladoceran and copepod crustaceans is also an important issue. Toxins produced by cyanobacteria have been widely suggested to pose a number of health risks for humans (Codd, 1995; Kuiper-Goodman et al., 1999; White et al., 2005). Some studies have examined the potential transfer of cyanotoxins into the food web (Kotak et al., 1996; Ibelings et al., 2005), and their accumulation in mollusks (Negri and Jones, 1995; Prepas et al., 1997; Williams et al., 1997; Dionisio Pires et al., 2004; Saker et al., 2004) and fish (De Magalha˜es et al., 2001, 2003; Ernst et al., 2001). De Magalha˜es et al. (De Magalha˜es et al., 2001, 2003) found microcystins (MCYs) in the muscle tissue of fish, suggesting a vector to human consumption, but few studies of this nature have been performed, especially with Planktothrix. Thus, although health risks have been widely evaluated for drinking water containing cyanotoxins, less is known about health risks related to fish consumption. Some studies focusing on MCY accumulation and transfer by zooplankton, a key link in the transfer of MCY to fish

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and humans exist (Thostrup and Christoffersen, 1999; Ferra˜o-Filho et al., 2000), but these also remain scarce. The main objectives of this study were 2-fold. The first was to elucidate grazing interactions between the filamentous cyanobacteria Planktothrix and the zooplankton herbivore Daphnia, a key component of plankton food webs, and to determine the potential for grazer control of these cyanobacterial blooms. The second was to examine the potential for transfer of MCYs produced by Planktothrix in the pelagic food chain, via grazing by Daphnia. A large number of studies have attempted to examine grazing by zooplankton of the colonial cyanobacterium Microcystis aeruginosa (DeMott, 1999; Rohrlack et al., 1999b; Ghadouani et al., 2004). Although some studies have examined grazing of filamentous cyanobacteria (Aphanizomenon flos-aquae, Anabaena flos-aquae and Anabaena wisconsinense) (Epp, 1996), very few studies have focused on the filamentous cyanobacteria of the genus Planktothrix despite their frequent occurrence in freshwater lakes and ponds throughout temperate regions.

METHOD Phytoplankton and Daphnia cultures Strains of P. rubescens (Thonon Culture Collection reference 29-1, isolated from Lake Bourget, Savoie, France) and P. agardhii (TCC ref. 83-2, isolated from Lake Nantua, Ain, France) were raised in batch cultures using Z medium (Zehnder in Staub, 1961), at 208C and on a 16:8 h light:dark cycle. Scenedesmus acutus (TCC 141-4, isolated from the Bultie`re Reservoir, Vende´e, France) was raised in batch cultures using L-C medium (Leboulanger et al., 2001) without soil or moss extracts, at 258C on the same diurnal cycle. Scenedesmus acutus was used as food for the Daphnia cultures and as a control for the grazing experiments. All batch cultures were renewed regularly to maintain exponential growth. Planktothrix filaments were measured at experiment onset (n = 2250) and showed lengths ranging from 0.003 to 0.984 mm for P. rubescens (mean = 0.15 mm, mode = 0.026 mm), and from 0.003 to 1.79 mm for P. agardhii (mean = 0.21 mm, mode = 0.026 mm). Earlier measurements of these strains established average filament widths of P. rubescens and P. agardhii to be 6 and 4 mm, respectively (unpublished data). The P. rubescens TCC 29-1 strain is known to produce [D-Asp3] MC-RR, and MCY cell content in laboratory cultures was found to range from 0.3 to 0.7 pg cell21, depending on growth rate (Briand et al., 2005). HPLC analysis has shown that the P. agardhii TCC 83-2 strain can produce two variants of MCY (Humbert, INRA

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Thonon, unpublished data), which have not yet been identified by mass spectrometry analysis. Daphnia pulicaria were collected at the end of June, 2004, from Lake St Ade`le (458950 N, 748140 W, Quebec, Canada), where they are the largest, and the dominant, zooplankton species (Pinel-Alloul and Ge´linas, Universite´ de Montre´al, unpublished data), and where blooms of cyanobacteria are not known to occur. A conical plankton net with a 1 mm mesh size was used for collection to avoid contamination with smaller zooplankton species present in the lake. Daphnia pulicaria were transferred individually to glass aquariums filled with Whatmanfiltered water from Lake St Ade`le, using a pipette with a large tip opening. The species was verified by microscope at 100 total magnification (Leica MZ12) using the Pictoral Catalogue of North American Daphnia (Hebert, 2001). Individual clones were not separated, nor isofemale lines established, in order to study grazing on a population level. Daphnia pulicaria cultures were raised for several weeks prior to grazing experiments, at the same temperature and light cycle as S. acutus cultures, and were fed S. acutus every 2 days, at an approximate concentration of 10 000– 17 000 cells mL21 (1.1–1.9  106 mm3 mL21).

