Microzooplankton grazing experiments in the subtropical Indian River Lagoon, Florida challenge assumptions of the dilution technique

Journal of Experimental Marine Biology and Ecology 465 (2015) 1–10

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Microzooplankton grazing experiments in the subtropical Indian River Lagoon, Florida challenge assumptions of the dilution technique Nicole Dix ⁎, M. Dennis Hanisak Florida Atlantic University, Harbor Branch Oceanographic Institute, 5600 US 1 North, Ft. Pierce, FL 34946, USA

a r t i c l e

i n f o

Article history: Received 30 July 2014 Received in revised form 19 December 2014 Accepted 20 December 2014 Available online xxxx Keywords: Dilution Estuaries Grazing Microzooplankton Positive

a b s t r a c t The most widely accepted method for measuring microzooplankton grazing rates, the dilution technique, was developed in oligotrophic, open-ocean environments. Applications of the technique in productive waters have resulted in varied responses that often complicate the interpretation of results in light of potential violations of assumptions. Grazing experiments using the dilution technique were carried out for 12 months at two sites in the Indian River Lagoon (IRL), Florida, an underrepresented ecosystem type with regard to global knowledge of microzooplankton grazing. Positive slopes were observed in the majority of experiments implying that grazing activity actually enhanced phytoplankton growth. In two such experiments, plankton community analysis supported the hypothesis that grazer abundances were not constant over the incubation period and net grazer growth rates were related to dilution level, i.e., an important assumption was violated. Trophic cascades (i.e., grazers eating grazers) are the most likely explanation for these observations. The frequency of positive slopes found in this study highlights the need for more detailed studies of trophic interactions within plankton communities of the IRL, and estuarine systems in general. The IRL has experienced persistent harmful algal blooms, the causes of which are currently under investigation; however, the impact of microbial grazers on harmful algal species cannot be quantified until appropriate methods for resolving complex trophic interactions are identified. Published by Elsevier B.V.

1. Introduction Microzooplankton (b200 μm) grazers are thought to consume the majority of pelagic primary production in marine environments, including estuaries (Calbet and Landry, 2004; Schmoker et al., 2013; Sherr and Sherr, 2002). Hence, microzooplankton represent a critical ecosystem component, influencing energy flow and community structure of flora and fauna. Most of the evidence concerning the role of microzooplankton is from oligotrophic, open-ocean environments because the method most commonly used to measure microzooplankton grazing rates, the dilution technique (Landry and Hassett, 1982), was developed in such areas. While advancements have been made in procedures of the dilution technique and interpretation of its results, much is yet to be understood about its application in estuaries (Schmoker et al., 2013). For that reason, and to address regional knowledge gaps, this study was undertaken to examine the role of microzooplankton grazing in the Indian River Lagoon, Florida, a shallow, subtropical estuary. The dilution technique is based on manipulation of encounter rates between predator and prey using a serial dilution of whole water with ⁎ Corresponding author at: Guana Tolomato Matanzas National Estuarine Research Reserve, 505 Guana River Road, Ponte Vedra Beach, FL 32082, USA. Tel.: +1 904 823 4519. E-mail address: [email protected].fl.us (N. Dix).

http://dx.doi.org/10.1016/j.jembe.2014.12.010 0022-0981/Published by Elsevier B.V.

particle-free water (Landry and Hassett, 1982). After an incubation period, the negative slope of the regression line between growth rate of prey and dilution level represents the microzooplankton grazing rate. The y-intercept of the regression line estimates the instantaneous growth rate of prey without the influence of grazing. The technique relies on three main assumptions. The first assumption is that growth of prey is not density dependent. In other words, instantaneous growth rate of the prey community is assumed to remain constant throughout the dilution series. The technique also assumes that photosynthetic organisms grow exponentially and will not become nutrient-limited during the course of the experiment. For that reason, nutrients are often added to experimental containers. Control treatments without nutrient amendments are used to correct for nutrient-replete growth rates. The final assumption is that grazer community consumption rates (individual clearance rates ∗ grazer abundance) are linear with respect to dilution level. This implies that predators do not become food-saturated at higher prey concentrations and that, at low prey concentrations, no threshold exists below which predators do not feed (Evans and Paranjape, 1992; Gallegos, 1989; Lessard and Murrell, 1998). Another implication of the final assumption is that grazer abundance relative to dilution level does not change over the incubation period. Violations of this implication have been given some attention (Agis et al., 2007; Berninger and Wickham, 2005; Calbet et al., 2011; Dolan et al., 2000; Teixeira and Figueiras, 2009), but more work is

