The influence of resource limitation on the allelopathic effect of Chlamydomonas reinhardtii on other unicellular freshwater planktonic organisms

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1 JPR Advance Access published August 21, 2013 Journal of Plankton Research plankt.oxfordjournals.org J. Plankton Res. (2013) 0(0): 1 6. doi: /plankt/fbt080 SHORT COMMUNICATION The influence of resource limitation on the allelopathic effect of Chlamydomonas reinhardtii on other unicellular freshwater planktonic organisms ALDO BARREIRO * AND NELSON G. HAIRSTON JR DEPARTMENT OF ECOLOGY AND EVOLUTIONARY BIOLOGY, CORNELL UNIVERSITY, CORSON HALL, ITHACA 14850, NY, USA PRESENT ADDRESS: LABORATORY OF ECOTOXICOLOGY, GENOMICS AND EVOLUTION (LEGE). CIIMAR, PORTO. PORTUGAL. *CORRESPONDING AUTHOR: aldo.barreiro@gmail.com Received April 24, 2013; accepted July 29, 2013 Corresponding editor: Beatrix E. Beisner Because we found previously that Chlamydomonas reinhardtii produces allelochemicals to which the rotifer Brachionus calyciflorus is sensitive, we explored its effects on other freshwater plankton. We used Chlamydomonas under light-, nitrogen- and phosphorus-limitation to test its allelopathic effect on Microcystis aeruginosa, Cryptomonas ozolinii, Ochromonas danica, Tetrahymena thermophila and Paramecium aurelia. Allelopathy depended strongly on the target organism. Only Cryptomonas suffered a marked negative effect. Among the resource limiting regimes, light limitation exerted the greatest effect. KEYWORDS: allelopathy; resource limitation; phytoplankton; chlorophytes available online at # The Author Published by Oxford University Press. All rights reserved. For permissions, please journals.permissions@oup.com

2 JOURNAL OF PLANKTON RESEARCH j VOLUME 0 j NUMBER 0 j PAGES 1 6 j 2013 Although allelopathy by phytoplankton has been known since the middle of the twentieth century (Pratt and Fong, 1940; Lefèvre et al., 1950; Keating, 1977), interest in chemical interactions in planktonic communities increased recently because of their ecological importance (Legrand et al., 2003; Hulot and Huisman, 2004; Johnson et al., 2009), and potential importance for pharmacology (Borowitzka, 1995). Most allelopathic compounds are secondary metabolites (Legrand et al., 2003), which have been shown to be produced by a diversity of phytoplankton taxa including cyanobacteria, prymnesiophytes, dinoflagellates, diatoms and chlorophytes (see Legrand et al., 2003). Among chlorophytes, Proctor (Proctor, 1957)foundthat Chlamydomonas reinhardtii, a ubiquitous species in freshwater ecosystems, produced compounds inhibitory to other microalgae, later characterized as long chain fatty acids (McCrackan et al., 1979). It has further been shown that nutrient limitation in microalgae can affect the production of secondary metabolites, which can function either as allelochemicals (Johansson and Granéli, 1999; John and Flynn, 2000) or as toxins that suppress the growth of a rotifer consumer, as our own research has shown for C. reinhardtii (Barreiro and Hairston, 2013). Our goal in this study was to analyze the effect of resource-limited growth in C. reinhardtii (hereafter Chlamydomonas) on its allelopathic effect on other freshwater plankton of two functional groups: photosynthetic competitors and osmotrophic or phagotrophic consumers. Exploring these relationships is important for understanding their effects on ecological dynamics. Nutrients have a quantitative influence on phytoplankton population dynamics, but if the nutrient limiting growth also affects phytoplankton qualitatively by stimulating or inhibiting the production of secondary metabolites, this would complicate the relationship between nutrient and community dynamics by creating indirect bottom-up effects. Our specific aims were: (i) to establish, using chemostats, Chlamydomonas cultures at equilibrium under light-, nitrogen- or phosphorus-limitation, and (ii) to