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J. Plankton Res. (2017) 39(3): 450–462. First published online April 13, 2017 doi:10.1093/plankt/fbx020 Downloaded from https://academic.oup.com/plankt/article/39/3/450/3610955 by guest on 30 September 2021 Diel variation of grazing of the dinoﬂagellate Lepidodinium sp. and ciliate Euplotes sp. on algal prey: the effect of prey cell properties

WAI HO ALBERT NG, HONGBIN LIU* AND SHUWEN ZHANG DIVISION OF LIFE SCIENCE, HONG KONG UNIVERSITY OF SCIENCE AND TECHNOLOGY, CLEAR WATER BAY, KOWLOON, HONG KONG, CHINA

*CORRESPONDING AUTHOR: [email protected]

Received October 24, 2016; editorial decision March 23, 2017; accepted March 29, 2017

Corresponding editor: John Dolan

The effect on protist grazing of diel variation of carbon to nitrogen ratio (C:N) in algal prey was investigated using the dinoﬂagellate Lepidodinium sp. and the ciliate Euplotes sp. as predators and the green algae Dunaliella salina and Chlorella autotrophica as respective prey. Both predator and prey cultures were maintained in light:dark cycle, with an additional set of prey cultures in a reversed light:dark cycle to that of predator cultures. Grazing experiments were conducted near the end of light (light experiment) and dark (dark experiment) phase with the algal prey in the same and opposite phases provided as mono-diets. In all experiments, prey at the end of light phase (day prey) possessed higher C:N than prey at the end of dark phase (night prey). Grazing rates in the light experiments were higher than in the dark experiments for both predators. Grazing rates and C ingestion rates (IRs) on day prey were higher than that on night prey for both predators. However, similar N IRs on day and night prey were observed in Lepidodinium sp., suggesting a compensatory feeding response of the predator by taking extra C for sufﬁcient acquisition of N from the prey with relatively high C:N.

KEYWORDS: diel periodicity; grazing; herbivory; microzooplankton; prey C:N ratio; protist

INTRODUCTION as a key component of carbon ﬂux in marine planktonic Microzooplankton (<200 μm, mainly protists) are the food webs (Calbet and Landry, 2004). In addition to dominant herbivores in marine ecosystems consuming an imposing a top-down control on phytoplankton biomass, average of 75% of primary production, hence regarded microzooplankton grazing plays a role in shaping the

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composition of the phytoplankton community (Landry C:N prey (John and Davidson, 2001; Shannon et al., et al., 2000; Strom et al., 2007; Guo et al., 2014). Further, 2007). However, other studies reported opposite findings microzooplankton grazing is regarded as an important (Grover and Chrzanowski, 2009; Siuda and Dam, 2010; mechanism of dissolved organic carbon (DOC) produc- Chrzanowski and Foster, 2014).Forexample,tworecent tion and nutrient regeneration (Nagata, 2000). For studies have demonstrated that the ciliate Strombidinopsis sp. example, it was observed that the production of DOC and flagellate O. danica exhibited higher feeding rates on a by the grazing of protists and copepods was higher than mono-diet of N-limited (high C:N) than on N-replete (low that from direct algal release, signifying the importance C:N) algal and bacterial prey, respectively, (Grover and of the grazing processes in stimulating bacterial growth Chrzanowski, 2009; Siuda and Dam, 2010). A similar and biogeochemical cycling of carbon and nutrients result was observed in another recent study on the flagel- (Strom et al., 1997). late O. danica with bacterial prey of different C:N prepared Downloaded from https://academic.oup.com/plankt/article/39/3/450/3610955 by guest on 30 September 2021 Protists are reported to exhibit feeding preference based from a matrix of 15 species and two growth phases (mid- on various prey properties including size (Šimek and exponential and late-stationary) (Chrzanowski and Foster, Chrzanowski, 1992; Wilks and Sleigh, 1998), motility 2014). This increasingly observed higher protist ingestion (Gonzalez et al.,1993; Boenigk et al., 2001; Matz and on prey with lower food quality when they were provided Jürgens, 2005), chemical composition (John and Davidson, as the only food source is in agreement to that observed in 2001; Siuda and Dam, 2010), cell surface properties mesozooplankton (Hillebrand et al., 2009), where compen- (Monger et al., 1999) and chemical release (Breckels et al., satory feeding has often been suggested as the mechanism 2010). Food selectivity of protists is often concentration- of such feeding response (Plath and Boersma, 2001; dependent, i.e. the level of selectivity depends on the prey Augustin and Boersma, 2006). abundance (Jurgens and DeMott., 1995; Boenigk et al., Diel variation in microzooplankton grazing has been 2002). Furthermore, different behavior of selectivity reported (Wikner et al., 1990; Christoffersen, 1994; Liu among protists with different feeding mechanisms has et al., 1997; Dolan and Šimek, 1999; Binder and been reported (Boenigk and Arndt, 2000). Among the DuRand, 2002; Jakobsen and Strom, 2004) and a few prey properties that affect protist grazing preference, size is theoretical bases have been suggested. For example, a regarded as a primary physical constraint deciding the mechanism of light-aided digestion of algal prey was sug- capability and efficiency of prey consumption for a specific gested to enhance the feeding rate of protists on phyto- predator. For example, bacteria are usually too small to be plankton prey under conditions of food saturation where captured efficiently by the maxillae of filter-feeding cope- prey digestion becomes a rate-limiting step in the prey pods. Phagotrophic protists are generally only able to consumption process (Strom, 2001). In addition, a circa- ingest prey particles smaller than themselves (Hansen et al., dian rhythm of grazing activity was observed in several 1994), though exceptions exist due to the specific feeding species of ciliate and dinoflagellate, where a diel rhythm strategies of certain species (Sherr and Sherr, 2009). of feeding was observed in 24 h darkness after previous Specific size relationships of predator and prey, i.e. ranges exposure to diel light-dark cycle (Jakobsen and Strom, of the predator–prey size ratios, have been suggested for 2004). Less studied has been the potential effect of diel optimal feeding efficiency of different predator groups variation of the algal prey properties on the diel rhythmic (Hansen et al., 1994). grazing of protists (Ng and Liu, 2015). Diel periodicity in Comparing to the effect of prey size, the effect of food phytoplankton physiology has been well documented nutritional quality on protistan grazing is being increas- (Prezelin, 1992; Vaulot and Marie, 1999). Among various ingly studied (John and Davidson, 2001; Shannon et al., physiological characteristics, the respective increasing 2007; Grover and Chrzanowski, 2009; Siuda and Dam, and decreasing of cellular C:N during day and night was 2010; Chrzanowski and Foster, 2014). Studies have con- reported in some phytoplankton species (Stramski and sistently suggested selectivity toward prey of higher food Reynolds, 1993; Clark et al., 2002; Jauzein et al., 2011; quality, those possessing lower carbon to nitrogen ratio (C: Ng and Liu, 2015). This interspecific characteristic in N) or supporting higher yield of predator, when protists phytoplankton is based on the strong diel variation of C were provided with a mixture of prey (John and Davidson, metabolism, with increase of algal C during day due to 2001; Matz and Jürgens, 2003; Thurman et al., 2010). On photosynthetic C fixation and decrease during night due the contrary, inconsistent results have been reported to respiration, in contrast to the relatively small diel vari- among studies that used mono-diets of an algal prey of ation in assimilation of N (DiTullio and Laws, 1986; contrasting C:N. It was observed in two previous studies Jauzein et al., 2011). While a feeding response to C:N of that the flagellates Paraphysomonas vestita and Ochromonas prey in protists has been reported in various studies (John danica exhibited higher grazing activity on low C:N prey and Davidson, 2001; Shannon et al., 2007; Siuda and when they were provided with mono-diets of contrasting Dam, 2010), it can be hypothesized that the diel variation