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needed to understand the range of predator–prey interactions that may affect dilution experiment results (Schmoker et al., 2013). In the few studies that have examined grazer community dynamics in dilution experiments, differences in grazer growth and mortality among dilution levels has been a common finding (Calbet et al., 2011; Dolan et al., 2000; First et al., 2007; First et al., 2009; Modigh and Franzè, 2009). As detailed below, five response types of apparent growth rate versus dilution level have been described in the past: insignificant, negative linear, negative saturated, saturated increasing, and positive linear (Fig. 1). The three latter response types are associated with assumption violations, but teasing apart the causes of varied response types given complex community interactions can be difficult. Negative saturated responses are believed to be the result of saturated feeding (Chen et al., 2009; Gallegos, 1989; Redden et al., 2002), while saturated increasing responses may involve a combination of saturated feeding and prey selectivity or nutrient regeneration (Teixeira and Figueiras, 2009). Positive linear responses have been attributed to changes in phytoplankton intrinsic growth rates among dilution levels, i.e., from toxic contaminants in the particle-free seawater used for dilutions (Landry et al., 1995) or from elevated nutrients in less-dilute samples via grazing-induced nutrient regeneration (Modigh and Franzè, 2009). While more difficult to test, positive linear responses may also result from complexities in trophic relationships such as mixotrophy (Calbet, 2008) and trophic cascades (Calbet and Saiz, 2013; Calbet et al., 2011). If the main grazers in an experiment themselves contain chlorophyll, then their relative success in the least dilute samples could result in a positive, chlorophyll-based slope. Alternatively, if grazers are prey for other grazers in an experiment, phytoplankton may be released from grazing pressure in relative proportion to dilution level. Positive regression slopes were observed in the majority of 24 experiments performed in the Indian River Lagoon, suggesting that grazing actually stimulated prey growth. In two experiments with strong positive slopes, the plankton community was examined to test the hypothesis that grazer net growth rates were significantly related to dilution level, i.e., the final assumption was violated. Results support the hypothesis, although data were somewhat limited, and point to the need for more detailed studies of trophic interactions within estuarine plankton communities. 2. Material and methods 2.1. Site description The Indian River Lagoon (IRL) is a shallow, bar-built estuary on the east coast of Florida, USA. Massive blooms of picoplanktonic eukaryotes and the brown tide species Aureoumbra lagunensis have plagued

Apparent Growth Rate

E B

C

D

A 0

1

Fraction Unfiltered Seawater Fig. 1. A) Insignificant, B) negative linear, C) negative saturated, D) saturated increasing, and E) positive linear apparent growth rate responses previously observed from microzooplankton dilution experiments.

