TY - JOUR
AU1 - Lin, Chih-Hsien, (Michelle)
AU2 - Glibert, Patricia, M
AB - Abstract Grazing studies were conducted on the mixotrophic dinoflagellate, Karlodinium veneficum, in the presence of single prey species, the cryptophyte Rhodomonas salina, and a phycocyanin-containing strain of the cyanobacterium, Synechococcus sp., and when prey were mixed in varying proportions. A multiwavelength PAM fluorometer was used for non-invasive biomass estimates and to detect changes in photophysiology. Growth and grazing rates of K. veneficum increased as the function of increasing prey concentrations of R. salina, regardless of whether the prey were provided as a single prey item or in a mixed prey assemblages. With Synechococcus as the prey, in single or mixed prey assemblages, it was poorly grazed and growth rates of the mixotroph declined. The maximal quantum yields of PSII fluorescence (Fv/Fm) of the mixotroph declined when Synechococcus was the prey but remained high when fed on R. salina. The Fv/Fm of R. salina declined by about 60% as it was consumed, while that of Synechococcus sp. increased as mixotroph growth declined. Robust relationships were established between flow cytometry-based cell counts and PAM fluorometry-based chlorophyll measurements, validating the usefulness of this rapid and non-intrusive quantification approach for measuring mixotrophy in predator–prey interactions among multiple differently pigmented species. INTRODUCTION Mixotrophic nutrition (i.e. the combination of autotrophy and phagotrophy) is widespread among many photosynthetic algal species, in particular, phototrophic chrysophytes, dinoflagellates and haptophytes (Jeong et al., 2005a; 2005b; Burkholder et al., 2008; Flynn et al., 2013). Mixotrophs are known to play important roles in plankton dynamics (Stickney et al., 2000; Tittel et al., 2003) and both experimental and numerical studies have indicated that there are growth advantages to being mixotrophic in dynamic environmental conditions (Sanders, 1991; Stoecker, 1999; Jeong et al., 2005a; Glibert et al., 2009; Stoecker et al., 2017). For instance, planktonic mixotrophy has been found in oligotrophic habitats where limiting nutrients are often concentrated in microbial prey compared to the water column (Jones, 1994) and eutrophic estuaries where light can be limiting and/or where nutrients are sufficient but not in balanced proportions (Burkholder et al., 2008; Glibert and Burford, 2017; Millette et al., 2017). Phagotrophy in algae can provide an alternative or supplement to photosynthesis as sources of carbon (C), or dissolved substrates, nitrogen (N) and/or phosphorus (P), and through the acquisition of food, particularly high-quality food, mixotrophs can enhance their growth rates relative to their autotrophic growth (Li et al., 2000; Jeong et al., 2005a; 2005b; Adolf et al., 2008; Glibert et al., 2009; Lin et al., 2017). There are several approaches for the measurement of mixotrophy and most of these involve manipulation with a tracer. Prey may be labeled using an isotope (14C, 15N), or artificial prey (for example, beads) may be added and over time the accumulation of tracer in the mixotroph is measured (e.g. Hall et al., 1993; Smalley et al., 1999; Adolf et al., 2006; Lundgren et al., 2016). Alternatively, enumerations of changes in predator and/or prey may be made through microscopy or other enumeration approaches (Carvalho and Granéli, 2006; Lin et al., 2017). Tracer labeling techniques and/or feeding trials with artificial substrates (beads) may lead to artifacts due to various manipulations or artificial food required. Pulse-amplitude-modulated (PAM) fluorescence is a widely used tool for determining the maximum quantum efficiency of photosystem (PS) II fluorescence (Fv/Fm), a commonly reported measure of phytoplankton physiological state (Kolber et al., 1988; Geider et al., 1993; Goto et al., 2008). The Phyto-PAM II (Walz, Germany) incorporates multiple modulating beams set to different excitation spectra and therefore can measure different groups of phytoplankton simultaneously in the same solution. With appropriate calibration and deconvolution of signals, the dark-adapted, minimal fluorescent signal (F0) can also be used as a measure of biomass (chlorophyll) and thus this instrument allows the simultaneous measurement of the abundance of different phytoplankton groups in a mixed sample. Given the spectral composition of the five available excitation wavelengths, differentiation of phytoplankton groups is optimal between cyanobacteria, green algae, diatoms/dinoflagellates and phycoerythrin-containing cells such as cryptophytes. Here, the Phyto-PAM II was used to characterize the change in abundance of a mixotroph, the dinoflagellate Karlodinium veneficum, in the presence of prey, including the cryptophyte, Rhodomonas salina, and a phycocyanin-containing cyanobacterium, Synechococcus sp., that is also a prey of the cryptophyte (e.g. Urabe et al., 2000; Izaguirre et al., 2012). The mechanisms for feeding on cryptophytes by K. veneficum have been extensively examined in the field and laboratory (Li et al., 2000; Adolf et al., 2008), while some Synechococcus have been found to be readily grazed by other gymnodinoid dinoflagellates (Jeong et al., 2005a; Glibert et al., 2009). This study addressed the hypothesis that feeding by the mixotroph will differ depending on the type of prey provided, and that rates of grazing on individual prey—when provided together with other prey sources—may differ relative to rates when only single prey species are available due to the combination of altered prey concentrations and interactions among prey species. In addition to assessing rates of mixotrophy and physiological state of this important dinoflagellate in the presence of multiple prey species and in different proportions, a secondary goal of this effort was to demonstrate the effectiveness of this variable fluorescence approach. METHODS Phytoplankton cultures Non-axenic cultures of the dinoflagellate Karlodinium veneficum (CCMP1975) and the cryptophyte Rhodomonas salina, provided by the National Center for Marine Algae and Microbiota and the Oyster Hatchery of Horn Point Laboratory, respectively, were grown in f/2 media (Guillard, 1975) with a light intensity of ~184 μmol photons m−2 s−1. A Chesapeake isolate, CB0101, of a phycocyanin (PC)-rich cyanobacteria Synechococcus sp., was obtained from Dr Feng Chen at University of Maryland Institute of Marine and Environmental Technology. It was subsequently grown in SN media (Waterbury et al., 1986) using the same light conditions as the other cultures. All culture media were prepared with 0.2 μm filtered Choptank River (a tributary of Chesapeake Bay) water (salinity of 10) and sterilized by autoclaving. All three species were maintained in batch cultures at 22°C on a 12 h light: 12 h dark cycle. Mixed-culture experimental design Multiple, mixed-culture experiments were conducted to examine the feeding and growth responses of K. veneficum to two different types of prey in different proportions. Two-species mixtures with K. veneficum (as a predator) and R. salina or Synechococcus sp. (as prey) were conducted in 250 mL flasks. Experimental cultures were derived from stock cultures of individual taxa in the same physiological state and were incubated under the same environmental conditions (see “Phytoplankton cultures” above). For convenience, the experimental treatments are referred to herein by culture volume: volume ratios (see Table I for cell concentrations). For the two-species mixtures, experiments were conducted at predator-to-prey ratios of 3:1, 2:1, 1:1 and 1:2, and for three-species mixtures experiments were conducted at predator-to-prey ratios of 3:1:1, 2:1:1, 1:1:1 and 1:2:1 for K. veneficum, R. salina, and Synechococcus, respectively. When cultures with Synechococcus were combined with other taxa, this necessitated a mixture of different culture media. Together with controls of individual species for each set of mixtures, this study thus consisted of 19 treatments, in triplicate, with a total of 57 culture flasks. The mixed-cultured treatments were sampled at 12 h intervals over 96 h, with each sampling period being 1–2 h after lights on/lights off. During the incubation period, the cells were frequently mixed to maintain a homogeneous cell suspension in culture flasks. In order to validate the reproducibility of this method, some treatments were replicated in a separate experiment (see Table I) and incubated over 72 h. Table I: Summary of cell conversion based on culture volume: volume ratios in mixed culture experiments Species composition Treatments Initial cell densities (×108 cell L−1) R. salina: K. veneficum Synechococcus: K. veneficum Synechococcus: R. salina K. veneficum R. salina Synechococcus cell:cell ratio cell:cell ratio cell:cell ratio Exp1 Exp2 Exp1 Exp2 Exp1 Exp2 Exp1 Exp2 Exp1 Exp2 Exp1 Exp2 Monoculture Control 1.5 0.7 9.7 7.5 419 469 Two-species mixtures: 1:2 0.5 0.2 8.1 4.0 15.4 16.0 Kv:Rs ratio 1:1 0.7 0.4 6.3 4.0 8.7 10.5 2:1 