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of C:N in algal prey would have an effect on the diel green alga D. salina as food. Predator cultures were sub- feeding behavior of protists. This study aims at investigat- cultured to fresh autoclaved seawater every month, with ing the effect of the varying C:N in algal prey in the diel algal food replenished every week. Species identification cycle on the diel grazing rhythm of protists, using the was carried out microscopically together with 18 s dinoflagellate Lepidodinium sp. and ciliate Euplotes sp. iso- rDNA sequencing. Briefly, for 18 s rDNA sequencing, lated from Hong Kong coastal water as model organisms. predator cultures were left unfed for a week to minimize Both dinoflagellates and ciliates are regarded as major the presence of prey. Predator cultures were then fil- grazers in microbial food webs in marine systems tered onto a 10 μm polycarbonate (PC) membrane (GE (Levinsen and Nielsen, 2002; Calbet, 2008; Jeong et al., Water & Process Technologies). DNA was extracted 2010). Although Euplotes sp. is not a typical planktonic from the filters and the 18 s rDNA fragments were amp- ciliate, it is abundant in Hong Kong coastal waters and lified by polymerase chain reaction using the universal Downloaded from https://academic.oup.com/plankt/article/39/3/450/3610955 by guest on 30 September 2021 has been used in various studies of protist grazing (Wilks eukaryote primers Euk82f (50 GAA ACT GCG AAT and Sleigh, 1998; Tso and Taghon, 1999; Liu and Buskey, GGT TCA TTA AAT CAG 30) and EUK516r (50- 2000). ACC AGA CTT GCC CTC C-30) (Casamayor et al., 2002; Lepère et al., 2006). Lepidodinium sp. was identified using BLAST search which showed 99% similarity to Lepidodinium sp. (MH 360) and Lepidodinium chlorophorum METHOD and Euplotes sp. was identified with phylogenetic method (Rocke and Liu, 2014). The 18 s rDNA sequences of the Culture maintenance predators have been filed in the NCBI Genbank under Adinoflagellate Lepidodinium sp. and a ciliate Euplotes sp. the accession number KU156670 (Lepidodinium sp.) and were used as predators and the green algae Dunaliella salina KJ754150 (Euplotes sp.) (Rocke and Liu, 2014). [strain obtained from the Culture Collection of Algae and Protozoa, estimated spherical diameter (ESD) ca. 6 μm] and Chlorella autotrophica (strain obtained from the National Experimental setup Center for Marine Algae and Microbiota, CCMP 243, Diel grazing experiments were conducted with the two ESD ca. 5 μm) were used as respective prey. The green predator–prey pairs, namely Lepidodinium sp. with D. salina algae were maintained in f/2 medium prepared with auto- and Euplotes sp. with C. autotrophica. Euplotes sp. was adapted claved seawater that was gravity filtered through a 0.2 μm to the prey C. autotrophica 1 week before each experiment. capsule (Pall). Cultures of predators and prey were main- Three experiments were carried out for each predator– tained at 24°C in a 14:10 light-dark cycle at light inten- prey pair with identical experimental procedures. From 1 − sities of 50 and 120 μmol photons m2 s 1,respectively.An week before each experiment, frequency of food replenish- additional set of prey cultures was maintained under the ment to the predator cultures was increased to once per 1 same growing conditions as described above but in a or2daystoensuresufficient food supply for the exponen- reversed 14:10 light-dark cycle, i.e. when the first set of prey tial growth of the predators. Each diel grazing experiment cultures was at the mid-time of the light period, this set of included two sections, one during the last hours of the light prey cultures was at the mid-time of the dark period. This period (light experiment) and the other during the last additional set of prey cultures was adapted to the reversed hours of the dark period (dark experiment). The timing of light-dark cycle for at least 8 weeks before the experiments the experiments were chosen as such so that prey cells with were conducted. To characterize the predator–prey rela- the highest contrast of C:N in the diel cycle could be simul- tionship of the two pairs of predators and prey, a curve taneously available (Ng and Liu, 2015). of predator growth rate against different prey concentra- Before carrying out each grazing experiment (i.e. light tion was obtained for each pair. Briefly, the predators or dark experiment), the algal prey that remained in the were incubated with different initial prey concentrations predator cultures was removed by a series of gravity filtra- for 2 days with the predators acclimated to the respective tion through a 10 μmPCmembrane.Afterthefiltration prey 1 week before the incubation. Predators were counted process, microscopic examination was carried out to verify at the beginning and end of incubation to obtain the preda- that the swimming behavior and motion of predators were tor growth rates. normal. Prior testing has also shown that the growth of The predators Lepidodinium sp. (ESD ca. 14 μm) and predators resumed upon replenishment of maintenance Euplotes sp. (ca. 50 × 30 μm) were isolated in Hong prey after the filtration treatment, hence ensuring the fil- Kong coastal water. Pure cultures of the two predators tration process did not affect the grazing capability of the were obtained from single cell isolation and were main- predators. The relatively prey-free predator cultures were tained in autoclaved filtered (0.2 μm) seawater with the then used for the experimental setup.