Grazing experiments Adult (length range: 1.2– 1.7 mm) and juvenile (length range: 0.7– 1.2 mm) D. pulicaria were separated, and about 10 individuals were combined with 17 mL of filtered water from Lake St Ade`le (Millipore, 0.8 mm). After a 24 h acclimation and gut-emptying period, one of four types of food was added to each vial: the toxic cyanobacteria P. rubescens or P. agardhii, the control chlorophyte S. acutus or a mixture of S. acutus cells re-suspended in P. rubescens culture filtrate, to test for the presence of anti-grazer substances (including but not limited to extracellular MCYs). Estimated average initial concentrations were: 860 000 cells mL21 (54.0  106 mm3 mL21) for P. agardhii, 280 000 cells mL21 (39.6  106 mm3 mL21) for P. rubescens, 97 000 cells mL21 (11.0  106 mm3 mL21) for S. acutus and 100 000 cells mL21 (11.3  106 mm3 mL21) for the S. acutus/ P. rubescens “Mixture”. On the basis of previous observations (Humbert, INRA Thonon, unpublished data), Planktothrix filaments of 100 mm in length were estimated to contain 20 cells. Scenedesmus acutus cells were assumed to have an average equivalent diameter of 6 mm (Kreutzer and Lampert, 1999). Experiments were carried out in glass scintillation vials, in triplicate, at a temperature of 258C and on a 16:8 h light:dark cycle. Experimental vials were placed on an orbital shaker table set to 0.7 rpm which was activated for 30 min of every hour. Fluorescence was measured non-sacrificially

at intervals of 12 h or less for 72 h using a Perkin-Elmer Fluorescence 204 Spectrophotometer, set to the maximum excitation and emission wavelengths for each phytoplankton species (P. rubescens: excitation at 470 nm, emission at 580 nm; P. agardhii: ex. 470 nm, em. 650 nm; S. acutus: ex. 470 nm, em. 685 nm). This was done for experimental cultures containing animals as well as for control cultures, in which animals were absent. Clearance rates (CRs, mL individual21 h21) were calculated from blank-adjusted fluorescence measurements, according to Coughlan (Coughlan, 1969):

CR ¼

  V C0 C0c ln  ln Ct Ctc nt

ð1Þ

where V is the volume of the food suspension (mL), n the average number of D. pulicaria per vial over the time interval, t the duration of the time interval (h), C0 the phytoplankton concentration at the start of the time interval, Ct the phytoplankton concentration at the end of the time interval, C0c the phytoplankton concentration at the start of the time interval in the control vials and Ctc the phytoplankton concentration at the end of the time interval in the control vials. CRs were calculated between each time interval for which fluorescence measurements existed (0–6, 6–12 h, etc.), as well as for the entire duration of the experiment (0–72 h). CRs for different time intervals were Log10 transformed to attain normality and homogeneity of variances (Kolmogorov–Smirnov, P . 0.05). Differences in these CRs were tested using an ANCOVA with food type (P. agardhii, P. rubescens, S. acutus and the Mixture) and life stage (adults and juveniles) as factors, and time (0–6, 6–12, 12–24, 24–48, 48–60 and 60–72 h) as a covariate. The probability value for the interaction between food type and life stage using time as the covariate (0.51, not shown) indicated a positive assumption of parallel slopes. CRs for the seven time intervals were also tested for their difference from zero using a Student’s t-test (Sokal and Rohlf, 1995; implemented on Microsoft Excel software). In a second analysis, CRs calculated for 0–72 h showed a normal distribution and homogeneity of variances (Kolmogorov– Smirnov, P . 0.05) and were analyzed using a two-way ANOVA to test the influence of food type and life stage, eliminating time as a main effect. Significant differences among groups were tested using Tukey HSD multiple comparison tests for both analyses. Daphnia survivorship was assessed at each fluorescence measurement. Survivorship data (number of surviving Daphnia) were normally distributed and had homogenous variances (Kolmogorov – Smirnov, P . 0.05). Kaplan – Meier survival functions were used to assess Daphnia