0.7 0.5 3.4 2.3 4.9 4.2 3:1 0.8 2.4 3.1 Two-species mixtures: 1:2 0.1 0.1 337 467 2903 3720 Kv:Syn ratio 1:1 0.3 0.3 285 455 1110 1445 2:1 0.4 0.4 201 346 478 855 3:1 0.5 142 300 Two-species mixtures: 1:2 3.7 2.9 326 500 88 173 Rs:Syn ratio 1:1 5.7 4.6 344 425 60 92 2:1 6.8 5.0 173 307 25 61 3:1 8.5 171 20 Three-species mixtures: 1:2:1 0.2 0.1 3.8 2.0 136 256 15.9 14.2 566 1800 36 127 Kv:Rs:Syn ratio 1:1:1 0.3 0.2 2.9 2.6 164 355 10.3 10.6 588 1462 57 138 2:1:1 0.4 0.3 1.7 1.4 136 251 4.8 5.5 379 956 79 174 3:1:1 0.5 1.8 135 3.5 265 76 Species composition Treatments Initial cell densities (×108 cell L−1) R. salina: K. veneficum Synechococcus: K. veneficum Synechococcus: R. salina K. veneficum R. salina Synechococcus cell:cell ratio cell:cell ratio cell:cell ratio Exp1 Exp2 Exp1 Exp2 Exp1 Exp2 Exp1 Exp2 Exp1 Exp2 Exp1 Exp2 Monoculture Control 1.5 0.7 9.7 7.5 419 469 Two-species mixtures: 1:2 0.5 0.2 8.1 4.0 15.4 16.0 Kv:Rs ratio 1:1 0.7 0.4 6.3 4.0 8.7 10.5 2:1 0.7 0.5 3.4 2.3 4.9 4.2 3:1 0.8 2.4 3.1 Two-species mixtures: 1:2 0.1 0.1 337 467 2903 3720 Kv:Syn ratio 1:1 0.3 0.3 285 455 1110 1445 2:1 0.4 0.4 201 346 478 855 3:1 0.5 142 300 Two-species mixtures: 1:2 3.7 2.9 326 500 88 173 Rs:Syn ratio 1:1 5.7 4.6 344 425 60 92 2:1 6.8 5.0 173 307 25 61 3:1 8.5 171 20 Three-species mixtures: 1:2:1 0.2 0.1 3.8 2.0 136 256 15.9 14.2 566 1800 36 127 Kv:Rs:Syn ratio 1:1:1 0.3 0.2 2.9 2.6 164 355 10.3 10.6 588 1462 57 138 2:1:1 0.4 0.3 1.7 1.4 136 251 4.8 5.5 379 956 79 174 3:1:1 0.5 1.8 135 3.5 265 76 All the culture flasks were derived from the stock cultures of individual taxa at the same physiological status. n = 3. Exp1 represents the original experimental set and Exp2 is the repeated experimental set. Table I: Summary of cell conversion based on culture volume: volume ratios in mixed culture experiments Species composition Treatments Initial cell densities (×108 cell L−1) R. salina: K. veneficum Synechococcus: K. veneficum Synechococcus: R. salina K. veneficum R. salina Synechococcus cell:cell ratio cell:cell ratio cell:cell ratio Exp1 Exp2 Exp1 Exp2 Exp1 Exp2 Exp1 Exp2 Exp1 Exp2 Exp1 Exp2 Monoculture Control 1.5 0.7 9.7 7.5 419 469 Two-species mixtures: 1:2 0.5 0.2 8.1 4.0 15.4 16.0 Kv:Rs ratio 1:1 0.7 0.4 6.3 4.0 8.7 10.5 2:1 0.7 0.5 3.4 2.3 4.9 4.2 3:1 0.8 2.4 3.1 Two-species mixtures: 1:2 0.1 0.1 337 467 2903 3720 Kv:Syn ratio 1:1 0.3 0.3 285 455 1110 1445 2:1 0.4 0.4 201 346 478 855 3:1 0.5 142 300 Two-species mixtures: 1:2 3.7 2.9 326 500 88 173 Rs:Syn ratio 1:1 5.7 4.6 344 425 60 92 2:1 6.8 5.0 173 307 25 61 3:1 8.5 171 20 Three-species mixtures: 1:2:1 0.2 0.1 3.8 2.0 136 256 15.9 14.2 566 1800 36 127 Kv:Rs:Syn ratio 1:1:1 0.3 0.2 2.9 2.6 164 355 10.3 10.6 588 1462 57 138 2:1:1 0.4 0.3 1.7 1.4 136 251 4.8 5.5 379 956 79 174 3:1:1 0.5 1.8 135 3.5 265 76 Species composition Treatments Initial cell densities (×108 cell L−1) R. salina: K. veneficum Synechococcus: K. veneficum Synechococcus: R. salina K. veneficum R. salina Synechococcus cell:cell ratio cell:cell ratio cell:cell ratio Exp1 Exp2 Exp1 Exp2 Exp1 Exp2 Exp1 Exp2 Exp1 Exp2 Exp1 Exp2 Monoculture Control 1.5 0.7 9.7 7.5 419 469 Two-species mixtures: 1:2 0.5 0.2 8.1 4.0 15.4 16.0 Kv:Rs ratio 1:1 0.7 0.4 6.3 4.0 8.7 10.5 2:1 0.7 0.5 3.4 2.3 4.9 4.2 3:1 0.8 2.4 3.1 Two-species mixtures: 1:2 0.1 0.1 337 467 2903 3720 Kv:Syn ratio 1:1 0.3 0.3 285 455 1110 1445 2:1 0.4 0.4 201 346 478 855 3:1 0.5 142 300 Two-species mixtures: 1:2 3.7 2.9 326 500 88 173 Rs:Syn ratio 1:1 5.7 4.6 344 425 60 92 2:1 6.8 5.0 173 307 25 61 3:1 8.5 171 20 Three-species mixtures: 1:2:1 0.2 0.1 3.8 2.0 136 256 15.9 14.2 566 1800 36 127 Kv:Rs:Syn ratio 1:1:1 0.3 0.2 2.9 2.6 164 355 10.3 10.6 588 1462 57 138 2:1:1 0.4 0.3 1.7 1.4 136 251 4.8 5.5 379 956 79 174 3:1:1 0.5 1.8 135 3.5 265 76 All the culture flasks were derived from the stock cultures of individual taxa at the same physiological status. n = 3. Exp1 represents the original experimental set and Exp2 is the repeated experimental set. For each sampling, flasks were gently mixed, and 3 mL aliquots were withdrawn and analyzed, after 30 min of dark adaptation, with the Phyto-PAM II to yield minimal fluorescence (F0) and maximal fluorescence (Fm) for each taxa. Taxon-specific variable fluorescence (Fv = Fm − F0) and its yield (Fv/Fm) parameters were also obtained. At each time point, samples (0.5 mL) were also preserved in 1% paraformaldehyde and held at 4°C for later cell enumeration via flow cytometry. Chlorophyll a fluorescence validation/calibration The F0-based chlorophyll (Chl) a concentrations were validated with the acetone-extraction method using single, and two- and three-species in mixed cultures in preliminary trials. Firstly, the acetone-based Chl a measurements were used to establish the reference of each algal strain for the purpose of instrument calibration. Secondly, mixtures of each algal group were made in 1:1 and/or 1:1:1 stock volume proportions. While fully aware of the differences in cell size, and potential for Chl a:cell and relative contribution of Chl a to vary in different taxa in those mixtures, as a first order assumption for cultures in the same state of growth, 1:1 mixtures were presumed to reflect 50% of the Chl a from each algal group, while those in the 1:1:1 mixtures were presumed to reflect 33% of the Chl a from each species. Finally, filtered Choptank River water was added to dilute the single- and mixed-culture stock cultures by 80%, 60%, 40% and 20% prior to extraction to obtain a concentration gradient. Samples were extracted with acetone for 12 h at 4°C following the method of Arar and Collins (1997) and measured with a calibrated Turner Designs AU-10 fluorometer. Flow cytometry comparison/validation The F0-based Chl a concentrations of the mixed species cultures were also compared with flow cytometry-based cell counts using combinations of two- and three-species (see Mixed-culture experimental design section). The preserved samples collected at each time point were analyzed within a week using a BD Accuri C6 flow cytometry with dual excitation: 488 nm (blue laser) and 640 nm (red laser). The cells were identified and gated based on their size, shapes and structural complexity using forward scatter (FSC) and side scatter (SSC) light threshold. In addition, four photomultipliers including FL1 (533/30 nm; green fluorescence), FL2 (585/40 nm; phycoerythrin, PE fluorescence) and red fluorescence (FL3 > 670 nm and FL4 at 675/25 nm) filters were applied for the direct identification of different taxa. Absolute cell counts were determined by volume flow rates, which were fixed at 14 μL min−1. The bi-plots of FL3-H vs. SSC allowed for the optimal gating design and the absolute estimation of the three pigmented algal taxa (Supplementary Fig. 1). The accuracy of flow cytometric counts relative to microscopy for the mixed-culture samples was previously evaluated (Lin et al., 2017). Those results indicated that estimates of flow cytometry are generally robust for these algal species (i.e. K. veneficum r2 = 0.74, R. salina r2 = 0.81). Photosynthetic activities To assess physiological states of K. veneficum and its prey, Fv/Fm values were measured using the Phyto-PAM II. Fluorescence signals from five color wavelengths (440, 480, 540, 590 and 625 nm) were deconvoluted to the three algal groups by calibrating Chl a fluorescence for each taxa (Schreiber, 1998). Calculations and statistical analyses In separate calculations, changes in taxon-specific biomass based on PAM-derived F0 values, calibrated with extracted Chl a, and those derived by flow cytometry, were used to calculate the growth of the mixotroph and death rates of its prey. Cell-specific growth rates (d−1) of K. veneficum were determined based on the slopes of the regression of natural log-transformed biomass data for the experimental periods (72 or 96 h). Cell-specific death rates of R. salina (Rs cells K. veneficum−1 d−1) were calculated as the difference between growth rates of prey in the control and mixed-cultures flasks, based on the equations of Frost (1972) and Heinbokel (1978) to account for grazer growth. Death rates of the prey are reported rather than ingestion rates because of the difficulty in differentiating between cells that were actually grazed and those cells that may have burst due to putative toxic effects (e.g. Lin et al., 2017). Low grazing rates on Synechococcus precluded the comparable calculation of death rates of this prey species. All statistical analyses were performed with R. The Shapiro-Wilks test and Q-Q plot were used to check for, and to visualize, normality of data presented in regression analyses, while the Levene’s test was used to assess the equality of variance. Comparisons of the methods of measurement for Chl a (PAM fluorometry and acetone extraction), and rates of growth and death determined by Chl a fluorescence from PAM fluorometry and cell density by flow cytometry were conducted using coefficient of determination, r2. Analysis of covariance (ANCOVA) was used to compare the statistical differences in slopes of regression