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The experimental setup consisted of grazing treatments For determination of the carbon and nitrogen con- (triplicate) with the predators provided with mono-diets of tents of prey and predators, duplicate 10 mL subsamples exponentially growing prey in late light (day prey) and of prey cultures and triplicate 50 mL subsamples of late dark (night prey) phases, together with the respective prey-free predator cultures were filtered onto pre- prey-only control treatments. The predators were distrib- combusted (550°C) GF/C filters (Whatman). Samples uted into 50 mL polypropylene centrifuge tubes (Falcon) were stored at −80°C until analysis with a CHN elem- and autoclaved filtered (0.2 μm) seawater was added when ental analyzer (PerkinElmer model 2400 CHNS). appropriate to achieve targeted predator concentrations Assuming exponential growth, apparent growth rate − − of 2500 and 150 cell mL 1 for Lepidodinium sp. and Euplotes (k,h 1) of prey in each experimental tube was determined sp., respectively. An aliquot of typically <1mLofprey by the formula: k = ln (Pt/Po)/t,wherePo and Pt are the ini-

− Downloaded from https://academic.oup.com/plankt/article/39/3/450/3610955 by guest on 30 September 2021 culture was added into each tube to achieve the targeted tial and final prey abundances (cell mL 1)andt is the incu- prey concentrations of 10 000–15 000 and 20 000–40 000 bation duration (h). Initial prey abundances were − cell mL 1 for D. salina and C. autotrophica, respectively. calculated by multiplying the cell concentration of the prey Rough estimation of prey and predator abundances for culture sample for each experiment with respective volu- the experimental setup was achieved with a flow cyt- metric proportion of added prey culture in each experi- ometer (FCM) and a Coulter Counter, respectively (see mental tube. The apparent growth rates of prey in the below for details). The experimental tubes were then incu- prey-only control treatments were regarded as the growth − − bated for 3 h under the same conditions (i.e. at 24°Cand rates (μ,h 1) of prey. The grazing mortality rates (m,h 1) − light intensity of 50 μmol photons m2 s 1) and phases of of prey in the grazing treatments were calculated with the light-dark cycle under which the predator cultures were formula m = μ–kG,wherekG are the apparent growth rates maintained. Samples (1 mL) for FCM analysis were taken of prey in the grazing treatments. Clearance rates (CRs, − − at the beginning and end of the experiments for prey enu- unit vol predator 1 h 1)werecalculatedasm divided by − meration. Samples for determination of cellular carbon predator abundances (predator unit vol 1), and ingestion − − and nitrogen contents of prey and predators were pre- rates (IRs, cell predator 1 h 1)werecalculatedasCRs − pared at the beginning of the experiments. multiplied by prey abundances (cell unit vol 1). The C and − − Three additional batch cultures in exponential phase of NIRs(pmolpredator 1 h 1) were calculated as IRs multi- − each alga were prepared in tandem with the feeding experi- plied by the prey cellular C and N contents (pmol cell 1), ments to monitor whether the C:N of the prey changed sig- respectively. Statistical analysis with a mixed effect model nificantly during the 3 h incubation under opposite phasing and analysis of variance (ANOVA) was conducted with condition. Samples for determination of cellular carbon the software R (version 2.11.1) to examine the effects of and nitrogen contents of the algae were prepared at the light/dark period and C:N of prey on protist grazing, beginning and end of incubation. with the assumptions of ANOVA validated by examin- ing the diagnostic plots of the residuals (Galwey 2014). Sample and data analysis – Prey abundances were analyzed with a Becton Dickinson RESULTS FACSCalibur cytometer. Yellow-green fluorescent beads (1 μm, Polysciences) were added as internal standard. From the plot of predator growth rate versus prey con- Cytograms were analyzed with the CellQuest software centration, the growth of Lepidodinium sp. feeding on (version 6.0, Becton-Dickinson). Prey cell sizes were esti- D. salina approached saturation at prey concentration of − mated based on FCM side scatter signal calibrated with 20 000 cell mL 1 and that of Euplotes sp. feeding on the ESD of prey measured by a Coulter Counter C. autotrophica appears to approach saturation at prey con- − (Beckman Coulter, Z2 Coulter Particle Count and Size centration of 80 000 cell mL 1 (Fig. 1). The initial prey Analyzer) at the beginning of the experiments. For enu- abundances in the grazing experiments were below the meration of predators for rate determination, triplicate ali- growth saturating prey concentrations for both predator– quots of 50 μL(Lepidodinium sp.) or 0.25 mL (Euplotes sp.) of prey pairs (Table I). The variations of initial prey abun- predator cultures were spiked into wells of a tissue culture dances among different experimental conditions and prey plate (24 well, Falcon). Samples were diluted with filtered types within each experiment were small but exhibited (0.2 μm) seawater as appropriate and fixedwithacid up to 2-fold difference across experiments for the preda- Lugol’ssolution(5%final concentration). After an over- tor–prey pair of Euplotes sp. with C. autotrophica (Table I). night settling in darkness, all cells in each well were The C:N of predator Lepidodinium sp. and Euplotes sp. counted with an Olympus CK30 microscope at a ×100 were around five, with that of Lepidodinium sp. (mean ± magnification. SE = 5.33 ± 0.09) slightly higher than that of Euplotes