northern reaches of the IRL in recent years (Phlips et al., 2014) and, although observational data on zooplankton abundances have been examined, the role of microzooplankton grazing in structuring plankton communities in the IRL is unclear. Experiments were carried out with water from two sites in the south-central region of the IRL (Fig. 2). South Canal (SC) is located 28 km south of the Sebastian Inlet, 17 km north of the Fort Pierce Inlet, and near the outfall of the southernmost of 3 discharge canals in Vero Beach. Link Port (LP) is 9 km north of the Fort Pierce Inlet and surrounded by a relatively less-developed watershed. Proximity of the two inlets causes short water residence times (R50 on the order of days to weeks) (Smith, 2001; Smith, 1993). Florida's subtropical climate is characterized by a warm wet season from May to October and a cool dry season from November to April (Chen and Gerber, 1990). Phytoplankton biomass (as chlorophyll a) at SC is typically higher in the warm season than that in the cool season (Mahoney and Gibson, 1983; Phlips et al., 2002; Youngbluth et al., 1977). Plankton communities have been examined at both sites on different occasions. In the first comprehensive study of plankton in the IRL, Youngbluth et al. (1977) sampled at SC and LP weekly from January to September 1976. On average, in terms of cell numbers, phytoplankton (N10 μm), microzooplankton (28–202 μm), and mesozooplankton (N202 μm) were dominated by diatoms (65%), tintinnids (62%), and copepods (71%), respectively. It is possible, however, that their methods underestimated the number of athecate dinoflagellates and ciliates, which typically dominate the microzooplankton in estuaries. Microzooplankton community composition in the IRL has not been evaluated since that study. The prevalence of diatoms in the N 20 μm phytoplankton community has been corroborated in subsequent studies (Badylak, 2004; Mahoney and Gibson, 1983). At SC, the majority of total phytoplankton biomass (as chlorophyll a) and biovolume (from microscopic counts) consists, on average, of cells b20 μm (Hargraves and Hanisak, 2011; Phlips et al., 2010; Youngbluth et al., 1977). 2.2. Grazing experiments Water was collected monthly at LP and SC on flooding tides with a water column integrating tube made of 1¼″ (3.2 cm) Schedule 40 PVC tube with a rubber stopper closing device. Tube samples were combined in a bucket, and multiple buckets were collected. Bucket water was gently poured into a high-density polyethylene tank over a submerged 210-μm nylon mesh to remove mesozooplankton. Tanks with at least 30 L of screened water (henceforth termed “whole water”) from each site were transported to Harbor Branch Oceanographic Institute, Fort Pierce, FL for the experiments. Water to make diluent for the experiments was collected a day prior to experimental set-up to allow time for filtration. The same methods were used to collect and transport 30 L of whole water from each site. Water was then filtered sequentially through 0.7-μm glass fiber filters and 0.2-μm membrane filters using low vacuum pressure (10–15 in Hg). Diluent was stored in plastic jugs covered with white garbage bags and placed in outdoor baths with running seawater for approximately 24 h. To set up the experiments, whole water and diluent were combined in various proportions to create five dilution treatments in duplicate or triplicate. Water was transferred gently with silicone tubing into 2-L polypropylene incubation containers. Duplicate containers for initial samples and duplicate (January–March) or triplicate (April–December) containers for nutrient controls were filled with 100% whole water. One container of 100% diluent from each site acted as a control for growth in the b0.2-μm fraction (Li, 1990). From January to March, dilution levels in the experiments were 10%, 25%, 50%, 75%, and 100%. From April to December, in anticipation of saturated feeding, dilution levels were adjusted to 10%, 20%, 30%, 65%, and 100% so that regression slopes from more dilute treatments would be as informative as possible (Gallegos, 1989).

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Fig. 2. Grazing experiment sampling locations in the south-central Indian River Lagoon, South Canal (SC; 27°36′40″ N, 80°22′5″ W) and Link Port (LP; 27°32′27″ N, 80°20′41″ W).

Nutrients were added to all treatment groups except nutrient controls in final concentrations of 440 μg NO3–N L− 1, 810 μg Si L− 1, and 60 μg PO4–P L−1 to allow for two doublings day−1. Nutrient amendment concentrations were derived by assuming an average in situ chlorophyll a (CHL) of 10 μg L− 1 (MD Hanisak, unpublished data), a 1:1 relationship between CHL and P (Reynolds, 2006), and a 13.5:7.2:1 Si: N:P mass ratio (Redfield et al., 1963). To simulate in situ conditions, incubation containers were placed in outdoor running seawater baths and spun continuously with magnetic stir bars. Samples were acclimated for 1 h before sampling the initial (t0) containers so that any effects of experimental set-up and sample spinning on sensitive protozoans would have minimal impact on model results (Gifford, 1993). Plankton were incubated for 24 h, during which time water baths were covered with one layer of window screening to reduce incident irradiance. Transparent plastic tarps were placed on top of screens to protect samples from frequent and unpredictable rain events. CHL concentration was used as a proxy for phytoplankton biomass. At least 300 mL was filtered onto 0.7-μm glass fiber filters in duplicate. Filters were wrapped in aluminum foil and stored at − 80 °C for less than three months. CHL was extracted with hot ethanol (Sartory and Grobbelaar, 1984) and determined spectrophotometrically, correcting for pheophytin (Eaton et al., 2005). Net phytoplankton growth rates were calculated as (ln (CHLt / CHL0)) / t, where t = 1 day (24 h), CHLt = final CHL and CHL0 = initial CHL. CHL0 for each treatment group was calculated by multiplying the average CHL from duplicate undiluted initial containers by the percentage of whole water in that treatment. CHL from initial duplicates had coefficients of variation ranging from 1 to 7%. Microzooplankton grazing rates (g) were calculated as the slope of the relationship between net