of natural log-transformed data among the variables measured in different treatments for predator growth conditions, as well as slopes of the regression line of Chl a fluorescence vs. cell density measurements between the two experiment sets. Correlations between two variables were estimated by the Pearson’s product moment coefficients. The significance of the differences of two correlations that were determined by Chl a fluorescence from PAM fluorometry and cell density by flow cytometry were assessed using Fisher r-to-z transformation. Differences in cell-specific growth rates of K. veneficum, as well as death rates of prey and Fv/Fm of individual species (e.g. predator and two-prey species) in the monocultures and two- and three-species mixed cultures, were analyzed using ANOVA tests followed by Tukey’s HSD tests for pairwise comparisons. RESULTS Calibration and verification of chlorophyll a Values of Chl a based on F0 (hereafter ChlF0) agreed well with those of extracted Chl a. The coefficient of determination between the combined values for all treatments was 0.83 (P < 0.001, n = 35), but the slope did deviate from 1.0 and there was a positive intercept (y = 0.68x + 37.01; Table II, Fig. 1). Total Chl a concentrations up to ~220 μg L−1 for single K. veneficum and R. salina algal cultures were reliably determined by ChlF0. The monoculture of Synechococcus sp. also yielded a sufficiently reliable determination using F0, but total Chl a concentrations were at the lower end of the calibration curve, 120 μg L−1. Values of ChlF0 in two or three species mixed-cultures deviated from the 1:1 line at Chl a concentrations >200 μg L−1 and <20 μg L−1 (Fig. 1). When treatments were compared independently, all seven-treatment conditions yielded comparable estimates between the two methods with slope values of ~0.8, except for the two-species mixed cultures that included Synechococcus sp. (Table II). In two-species mixtures, an approximately equal amount of total Chl a was attributed to K. veneficum (52 ± 3%) and R. salina (48 ± 3%). Synechococcus sp. was generally underestimated when mixed with K. veneficum and/or R. salina. When mixed with K. veneficum, it accounted for 41 ± 3% of total Chl a and K. veneficum accounted for 59 ± 3% of total Chl a. When the two prey were mixed, R. salina represented 67 ± 2% of total Chl a, whereas Synechococcus sp. represented 33 ± 2% of the Chl a. In the three-species mixtures, K. veneficum, R. salina and Synechococcus sp. contributed 36 ± 5%, 41 ± 5% and 23 ± 5% of the Chl a, respectively. Given the large disparity in cell size between Synechococcus sp. and the other taxa, the strength of these relationships is surprisingly strong. Table II: Summary of linear regression equations of Phyto-PAM II chlorophyll a autofluorescence (ChlF0) against acetone-extracted chlorophyll a concentrations (μg L-1) over serial (0, 20, 40, 60 and 80%) diluted conditions for single algal culture, and two-species and/or three-species mixed cultures Species compositions Regression analysis Slope ± SE y-intercept n r2 P-value Karlodinium veneficum (Kv) 0.74 ± 0.11 37.43 5 0.94 0.005 Rhodomonas salina (Rs) 0.81 ± 0.05 48.82 5 0.99 <0.001 Synechococcus sp. (Syn) 0.76 ± 0.06 30.29 5 0.98 <0.001 Two-species mixtures (Kv + Rs) 0.72 ± 0.08 38.12 5 0.96 0.003 Two-species mixtures (Kv + Syn) 1.15 ± 0.10 13.68 5 0.98 0.001 Two-species mixtures (Rs + Syn) 0.47 ± 0.03 37.36 5 0.98 <0.001 Three species mixtures (Kv + Rs + Syn) 0.78 ± 0.12 1.53 5 0.94 0.006 Full model 0.68 ± 0.05 37.01 35 0.83 <0.001 Species compositions Regression analysis Slope ± SE y-intercept n r2 P-value Karlodinium veneficum (Kv) 0.74 ± 0.11 37.43 5 0.94 0.005 Rhodomonas salina (Rs) 0.81 ± 0.05 48.82 5 0.99 <0.001 Synechococcus sp. (Syn) 0.76 ± 0.06 30.29 5 0.98 <0.001 Two-species mixtures (Kv + Rs) 0.72 ± 0.08 38.12 5 0.96 0.003 Two-species mixtures (Kv + Syn) 1.15 ± 0.10 13.68 5 0.98 0.001 Two-species mixtures (Rs + Syn) 0.47 ± 0.03 37.36 5 0.98 <0.001 Three species mixtures (Kv + Rs + Syn) 0.78 ± 0.12 1.53 5 0.94 0.006 Full model 0.68 ± 0.05 37.01 35 0.83 <0.001 For the full model, all data were considered. Measurement of ChlF0 excitation and/or emission at multiple wavelengths (440, 480, 540, 590 and 625 nm) for monoculture cultures of Synechococcus sp., Karlodinium veneficum and Rhodomonas salina were recorded as reference spectra. For the two- and three-species mixtures, the ChlF0 values were deconvoluted into the contribution of the corresponding algal groups based on previously recorded reference. Table II: Summary of linear regression equations of Phyto-PAM II chlorophyll a autofluorescence (ChlF0) against acetone-extracted chlorophyll a concentrations (μg L-1) over serial (0, 20, 40, 60 and 80%) diluted conditions for single algal culture, and two-species and/or three-species mixed cultures Species compositions Regression analysis Slope ± SE y-intercept n r2 P-value Karlodinium veneficum (Kv) 0.74 ± 0.11 37.43 5 0.94 0.005 Rhodomonas salina (Rs) 0.81 ± 0.05 48.82 5 0.99 <0.001 Synechococcus sp. (Syn) 0.76 ± 0.06 30.29 5 0.98 <0.001 Two-species mixtures (Kv + Rs) 0.72 ± 0.08 38.12 5 0.96 0.003 Two-species mixtures (Kv + Syn) 1.15 ± 0.10 13.68 5 0.98 0.001 Two-species mixtures (Rs + Syn) 0.47 ± 0.03 37.36 5 0.98 <0.001 Three species mixtures (Kv + Rs + Syn) 0.78 ± 0.12 1.53 5 0.94 0.006 Full model 0.68 ± 0.05 37.01 35 0.83 <0.001 Species compositions Regression analysis Slope ± SE y-intercept n r2 P-value Karlodinium veneficum (Kv) 0.74 ± 0.11 37.43 5 0.94 0.005 Rhodomonas salina (Rs) 0.81 ± 0.05 48.82 5 0.99 <0.001 Synechococcus sp. (Syn) 0.76 ± 0.06 30.29 5 0.98 <0.001 Two-species mixtures (Kv + Rs) 0.72 ± 0.08 38.12 5 0.96 0.003 Two-species mixtures (Kv + Syn) 1.15 ± 0.10 13.68 5 0.98 0.001 Two-species mixtures (Rs + Syn) 0.47 ± 0.03 37.36 5 0.98 <0.001 Three species mixtures (Kv + Rs + Syn) 0.78 ± 0.12 1.53 5 0.94 0.006 Full model 0.68 ± 0.05 37.01 35 0.83 <0.001 For the full model, all data were considered. Measurement of ChlF0 excitation and/or emission at multiple wavelengths (440, 480, 540, 590 and 625 nm) for monoculture cultures of Synechococcus sp., Karlodinium veneficum and Rhodomonas salina were recorded as reference spectra. For the two- and three-species mixtures, the ChlF0 values were deconvoluted into the contribution of the corresponding algal groups based on previously recorded reference. Fig. 1. View largeDownload slide Relationship between fluorescence-based Chl a (Phyto-PAM II) and acetone-extracted Chl a concentrations (μg L−1) in single cultures of individual algal strains (open symbols; Karlodinium veneficum, circles; Rhodomonas salina, squares; Synechococcus sp., triangles), and in mixed cultures of two species (solid symbols; K. veneficum plus R. salina, circles; K. veneficum plus Synechococcus sp., squares; R salina plus Synechococcus sp., triangles) and/or three species (cross wheel symbols). The overall regression line is shown; regression statistics of individual mixtures are given in Table II. Dashed line represents the 1:1 relationship. Note that the Phyto-PAM II overestimates that measured by acetone extraction at low chlorophyll levels and underestimates it at values >100 μg L−1. Fig. 1. View largeDownload slide Relationship between fluorescence-based Chl a (Phyto-PAM II) and acetone-extracted Chl a concentrations (μg L−1) in single cultures of individual algal strains (open symbols; Karlodinium veneficum, circles; Rhodomonas salina, squares; Synechococcus sp., triangles), and in mixed cultures of two species (solid symbols; K. veneficum plus R. salina, circles; K. veneficum plus Synechococcus sp., squares; R salina plus Synechococcus sp., triangles) and/or three species (cross wheel symbols). The overall regression line is shown; regression statistics of individual mixtures are given in Table II. Dashed line represents the 1:1 relationship. Note that the Phyto-PAM II overestimates that measured by acetone extraction at low chlorophyll levels and underestimates it at values >100 μg L−1. Growth and death rates of the mixotroph and prey: methods comparison and validation In the first experiment, cell-specific growth rates of K. veneficum with additions of R. salina and/or Synechococcus, and death rates of R. salina based on the changes in ChlF0, were in good agreement with those based on algal cell densities (Fig. 2). The regression line for K. veneficum growth rates based on cell numbers and ChlF0 has a slope of 0.94, indicating that rate estimates using Phyto-PAM II were slightly lower than those calculated by cell numbers (Table III). The observed results from mixed-culture treatments with R. salina clearly deviated from the 1:1 line, but results were closer to the 1:1 line when three-species mixtures were compared (Fig. 2A). Although not significant, rates of grazing for K. veneficum on R salina based on ChlF0 were less than those measured by flow cytometry. On the other hand, death rates of R. salina showed a slope of >1 with an r2 of 0.96 (Fig. 2B) and the highest death rate values (6.49 ± 1.22 Rs cells K. veneficum−1 