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those of D. salina were similar between day and night prey (mean ± SE = 0.397 ± 0.009 and 0.395 ± 0.015, respectively) (Fig. 3). The initial C:N of day prey (mean ± SE = 7.48 ± 0.27 and 8.39 ± 0.19 for D. salina and C. autotrophica, respectively) were higher (mixed model ANOVA, time of day of prey as fixed factor, between-experiment variation as random factor, P < 0.01, Table II) than those of night prey (mean ± SE = 6.63 ± 0.27 and 7.36 ± 0.2 for D. salina and C. autotrophica, respectively) for both prey species in all diel grazing experiments (Fig. 2A and B). For the additional Downloaded from https://academic.oup.com/plankt/article/39/3/450/3610955 by guest on 30 September 2021 batch cultures for testing the response of prey C:N to incubation, the C:N calculated as the averages of the initial and final values of each incubation of day prey Fig. 1. Growth rate of Lepidodinium sp. and Euplotes sp. versus initial (mean ± SE = 7.91 ± 0.03 and 8.26 ± 0.03 for D. salina prey concentration. and C. autotrophica, respectively) were also higher (mixed model ANOVA, time of day of prey as fixed factor, Table I: Initial concentrations of day prey between-culture variation as random factor, P < 0.01, and night prey in light and dark grazing Table II) than those of night prey (mean ± SE = 7.02 ± experiments of the predator–prey pairs 0.03 and 7.25 ± 0.03 for D. salina and C. autotrophica, Lepidodinium sp. with D. salina and respectively) (Fig. 2C and D). Contrary to C:N, the diel Euplotes sp. with C. autotrophica patterns of cell sizes, calculated as the averages of the initial and final values in each experiment, of day and night Concentration prey were different between the two species. While the ± = Exp Condition Prey D. salina C. autotrophica cell volumes of D. salina during the day (mean SE 95.77 ± 3.31 μm3)andnight(mean± SE = 95.85 ± 1 Light Day 9553 40 194 μ 3 Night 9824 41 804 2.72 m ) were similar (Fig. 4A, Table II), the cell volume Dark Day 9713 41 053 of C. autotrophica during the day (mean ± SE = 53.47 ± Night 9354 39 713 2.67 μm3) was larger (mixed model ANOVA, time of day 2 Light Day 8627 19 847 fi Night 9153 18 812 of prey as xed factor, between-experiment variation as Dark Day 8842 19 362 random factor, P < 0.01, Table II) than during night Night 8210 19 692 (mean ± SE = 35.06 ± 1.03 μm3)(Fig.4B). 3 Light Day 14 337 19 622 – Night 14 285 20 914 For diel grazing experiments of the predator prey Dark Day 14 666 21 274 pair of Lepidodinium sp. and D. salina, the CRs varied 2- Night 14 555 19 342 fold among the three experiments, with the average CR in Experiment 2 (mean ± SE = 42.2 ± 4.5 nL dinofla- − − gellate 1 h 1) double that of Experiment 1 (mean ± SE = − − sp. (mean ± SE = 4.89 ± 0.11) but the difference was 21.8 ± 2.1 nL dinoflagellate 1 h 1)(Fig.5A). A smaller not significant (t-test, P > 0.05). The C:N of D. salina variation was found in the IRs of the dinoflagellate gra- and C. autotrophica were higher than those of predators, zer, ranging from an average of 0.19 ± 0.01 (mean ± SE) − − ranging from 6 to 9 (Fig. 2A and B). For both prey spe- cell dinoflagellate 1 h 1 in Experiment 1 to 0.33 ± 0.08 − − cies, cellular C content of day prey (mean ± SE = 2.97 ± (mean ± SE) cell dinoflagellate 1 h 1 in Experiment 3 0.16 and 0.64 ± 0.06 for D. salina and C. autotrophica, (Fig. 5B). Both the light/dark period and C:N of prey respectively) were higher (mixed model ANOVA, time of had a significant effect (mixed model ANOVA, light/ day of prey as fixed factor, between-experiment variation dark period and C:N of prey as fixed factors, between- as random factor, P < 0.05, Table II) than those of night experiment variation as random factor; P < 0.05, prey (mean ± SE = 2.62 ± 0.16 and 0.49 ± 0.05 for D. Table III) on the CRs of the dinoflagellate grazer, salina and C. autotrophica, respectively). Cellular N of C. with a higher average in the light than in the dark per- autotrophica was also higher (mixed model ANOVA, time iod and a higher average on prey with higher C:N of day of prey as fixed factor, between-experiment vari- (dayprey)thanonpreywithlowerC:N(nightprey) ation as random factor, P < 0.05, Table II) in day prey (Table IV,Fig.5A). The same pattern was observed than night prey (mean ± SE = 0.076 ± 0.007 and 0.067 ± for IRs (Table IV,Fig.5B), but the effects of both fac- 0.007 for day prey and night prey, respectively) but tors of light/dark period and C:N of prey were not