phytoplankton growth rate and fraction whole water. The y-intercept of that relationship was assumed to be the nutrient-replete phytoplankton growth rate in the absence of grazing (μn). When a regression slope was positive, however, the y-intercept was more difficult to interpret. Therefore, net phytoplankton growth rates observed in the undiluted, nutrient-amended (kn) and nutrient control (k0) treatment groups were also calculated. The degree of nutrient limitation was assessed via a nutrient limitation index (kn − k0). In cases where significant negative slopes were observed, the percent standing crop grazed per day (Pi = 1 − exp (−g)) was determined (Murrell et al., 2002). The LP February regression slope was nonlinear (lack of fit p = 0.030), so the most dilute treatments that yielded a linear regression (10%, 25%, and 50%) were used to calculate growth and grazing rates as in Teixeira and Figueiras (2009). Realizing that transparent tarps had the potential to affect plankton growth and grazing rates by altering the light regime, two extra experiments were conducted to test for differences in growth and grazing rates between covered and uncovered treatments, one in August 2012 and one in January 2013. Water for the extra experiments was collected from a jetty-like structure in the IRL 0.7 km south–southeast of LP. Experimental methods were identical to regular monthly experiments except that one set of dilution treatments was covered with a screen and a tarp, while the other set was covered with two screens (approximately 60% light reduction) to mimic natural conditions. Additionally, in August 2012, to test for a potential toxic effect of the plastic jugs used for diluent storage, a dilution series with diluent stored in a high-density polyethylene tank was incubated with the tarped treatment dilution series. Differences in regression slopes and y-intercepts among treatment groups were tested with analysis of covariance.

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SAS software (Version 9.1.3, Cary, NC) was used to perform all statistical analyses. Some variables were not normally distributed, so non-parametric Spearman correlation analysis was used to explore relationships among all variables. Values for g that were not statistically significant in the regressions were set to zero for correlation analysis. 2.3. Plankton dynamics in July experiments Plankton samples were collected from all 24 experiments and a subset was analyzed to estimate net growth rates at two dilution levels. July samples from South Canal and Link Port were chosen to compare because CHL-based regression lines were strongly positive and had high r2 values (Table 1). Approximately 0.9 L of each duplicate initial 100% (undiluted), final 20%, and final 100% treatment samples were preserved with 1% acid Lugol's and stored in amber glass bottles. Larger plankton were concentrated by filtering through a 41-μm nylon mesh between four and eleven days after collection. Contents on the mesh were rinsed with filtered seawater from the back into an amber vial and stored in 25–100 mL with 8% Lugol's until analyzed. The b 41-μm filtrate was preserved and analyzed for smaller plankton abundance. Sample aliquots were placed in a settling chamber and analyzed with an Olympus CK2 inverted microscope at 100 × and 400 ×. At 100 ×, multiple aliquots were analyzed in their entirety until at least 100 dominant microzooplankton individuals were counted. At 400×, counts were completed within 1-mm grids until a minimum of 100 cells of a single taxon and 50 grids were counted. In cases where 100 dominant individuals could not be reached (some of the highly diluted samples), at least 30% of the sample volume was analyzed. Upon microscopic examination of b 41-μm filtrate, it was clear that the 41-μm filter captured the majority of organisms, especially those N30 μm. Apparently, the openings in the 41-μm mesh were functionally smaller than 41 μm, perhaps due to clogging. Therefore, ≥30-μm organisms (large diatoms, ciliates, dinoflagellates, and crustaceans) were counted from concentrated samples at 100 ×. Organisms b30 μm