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survivorship in each treatment (Kaplan and Meier, 1958). The differences in Daphnia survivorship in each of the three treatments (Mixture, P. rubescens, P. agardhii) were compared against the control (S. acutus) with a non-parametric Log-rank test included in the Kaplan – Meier module available in the statistical package JMP IN version 3.2.6 (SAS Institute Inc.). After 72 h of exposure, lengths of Daphnia individuals were measured from the head to the base of the caudal tail spine, and gut contents were observed under a microscope (Leica MZ12, 100 magnification). Neonates born during the experiment were also measured and recorded separately. Individuals were then rinsed with distilled water and frozen at 2208C for toxin analysis. A second set of replicates was fasted in cyanobacteriafree, 0.8 mm (Millipore) filtered lake water for 24 h prior to removal, observation, rinsing and freezing. Three milliliters of algal suspension were removed from all vials at experiment onset (0 h), and again at experiment end (72 h). Samples from vials containing Planktothrix cultures were conserved for filament length measurements using formaldehyde. After at least 12 h of sedimentation of a 1 mL sub-sample, lengths of 250 randomly selected filaments were measured for all replicates using an image analyzer (Leica MZ20 microscope and Image-Pro Plus software) at 100. Significant differences in filament lengths between control and grazing experiments were determined with a two-sample x 2 test (PAST software).

Extraction and analysis of MCYs in Daphnia Methods for extraction of MCYs were adapted from Vincent (Vincent, 2003). Conserved Daphnia were thawed and toxins extracted in 5 mL of 75% methanol. Each thawed D. pulicaria replicate sample was sonicated in an icewater ultrasound bath (Elma T490DH, 85 W) for a minimum duration of 50 min, and vortexed at 10 min intervals, until no remaining tissue could be observed. This was followed by centrifugation at 3220g for 1.5 h at 48C. This was done for Daphnia exposed to the two Planktothrix species and S. acutus. Starved replicates were combined for extraction, in order to increase the probability of detection of any MCYs present. The supernatant of extracted Daphnia samples was recovered and evaporated overnight by vacuum centrifuge at 308C. The remaining material was redissolved in 50 mL of 50% methanol, in an effort to dilute samples as little as possible and to improve detection of very small MCY quantities. Potential toxicity of MCYs was analyzed using the method of Tubaro et al. (Tubaro et al., 1996) for the Protein Phosphatase-2A (PP-2A)

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Inhibition Assay, chosen for its greater sensitivity than HPLC methods and decreased cost compared with ELISA. After incubation for 1 h at 378C in 96-well plates, the percentage of enzyme inhibition by MCYs in each well was measured by a spectrophotometer set to 405 nm. The detection limit for experiments was 25 pg MCY-LR mL21. Average individual dry weight (g DW) was calculated for each age class from measured body lengths (Lynch et al., 1986). The average DWs were multiplied by the number of individuals present in each sample to obtain the total DW for that extracted sample. The quantity of MCY-LR equivalents was calculated from spectrophotometer measurements and a standard curve, and then estimated per calculated g DW of Daphnia present in extracted samples. Two-sample F and Permutation t-tests were performed on results to trace significant differences between main groups (PAST software).

R E S U LT S Grazing experiments Overall, mean CRs calculated for each of the seven time intervals (Fig. 1) ranged from 20.019 (P. agardhii, juveniles, 24– 36 h) to 0.31 mL ind21 h21 (S. acutus, adults, 60– 72 h). Using all replicate values for each treatment, Student’s t-tests showed CRs to be significantly different from zero for adults and juveniles exposed to S. acutus (P , 0.001) and the Mixture (P , 0.001). Although adults exposed to P. agardhii and P. rubescens showed CRs significantly different from zero (P , 0.05 and P , 0.05), juvenile CRs were not statistically significant (P = 0.53 for P. agardhii and P = 0.89 for P. rubescens). Food type, life stage and time each had a significant influence on CRs of D. pulicaria (Table I). The interaction between the two factors (food type and life stage) was not statistically significant. CRs of the cultures containing S. acutus were greater than those of the two Planktothrix strains (Tukey HSD, P , 0.05, Fig. 1), although CRs did not differ significantly between P. rubescens and P. agardhii (Tukey HSD, P . 0.05), nor between S. acutus and the Mixture (Tukey HSD, P . 0.05). Adult D. pulicaria showed greater CRs than juveniles for each food type (Tukey HSD, P , 0.05, Fig. 1). Mean CRs calculated for the 60– 72 h time interval, which ranged from 0.07 + 0.009 to 0.31 + 0.09 mL ind21 h21 for adults and from 20.007 + 0.01 to 0.09 + 0.01 mL ind21 h21 for juveniles, were greater than those calculated for all others (Tukey HSD, P , 0.05, Fig. 1), which did not differ significantly from each other (Tukey HSD, P . 0.05,