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Fig. 2. Variation of initial cellular carbon to nitrogen molar ratio (C:N) in the grazing experiments (A, B) and average C:N during incubation of cells from the additional batch cultures (C, D) of the green algae D. salina (Duna) (A, C) and C. autotrophica (Chl) (B, D). Labels on the x-axis indicate the experiment treatments. Preﬁx “Light” and “Dark” refer to the light and dark experiments respectively and sufﬁx “Day” and “Night” refer to day and night prey respectively. Error bars indicate standard errors.

Table II: Effect of time of day (day or night) than that on prey with lower C:N (night prey) on cellular properties of the green algae D. sal- (Table IV), the effect was not significant (Table III)on ina and C. autotrophica N IRs, with similar averages on high (day prey) and low (night prey) C:N prey (Table IV). For all of the graz- D. salina C. autotrophica ing parameters (i.e. CRs, IRs, C and N IRs), the inter- FPFPaction between the factors of light/dark period and the fi Init C 5.68 0.04* 23.89 <0.01** C:N of prey was not signi cant (mixed model ANOVA, Init N 0.01 0.93 7.05 0.03* factor assignment as above; P > 0.05, Table III) for the Init C:N 55.14 <0.01** 109.1 <0.01** dinoflagellate grazer, suggesting the two factors were Ave C:N 481.3 <0.01** 619.5 <0.01** Cell vol 4 × 10−4 0.98 41.37 <0.01** not synergistic. As for the diel grazing experiments of the predator–prey The effect was assessed for the initial cellular carbon (Init C, pmol cell−1), −1 pair of Euplotes sp. and C. autotrophica, the CRs and IRs var- cellular nitrogen (Init N, pmol cell ), carbon to nitrogen molar ratio (C:N) ± ± μ −1 −1 in the grazing experiments (Init C:N), average C:N during incubation of ied from 0.68 0.22 (mean SE) L ciliate h and −1 −1 cells from the additional batch cultures (Ave C:N) and average cell volume 12.4 ± 3.2 (mean ± SE) cell ciliate h ,respectivelyin during incubation in the grazing experiments (Cell vol). F and P indicate ± ± μ −1 −1 F P experiment2to1.16 0.18 (mean SE) L ciliate h 1,8 and values from the mixed model ANOVA analysis, respectively. ± ± −1 −1 “*” indicates P < 0.05; “**” indicates P < 0.01. and 50.0 6.6 (mean SE) cell ciliate h , respectively in experiment 1 (Fig. 5C and D). Both factors of light/dark period and C:N of prey had a significant statistically significant (mixed model ANOVA, factor effect (mixed model ANOVA, light/dark period and C: assignment as above; P > 0.05, Table III). As for the N of prey as fixed factors, between-experiment variation IRsonpreyCandN,bothrateswerehigher(mixed as a random factor; P < 0.05, Table III) on all grazing model ANOVA, factor assignment as above; P < 0.05, parameters (i.e. CRs, IRs, C and N IRs), with higher Table III) in the light than in the dark period averages during the day than during the night and on (Table IV,Fig.6A and B). While the prey C:N had a prey with higher C:N (day prey) than on that with lower significant effect (mixed model ANOVA, factor assign- C:N (night prey) (Table V, Fig. 5C and D, 6C and D). ment as above; P < 0.05, Table III)onCIRs,with Similar to the results from the predator–prey pair of higher average on prey with higher C:N (day prey) Lepidodinium sp. and D. salina, the interaction between

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Fig. 3. Variation of initial cellular carbon (A, C) and nitrogen (B, D) in the grazing experiments of algal prey D. salina (Duna) (A, B) and C. autotrophica (Chl) (C, D). See caption of Fig. 2 for explanation of the labels on x-axis. Error bars indicate standard errors.

Fig. 4. Variation of average cell volume during incubation of the green algae D. salina (Duna) (A) and C. autotrophica (Chl) (B) in the grazing experiments. See caption of Fig. 2 for explanation of the labels on x-axis. Error bars indicate standard errors. the factors of light/dark period and C:N of prey was not period which was verified by the measurements from the significant (mixed model ANOVA, factor assignment as additional algal batch cultures. As the diel variation of C: above; P > 0.05, Table III) for all grazing parameters N in phytoplankton is caused by the uncoupling of C and for the ciliate grazer. N metabolism through the day, it proceeds at a slow pace (Ng and Liu, 2015). Therefore, the contrasting cellular C:N between day and night prey in the experiments DISCUSSION was likely to sustain through the short incubation period. It was also due to the short incubation period that the Effect of prey C:N on feeding effect of enhanced growth of prey by the recycled nutri- In the present study, the cellular contents of carbon and ents from grazing was assumed to be not significant. nitrogen of prey were only determined at the beginning Therefore, nutrients were not added in either the grazing of the grazing experiments. They were assumed not to or control experimental setups in an attempt to address change substantially during the relatively short incubation the effect of recycled nutrients from grazers.