use cellfree filtrates from these cultures to test their effect on the growth of other microplanktonic taxa. We obtained C. reinhardtii from the University of Texas culture collection, (UTEX no. 89) and cultured it in 350 ml glass chemostats with constant air bubbling, 24 h light from daylight fluorescent lamps at saturating intensity ( 60 mmols photons m 21 s 21 ) and a dilution rate of 0.3 day 21. To induce light-limited growth, we supplied nitrate and phosphate at 3200 mm NO 3 2 and 200 mm PO 4 32 (N:P ¼ 16:1) so that chemostat cell densities were extremely high, markedly reducing light penetration into the cultures. To produce nitrogen-limited growth, we supplied 160 mm NO 3 2 and 200 mm PO 4 32 (N:P ¼ 0.8:1), and for phosphorus-limited growth, 3200 mm NO 3 2 and 10 mm PO 4 32 (N:P ¼ 320:1). The rest of the nutrient medium had constituents provided in excess; see Barreiro and Hairston (2013) for a list of components and concentrations. The ph of the chemostats (measured daily) was always,9, indicating CO 2 availability was never limiting. Chlamydomonas abundance and cell size were monitored daily for each chemostat ensuring that cultures were at steady state. Phytoplankton employed as targets for allelopathy, Microcystis aeruginosa (culture from MPIL; see Hairston et al., 2001), Cryptomonas ozolinii (UTEX no. 2782) and Ochromonas danica (Science Kit and Boreal Laboratories, Tonawanda, NY, USA), were cultured in batch with Bold s basal medium (Bold, 1949). For Ochromonas, peptone (0.2 g L 21 ) and yeast extract (0.05 g L 21 ) were added to the medium, because as a mixotroph it was not able to grow solely autotrophically. The protozoans employed as targets were Tetrahymena thermophila (ATCC no ) which can be fed exclusively osmotrophically (Curds and Cockburns, 1968) and was batch cultured with peptone (1 gl 21 )andyeast extract (0.5 g L 21 ), and Paramecium aurelia (Carolina Biological Supply) which consumes bacteria and detritus (Barna and Weis, 1973) and was batch cultured with autoclaved (heat-killed) cells of Cryptomonas. All taxa are hereafter referred to by their generic names (Tables I and II). Our experimental tests of allelopathy on phytoplankton were performed using 5 ml transparent polycarbonate vials. Each species was exposed to five different amounts of cell-free Chlamydomonas filtrate from each resource regime: 0, 5, 10, 25 and 50% of total vial volume. Cell-free filtrates were obtained by centrifugation of chemostat cultures followed by filtration of the supernatant through a cellulose acetate 0.2-mm filter (Cole-Parmer). The filtrate was diluted with sterile medium in order to establish a constant ratio of Chlamydomonas biovolume per cell-free filtrate volume in each treatment. This ratio was set to be that of the chemostat culture with the lowest biovolume density with cell-free filtrate from this chemostat used undiluted. The specific dilutions performed were 3.6 for the filtrate from the light-limited chemostat and 1.05 for the filtrate from the phosphorus limited treatment. In addition to cell-free filtrate, each experimental vial was supplied with culture medium with the same final nutrient concentrations (640 mmno 3 2 þ 200 mmpo 4 32 for Cryptomonas and Microcystis aeruginosa, and 1 g L 21 of peptone þ 0.5 g L 21 of yeast extract for Ochromonas) to ensure the absence of nutrient limitation during the experiment. The following numbers of cells were added to the corresponding treatments: , and cells ml 21 for Cryptomonas, Ochromonas and Microcystis, respectively, so that the total phytoplankton biovolume was approximately the same in each vial. To 2