d−1) were found when values were based on ChlF0 (Table IV). The points deviating from the line were found in the mixed cultures with proportionally higher prey R. salina culture volume at a predator-to-prey ratios of 1:2 and/or 1:2:1. Fig. 2. View largeDownload slide Relationships between cell-specific growth rates of Karlodinium veneficum (A, C) and death rates of prey, Rhodomonas salina (B, D) in Experiments 1 and 2, respectively. The regression lines are based on minimal fluorescence yield using Phyto-PAM II vs. cell densities (from flow cytometry) over 96 h (Experiment 1) or 72 h (Experiment 2). Fig. 2. View largeDownload slide Relationships between cell-specific growth rates of Karlodinium veneficum (A, C) and death rates of prey, Rhodomonas salina (B, D) in Experiments 1 and 2, respectively. The regression lines are based on minimal fluorescence yield using Phyto-PAM II vs. cell densities (from flow cytometry) over 96 h (Experiment 1) or 72 h (Experiment 2). Table III: Specific growth rates (μ, d−1) calculated from the slopes of the regressions of the changes in log-transformed Phyto-PAM II chlorophyll a and flow cytometric measurements of cell density with time for Karlodinium veneficum growing in monocultures, two-species and three-species mixed cultures over multiple treatments at varying predator-to-prey volume ratios Gp Species composition Treatments Flow cytometry (cell) Phyto-PAM II (ChlF0) Growth rates of K. veneficum (d-1) Growth rates of K. veneficum (d-1) Exp1 Exp2 Exp1 Exp2 GR ± SE Effect GR ± SE Effect GR ± SE Effect GR ± SE Effect I. Monoculture Control 0.15 ± 0.03 Gp: I > II > IV > III F = 81.42 P < 0.001 0.19 ± 0.05 Gp: I > II > IV > III F = 20.04 P < 0.001 0.12 ± 0.02 Gp: I > II > IV > III F = 15.04 P < 0.001 0.08 ± 0.04 Gp: I > II > IV > III F = 20.53 P < 0.001 II. Two-species mixtures: 1:2 0.37 ± 0.03 0.37 ± 0.08 0.22 ± 0.03 0.20 ± 0.03  Kv:Rs ratio 1:1 0.31 ± 0.04 0.29 ± 0.05 0.16 ± 0.02 0.17 ± 0.02 2:1 0.32 ± 0.03 0.23 ± 0.03 0.20 ± 0.02 0.12 ± 0.02 3:1 0.30 ± 0.05 0.17 ± 0.03 III. Two-species mixtures: 1:2 −1.03 ± 0.17 −1.19 ± 0.21 −1.22 ± 0.18 −1.46 ± 0.11  Kv:Syn ratio 1:1 −0.84 ± 0.13 −1.10 ± 0.16 −0.65 ± 0.11 −1.34 ± 0.06 2:1 0.09 ± 0.05 −1.08 ± 0.27 0.04 ± 0.04 −1.20 ± 0.08 3:1 0.12 ± 0.06 0.06 ± 0.03 IV. Three-species mixtures: 1:2:1 −0.54 ± 0.07 −0.36 ± 0.12 −0.63 ± 0.11 −0.35 ± 0.09  Kv:Rs:Syn ratio 1:1:1 0.25 ± 0.06 −0.18 ± 0.05 0.17 ± 0.04 −0.26 ± 0.22 2:1:1 0.30 ± 0.06 0.29 ± 0.06 0.19 ± 0.03 0.22 ± 0.08 3:1:1 0.27 ± 0.04 0.19 ± 0.03 Gp Species composition Treatments Flow cytometry (cell) Phyto-PAM II (ChlF0) Growth rates of K. veneficum (d-1) Growth rates of K. veneficum (d-1) Exp1 Exp2 Exp1 Exp2 GR ± SE Effect GR ± SE Effect GR ± SE Effect GR ± SE Effect I. Monoculture Control 0.15 ± 0.03 Gp: I > II > IV > III F = 81.42 P < 0.001 0.19 ± 0.05 Gp: I > II > IV > III F = 20.04 P < 0.001 0.12 ± 0.02 Gp: I > II > IV > III F = 15.04 P < 0.001 0.08 ± 0.04 Gp: I > II > IV > III F = 20.53 P < 0.001 II. Two-species mixtures: 1:2 0.37 ± 0.03 0.37 ± 0.08 0.22 ± 0.03 0.20 ± 0.03  Kv:Rs ratio 1:1 0.31 ± 0.04 0.29 ± 0.05 0.16 ± 0.02 0.17 ± 0.02 2:1 0.32 ± 0.03 0.23 ± 0.03 0.20 ± 0.02 0.12 ± 0.02 3:1 0.30 ± 0.05 0.17 ± 0.03 III. Two-species mixtures: 1:2 −1.03 ± 0.17 −1.19 ± 0.21 −1.22 ± 0.18 −1.46 ± 0.11  Kv:Syn ratio 1:1 −0.84 ± 0.13 −1.10 ± 0.16 −0.65 ± 0.11 −1.34 ± 0.06 2:1 0.09 ± 0.05 −1.08 ± 0.27 0.04 ± 0.04 −1.20 ± 0.08 3:1 0.12 ± 0.06 0.06 ± 0.03 IV. Three-species mixtures: 1:2:1 −0.54 ± 0.07 −0.36 ± 0.12 −0.63 ± 0.11 −0.35 ± 0.09  Kv:Rs:Syn ratio 1:1:1 0.25 ± 0.06 −0.18 ± 0.05 0.17 ± 0.04 −0.26 ± 0.22 2:1:1 0.30 ± 0.06 0.29 ± 0.06 0.19 ± 0.03 0.22 ± 0.08 3:1:1 0.27 ± 0.04 0.19 ± 0.03 ANCOVA was used to compare statistical differences in slopes for Group (Gp) I, II, III and IV of each predator growth condition. Exp1 represents the original experimental set and Exp2 is the repeated experimental set. View Large Table III: Specific growth rates (μ, d−1) calculated from the slopes of the regressions of the changes in log-transformed Phyto-PAM II chlorophyll a and flow cytometric measurements of cell density with time for Karlodinium veneficum growing in monocultures, two-species and three-species mixed cultures over multiple treatments at varying predator-to-prey volume ratios Gp Species composition Treatments Flow cytometry (cell) Phyto-PAM II (ChlF0) Growth rates of K. veneficum (d-1) Growth rates of K. veneficum (d-1) Exp1 Exp2 Exp1 Exp2 GR ± SE Effect GR ± SE Effect GR ± SE Effect GR ± SE Effect I. Monoculture Control 0.15 ± 0.03 Gp: I > II > IV > III F = 81.42 P < 0.001 0.19 ± 0.05 Gp: I > II > IV > III F = 20.04 P < 0.001 0.12 ± 0.02 Gp: I > II > IV > III F = 15.04 P < 0.001 0.08 ± 0.04 Gp: I > II > IV > III F = 20.53 P < 0.001 II. Two-species mixtures: 1:2 0.37 ± 0.03 0.37 ± 0.08 0.22 ± 0.03 0.20 ± 0.03  Kv:Rs ratio 1:1 0.31 ± 0.04 0.29 ± 0.05 0.16 ± 0.02 0.17 ± 0.02 2:1 0.32 ± 0.03 0.23 ± 0.03 0.20 ± 0.02 0.12 ± 0.02 3:1 0.30 ± 0.05 0.17 ± 0.03 III. Two-species mixtures: 1:2 −1.03 ± 0.17 −1.19 ± 0.21 −1.22 ± 0.18 −1.46 ± 0.11  Kv:Syn ratio 1:1 −0.84 ± 0.13 −1.10 ± 0.16 −0.65 ± 0.11 −1.34 ± 0.06 2:1 0.09 ± 0.05 −1.08 ± 0.27 0.04 ± 0.04 −1.20 ± 0.08 3:1 0.12 ± 0.06 0.06 ± 0.03 IV. Three-species mixtures: 1:2:1 −0.54 ± 0.07 −0.36 ± 0.12 −0.63 ± 0.11 −0.35 ± 0.09  Kv:Rs:Syn ratio 1:1:1 0.25 ± 0.06 −0.18 ± 0.05 0.17 ± 0.04 −0.26 ± 0.22 2:1:1 0.30 ± 0.06 0.29 ± 0.06 0.19 ± 0.03 0.22 ± 0.08 3:1:1 0.27 ± 0.04 0.19 ± 0.03 Gp Species composition Treatments Flow cytometry (cell) Phyto-PAM II (ChlF0) Growth rates of K. veneficum (d-1) Growth rates of K. veneficum (d-1) Exp1 Exp2 Exp1 Exp2 GR ± SE Effect GR ± SE Effect GR ± SE Effect GR ± SE Effect I. Monoculture Control 0.15 ± 0.03 Gp: I > II > IV > III F = 81.42 P < 0.001 0.19 ± 0.05 Gp: I > II > IV > III F = 20.04 P < 0.001 0.12 ± 0.02 Gp: I > II > IV > III F = 15.04 P < 0.001 0.08 ± 0.04 Gp: I > II > IV > III F = 20.53 P < 0.001 II. Two-species mixtures: 1:2 0.37 ± 0.03 0.37 ± 0.08 0.22 ± 0.03 0.20 ± 0.03  Kv:Rs ratio 1:1 0.31 ± 0.04 0.29 ± 0.05 0.16 ± 0.02 0.17 ± 0.02 2:1 0.32 ± 0.03 0.23 ± 0.03 0.20 ± 0.02 0.12 ± 0.02 3:1 0.30 ± 0.05 0.17 ± 0.03 III. Two-species mixtures: 1:2 −1.03 ± 0.17 −1.19 ± 0.21 −1.22 ± 0.18 −1.46 ± 0.11  Kv:Syn ratio 1:1 −0.84 ± 0.13 −1.10 ± 0.16 −0.65 ± 0.11 −1.34 ± 0.06 2:1 0.09 ± 0.05 −1.08 ± 0.27 0.04 ± 0.04 −1.20 ± 0.08 3:1 0.12 ± 0.06 0.06 ± 0.03 IV. Three-species mixtures: 1:2:1 −0.54 ± 0.07 −0.36 ± 0.12 −0.63 ± 0.11 −0.35 ± 0.09  Kv:Rs:Syn ratio 1:1:1 0.25 ± 0.06 −0.18 ± 0.05 0.17 ± 0.04 −0.26 ± 0.22 2:1:1 0.30 ± 0.06 0.29 ± 0.06 0.19 ± 0.03 0.22 ± 0.08 3:1:1 0.27 ± 0.04 0.19 ± 0.03 ANCOVA was used to compare statistical differences in slopes for Group (Gp) I, II, III and IV of each predator growth condition. Exp1 represents the original experimental set and Exp2 is the repeated experimental set. View Large Table IV: Death rates of prey, Rhodomonas salina (DR: Rs cell Karlodinium veneficum−1 d−1), estimated based on equations of Frost (1972) and Heinbokel (1978) using Phyto-PAM II chlorophyll a measurements applied with cell-to-chlorophyll correction factors vs. flow cytometric assay of cell density for two- and/or three-species mixtures in multiple treatments at predator-to-prey volume ratio Gp Species composition Treatments Flow cytometry (cell) Phyto-PAM II (ChlF0) Death rates of R. salina (d−1) Death rates of R. salina (d−1) Exp1 Exp2 Exp1 Exp2 DR ± SE Effect DR ± SE Effect DR ± SE Effects DR ± SE Effects I. Two-species mixtures: 1:2 4.83 ± 0.30 Gp: I > II F = 6.67 P = 0.016 3.58 ± 0.73 Gp: I > II F = 14.44 P = 0.002 6.49 ± 1.22 Gp: I > II F = 8.48 P < 0.001 6.29 ± 0.29 Gp: I > II F = 12.28 P = 0.003  Kv:Rs ratio 1:1 3.27 ± 0.03 2.28 ± 0.13 3.89 ± 0.26 1.66 ± 0.27 2:1 1.76 ± 0.15 1.06 ± 0.03 1.37 ± 0.36 0.81 ± 0.10 3:1 1.28 ± 0.05 0.68 ± 0.04 II. Three-species mixtures: 1:2:1 −4.41 ± 0.15 −3.52 ± 1.16 −4.64 ± 0.85 −6.99 ± 0.60  Kv:Rs:Syn ratio 1:1:1 2.33 ± 0.17 −2.98 ± 0.04 1.80 ± 0.08 −2.31 ± 0.14 2:1:1 1.94 ± 0.43 0.83 ± 0.13 1.77 ± 0.50 0.53 ± 0.35 3:1:1 1.42 ± 0.12 1.42 ± 0.03 Gp Species composition Treatments Flow cytometry (cell) Phyto-PAM II (ChlF0) Death rates of R. salina (d−1) Death rates of R. salina (d−1) Exp1 Exp2 Exp1 Exp2 DR ± SE Effect DR ± SE Effect DR ± SE Effects DR ± SE Effects I. Two-species mixtures: 1:2 4.83 ± 0.30 Gp: I > II F = 6.67 P = 0.016 3.58 ± 0.73 Gp: I > II F = 14.44 P = 0.002 6.49 ± 1.22 Gp: I > II F = 8.48 P < 0.001 6.29 ± 0.29 Gp: I > II F = 12.28 P = 0.003  Kv:Rs ratio 1:1 3.27 ± 0.03 2.28 ± 0.13 3.89 ± 0.26 1.66 ± 0.27 2:1 1.76 ± 0.15 1.06 ± 0.03 1.37 ± 0.36 0.81 ± 0.10 3:1 1.28 ± 0.05 0.68 ± 0.04 II. Three-species mixtures: 1:2:1 −4.41 ± 0.15 −3.52 ± 1.16 −4.64 ± 0.85 −6.99 ± 0.60  Kv:Rs:Syn ratio 1:1:1 2.33 ± 0.17 −2.98 ± 0.04 1.80 ± 0.08 −2.31 ± 0.14 2:1:1 1.94 ± 0.43 0.83 ± 0.13 1.77 ± 0.50 0.53 ± 0.35 3:1:1 1.42 ± 0.12 1.42 ± 0.03 Differences in prey death rates between Group (Gp) I and II are compared (ANOVA F-test). Exp1 represents the original experimental set and Exp2 is the repeated experimental set. View Large Table IV: Death rates of prey, Rhodomonas salina (DR: Rs cell