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Fig. 5. Variation of CRs (A, C) and IRs (B, D) of the dinoﬂagellate grazer Lepidodinium sp. (A, B) and ciliate grazer Euplotes sp. (C, D). See caption of Fig. 2 for explanation of the labels on x-axis. Error bars indicate standard errors.

Table III: Analytical results of mixed model ANOVA of CR, IR, carbon ingestion (C IR) and nitrogen ingestion (N IR) rates in the diel grazing experiments with the predators Lepidodinium sp. and Euplotes sp.

Lepidodinium sp. Euplotes sp.

Time C:N Time × C:N Time C:N Time × C:N

F P FP FPFP FP FP

CR 18.77 0.01** 8.73 0.03* 0.51 0.50 29.9 <0.01** 97.8 <0.01** 1.71 0.24 IR 3.97 0.09 2.94 0.14 0.06 0.82 13.5 0.01** 20.3 <0.01** 0.02 0.90 C IR 8.69 0.03* 6.97 0.04* 0.16 0.70 25.2 <0.01** 78.2 <0.01** 2.01 0.21 N IR 6.20 0.05* 2.13 0.20 0.07 0.81 44.7 <0.01** 94.7 <0.01** 2.18 0.19

Fixed factors examined include the time of experiment (Time) (light or dark) and carbon to nitrogen molar ratio of prey (C:N). The columns Time, C:N and Time × C:N indicate the effects of Time, C:N and the interaction between both factors, respectively. F and P indicate F1,6 and P values from the ANOVA analysis, respectively. “*” indicates P < 0.05, “**” indicates P < 0.01.

The grazing rates varied considerably among experi- the interactive effect of the two factors was absent prob- ments for each predator–prey pair. In addition to the ably due to the distinct underlying mechanisms of the up to 2-fold difference in initial prey concentrations two factors: the former being the effect of the physio- across experiments, the large inter-experimental vari- logical character of the grazers, and the latter being the ation in grazing rates could also have resulted from the effect of prey properties. inconsistent food concentrations in the grazer cultures Under the framework of ecological stoichiometry before each experiment, which might lead to different (Sterner and Elser, 2002), the elemental ratios along the degrees of food limitation of the grazers (Zubkov and marine planktonic food chain depart from the Redﬁeld Sleigh, 1996; Christaki et al., 1998). Nevertheless, the molar ratio of 106 C to 16 N in phytoplankton to lower patterns of diel grazing activities were consistent among ratios in herbivores (Koski, 1999; Pertola et al.,2002). the experiments. While the effects of period of the day This generally observed C:N imparity between phyto- and prey C:N on grazing were evident in both protists, plankton and herbivores is supported by the lower C:N of

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− − Table IV: Averages (SE in parentheses) of clearance (CR, nL predator 1 h 1), ingestion (IR, cell − − − − predator 1 h 1), carbon ingestion (C IR, pmol predator 1 h 1) and nitrogen ingestion (N IR, pmol − − predator 1 h 1) rates in the light and dark experiments and on day and night prey and their percentage differences (% Diff) in the grazing experiments of Lepidodinium sp. feeding on D. salina

Exp Prey

Light Dark % Diff Day Night % Diff

CR 35.7 (4.8) 23.9 (4.4) 49% 33.79 (5.71) 25.76 (4.23) 31% IR 0.33 (0.1) 0.23 (0.03) 43% 0.32 (0.05) 0.24 (0.03) 33% C IR 0.97 (0.2) 0.61 (0.10) 59% 0.95 (0.15) 0.63 (0.10) 51%

N IR 0.13 (0.02) 0.09 (0.01) 44% 0.13 (0.02) 0.10 (0.02) 30% Downloaded from https://academic.oup.com/plankt/article/39/3/450/3610955 by guest on 30 September 2021

Fig. 6. Variation of carbon IRs (A, C) and nitrogen IRs (B, D) of the dinoﬂagellate grazer Lepidodinium sp. (A, B) and ciliate grazer Euplotes sp. (C, D). See caption of Fig. 2 for explanation of the labels on x-axis. Error bars indicate standard errors.

− − Table V: Averages (SE in parentheses) of clearance (CR, μL predator 1 h 1), ingestion (IR, cell − − − − predator 1 h 1), carbon ingestion (C IR, pmol predator 1 h 1) and nitrogen ingestion (N IR, pmol − − predator 1 h 1) rates in the light and dark experiments and on day and night prey and their percentage differences (% Diff) in the grazing experiments of Euplotes sp. feeding on C. autotrophica

Exp Prey

Light Dark % Diff Day Night % Diff

CR 1.07 (0.17) 0.75 (0.15) 43% 1.20 (0.11) 0.62 (0.13) 94% IR 30.62 (8.84) 21.37 (7.34) 43% 31.45 (8.53) 20.54 (7.48) 53% C IR 15.78 (3.60) 10.50 (2.87) 50% 17.78 (3.19) 8.50 (2.27) 109% N IR 1.96 (0.40) 1.29 (0.32) 52% 2.11 (0.36) 1.14 (0.29) 85% both predators compared to their algal prey in the present of the dinoﬂagellate, this grazer was found not able to study. Although pigments can be observed in Lepidodinium grow in f/2 medium without prey but can grow with prey sp., indicating the potential mixotrophic nutritional mode in the dark. The requirement of prey ingestion for