3 A. BARREIRO AND N.G. HAIRSTON j INFLUENCE OF RESOURCE LIMITATION IN PHYTOPLANKTON ALLELOPATHY Table I: Results of linear regressions of growth rate as a function of the percentage of cell-free filtrate from Chlamydomonas cultured in three different nutrient regimes: light limitation (L), nitrogen limitation (N) and phosphorus limitation (P) ANOVA Organism and treatment F df total P-value Slope r 2 Cryptomonas ozolinii L , ; P, N ; P ¼ P , ; P, Microcystis aeruginosa L ; P ¼ N ; P ¼ P ; P ¼ Ochromonas danica L , ; P, N , ; P, P , ; P, Tetrahymena thermophila L , ; P, N ; P ¼ P P ¼ Paramecium aurelia L ; P ¼ N ; P ¼ P ; P ¼ Brachionus plicatilis L ; P ¼ N , ; P, P ; P ¼ Organism Table II: Comparison of the effects of Chlamydomonas reinhardtii cell-free filtrate on phytoplankton, protozoans and rotifers ANCOVA Tukey post hoc F df total P-value N-L P-L P-N Cryptomonas ozolinii P ¼ 0.78 P ¼ 0.85 P ¼ 0.46 Microcystis aeruginosa P ¼ 0.06 P ¼ 0.39 P ¼ 0.59 Ochromonas danica P ¼ 0.10 P ¼ 0.13 P ¼ 0.99 Tetrahymena thermophila ,0.001 P, P ¼ P ¼ 0.13 Paramecium aurelia P ¼ 0.54 P ¼ 0.69 P ¼ 0.97 Brachionus plicatilis ,0.001 P, P ¼ 0.02 P ¼ 0.65 Results of the linear model and post hoc comparisons performed using target species growth rate as the dependent variable, log % of filtrate as the covariate and limiting resource as the factor: light limitation (L), nitrogen limitation (N) and phosphorus limitation (P). adjust final vial volume to 5 ml, we used sterile medium without macronutrients. Triplicate vials were run for each cell-free filtrate concentration for each phytoplankton species. Vials were placed in controlled environment chambers with temperature and light regimes the same as for the chemostat cultures. Initial cell counts were made using samples from five randomly chosen vials. Final samples were taken from each vial after 24 h. Samples consisted of small aliquots from experimental vials and were counted using Neubauer chambers for the higher abundances or Sedgewick-Rafter chambers for the lower abundances. Experiments were not run simultaneously for all target species. Cryptomonas and Ochromonas cells were easily disrupted by fixatives, so samples for these species were preserved with highly dilute Lugol s iodine solution (a small drop from a Pasteur pipette in the 5 ml vials) and counted within 6 h of preservation. Microcystis cells could be preserved with Lugol s solution and were counted within several days of preservation. The strain employed here grows as single cells with a mucilage cover (Hairston et al., 2001). Our experimental tests of allelopathy on Tetrahymena and Paramecium were run using 12-well tissue culture plates, each well filled with 3 ml of test medium. 3

4 JOURNAL OF PLANKTON RESEARCH j VOLUME 0 j NUMBER 0 j PAGES 1 6 j 2013 Cell-free filtrate was obtained as for the phytoplankton experiments. Food for Tetrahymena was provided at a final concentration of 1 g L 21 of peptone þ 0.5 g L 21 of yeast extract. Food for Paramecium was 0.5 ml of autoclaved (heat killed) Cryptomonas. The protozoans were introduced to each well by pipetting in either 100 ml oftetrahymena or 0.5 ml of Paramecium batch culture, final volume adjusted to 3 ml with sterile mineral medium. Tissue culture plates were maintained at the same temperature and light conditions as the chemostat cultures. Initial samples from each species were counted for five random replicates. Final abundances of Tetrahymena were counted after 24 h and Paramecium after 48 h. To compare these results with others obtained for the rotifer Brachionus plicatilis, we include here data from an experiment described in Barreiro and Hairston (Barreiro and Hairston, 2013). The protocol of this experiment differed substantially from the current one, and consisted of 24 h rotifer growth rate estimates (as N t N 21 t2 1 ) during 8 days under constant food conditions, with constant initial rotifer densities (see Barreiro and Hairston, 2013). Chlamydomonas chemostat growth differed by nutrient regime. Under light limitation steady state was reached after 8 days and at a density cells ml 21. Nitrogen-limited cells reached steady state after 7 days with a density overshoot before stabilizing at cells ml 21. Phosphorus-limited cells