Karlodinium veneficum−1 d−1), estimated based on equations of Frost (1972) and Heinbokel (1978) using Phyto-PAM II chlorophyll a measurements applied with cell-to-chlorophyll correction factors vs. flow cytometric assay of cell density for two- and/or three-species mixtures in multiple treatments at predator-to-prey volume ratio Gp Species composition Treatments Flow cytometry (cell) Phyto-PAM II (ChlF0) Death rates of R. salina (d−1) Death rates of R. salina (d−1) Exp1 Exp2 Exp1 Exp2 DR ± SE Effect DR ± SE Effect DR ± SE Effects DR ± SE Effects I. Two-species mixtures: 1:2 4.83 ± 0.30 Gp: I > II F = 6.67 P = 0.016 3.58 ± 0.73 Gp: I > II F = 14.44 P = 0.002 6.49 ± 1.22 Gp: I > II F = 8.48 P < 0.001 6.29 ± 0.29 Gp: I > II F = 12.28 P = 0.003  Kv:Rs ratio 1:1 3.27 ± 0.03 2.28 ± 0.13 3.89 ± 0.26 1.66 ± 0.27 2:1 1.76 ± 0.15 1.06 ± 0.03 1.37 ± 0.36 0.81 ± 0.10 3:1 1.28 ± 0.05 0.68 ± 0.04 II. Three-species mixtures: 1:2:1 −4.41 ± 0.15 −3.52 ± 1.16 −4.64 ± 0.85 −6.99 ± 0.60  Kv:Rs:Syn ratio 1:1:1 2.33 ± 0.17 −2.98 ± 0.04 1.80 ± 0.08 −2.31 ± 0.14 2:1:1 1.94 ± 0.43 0.83 ± 0.13 1.77 ± 0.50 0.53 ± 0.35 3:1:1 1.42 ± 0.12 1.42 ± 0.03 Gp Species composition Treatments Flow cytometry (cell) Phyto-PAM II (ChlF0) Death rates of R. salina (d−1) Death rates of R. salina (d−1) Exp1 Exp2 Exp1 Exp2 DR ± SE Effect DR ± SE Effect DR ± SE Effects DR ± SE Effects I. Two-species mixtures: 1:2 4.83 ± 0.30 Gp: I > II F = 6.67 P = 0.016 3.58 ± 0.73 Gp: I > II F = 14.44 P = 0.002 6.49 ± 1.22 Gp: I > II F = 8.48 P < 0.001 6.29 ± 0.29 Gp: I > II F = 12.28 P = 0.003  Kv:Rs ratio 1:1 3.27 ± 0.03 2.28 ± 0.13 3.89 ± 0.26 1.66 ± 0.27 2:1 1.76 ± 0.15 1.06 ± 0.03 1.37 ± 0.36 0.81 ± 0.10 3:1 1.28 ± 0.05 0.68 ± 0.04 II. Three-species mixtures: 1:2:1 −4.41 ± 0.15 −3.52 ± 1.16 −4.64 ± 0.85 −6.99 ± 0.60  Kv:Rs:Syn ratio 1:1:1 2.33 ± 0.17 −2.98 ± 0.04 1.80 ± 0.08 −2.31 ± 0.14 2:1:1 1.94 ± 0.43 0.83 ± 0.13 1.77 ± 0.50 0.53 ± 0.35 3:1:1 1.42 ± 0.12 1.42 ± 0.03 Differences in prey death rates between Group (Gp) I and II are compared (ANOVA F-test). Exp1 represents the original experimental set and Exp2 is the repeated experimental set. View Large The second experiment revealed similar relationships. Similar slopes in both growth rates of K. veneficum and death rates of R. salina were consistently obtained between both experiments; there were no statistically significant differences between the data sets (P > 0.05; ANCOVA test). The relationship between cell-specific growth rates based on the changes in ChlF0 vs. those based on algal cell densities was stronger in the second experiment compared to the first (r2 = 0.98, P < 0.001, n = 30; Fig. 2C). On the other hard, similar results were obtained with respect to death rates of R. salina between the two experimental data sets (Fig. 2B and D). Two-species mixtures: Karlodinium and Rhodomonas Karlodinium veneficum consistently grazed Rhodomonas salina, and consistently grew at a rate approaching double the rate in the monoculture without prey. Rates of grazing were independent of the amount of prey provided, suggesting that even the lowest proportion of prey added was sufficient to saturate the rate of feeding by the mixotroph (Table III, Fig. 3 and Supplementary Fig. 2). Fig. 3. View largeDownload slide Changes over time in chlorophyll a based on minimal fluorescence yield (A and B), and cell densities based on flow cytometric measurements (C and D) of Karlodinium veneficum (circles) mixed with Rhodomonas salina (triangles) in monocultures (open symbols), and two-species mixed cultures (filled symbols). The intensity of the shading of the symbols indicates increasing predator: prey proportions. For calibration purposes, the chlorophyll a concentrations and cell densities in mixed-cultures were multiplied by the dilution factors (culture volume: volume ratios) to allow comparison between those measurements in monocultures. Data shown are for Experiment 1; see Supplementary Fig. 2 for comparable results from Experiment 2. Fig. 3. View largeDownload slide Changes over time in chlorophyll a based on minimal fluorescence yield (A and B), and cell densities based on flow cytometric measurements (C and D) of Karlodinium veneficum (circles) mixed with Rhodomonas salina (triangles) in monocultures (open symbols), and two-species mixed cultures (filled symbols). The intensity of the shading of the symbols indicates increasing predator: prey proportions. For calibration purposes, the chlorophyll a concentrations and cell densities in mixed-cultures were multiplied by the dilution factors (culture volume: volume ratios) to allow comparison between those measurements in monocultures. Data shown are for Experiment 1; see Supplementary Fig. 2 for comparable results from Experiment 2. A number of patterns were revealed by comparison of the culture mixtures. The highest cell density and ChlF0 concentrations of K. veneficum cultures were reached when it was mixed with R. salina at a 1:2 volume ratio (Fig. 3A and C and Supplementary Fig. 2A and C). The time period at which ChlF0 of R. salina became undetectable due to grazing was 24 h in the mixed culture with predators at a 3:1 volume ratio but was proportionately longer, 48, 72 and 96 h, respectively, when predators were in volume ratios of 2:1, 1:1 and 1:2 (Fig. 3B). In terms of cell densities of R. salina, values fell to near zero after 36 and 60 h in the first experiment when it was mixed with predators in volume ratios of 3:1 and 2:1, respectively, but were maintained at detectable levels over the entire experimental time course with higher prey abundance (1:1 and 1:2 volume ratios; Fig. 3D). As for the ChlF0 and cell densities of R. salina in the second experiment, values fell to undetectable levels after 72 h with higher predator abundance (2:1 volume ratios; Supplementary Fig. 2B and D). Two-species mixtures: Karlodinium and Synechococcus Overall, K. veneficum did not grow well on Synechococcus sp. in either experiment (Table III). There were, however, several trends revealed in the time series (Fig. 4 and Supplementary Fig. 3). In all treatments, cell densities and/or ChlF0 of K. veneficum increased during the first 24−48 h of incubations and then declined in the first experiment, while cell densities and/or ChlF0 of K. veneficum consistently decreased over time in the second experiment. For the treatments with the highest prey proportions (1:1 and 1:2 predator: prey), both abundance and ChlF0 of the mixotroph declined to very low levels, suggesting substantial cell stress, but those mixotrophs with lower prey abundance (2:1 and 3:1) did not decline to the same degree, at least maintaining their abundance and ChlF0 at levels comparable to controls without prey (Fig. 4A and C and Supplementary Fig. 3A and C). In the original experiments, cell densities of Synechococcus sp. increased in the treatments with the highest predator proportions through 48 h and then remained relatively stable during 48−96 h, while ChlF0 of Synechococcus sp. consistently increased through the entire time series in all treatments except the control for which increases were seen only in the last 12 h (Fig. 4B and D). In contrast, cell densities and ChlF0 of Synechococcus sp rose slightly or remained unchanged over the entire incubation period (72 h) in the second experiment (Supplementary Fig. 3B and D). The difference between ChlF0 and cell abundance suggests that photoacclimation of Chl a likely occurred as culture densities increased. Fig. 4. View largeDownload slide Changes over time in chlorophyll a based on minimal fluorescence yield (A and B), and cell densities based on flow cytometric measurements (C and D) of Karlodinium veneficum (circles) mixed with Synechococcus sp. (squares) in monocultures (open symbols), and two-species mixed cultures (filled symbols). The intensity of the shading of the symbols indicates increasing predator: prey proportions. For calibration purposes, the chlorophyll concentrations and cell densities in mixed-cultures were multiplied by the dilution factors (culture volume: volume ratios) to allow comparison between those measurements in monocultures. Data shown are for Experiment 1; see Supplementary Fig. 3 for comparable results from Experiment 2. Fig. 4. View largeDownload slide Changes over time in chlorophyll a based on minimal fluorescence yield (A and B), and cell densities based on flow cytometric measurements (C and D) of Karlodinium veneficum (circles) mixed with Synechococcus sp. (squares) in monocultures (open symbols), and two-species mixed cultures (filled symbols). The intensity of the shading of the symbols indicates increasing predator: prey proportions. For calibration purposes, the chlorophyll concentrations and cell densities in mixed-cultures were multiplied by the dilution factors (culture volume: volume ratios) to allow comparison between those measurements in monocultures. Data shown are for Experiment 1; see Supplementary Fig. 3 for comparable results from Experiment 2. Two-species mixtures: Rhodomonas and Synechococcus In the first experiment with mixtures