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Fig. 1. CRs [as calculated from equation (1)] of D. pulicaria for each time interval in the presence of P. rubescens (a), P. agardhii (b), S. acutus (c) and a mixture of S. acutus cells and P. rubescens filtrate (d). Filled bars represent CRs for adults and striped bars those for juveniles. Error bars represent the standard deviation for three experimental replicates.

Table I: Results of an ANCOVA, with time as a covariate, performed on CRs by time interval Factors

d.f.

MS

F-value

P value

Food type Life stage Food type  life stage Time Error

3 1 3 1 159

0.006 0.010 0.001 0.008 ,0.001

13.8 23.3 2.2 19.7

,0.001 ,0.001 0.080 ,0.001

Fig. 1). Considering the interaction between food type and life stage, the greatest CRs could be attributed to adult D. pulicaria grazing on S. acutus (overall mean 0.1 + 0.09, max 0.31 + 0.09 mL ind21 h21, Tukey HSD, P , 0.05, Fig. 1c), followed by those of juvenile Daphnia grazing on S. acutus (overall mean 0.03 + 0.03, max 0.06 + 0.01 mL ind21 h21) and adults grazing on the Mixture (mean 0.08 + 0.08, max 0.17 + 0.15 mL ind21 h21), although these two did not differ significantly (Tukey HSD, P . 0.05, Fig. 1). All other experimental groups (adults and juveniles grazing on Planktothrix, and juveniles grazing on the Mixture)

Fig. 2. CRs of adult (filled bars) and juvenile (striped bars) D. pulicaria in the presence of P. rubescens, P. agardhii, S. acutus and a mixture of S. acutus cells and P. rubescens filtrate, calculated for the duration of the experiment (0– 72 h).

showed similar CRs among themselves (Tukey HSD, P . 0.05, Fig. 1). CRs calculated over the duration of the experiment (0 –72 h, Fig. 2) ranged from 0.001 ( juveniles grazing on P. rubescens) to 0.08 mL ind21 h21 (adults grazing on the S. acutus/P. rubescens mixture). Differences in CRs calculated from 0 to 72 h were significant for the individual effects of food type and life stage, but not for their interaction (Table II). Juvenile CRs for 0 –72 h were significantly lower than those of adults (Tukey HSD, P ,

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Table II: Results of a two-factor ANOVA on overall CRs (0– 72 h) Factors

d.f.

MS

F-value

P value

Food type Life stage Food type  Life stage Error

3 1 3 16

0.003 0.006 0.000 0.000

9.6 18.9 0.95

0.001 ,0.001 0.439

0.05, Fig. 2). Food type influenced CRs however, which were always greater on cultures containing S. acutus (Tukey HSD, P , 0.05, Fig. 2), regardless of age class. End-experiment microscope observation of adult D. pulicaria confirmed ingestion of S. acutus, the Mixture, P. agardhii and P. rubescens. Observation of juveniles confirmed ingestion of S. acutus and the Mixture, but gut fullness and coloration varied among individuals exposed to P. agardhii and P. rubescens. The comparison of Kaplan – Meier survival functions of the control (S. acutus) against the three treatments (Mixture, P. rubescens and P. agardhii) did not reveal any statistically significant differences (Log-rank, P . 0.05). In addition, there was no statistically significant effect of life stage, as the response of adults was similar to that of juveniles in all treatments (Log-rank, P . 0.05).