took 12 days to reach steady state cells ml 21 with an overshoot more prolonged than that under nitrogen limitation. Cell sizes (radius in mm + SD) and their corresponding biovolumes (in mm 3, assuming spherical cells) at steady state differed among treatments: mm; 48 mm 3 for light-limited, mm; 333 mm 3 for nitrogen-limited and mm; 697 mm 3 for phosphorus-limited chemostats. The effects of cell-free filtrate differed markedly among test organisms. The only phytoplankton species that showed negative growth rates was Cryptomonas, which was significantly affected by filtrate from light- and phosphorus-limited Chlamydomonas, and marginally significantly affected by nitrogen-limited filtrate (Fig. 1; Table I). Although the negative effect appeared to be strongest for filtrate from light-limited Chlamydomonas, there were no significant differences among treatments (ANCOVA; Table II). Microcystis was not affected at all by filtrate (Fig. 1): regressions were not statistically significant, and slopes did not differ from 0 (Table I) or form each other (ANCOVA; Table II). The results for Ochromonas were qualitatively different from the other two phytoplankton taxa with cell-free Chlamydomonas filtrate having a positive effect on population growth rate (Fig. 1). Data fitted by linear regressions (after log-transforming the independent variable) (Table I) showed no statistically significant differences among treatments for this species (Table II). Of the protozoans, only Tetrahymena was affected, and only in the light-limited cell-free filtrate treatment (Fig. 1; Table I): this treatment differed significantly from the other two (ANCOVA; Table II). In this treatment, concentrations of 10 and 25% filtrate showed larger deviations due to the fact that some replicates had negative growth and others positive growth, which results in a high variation in the values of r due to the different rates at which growth and death occur. Paramecium was unaffected by any of the treatments (Fig. 1; Tables I and II). The allelopathic effect of Chlamydomonas exudate differed markedly both among target species and as a function of Chlamydomonas culture conditions. The sensitivity of target species is likely to depend upon morphological characteristics such as cell-wall thickness or presence of mucilage in phytoplankton, or the proteinaceous cover of protozoans. In addition, the mode of target cell nutrition may play an important role: osmotrophs might take up organic allelochemicals more readily than autotrophs or detritivores. For example, the mucilaginous matrix surrounding Microcystis may explain its lower sensitivity to allelochemicals in comparison with Cryptomonas, which lacks mucilage. Ochromonas, also a naked flagellate, is an obligate osmotroph (Pringsheim, 1952), and presumably able to use organic allelochemicals as a resource. In addition, because Ochromonas produces its own toxic allelochemicals (Hiltunen et al., 2012), it may simply be resistant to those produced by other phytoplankton. Similarly, the different effects Chlamydomonas allelochemicals on the protozoans may result from their distinct modes of nutrition. Tetrahymena, both an osmotroph and bacterivore (Curds and Cockburns, 1968), was more sensitive than Paramecium which consumes primarily larger particles, though is also somewhat osmotrophic (Wichterman, 1986). Our results emphasize the importance of both the source species and the target organism when assessing ecological effects of allelochemicals. The range of outcomes we observed in a small subset of target species covers essentially all possible effects: strongly negative (Cryptomonas), intermediately negative (Tetrahymena), no effect (Microcystis and Paramecium) and positive (at least of the cell-free filtrate for Ochromonas). We know of no previous study showing an effect of resource limitation on the production of allelopathic compounds by Chlamydomonas, though we previously showed that nitrogen-limited growth can result in a level of toxicity to rotifers comparable with that which we document here for Cryptomonas (Barreiro and Hairston, 2013). The negative effect on rotifer population growth occurs both when they are fed nitrogen-limited cells and when exposed to cell-free exudate (Fig. 1). 4