of the two-prey species, R. salina grew very well with the presence of Synechococcus sp., and the growth rates (0.17–0.23 d−1) were higher compared to monoculture growth (0.10 d−1). Similarly, the subsequent experiments showed enhanced growth rates of R. salina (0.18–0.33 d−1) with the addition of Synechococcus. In all treatments, cell densities and/or ChlF0 of R. salina remained unchanged during the first 24 h or 36 h and then dramatically increased, except for treatment with the highest Synechoccoccus proportions in the original experiments (1:2 Rhodomonas: Synechococcus; Fig. 5A and C and Supplementary Fig. 4A and C). In contrast, both abundance and ChlF0 of Synechococcus consistently declined after 24 or 48 h, suggesting potential grazing pressure by R. salina in all treatments (Fig. 5B and D and Supplementary Fig. 4B and D). Fig. 5. View largeDownload slide Changes over time in chlorophyll a based on minimal fluorescence yield (A and B) and cell densities based on flow cytometric measurements (C and D) of Rhodomonas salina (triangles) mixed with Synechococcus sp. (squares) in monocultures (open symbols), and two-species mixed cultures (filled symbols). The intensity of the shading of the symbols indicates increasing predator: prey proportions. For calibration purposes, the chlorophyll concentrations and cell densities in mixed-cultures were multiplied by the dilution factors (culture volume: volume ratios) to allow comparison between those measurements in monocultures. Data shown are for Experiment 1; see Supplementary Fig. 4 for comparable results from Experiment 2. Fig. 5. View largeDownload slide Changes over time in chlorophyll a based on minimal fluorescence yield (A and B) and cell densities based on flow cytometric measurements (C and D) of Rhodomonas salina (triangles) mixed with Synechococcus sp. (squares) in monocultures (open symbols), and two-species mixed cultures (filled symbols). The intensity of the shading of the symbols indicates increasing predator: prey proportions. For calibration purposes, the chlorophyll concentrations and cell densities in mixed-cultures were multiplied by the dilution factors (culture volume: volume ratios) to allow comparison between those measurements in monocultures. Data shown are for Experiment 1; see Supplementary Fig. 4 for comparable results from Experiment 2. Comparisons of growth and death rates in two and three-species mixed cultures For three-species mixed cultures, there were some parallel responses to those observed in the two-species treatments, and also some differences (Fig. 6 and Supplementary Fig. 5). In the two-species mixtures with R. salina, ChlF0 and cell abundances of K. veneficum consistently increased, and the same pattern was seen with the three species combined, except in the presence of Synechococcus at a 1:2:1 volume ratio in the first experiment (Fig. 6A and D) and at 1:2:1 and 1:1:1 volume ratio in the second experiment (Supplementary Fig. 5A and D). In all of the mixtures, growth rates of K. veneficum, when feeding on R. salina, ranged from 0.25 d−1 to 0.37 d−1 (based on changes in cell densities) and from 0.12 d−1 to 0.22 d−1 (based on changes in ChlF0; Table III). The relationship between Rhodomonas cell density and K. veneficum growth rate based on cell densities were not significantly different from those based on by ChlF0 (z = 0.3, P = 0.77; Fig. 8A). Fig. 6. View largeDownload slide Changes over time in chlorophyll a based on minimal fluorescence yield (A–C) and cell densities based on flow cytometric measurements (D–F) of Karlodinium veneficum (circles) mixed with Rhodomonas salina (triangles) and Synechococcus sp. (squares) in monocultures (open symbols), and three-species mixed cultures (filled symbols). The intensity of the shading of the symbols indicates increasing predator: prey proportions. For calibration purposes, the chlorophyll concentrations and cell densities in mixed-cultures were multiplied by the dilution factors (culture volume: volume ratios) to allow comparison between those measurements in monocultures. Data shown are for Experiment 1; see Supplementary Fig. 5 for comparable results from Experiment 2. Fig. 6. View largeDownload slide Changes over time in chlorophyll a based on minimal fluorescence yield (A–C) and cell densities based on flow cytometric measurements (D–F) of Karlodinium veneficum (circles) mixed with Rhodomonas salina (triangles) and Synechococcus sp. (squares) in monocultures (open symbols), and three-species mixed cultures (filled symbols). The intensity of the shading of the symbols indicates increasing predator: prey proportions. For calibration purposes, the chlorophyll concentrations and cell densities in mixed-cultures were multiplied by the dilution factors (culture volume: volume ratios) to allow comparison between those measurements in monocultures. Data shown are for Experiment 1; see Supplementary Fig. 5 for comparable results from Experiment 2. With Synechococcus alone, growth rates of K. veneficum remained unchanged and/or declined (Table III). In three-species mixtures, cell densities of Synechococcus increased through the entire time series in the presence of R. salina and predator when in proportions of 2:1:1 and 3:1:1 (Fig. 6C and F and Supplementary Fig. 5C and F). At a Synechococcus prey abundance of >270 × 108 cells L−1, or of biomass >25 ChlF0 μg L−1, K. veneficum exhibited negative growth (Fig. 7B). The relationship between Synechococcus cell density and K. veneficum growth rate determined based on cell numbers was not significantly different from that estimated by ChlF0 (z = 0.52, P = 0.60; Fig. 7B). When specific growth rates between mixed culture treatments of two- and three-species conditions were compared, there were significant increases in growth based on cell densities and/or ChlF0 when R. salina was the only prey compared with when it was combined with Synechococcus (Table III). Fig. 7. View largeDownload slide Cell-specific growth rates of Karlodinium veneficum (A and B) and death rates of Rhodomonas salina (C) as a function of prey concentrations of R. salina (circles) and Synechococcus sp. (triangles) based on minimal fluorescence yield using Phyto-PAM II (open symbols) vs. cell densities using flow cytometry (filled symbols). The cell-to-chloorphyll a correction factors are applied to the calculation of death rates in fluorescence-based chlorophyll a (ChlF0) measurements. Data include results from both experiments and error bars are standard deviations. Fig. 7. View largeDownload slide Cell-specific growth rates of Karlodinium veneficum (A and B) and death rates of Rhodomonas salina (C) as a function of prey concentrations of R. salina (circles) and Synechococcus sp. (triangles) based on minimal fluorescence yield using Phyto-PAM II (open symbols) vs. cell densities using flow cytometry (filled symbols). The cell-to-chloorphyll a correction factors are applied to the calculation of death rates in fluorescence-based chlorophyll a (ChlF0) measurements. Data include results from both experiments and error bars are standard deviations. In contrast to growth rates of K. veneficum, death rates of R. salina showed no difference between two- and three-species mixed cultures, suggesting effective feeding of R. salina was sustained despite the presence of Synechococcus sp. (Table IV). Across all treatments, death rates of R. salina caused by feeding of K. veneficum increased with increasing prey concentrations and the correlation coefficients were not different for rates determined with the cell-based and fluorescence-based methods (z = 0.31, P = 0.76; Fig. 7C). Quantum yields of Karlodinium, Rhodomonas, and Synechococcus Values of the Fv/Fm appeared to be species-specific for each taxa in monoculture (ANOVA, P < 0.01, Fig. 8; Table V). The measured Fv/Fm for K. veneficum was about 0.55 over the first 72 h and decreased to 0.41 at the end of the experiment. By comparison, the prey species R. salina and Synechococcus sp. in monocultures showed no significant changes in Fv/Fm and remained about 0.60 and 0.20, respectively. Fig. 8. View largeDownload slide Changes in maximal quantum yield (Fv/Fm) of Karlodinium veneficum (circles) mixed with Rhodomonas salina (triangles) and Synechococcus sp. (squares) in monocultures (open symbols) and two-species mixed cultures (filled symbols) with time. The intensity of the shading of the symbols indicates increasing predator: prey proportions. Data shown are for Experiment 1; see Supplementary Fig. 6 for comparable results from Experiment 2. Fig. 8. View largeDownload slide Changes in maximal quantum yield (Fv/Fm) of Karlodinium veneficum (circles) mixed with Rhodomonas salina (triangles) and Synechococcus sp. (squares) in monocultures (open symbols) and two-species mixed cultures (filled symbols) with time. The intensity of the shading of the symbols indicates increasing predator: prey proportions. Data shown are for Experiment 1; see Supplementary Fig. 6 for comparable results from Experiment 2. Table V: Measurements of the maximum quantum yield