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levels to 599 mg MCY-LR equivalents g DW21. In cases of large variation among three experimental replicates, “extreme values” were identified as those showing the greatest distance from the nearest data point (Fig. 4). Inhibition of PP-2A activity by MCYs was found in most Daphnia having been in contact with the two Planktothrix species, regardless of age class (Fig. 4). Differences in detected MCYs between Planktothrix and S. acutus were significant (F and Permutation t-tests, P , 0.05), with overall means (excluding extreme values) of 340, 240 and 0.01 mg MCY-LR equivalents g DW21 for P. agardhii, P. rubescens and S. acutus, respectively. Differences between adult and juvenile samples were not found to be significant (F and Permutation t-tests, P = 0.852). However, significantly greater concentrations of MCYs were detected in Daphnia that were exposed to Planktothrix and were not starved before conservation for toxin analysis (74 to 1099 mg MCY-LR equivalents g DW21) than in those starved for 24 h prior to freezing (undetectable levels to 221 mg MCY-LR equivalents g DW21) (F and Permutation t-tests, P , 0.05, with and without extreme values). MCYs were not detected in starved or unstarved Daphnia exposed to S. acutus (excluding one extreme value), or in starved juveniles previously exposed to P. rubescens.

DISCUSSION

Planktothrix f ilament lengths Filaments measured during D. pulicaria experiments ranged in size from less than 0.05 to 2.0 mm for both P. rubescens and P. agardhii (Fig. 3). No significant differences were found between filament lengths at experiment onset (T0) and experiment end (T72) for both cyanobacteria exposed to juvenile Daphnia (x 2 test, P. rubescens: P = 0.870, P. agardhii: P = 0.889) and in control experiments without Daphnia (x 2 test, P. rubescens: P = 0.990, P. agardhii: P = 0.773). Significant differences were however found for filament sizes measured before and after exposure to adult Daphnia, for both P. rubescens (x 2 test, P , 0.01) and P. agardhii (x 2 test, P , 0.001). When exposed to adult Daphnia, a large decrease in P. rubescens filaments of 0.05– 0.10 mm lengths was observed over time. For P. agardhii, a decrease in the number of larger filaments (.0.35 mm) was observed over the 72 h period.

MCY analysis and transfer to Daphnia MCYs could be detected in extractions from most Daphnia samples (Fig. 4), and concentrations ranged from undetectable levels to 1099 mg MCY-LR equivalents g DW21, while means varied from undetectable

Grazing and potential for control of Planktothrix blooms by Daphnia CRs calculated for our experiments initially seem somewhat low, for Daphnia in the presence of Planktothrix as well as S. acutus. A survey of the literature, however, shows a wide range of values in terms of clearance or grazing rates for zooplankton. Using radioisotopes in 10-min feeding experiments, DeMott and Moxter (DeMott and Moxter, 1991) found larger CRs for D. pulicaria grazing on Planktothrix, ranging from 0.05 – 1.0 for juveniles to 2.0 – 2.3 mL ind21 h21 for adults, when fed together with Oscillatoria tenuis. Using Chl a measurements (PhytoPAM) and a calculation method that differs from ours, Lu¨rling (Lu¨rling, 2003) found values closer to ours for D. magna (mean body length of 1.63 mm) in 2 h feeding experiments, with CRs of 0.30 mL ind21 h21 on cultures of 100% S. obliquus, and of 0.02– 0.03 on 75:25 toxic M. aeruginosa: S. obliquus mixtures. Also using Chl a measurements, as well as the same method of calculation, Dionisio Pires et al. (Dionisio Pires et al., 2003) found CRs for Dreissena polymorpha larvae (also zooplankton) of 0.05– 0.1 mL larvae21 h21. The wide range of methods

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Fig. 3. Distributions of filament lengths of P. agardhii (left column) and P. rubescens (right column) in the presence of D. pulicaria adults (top row), juveniles (middle row) and without Daphnia (bottom row), at experiment onset (white bars) and after 72 h (black bars). Error bars represent the standard deviation for three experimental replicates.

present in the literature makes comparisons with our results somewhat difficult; however, due to the somewhat high concentrations of Planktothrix used in our experiments (Geller, 1975 in Lampert, 1987; DeMott, 1995), our values may not represent maximum CRs of D. pulicaria on these strains.