5 A. BARREIRO AND N.G. HAIRSTON j INFLUENCE OF RESOURCE LIMITATION IN PHYTOPLANKTON ALLELOPATHY Fig. 1. Effect of Chlamydomonas reinhardtii cell-free filtrate on the population growth rate (+1 SD) of the species under study. The fitted line for O. danica in logarithmic while the statistics presented in Tables I and II are for linear fits. Some data points were slightly shifted in the OX axis in order to avoid overlay. The plot of Brachionus plicatilis results was modified from Barreiro and Hairston (Barreiro and Hairston, 2013). We found distinct effects of nutrient-limitation treatment on the two sensitive species: Cryptomonas was negatively affected by cell-free Chlamydomonas filtrate from all three resource-limitation regimes (Tables I and II), whereas Tetrahymena was only affected by filtrate from the light-limited treatment (Tables I and II; Fig. 1). Based on our experimental design, there are two possible explanations for this observed stronger effect of light-limited cellfree filtrate. Light limitation may have either induced higher per-cell allelochemical production compared with the other treatments, or distinct secondary metabolites may have been produced in this treatment, or both. However, another less interesting possibility is that the density of Chlamydomonas cells was greater in light-limited chemostats since light limitation was created by growing the alga at very high nutrient concentrations. Although we diluted cell-free filtrate to provide the same ratio of Chlamydomonas biovolume per target cell, it might have been that the rate of allelochemical production was density dependent so that standardizing the treatment for cell biovolume might not have been fully effective. Although we did not address this aspect of density dependence in our experimental protocol, we can speculate that it is not likely to have been important. The only treatments that showed an allelopathic effect in any of the target taxa were those with light limitation (C. ozolinii, T. termophila) and phosphorus limitation (C. ozolonii). Because phosphorus limitation showed significantly lower cell densities at steady state (see above), a densitydependent effect is not suggested by our data. We note, however, that our experimental protocol was not designed to test this hypothesis. It might also be hypothetically possible that bacterial degradation could affect allelochemicals in the chemostats. Our bacterial counts during steady state showed bacterial abundances of the same order of magnitude in all chemostats: L-limited: cells ml 21, N-limited: cells ml 21 and P-limited: cells ml 21 (n ¼ 4 in all cases). These bacterial abundances correspond to very low biomass in relation to that of microalgae, and would seem to indicate that they would have had little degradation effect. However, because biomass is not a direct measure of activity, this aspect should remain as an open question. 5

6 JOURNAL OF PLANKTON RESEARCH j VOLUME 0 j NUMBER 0 j PAGES 1 6 j 2013 The allelochemicals in our study were produced in cultures without the presence of other competing species. This approach is the most common way of testing for the production of allelochemicals (Legrand et al., 2003), but it is possible that the anti-competitor inhibitory effects of these compounds might be secondary and so result from selection for competitive ability. Several studies have shown an effect of resource limitation on production of secondary metabolites with allelochemical properties (Von Elert and Jüttner, 1997; Johansson and Granéli, 1999; John and Flynn, 2000; Barreiro et al., 2005), and we find similar results