of PS II fluorescence (Fv/Fm) in monocultures, and two- and three-species mixed cultures of Karlodinium veneficum, Rhodomonas salina and Synechococcus Species composition Maximum quantum yield of PSII (Fv/Fm) Exp1 Exp2 K. veneficum R. salina Synechococcus K. veneficum R. salina Synechococcus Monocultures of each species 0.55 ± 0.06a 0.60 ± 0.04a 0.20 ± 0.05a 0.53 ± 0.04a 0.60 ± 0.03a 0.20 ± 0.05a (n = 24) (n = 24) (n = 24) (n = 21) (n = 21) (n = 21) Two-species mixtures: Kv:Rs ratio 0.54 ± 0.05a 0.38 ± 0.33b 0.50 ± 0.08a 0.51 ± 0.21b (n = 96) (n = 96) (n = 63) (n = 63) Two-species mixtures: Kv:Syn ratio 0.39 ± 0.24b 0.27 ± 0.09a 0.18 ± 0.22b 0.20 ± 0.13a (n = 96) (n = 96) (n = 63) (n = 63) Two-species mixtures: Rs:Syn ratio 0.58 ± 0.04a 0.13 ± 0.12b 0.58 ± 0.04a 0.17 ± 0.07a (n = 96) (n = 96) (n = 63) (n = 63) Three-species mixtures: Kv:Rs:Syn ratio 0.48 ± 0.13a,b 0.38 ± 0.27b 0.23 ± 0.10a 0.28 ± 0.24b 0.41 ± 0.23c 0.20 ± 0.09a (n = 96) (n = 96) (n = 96) (n = 63) (n = 63) (n = 63) ANOVA F 4.698 5.693 8.291 13.077 3.391 0.387 P-value 0.004 0.001 <0.001 <0.001 0.023 0.762 Species composition Maximum quantum yield of PSII (Fv/Fm) Exp1 Exp2 K. veneficum R. salina Synechococcus K. veneficum R. salina Synechococcus Monocultures of each species 0.55 ± 0.06a 0.60 ± 0.04a 0.20 ± 0.05a 0.53 ± 0.04a 0.60 ± 0.03a 0.20 ± 0.05a (n = 24) (n = 24) (n = 24) (n = 21) (n = 21) (n = 21) Two-species mixtures: Kv:Rs ratio 0.54 ± 0.05a 0.38 ± 0.33b 0.50 ± 0.08a 0.51 ± 0.21b (n = 96) (n = 96) (n = 63) (n = 63) Two-species mixtures: Kv:Syn ratio 0.39 ± 0.24b 0.27 ± 0.09a 0.18 ± 0.22b 0.20 ± 0.13a (n = 96) (n = 96) (n = 63) (n = 63) Two-species mixtures: Rs:Syn ratio 0.58 ± 0.04a 0.13 ± 0.12b 0.58 ± 0.04a 0.17 ± 0.07a (n = 96) (n = 96) (n = 63) (n = 63) Three-species mixtures: Kv:Rs:Syn ratio 0.48 ± 0.13a,b 0.38 ± 0.27b 0.23 ± 0.10a 0.28 ± 0.24b 0.41 ± 0.23c 0.20 ± 0.09a (n = 96) (n = 96) (n = 96) (n = 63) (n = 63) (n = 63) ANOVA F 4.698 5.693 8.291 13.077 3.391 0.387 P-value 0.004 0.001 <0.001 <0.001 0.023 0.762 Fv/Fm between groups are marked as different letters (ANOVA F-test with Tukey’s post hoc comparison). Exp1 represents the original experimental set and Exp2 is the repeated experimental set. The mean difference of footnote designators “a and b” is significant at the 0.05 level. View Large Table V: Measurements of the maximum quantum yield of PS II fluorescence (Fv/Fm) in monocultures, and two- and three-species mixed cultures of Karlodinium veneficum, Rhodomonas salina and Synechococcus Species composition Maximum quantum yield of PSII (Fv/Fm) Exp1 Exp2 K. veneficum R. salina Synechococcus K. veneficum R. salina Synechococcus Monocultures of each species 0.55 ± 0.06a 0.60 ± 0.04a 0.20 ± 0.05a 0.53 ± 0.04a 0.60 ± 0.03a 0.20 ± 0.05a (n = 24) (n = 24) (n = 24) (n = 21) (n = 21) (n = 21) Two-species mixtures: Kv:Rs ratio 0.54 ± 0.05a 0.38 ± 0.33b 0.50 ± 0.08a 0.51 ± 0.21b (n = 96) (n = 96) (n = 63) (n = 63) Two-species mixtures: Kv:Syn ratio 0.39 ± 0.24b 0.27 ± 0.09a 0.18 ± 0.22b 0.20 ± 0.13a (n = 96) (n = 96) (n = 63) (n = 63) Two-species mixtures: Rs:Syn ratio 0.58 ± 0.04a 0.13 ± 0.12b 0.58 ± 0.04a 0.17 ± 0.07a (n = 96) (n = 96) (n = 63) (n = 63) Three-species mixtures: Kv:Rs:Syn ratio 0.48 ± 0.13a,b 0.38 ± 0.27b 0.23 ± 0.10a 0.28 ± 0.24b 0.41 ± 0.23c 0.20 ± 0.09a (n = 96) (n = 96) (n = 96) (n = 63) (n = 63) (n = 63) ANOVA F 4.698 5.693 8.291 13.077 3.391 0.387 P-value 0.004 0.001 <0.001 <0.001 0.023 0.762 Species composition Maximum quantum yield of PSII (Fv/Fm) Exp1 Exp2 K. veneficum R. salina Synechococcus K. veneficum R. salina Synechococcus Monocultures of each species 0.55 ± 0.06a 0.60 ± 0.04a 0.20 ± 0.05a 0.53 ± 0.04a 0.60 ± 0.03a 0.20 ± 0.05a (n = 24) (n = 24) (n = 24) (n = 21) (n = 21) (n = 21) Two-species mixtures: Kv:Rs ratio 0.54 ± 0.05a 0.38 ± 0.33b 0.50 ± 0.08a 0.51 ± 0.21b (n = 96) (n = 96) (n = 63) (n = 63) Two-species mixtures: Kv:Syn ratio 0.39 ± 0.24b 0.27 ± 0.09a 0.18 ± 0.22b 0.20 ± 0.13a (n = 96) (n = 96) (n = 63) (n = 63) Two-species mixtures: Rs:Syn ratio 0.58 ± 0.04a 0.13 ± 0.12b 0.58 ± 0.04a 0.17 ± 0.07a (n = 96) (n = 96) (n = 63) (n = 63) Three-species mixtures: Kv:Rs:Syn ratio 0.48 ± 0.13a,b 0.38 ± 0.27b 0.23 ± 0.10a 0.28 ± 0.24b 0.41 ± 0.23c 0.20 ± 0.09a (n = 96) (n = 96) (n = 96) (n = 63) (n = 63) (n = 63) ANOVA F 4.698 5.693 8.291 13.077 3.391 0.387 P-value 0.004 0.001 <0.001 <0.001 0.023 0.762 Fv/Fm between groups are marked as different letters (ANOVA F-test with Tukey’s post hoc comparison). Exp1 represents the original experimental set and Exp2 is the repeated experimental set. The mean difference of footnote designators “a and b” is significant at the 0.05 level. View Large In the two-species mixed cultures with K. veneficum and R. salina, the value of Fv/Fm for K. veneficum remained at ~0.55 over entire experimental period (first experiment; Fig. 8A) and/or experienced an initial decrease over the first 24 h and rebounded to ~0.55 at the end of the experimental period (second experiment; Supplementary Fig. 6A). In the first 12 h of the mixed-culture experiments, Fv/Fm of R. salina increased up to ~0.75 and then rapidly decreased to 0 as cells were consumed over the next 24−48 h in the treatments with the highest predator proportion (2:1 and 3:1 predator: prey). In addition, a pattern of decrease-increase rapidly-decrease in Fv/Fm values for R. salina was found under the high prey condition in both experiments (Fig. 8B and Supplementary Fig. 6B). When mixed only with Synechococcus in either experiment, Fv/Fm of K. veneficum rapidly decreased after 24 h incubation in the 1:2 and 1:1 predator: prey mixtures (Fig. 8C and Supplementary Fig. 6C). In contrast, Synechococcus sp. displayed increasing Fv/Fm values, reaching ~0.40 after 36–48 h and then gradually declining to 0.12 at the end of the mixed culture experiment (Fig. 8D and Supplementary Fig. 6D). Overall, an average Fv/Fm value of 0.39 in the first experiment and an even lower value of 0.18 for K. veneficum in the second experiment was found in the two-species mixed cultures with Synechococcus, which were significantly different from the measured values in the monoculture and in the two-species mixed cultures with R. salina (Table V). In two-prey species mixed cultures in both experiments, the values of Fv/Fm for R. salina remained at ~0.6 over the entire experimental periods, comparable with initial values measured for the single species treatments (Fig. 8E and Supplementary Fig. 6E, Table V). In contrast, Synechococcus sp. showed declining Fv/Fm values after 12 h, reaching zero in all treatments with R. salina. In the first experiment, an average Fv/Fm value of 0.13 for Synechococcus was observed in the two-prey species mixed cultures (Fig. 8F), which was significantly different from the values in the monocultures and in the two- and three-species mixed cultures with K. veneficum (Table V). In the second experiment, an average Fv/Fm value of 0.17 for Synechococcus mixed with R. salina was obtained, slightly lower, but not significantly so, than those values in monocultures and mixed cultures with K. veneficum (Supplementary Fig. 6F and Table V). In the three-species mixed cultures in both experiments, the Fv/Fm values over time for Synechococcus and K. veneficum were mostly comparable to those in the respective two-species mixed cultures (Fig. 9 and Supplementary Fig. 7, Table V). No differences in averaged Fv/Fm values for Synechococcus sp. were found between with- and without-predator cultures. There was little variation in the measured values of Fv/Fm for K. veneficum, except that Fv/Fm decreased in the presence of Synechococcus when in the 1:2:1 volume ratio (first experiment; Fig. 9A) and at 1:2:1 and 1:1:1 volume ratio (second experiment; Supplementary Fig. 7A). Overall, the average values of Fv/Fm for K. veneficum showed significant differences among culture conditions, particularly in the presence of Synechococcus (Table V). Unlike in the two-species mixed culture, however, the Fv/Fm for R. salina was not enhanced but remained at ~0.60 throughout the original experiment when in proportions of 1:1:1 and 1:2:1 and in the second experiment when in proportions of 1:2:1 volume ratio (Fig. 9B and Supplementary Fig. 7B). The values of Fv/Fm in Synechococcus sp. increased up to ~0.4 in first 24 h of mixed cultures and gradually decreased to zero under higher R. salina proportions of 1:2:1 ratio. In both experiments, the Fv/Fm for R. salina on average declined consistently and significantly (by about 60%) in two- and three-species mixed cultures compared to the monocultures treatments. Fig. 9. View largeDownload slide Changes in maximal quantum yield (Fv/Fm) of Karlodinium veneficum (circles) mixed with Rhodomonas salina (triangles) and Synechococcus sp. (squares) in monocultures (open symbols), and three-species mixed cultures (filled symbols) with time. The intensity of the shading of the symbols indicates increasing predator: prey proportions. Data shown are for Experiment 1; see Supplementary Fig. 7 for comparable results from Experiment 2. Fig. 9. View