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sustained growth of Lepidodinium sp. suggested that it pos- revealed a positive relationship between the stoichiometric sesses a nutritional mode closer to heterotrophy than mismatch in terms of discrepancy in C:N or C to phos- autotrophy, which was supported by its low C:N similar phorus (P) ratio between prey and predator and the IR in to that of the strictly heterotrophic Euplotes sp. mesozooplankton (Hillebrand et al., 2009), indicating a The data of both predator–prey pairs indicated higher general phenomenon of higher ingestion on prey of lower grazing rates on algal prey in the late day period with nutritional quality when they are the only food source. higher C:N than those in their late night period with low- Compensatory feeding, where high food consumption er C:N. It was widely reported that protists are capable compensates for low food quality to satisfy elemental con- of differentiating prey of different nutritional qualities in tent demand, has been commonly attributed to the above food mixture (Christaki et al., 1998; Hamels et al.,2004) observed feeding response (Plath and Boersma, 2001; and exhibited preferential feeding toward prey of higher Augustin and Boersma, 2006; Siuda and Dam, 2010). Downloaded from https://academic.oup.com/plankt/article/39/3/450/3610955 by guest on 30 September 2021 nutritional quality, cells that possess a lower cellular C:N The above phenomenon agrees with the higher C inges- or support a higher yield of predator (Verity, 1991; John tion on higher C:N day prey but similar N ingestion on and Davidson, 2001; Thurman et al.,2010). However, day (higher C:N) and night (lower C:N) prey in the dino- the feeding response is more diverse when protists are flagellate grazer in this study. However, the feeding provided with mono-diets of prey of contrasting nutri- response observed in the ciliate grazer in the present study tional qualities (e.g. high and low C:N). Recent studies could not be explained by compensatory feeding for have demonstrated higher feeding activities on prey of acquisition of sufficient N element as the N ingestion of lower food quality (higher C:N) in ciliates provided with the higher C:N prey was also higher than that of the low- high and low C:N algal prey (Siuda and Dam, 2010), er C:N prey. Recent studies have also reported similar andinnanoflagellates provided with bacterial prey of dif- behavior in protistan grazers (Grover and Chrzanowski, ferent C:N (Grover and Chrzanowski, 2009). These 2009; Siuda and Dam, 2010). For example, Siuda and observations are not consistent to the higher grazing rates Dam (2010) observed that N ingestion was doubled when on lower C:N prey observed in flagellate predators in the ciliate Strombidinopsis sp.fedonN-limitedcomparedto previous studies (John and Davidson, 2001; Shannon N-replete diatom T. weissflogii. While such feeding behav- et al.,2007). It should be noted that the approaches to ior is apparently inefficient in terms of energy utilization, preparing the prey of contrasting C:N were different further investigation is warranted to fully understand the between the above two groups of studies. Specifically, the underlying mechanism of this feeding response. former group of studies maintained prey cultures in the Intuitively, the contrast in prey cell size could have same growing conditions and prepared prey of high and also contributed to the difference in grazing rates of low C:N by modifying elemental ratios in the media Euplotes sp. on C. autotrophica. However, it was observed (Grover and Chrzanowski, 2009; Siuda and Dam, 2010). in a previous study that E. mutabilis, with both cell length In contrary, the latter group obtained prey of contrasting and width double that of the Euplotes sp. used in the pre- C:N with a combination of achieving different growth sent study, exhibited the highest uptake rates on micro- rates and maintenance temperatures (Shannon et al., spheres of sizes 1.90 and 3.06 μm, suggesting highest 2007) or using prey cultures in different growth phases feeding efficiency on these sizes among the five tested (John and Davidson, 2001). The approaches adopted in sizes across 0.57–10.0 μm(Wilks and Sleigh, 1998). the latter group could have introduced contrasting cell While the optimal size of prey is closely related to the properties other than C:N. For example, study has shown predator:prey size ratio (Kirchman, 2012), given that that bacterial prey in different growth phases possess con- the grazer in the present study was smaller than that of trasting surface hydrophobicity which effectively affected the above study, we suggest the larger cell size of day nanoflagellate feeding rates (Monger et al.,1999). While prey than that of night prey of C. autotrophica played a further studies are needed to fully understand the rela- less important role in the higher grazing on day prey tionship between prey food quality in terms of elemental compared to the effect of prey C:N. ratio and protistan feeding behavior, the present study supports the observations in the former group of recent studies in which prey of different C:N were grown under Intrinsic diel feeding the same conditions with media of different elemental In the present study, both the ciliate and the phago- ratios. trophic dinoflagellate exhibited intrinsic diel variation of Comparing with protozoan grazers, the feeding grazing rates, with higher rates in light conditions than response to prey food quality in terms of stoichiometric in dark conditions. The above results agree with the ratio in a mono-diet is more established in mesozooplank- higher protistan grazing rates on algal prey during day- ton. Recent meta-analysis on data from the literature time compared to night-time observed in various studies