(small diatoms and “others”) were counted from both the concentrated samples and the b 41-μm filtrate at 400 × and added together. Flagellates, chlorophytes, and unidentified organisms were relatively rare and variable, so they were grouped as “other b30 μm”. Chaetoceros spp. were identified separately from other diatoms because, although the cells were generally b 30 μm, their setae made the entire organisms N30 μm. Due to light microscope limitations, organisms b5 μm were not included in identifications. Individual Protoperidinium cells that appeared to be senescing (i.e., cytoplasm not completely filling the cell) were categorized as such and were not included in density counts. Apparent growth rates of protists were calculated as (ln (Ct / C0)) / t, where t = 1 day (24 h), Ct = final abundance (# L−1) and C0 = initial abundance (# L−1). If no individuals were found in a particular sample, the value was set to 1 to avoid taking the natural log of 0. Such adjustments were made for aloricate ciliates in one of the SC 20% replicates, “other dinoflagellates” in one of the SC 100% replicates, and Protoperidinium sp. in one of the LP 20% replicates. Apparent growth rates of crustaceans were calculated as changes in carbon content. Length (L, prosome + urosome) of copepodites and adult copepods, henceforth grouped as copepodites, was measured for each individual counted. Because nauplii were more abundant, individuals were binned into one of three size categories (N 200 μm, 101–200 μm, and ≤100 μm). To estimate mean nauplii length (L) within each treatment group, nauplii densities for each size category were multiplied by 210 μm, 150 μm, and 90 μm, respectively, then divided by the total nauplii density. Nauplii and copepod lengths were converted to carbon via the following relationships (Sabatini and Kiorboe, 1994): −8

W ðμg CÞ ¼ 5:545  10

W ðμg CÞ ¼ 9:4676  10

L

−7

2:71

for nauplii

2:16

L

for copepodites:

Carbon content (μg C L−1) for each experimental unit was calculated as W ∗ crustacean abundance (# L−1). Sabatini and Kiorboe's equations

Table 1 Initial phytoplankton biomass as chlorophyll a (CHL), linear regression statistics of apparent growth rate versus fraction undiluted seawater, net growth rates with nutrient amendment (kn), net growth rates in nutrient controls (k0), and nutrient limitation index (kn − k0) from dilution experiments at Link Port (LP) and (SC) and from extra experiments testing tarp effects. Bold values are significant at α = 0.10. Site

Experiment start date

Initial CHL (μg L−1)

Model r2

Model p-value

Slope (g, d−1)

y-Intercept (μn, d−1)

kn (d−1)

k0 (d−1)

Nutrient limitation index

LP LP LP LP LP LP LP LP LP LP LP LP SC SC SC SC SC SC SC SC SC SC SC SC Tarp No tarp Tarp No tarp

11 Jan 12 7 Feb 12 7 Mar 12 24 Apr 12 22 May 12 19 Jun 12 18 Jul 12 15 Aug 12 18 Sept 12 16 Oct 12 14 Nov 12 12 Dec 12 11 Jan 12 7 Feb 12 7 Mar 12 24 Apr 12 22 May 12 19 Jun 12 18 Jul 12 15 Aug 12 18 Sept 12 16 Oct 12 14 Nov 12 12 Dec 12 2 Aug 12 2 Aug 12 15 Jan 13 15 Jan 13

5.3 8.6 3.9 1.9 2.0 3.4 3.2 3.2 10.3 6.8 7.8 5.2 7.1 7.9 5.2 5.2 7.6 12.1 7.9 5.6 14.0 4.1 6.1 6.2 1.3 1.3 2.4 2.3

0.55 0.69 0.61 0.23 0.26 0.15 0.77 0.63 0.90 0.88 0.74 0.42 0.01 0.78 0.09 0.36 0.55 0.84 0.91 0.45 0.74 0.47 0.44 0.01 0.70 0.71 0.50 0.45

0.014 0.042 0.007 0.118 0.093 0.240 0.0002 0.002 b0.0001 b0.0001 0.0003 0.023 0.765 b0.001 0.402 0.041 0.006 b0.0001 b0.0001 0.017 0.0004 0.014 0.018 0.806 0.003 0.009 0.022 0.033