largeDownload slide Changes in maximal quantum yield (Fv/Fm) of Karlodinium veneficum (circles) mixed with Rhodomonas salina (triangles) and Synechococcus sp. (squares) in monocultures (open symbols), and three-species mixed cultures (filled symbols) with time. The intensity of the shading of the symbols indicates increasing predator: prey proportions. Data shown are for Experiment 1; see Supplementary Fig. 7 for comparable results from Experiment 2. DISCUSSION In this study, different approaches were brought to bear in measuring grazing (calculated as prey death rates) and photophysiology of Karlodinium veneficum and its multiple prey when provided in different proportions. Multiwavelength PAM fluorometry proved to be an effective tool to rapidly assess both biomass changes and changes in photosynthetic physiological state. In keeping with previous findings (e.g. Jeong et al., 2005b; Adolf et al., 2008; Calbet et al., 2011; Lin et al., 2017), the mixotroph readily grazed the cryptophyte Rhodomonas salina, but in contrast to other studies of gymnodinoid dinoflagellates, K. veneficum did not appear to substantially graze Synechococcus. In contrast to more traditional methods of assessing mixotrophy, the photosynthetic efficiency of predator and prey was also assessed. Photosynthetic efficiency of the mixotroph differed on the different prey species, declining when Synechococcus was the prey but not when R. salina was the prey. The lack of substantial grazing on Synechococcus by K. veneficum is interesting given that other gymnodinoids have been reported to graze substantially on Synechococcus, albeit different strains (e.g. Jeong et al., 2005a; Glibert et al., 2009). There are several possible reasons why feeding on this species was not observed to any significant degree. First, the specific strain selected for use in these experiments may not be preferred. This strain was chosen because, as a PC-rich cyanobacterium, it could be differentiated using the multiwavelength PAM. It is possible that a PE-rich or other type of picoplankton may be grazed by K. veneficum, but there are no reports to date substantiating this. Second, the effects of predation on this prey may not have been effectively detected with PAM fluorometry because a relatively high abundance of prey is necessary for the minimal detection of F0 (16 Chl a μg L−1, equal to 135 × 108 cell L−1 is the minimum detection limit; Fig. 1). The Synechococcus prey: predator (cell: cell) ratios used herein, in general, were higher than in previous studies that had ranges of 180–500 (Jeong et al., 2005a) and 0.7–226 (Glibert et al., 2009). While high cell densities of Synechococcus were used, in Chesapeake Bay, from which Synechococcus was isolated and for which K. veneficum is a common bloom-former, these PC-containing picocyanobacteria can exceed 109 cells L−1 (e.g. Ray et al., 1989; Affronti and Marshall, 1994). These high Synechococcus cell abundances may have lead to C-limitation and high pH stress in these cultures. In contrast to K. veneficum that showed reduced growth when provided with Synechococcus, the cryptophyte R. salina appeared to readily feed on Synechococcus in two-species mixtures, but not in three-species mixtures. Many nano-planktonic cryptophyte species have been revealed to be mixotrophic, grazing on co-occurring cyanobacteria (e.g. Izaguirre et al., 2012; Yoo et al., 2017). These findings suggest that a complex trophic relationship exists between cyanobacteria, cryptophyte and dinoflagellates. Multiple stage trophic relationships have been reported for other mixotrophs, such as the heterotrophic dinoflagellate genus Dinophysis that feeds on the ciliate Myrionecta rubra which, in turn, feeds on cryptophytes (e.g. Park et al., 2006). Compared to Synechococcus, R. salina was readily consumed, as previously observed (Jeong et al., 2005b; Adolf et al., 2008; Calbet et al., 2011; Lin et al., 2017), and rates were within the medium-low range of previous investigations, which ranged from 0.17 to 0.50 d−1 (~3-fold variation; Lin et al., 2017) and/or from 0.16 to 0.40 d−1 (Calbet et al., 2011). In general, mixotrophic growth was lower than the average value of 0.42 ± 0.09 d−1 obtained from feeding experiments with 12 cryptophyte strains reported in Adolf et al. (2008). The magnitude of K. veneficum growth (~2-fold) observed here with varying prey quantity was lower than the magnitude of the change in growth rates of this same species when provided the same prey, R. salina, but in variable nutrient conditions (Lin et al., 2017). The nutritional quality of prey thus appears to outweigh prey quantity in regulating feeding rates. Interestingly, growth rates of K. veneficum with multiple species were comparable to those in the two-species mixtures as feeding was dominated by R. salina as prey. Although previous laboratory study suggests that Fv/Fm may not be a robust index for assessing physiological status (Kruskopf and Flynn, 2006), this index was insightful in these experiments, especially on a comparative basis. Significant changes in Fv/Fm of different taxa were found in response to changes in predation pressures and in presumed changes in nutrients. When feeding on R. salina, the Fv/Fm of K. veneficum did not change substantially, but it did decline with time when feeding on Synechococcus as the sole prey. On the other hand, the two types of prey species in both experiments showed different patterns in Fv/Fm when being fed upon (Fig. 8 and Supplementary Fig. 6). For R. salina, Fv/Fm varied between 0.6 and 0.8 but declined rapidly to zero or close to zero as it was consumed (Fig. 8B and Supplementary Fig. 6B), suggesting that plastids were not stable inside the prey cells. For most algal cells, the maximal Fv/Fm value usually remains at ~0.65–0.70, but can be significantly reduced when the cells encounter nutrient starvation or stress and/or cellular damage (Falkowski and Raven, 2007). A further independent experiment (not shown) confirmed that the Fv/Fm change in R. salina was a function of the degradation of its phycoerythrin in the presence of the predator, but not necessarily due to direct grazing; rather it appeared to be due to the possible presence of toxin, although toxin was not measured in the study herein or in this independent experiment. In the case of Synechococcus, the Fv/Fm remained in a range of 0.2–0.4 throughout the grazing period. The Fv/Fm values of Synechococcus, while substantially lower than those of R. salina, are in the range of previously reported cyanobacteria (Raateoja et al., 2004; Hung et al., 2013), which tend to be consistently lower than reported for other taxa in a physiologically healthy state. The increases of photosynthetic efficiency for Synechococcus sp. in the mixed cultures may have been due to increased availability of recycled nutrients when the predator K. veneficum cells were under stress conditions with a very dense prey abundance (Fig. 8C and D and Supplementary Fig. 6C and D). CONCLUSIONS Based on changes in photosynthetic efficiency and specific growth rates, the physiological state of K. veneficum has fundamentally different responses to individual prey species. The results of this study show that variable Chl fluorescence parameters can provide robust measures of the role of mixotrophy in predator–prey interactions with multiple prey species and in different proportions. A substantial increase in growth of K. veneficum was achieved with increasing prey concentrations of R. salina. While photosynthetic status of the mixotroph was not affected by feeding, that of its primary prey (e.g. cryptophyte R. salina) declined with substantial predation pressure. No substantial feeding by K. veneficum was detected on Synechococcus sp., and this prey did not alter its photosynthetic status in the presence of the predator K. veneficum. This picoplanktonic prey species was consumed by the primary prey, which was able to maintain its photosynthetic efficiency when acting as a predator. This study further advances the use of Phyto-PAM II, especially as a laboratory tool, for assessing rates of mixotrophy as well as in photosynthetic status of algal cells in multiple prey–predator interactions. ACKNOWLEDGMENTS The authors thank T. Kana and Bay Instruments LLC (Easton, Maryland) for advice and for use of the Phyto-PAM II. This is contribution number 5530 from the University of Maryland Center for Environmental Science and number 923 from the NOAA ECOHAB program. FUNDING C.-H.L. was supported by funding from the Taiwanese Government and the Bay and Rivers Fellowship from the Horn Point Laboratory. 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TI - Mixotrophy with multiple prey species measured with a multiwavelength-excitation PAM fluorometer: case study of Karlodinium veneficum
JF - Journal of Plankton Research
DO - 10.1093/plankt/fby049
DA - 2019-01-01
UR - https://www.deepdyve.com/lp/oxford-university-press/mixotrophy-with-multiple-prey-species-measured-with-a-multiwavelength-HLvwXVR1Cn
SP - 46
VL - 41
IS - 1
DP - DeepDyve
ER -