for Chlamydomonas. Whether secondary metabolites excreted by phytoplankton could have ecological consequences in lakes has been discussed by Johnson and Pavia (Johnson et al., 2009), Hiltunen et al. (Hiltunen et al., 2012) and Barreiro and Hairston (Barreiro and Hairston, 2013). If these chemicals do affect ecology, resource limitation will be an interesting and important interacting effect. ACKNOWLEDGEMENTS We are grateful to Colleen M. Kearns for technical assistance. FUNDING This work was funded by a grant from the James S. McDonnell Foundation to Stephen Ellner and N.G.H. and a postdoctoral fellowship from the Spanish Science and Technology Foundation Fulbright Commission to A.B. REFERENCES Barna, I. and Weis, D. S. (1973) The utilization of bacteria as food for Paramecium bursaria. Trans. Amer. Microb. Soc., 92, Barreiro, A., Guisande, C., Maneiro, I. et al. (2005) Relative importance of the different negative effects of the toxic haptophyte Prymnesium parvum on Rhodomonas salina and Brachionus plicatilis. Aquat. Microb. Ecol., 38, Barreiro, A. and Hairston, N. G. Jr. (2013) Indirect bottom-up control of consumer resource dynamics: Resource-driven algal quality alters grazer numerical response. Limnol. Oceanogr., 58, Bold, H. C. (1949) The morphology of Chlamydomonas chlamydogama sp. nov. Bull. Torrey Bot. Club., 76, Borowitzka, M. A. (1995) Microalgae as sources of pharmaceutical and other biologically active compounds. J. Appl. Phycol., 7, Curds, C. R. and Cockburns, A. (1968) Studies on the growth and feeding of Tetrahymena pyriformis in axenic and monoxenic culture. J. Gen. Microbiol., 54, Hairston, N. G. Jr, Holtmeier, C. L., Lampert, W. et al. (2001) Natural selection for grazer resistance to toxic cyanobacteria: evolution of phenotypic plasticity? Evolution, 55, Hiltunen, T. J., Barreiro, A. and Hairston, N. G. Jr. (2012) Mixotrophy and the toxicity of Ochromonas in a pelagic food web. Freshwater Biol., 57, Hulot, F. D. and Huisman, J. (2004) Allelopathic interactions between phytoplankton species: the roles of heterotrophic bacteria and mixing intensity. Limnol. Oceanogr., 49, Johansson, N. and Granéli, E. (1999) Influence of different nutrient conditions on cell density, chemical composition and toxicity of Prymnesium parvum (Haptophyta) in semi-continuous cultures. J. Exp. Mar. Biol. Ecol., 239, John, E. H. and Flynn, K. J. (2000) Growth dynamics and toxicity of Alexandrium fundyense (Dinophyceae): the effect of changing N:P supply ratios on internal toxin and nutrient levels. Eur. J. Phycol., 35, Johnson, P. R., Pavia, H. and Toth, G. (2009) Formation of harmful algal blooms cannot be explained by allelopathic interactions. Proc. Nat. Acad. Sci. USA, 106, Keating, K. I. (1977) Allelopathic influence on blue-green bloom sequence in a eutrophic lake. Science, 296, Lefèvre, M., Jakob, H. and Nisbet, M. (1950) Sur la sécrétion, par certaines Cyanophytes, de substances algostatiques dans les collections d eau naturelles. C.R. Acad. Sci., 230, Legrand, C., Rengefors, K., Fistarol, G. O. et al. (2003) Allelopathy in phytoplankton biochemical, ecological and evolutionary aspects. Phycologia., 42, McCrackan, M. D., Middaugh, R. E. and Middaugh, R. S. (1979) A chemical characterisation of an algal inhibitor obtained from Chlamydomonas. Hydrobiologia, 70, Pratt, D. M. and Fong, J. (1940) Studies on Chlorella vulgaris II. Further evidence that Chlorella cells form a growth: inhibiting substance. Am. J. Botany, 27, Pringsheim, E. G. (1952) On the nutrition of Ochromonas. Quart. J. Microscop. Sci., 93, Proctor, V. W. (1957) Studies of algal antibiosis using Haematococcus and Chlamydomonas. Limnol. Oceanogr., 2, Von Elert, E. and Jüttner, F. (1997) Phosphorus limitation and not light controls the extracellular release of allelopathic compounds by Trichormus doliolum (Cyanobacteria). Limnol. Oceanogr., 42, Wichterman, R. (1986) The Biology of Paramecium. Plenum Press, New York. 6