−0.68 −0.52 0.56 0.39 0.46 0.15 0.70 0.47 1.26 1.11 0.54 0.22 0.03 −0.59 −0.20 0.44 0.41 0.53 1.27 2.26 1.12 0.68 0.54 −0.03 0.80 0.51 0.32 0.21

0.09 0.27 0.23 −1.17 −1.02 −0.47 −1.05 −0.20 −0.04 0.10 0.02 0.21 0.03 −0.47 0.05 −0.72 −0.62 0.06 −0.92 −0.79 −0.62 0.05 −0.99 0.05 −1.07 −0.68 0.18 0.49

−0.52 0.15 0.89 −0.71 −0.50 −0.32 −0.35 0.28 0.79 1.17 0.51 0.43 0.07 −1.02 −0.17 −0.23 −0.18 0.55 0.34 1.28 0.42 0.83 −0.48 0.03 −0.24 −0.17 0.48 0.68

−0.39 −0.39 0.04 −1.00 −0.69 −0.67 −0.69 0.32 −0.85 −0.50 −0.33 0.47 −0.54 −1.00 −0.40 −0.78 −0.53 0.10 0.31 0.67 0.09 0.79 −0.59 0.02 −0.64 – 0.60 0.72

−0.13 0.53 0.85 0.29 0.20 0.35 0.33 −0.05 1.65 1.67 0.84 −0.05 0.62 −0.02 0.23 0.55 0.34 0.45 0.03 0.61 0.32 0.04 0.12 0.00 0.40 – −0.12 −0.04

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were developed from Oithona similis, a species that can live inshore or offshore and has a range including the southeastern United States. Although Oithona was the dominate copepod genus found in the July samples (see the Results section), it is possible that O. similis was not present. Also, copepodite lengths were measured as cephalothorax only by Sabatini and Kiorboe (1994), so present carbon estimates may be high. The intent of these estimates was to calculate the relative carbon contributions before and after the experiment, rather than absolute values. Apparent growth rates of crustaceans were calculated as ln (Ct / C0), where Ct = final carbon content (μg L− 1) and C0 = initial carbon content (μg L−1). Plankton abundances and carbon content were tested for significant differences between the initial and final replicate samples with analysis of variance. 3. Results Environmental conditions measured at Link Port (LP) and South Canal (SC) were relatively stable throughout 2012. Water temperature ranged from 21 to 31 °C. Salinity was slightly lower and more variable at SC (29.5 ± 6.3) than at LP (34.4 ± 3.9). Initial CHL in all experiments ranged from 1.3 to 14.0 μg L−1 (Table 1). In the majority of grazing experiments, regression slopes were positive (Fig. 3). At LP, significant grazing was observed in January and February (corresponding to 49% and 41% of the phytoplankton standing stock grazed, respectively); grazing was not detected in three experiments (April–June); and, slopes were positive in the remaining seven experiments (Table 1). At SC, there was one instance of significant grazing in February (corresponding to 45% of the standing stock grazed); eight slopes were positive, and three (January, March, and December) were undetectable (Table 1). In all 100% diluent water treatments, CHL was below detection limit after incubations. In the experiments testing for tarp effects, the regression slopes (g) of tarped and un-tarped treatments were positive (Table 1) and not significantly different from each other (p = 0.234 and 0.438 for the August and January experiments, respectively). The y-intercepts (μn) were higher in the un-tarped treatments than in the tarped (p = 0.022 and 0.003 for the August and January experiments, respectively). The slope and y-intercept of the treatment with diluent stored in a highdensity polyethylene tank were not different (p = 0.978 and 0.729 for the slopes and y-intercepts, respectively) than the parameters from the treatment with diluent stored in a plastic jug. The initial microzooplankton community in the July LP experiment was dominated by aloricate ciliates, tintinnid ciliates, and dinoflagellates in similar abundances (Table 2). Potential grazers in the SC July experiment were dominated by tintinnids, followed by crustacean nauplii and aloricate ciliates (Table 3). At both sites, the majority of dinoflagellates were members of the heterotrophic genus Protoperidinium. “Other dinoflagellates” included Gyrodinium spp. and Akashiwo sanguinea. Ciliates at both sites consisted mainly of Strobilidium, Strombidium, Strombidinopsis and Tintinnopsis species. The method used for concentrating plankton (filtering through 41-μm mesh) may have caused densities of delicate plankton such as athecate dinoflagellates and ciliates to be underestimated. Relative densities used to estimate net growth rates should not have been affected, however. Although samples were screened through 210-μm mesh, copepodites ranging from 170 to 600 μm (mean 335 μm) in length were common in experiment samples. Although it is possible that copepodites appeared in samples through a hole in the collection mesh, it is more likely that they entered the sample by washing through vertically since their carapace widths were b200 μm and the mesh was checked regularly for holes. The group was dominated by Oithona spp., comprising 67% and 88% of the initial copepodite density from LP and SC, respectively. Phytoplankton communities (≥ 5 μm) from both sites in July were dominated by small diatoms (Tables 2 and 3). Picoplankton densities were not estimated.