In our experiments, significantly lower grazing rates were measured for D. pulicaria in the presence of Planktothrix filaments, as compared with S. acutus cells. This is thus in agreement with previous studies showing evidence for the decreased grazing of cyanobacteria. Adult CRs were greater than those of juveniles for both

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Fig. 4. Results from the PP2A inhibition assay, in mg MCY-LR equivalents per g DW of extracted D. pulicaria. Open diamonds represent separate experimental replicates for which reliable toxin data could be obtained, with extreme values marked as open circles. Means for all data points appear as filled squares, and those that exclude extreme values appear as filled triangles. Lone filled squares represent samples for which replicates were combined during extraction. Results are presented for Daphnia exposed to P. rubescens (Pr), P. agardhii (Pa) and S. acutus (Sa).

Planktothrix and the two cultures containing S. acutus. Smaller animals may have smaller carapace gapes and mesh sizes, and may also depend more than adults on bacteria and other small particles for food (Geller and Mu¨ller, 1981). Owing to starvation conditions during the acclimation period, higher CRs may have been expected for the 0 – 6 h time interval. It is however possible that greater CRs actually occurred in the first part of this time interval (e.g. 0 –2 h), but were masked by lower CRs in the latter part (e.g. 2 –6 h). Although our observations showed that adult D. pulicaria did ingest the two Planktothrix strains, no acute toxic effect of the cyanobacteria was detected through the Kaplan – Meier survival function analyses over the 72 h duration of our experiments. It is however possible that more subtle toxic effect, such as those on fecundity or size and age at maturation, existed. These might be estimated over a longer study period but were not considered during our relatively short-term exposure experiments. Furthermore, a recent meta-analysis of laboratory experiments on interactions between bloomforming cyanobacteria and zooplankton showed that most examples of toxic cyanobacteria decreasing survival rates were attributable to the effects of a single Microcystis strain, PCC 7820, and that filamentous cyanobacteria were in general significantly better food for grazers than single-celled cyanobacteria (Wilson et al., 2006). Some studies have shown decreased survival of Daphnia grazing on Planktothrix when compared with non-toxic (Repka, 1996) and foodless (Kurmayer and

Ju¨ttner, 1999) controls, and with increasing proportions of Planktothrix in a mixture with non-toxic algae (Infante and Abella, 1985). Although it was not often clear if the cyanobacteria were ingested, survival response was often found to vary between Daphnia species and clones. Rohrlack et al. (Rohrlack et al., 2005) recently showed that many Planktothrix strains produce inhibitors of the Daphnia digestive enzyme trypsin, potentially causing their death, but that this production varies among Planktothrix strains. Thus a wider range of examples of the effects of Planktothrix on the survival of zooplankton may still be useful, but these would best be based on careful designs and hypotheses that aim to understand sources of varying responses between different strains, species and clones. Concerning filament lengths, adult D. pulicaria significantly removed the two smallest size classes of P. rubescens filaments from experimental cultures, corresponding to those of ,50 mm in length and those ranging from 50 to 100 mm in length, whereas insignificant changes in filament size classes over time were observed in juvenile and control experiments. Geller and Mu¨ller (Geller and Mu¨ller, 1981) found that adult D. pulicaria had the highest filtering efficiency on particles ranging in size from 1.2 to 30 mm. This range of sizes would be included in our smallest size class, but our data also indicate the disappearance of slightly larger P. rubescens filaments in the presence of adults. This may be due to easier breakage of 50– 100 mm filaments during ingestion as compared to other filament lengths. DeMott