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(Christoffersen, 1994; Strom, 2001; Jakobsen and González et al.,2012). Specifically, coupled diel patterns Strom, 2004). For example, higher grazing activities of between algal photosynthetic activity and bacterial meta- marine flagellates during daytime have been revealed bolic activities have been observed and photosynthetic from their feeding on fluorescent-labeled Synechococcus in release has been suggested to be a major source of nutri- a previous field study (Christoffersen, 1994). While the tion for heterotrophic bacteria (Fuhrman et al., 1985). application of analog prey has effectively eliminated the Meanwhile, excretion from microzooplankton grazing is effect of diel variation of prey properties, the study sug- regarded as a major mechanism producing dissolved gested an intrinsic diel variation of grazing of the flagel- organic matter from photosynthetic sources (Nagata, lates. An explanation for the higher protist feeding 2000). For example, a previous study has demonstrated a activity on algal prey in light conditions is that light can higher production of dissolved organic C from herbivor- promote digestion, hence enhance the consumption of ous grazing by heterotrophic protists and copepods than Downloaded from https://academic.oup.com/plankt/article/39/3/450/3610955 by guest on 30 September 2021 pigmented prey by the protistan grazers (Strom, 2001). from direct algal release, hence a stimulation of bacterial However, it should be noted that according to the the- growth by the grazing activity (Strom et al., 1997). Besides ory, this mechanism of light promoted ingestion is only the grazing intensity, the extent of production of DOC valid under circumstances of saturated feeding, where from grazing also depends on the elemental composition prey digestion becomes the rate-limiting step in the prey of prey. In order to maintain the homeostasis of elemen- consumption process. While the curves of predator tal ratio, herbivores must release the excessive C obtained growth versus food concentration and the varying CRs from algal prey through DOC excretion (Hessen and between experiments suggest the prey abundances were Anderson, 2008). For example, higher DOC excretion − under saturation for the grazers in the present study, the rate (13.4% of body C day 1) was observed in Daphnia mechanism of light-aided digestion might not be a suit- when fed on P-low algae than on P-rich algae (5.7% of − able explanation for the higher feeding rates observed in body C day 1)(Darchambeau et al., 2003). Because our light conditions. Rather, other mechanisms such as cir- study shows that protist grazing on phytoplankton would cadian rhythms (Jakobsen and Strom, 2004) and photo- increase with the increase in prey C:N through the day, sensory response in prey detection (Selbach et al., 1999; the same trend of increasing DOC production from graz- Tomaru et al., 2001; Hartz et al., 2011) in heterotrophic ing and hence a stimulation to bacterial growth during protists might play a role in the intrinsic diel feeding of daytime is also expected. Therefore, the diel rhythm of both grazers in this study. protist herbivory as extrapolated from the present study probably plays a significant role in facilitating the coupling of photosynthetic production and heterotrophic bacterial Ecological implication growth at the diel temporal scale. In this study, an intrinsic higher protistan grazing during day than during night and a higher grazing activity on day prey than on night prey, presumably due to the con- CONCLUSIONS trasting C:N of prey, were observed. In general, the diel variation of C:N of phytoplankton is caused by the The present study investigated the effect of C:N in algal photosynthetic C gain during daytime and net C loss due prey on protist grazing over a diel cycle and showed that to respiration during night while the N metabolism is both dinoflagellate Lepidodinium sp. and ciliate Euplotes sp. relatively stable through the diel cycle (Ng and Liu, exhibited higher grazing rates on algal prey (D. salina and 2015). Consequently, C:N maxima and minima of phyto- C. autotrophica, respectively) in the light phase than on prey plankton were generally observed at the end of the day in the dark phase. For Lepidodinium sp., C IRs were also and night periods, respectively (Stramski and Reynolds, higher on day prey than on night prey but N IRs were 1993; Clark et al., 2002; Jauzein et al.,2011). Results of similar on both prey suggesting a behavior of compensa- the present study suggest a possible influence of this diel tory feeding, a strategy of increasing ingestion of low C:N rhythm of phytoplankton C:N on protist herbivory at the prey to acquire sufficient N in the grazer (Plath and diel temporal scale, promoting an increase of protist her- Boersma, 2001; Augustin and Boersma, 2006; Siuda and bivory through the day and decrease during night. Dam, 2010). As for Euplotes sp., both C and N IRs were A possible ecological implication of the above pattern higher on day prey than on night prey and further investi- of diel protistan herbivory is the role it may play in the gation is warranted to fully understand the underlying diel variation in bacterial growth. Diel rhythms in bacter- mechanism of this feeding response. In addition, the pre- ial activities have been observed, although heterotrophic sent study demonstrated a higher intrinsic (i.e. after elim- bacteria do not directly utilize solar radiation for energy inating the effect of influence of prey properties) grazing or nutritional resource (Kuipers et al.,2000; Ruiz- activity during daytime than at night for both grazers

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which has also been reported in earlier studies. A possible effects of physiological state of the ciliate and particle quality. ecological implication of the pattern of diel protistian her- Limnol. Oceanogr., 43, 458–464. bivory observed in the present study is a facilitation of the Christoffersen, K. (1994) Variations of feeding activities of heterotrophic fl – coupling of photosynthetic production and heterotrophic nano agellates on picoplankton. Mar. Microb. Food Webs, 8, 111 123. bacterial growth at the diel temporal scale in aquatic Chrzanowski, T. H. and Foster, B. L. (2014) Prey element stoichiom- fi fl systems. etry controls ecological tness of the agellate Ochromonas danica. Aquat. Microb. Ecol., 71, 257–269. Clark, D. R., Flynn, K. J. and Owens, N. J. P. (2002) The large capacity for dark nitrate-assimilation in diatoms may overcome nitrate – ACKNOWLEDGEMENTS limitation of growth. New. Phytol., 155, 101 108. Darchambeau, F., Faerøvig, P. J. and Hessen, D. O. 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