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(DeMott, 1995) suggested that along with particle size, particle hardness is an important factor in determining Daphnia feeding on large prey, and showed that Daphnia can ingest large particles with softer cell walls but not harder polystyrene beads of the same size. Although experimental evidence on size-differentiated grazing of filaments is somewhat rare, other studies have observed the disappearance of filaments .1 mm in length in the presence of copepods (DeMott and Moxter, 1991, in DeMott, 1995). Although the “sieving” model seems largely accepted (Geller and Mu¨ller, 1981; Lampert, 1987), the Daphnia feeding mechanism appears complex and difficult to observe (Gerritsen et al., 1988; Hartmann and Kunkel, 1991), and its exact treatment of filamentous phytoplankton seems as yet to be completely clarified. Whether due to size restrictions and rejection (Webster and Peters, 1978; Geller and Mu¨ller, 1981; Gliwicz and Lampert, 1990) or excessive handling time and poor interception of filaments (Hartmann and Kunkel, 1991), grazing of filaments by Daphnia can take place, as we have shown here, but appears to be accompanied by decreased feeding efficiency when compared with non-filamentous phytoplankton. In our experiments with P. agardhii, a decrease in the largest filaments was not accompanied by a significant increase in smaller ones, also possibly because of their breakage by adult Daphnia, followed by consumption of the smaller, broken filaments. This difference from measured removal of P. rubescens filament sizes might be due to several things. Among them could be subtle variations in hardness or filament structure, or possibly the larger initial filament sizes and concentrations of P. agardhii. This higher ratio between filaments and the number of Daphnia individuals per volume may have especially contributed to the mechanical disruption of very long filaments, making them smaller and easier to graze. In light of filament length measurements, our observations of slower grazing of Planktothrix than of S. acutus seems at least partly due to the filamentous structure of the cyanobacteria. Since individual filaments appear to grow in length before increasing in number (Humbert, unpublished data), efficient control of a Planktothrix bloom by Daphnia may only be possible in its early stages, before greater filament lengths render grazing inefficient. Kurmayer and Ju¨ttner (Kurmayer and Ju¨ttner, 1999) concluded that grazing resistance shown by P. rubescens was due to chemical defenses rather than large size and rigidity of filaments. Although our observations of preferential grazing of smaller filaments suggest that large filament size can potentially decrease grazing efficiency, the question of chemical defenses remains a very interesting one; it is possible that a

combination of these two factors reduces grazability of Planktothrix by zooplankton. In our experiments, comparable grazing of the mixture of S. acutus cells and P. rubescens filtrate with that of S. acutus alone suggested that extracellular chemical deterrents, provided still present in the filtrate, were not influential.

Transfer of MCYs to Daphnia and in the food chain We can compare our maximum mean value of 599 mg MCY-LR equivalents g DW21 to literature values found for zooplankton. Using the same assay, Vincent (Vincent, 2003) found 0.5– 2.5 mg MCY-LR equivalents g DW21 in zooplankton sampled from Lake Bourget, whereas Ferra˜o-Filho et al. (Ferra˜o-Filho et al., 2000), using ELISA, reported values of 0.3– 16.4 mg g DW21 in zooplankton sampled during a M. aeruginosa bloom. Thostrup and Christoffersen (Thostrup and Christoffersen, 1999) reported a value of 24.5 mg g DW21 in D. magna incubated in the laboratory with M. aeruginosa, whereas Ibelings et al. (Ibelings et al., 2005) found a maximum value of 1000 mg g21 ash free dry weight in zooplankton (910 mg g DW21, based on other conversions in the paper). Watanabe et al. (Watanabe et al., 1992) estimated up to 1387 mg g DW21 in lake communities of zooplankton, though these samples also contained Microcystis. Kotak et al. (Kotak et al., 1996) found a maximum value of 67 mg g biomass21 in zooplankton sampled in situ. Sivonen and Jones (Sivonen and Jones, 1999) reported the highest published concentrations of MCYs from cyanobacterial bloom samples to be 7300 mg g DW21 (measured by HPLC, from China and Portugal). Our values are somewhat high compared with most of these and may indicate the presence of filaments in our Daphnia samples, such as those still present in digestive apparatus or, as was observed in a few cases, entangled near the post-abdominal claw. The high quantities detected in our samples may otherwise be due to the differences in potential toxin uptake by zooplankton in laboratory cultures, with a high surrounding concentration of cyanobacteria, versus conditions in natural environments, where Daphnia may be able to selectively feed on other phytoplankton species or to avoid cyanobacteria blooms by vertical migration. Moreover, MCY accumulation in zooplankton should be expected to vary with MCY production by cyanobacteria—the latter seems to depend on growth rate (Briand et al., 2005) among other possible factors, but factors regulating the production of MCYs by cyanobacteria have yet to be completely elucidated. Microscope observation before freezing showed phytoplankton in the gut of most unstarved adult

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individuals, and MCYs present would show up in results from PP2A analysis. MCY levels present in Daphnia exposed to Planktothrix and starved 24 h prior to freezing were significantly lower than those found in unstarved animals, presumably due to emptier guts. Detection of MCYs in starved animals may be indicative of accumulation, but even after 24 h in cyanobacteria-free water, pigments could sometimes still be observed in the gut content. It is possible that because of the filamentous structure of Planktothrix, evacuation was delayed, and that MCYs in the remaining cells were detected in these samples. But the significantly smaller quantities detected in starved Daphnia compared with those in their unstarved counterparts highlight the important contribution of gut content to MCY concentrations measured in zooplankton. Studies whose goal is to measure toxin quantities in tissues alone while using filamentous cyanobacteria must therefore allow for increased gut emptying time, since gut content may have an impact on voiding (Mitra and Flynn, 2007). It was interesting to find that, although differences between adult and juvenile CRs were found to be significant, differences in their MCY levels were not. Similar MCY levels in juveniles, which did not significantly graze Planktothrix, may be due to direct filtering of dissolved MCYs out of the water, for as daphnids filter for oxygen, they may also ingest these molecules. Another possible explanation might be the adhesion of extracellular MCY to small particles such as bacteria, which were subsequently ingested by juveniles, increasing their MCY content despite lesser grazing of cyanobacteria. The positive detection of MCYs originating from Daphnia in our study supports the possibility of transfer of cyanotoxins in the food chain. This is plausible through predation by fish of Daphnia having ingested cyanobacteria, including the cyanobacteria and toxins still present in the Daphnia digestive tract. Presence of MCYs in the muscle tissue of fish (De Magalha˜es et al., 2001, 2003) and mussels (Williams et al., 1997) exposed to cyanobacteria has been documented, and in Lake Ammersee (Germany), the presence of Planktothrix sp. producing cyanotoxins had negative impacts on whitefish, which ingested Planktothrix filaments during blooms, showing blue discoloration of gut content and MCY-protein adducts in the liver (Ernst et al., 2001). Ibelings et al. (Ibelings et al., 2005) also found evidence for transfer of MCYs from Daphnia to fish, with higher concentrations found in obligate planktivores, which showed signs of liver damage but no evidence for biomagnification. Further implications for transfer of toxins in the food chain remain to be investigated in light of such results, as they show the potential deleterious

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effects of cyanotoxins on fish species and their considerable importance for the fishing industry, as well as for possible effects on piscivorous birds and humans. Grazing rates of Planktothrix by D. pulicaria are confirmed to be slower than those of the non-filamentous and non-toxic S. acutus. Yet smaller filaments do seem to be preferentially grazed, and successful control of blooms by predation may be possible in their early stages, when a Planktothrix population just starts to form. This possibility should depend on the timing of zooplankton and cyanobacteria population increases, and perhaps also on the mono-specificity of the phytoplankton community. Despite the fact that MCYs were detected in zooplankton, these had no observed acute lethal effect on D. pulicaria. Although we could not clearly conclude on the matter from our study, cyanotoxins may have contributed to slower Daphnia grazing on Planktothrix but did not prevent it. Therefore, in agreement with other studies on zooplankton – cyanobacteria interactions, we found that Daphnia could be an important vector in the transfer of MCYs in the food chain, with a transfer potential that appears greater just after ingestion of toxin-containing cyanobacteria.

AC K N OW L E D G E M E N T S We thank C. Avois-Jacquet (INRA) for her important role in the conception of this project, G. Me´thot (Univ. Montre´al) for valuable assistance in the laboratory and helpful discussions and C. Bernard and A. Ledreux at the Muse´e National d’Histoire Naturelle (Paris) for materials and assistance with PP-2A analyses. We further thank G. Paolini, C. Leboulanger, L. Vincent and E. Menthon (INRA, France) for technical assistance with algal cultures. We also acknowledge the collaboration of N. Milot, L. Pelletier, H. Lavigne, L. Titley and D. Be´langer for technical assistance during sampling, image analysis, algal cultures and fluorescence spectrophotometry at the Universite´ de Montre´al. We gratefully acknowledge the valuable comments of two anonymous reviewers, which significantly improved an earlier version of this manuscript.

FUNDING NSERC (Canada) (225 – 2005) to B.P.A.; Fonds France-Canada pour la Recherche and the INSU program ECODYN (France); French Ministe`re de l’Education Nationale, de l’Enseignement Supe´rieur et de la Recherche to L.O.

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Feuillade, J. (1994) The cyanobacterium (blue-green alga) Oscillatoria rubescens D. C. Arch. Hydrobiol. Beih. Ergebn. Limnol., 41, 77– 93.

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