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Differences in plankton net growth rates among treatment groups (tested by regressions of growth rate versus dilution factor) were rarely statistically significant (Tables 2 and 3, Fig. 4). A number of relationships exhibited relatively low variance (r2 N 0.70), but did not yield significant conclusions due to small sample sizes. Three exceptions were the growth rate responses of aloricate ciliates, strongly negative in the LP experiment and strongly positive in the SC experiment, and the significantly positive regression slope for centric diatoms b30 μm in the SC experiment. Tintinnids experienced a net loss in abundance in both experiments and dilution levels (Tables 2 and 3). Empty tintinnid loricas increased over the same period. Also common among the two experiments was the absence of detectable changes in crustacean carbon content over the 24-hour incubation period, which may have been too short to detect such changes. Protoperidinium spp. exhibited similar growth rate responses in LP and SC experiments (Fig. 4) and there was some evidence to suggest that they may have not been able to survive low food densities in dilute treatments. At LP, 49–100% of cells were senescing in the final 20% treatments, compared to only 2–5% of cells senescing in the final undiluted treatments (data not shown). Protoperidinium spp. abundances were much lower at SC, but in 20% treatments, an average 50% of cells were senescing, while none were found senescing in initial or final undiluted samples (data not shown). Although the relative contribution of b5-μm phytoplankton is unknown, the positive CHL-based growth rate responses in July relative to fraction unfiltered seawater mirrored the combined changes in relative abundances of diatom groups (Fig. 5). Small pennates were the only diatoms that exhibited significant positive net growth. Differences in initial and final abundances of large diatoms were not statistically different from zero, suggesting that growth and mortality were approximately balanced. In LP samples, Chaetoceros spp. abundance decreased in both dilution treatments. Chaetoceros spp. was not present in SC samples. 4. Discussion The most significant finding from this study was the frequency of positive slopes observed (15 out of 24, or 63% of experiments). In a comprehensive literature review of nearly all published studies using the dilution technique, Schmoker et al. (2013) found negative values (positive slopes) for only 2% of the 1435 published grazing rates. The seeming disparity in frequencies of positive slopes between this study and the global compilation may be related to the biogeographical bias of microzooplankton grazing rate studies. Only 9% of the grazing rate values in Schmoker et al. (2013) were from productive, subtropical estuaries. In addition, positive slopes have often been viewed as experimental error or no grazing effect, and reported as zero (Lawerence and Menden-Deuer, 2012; Liu and Dagg, 2003; Strom et al., 2007; Zhang et al., 2001). By contrast, the spatiotemporal consistency of significant linear positive slopes in this study showed that they are not an anomaly. According to the assumptions of the dilution technique, positive linear slopes must be caused by phytoplankton instantaneous growth rates, individual grazer clearance rates, and/or grazer abundances differing among dilution treatments over the incubation period. Differences in phytoplankton instantaneous growth rates among dilution treatments could have been caused by changes in nutrient form. Regenerated nitrogen can be an important substrate for phytoplankton growth (Ferrier-Pagès and Rassoulzadegan, 1994) and, unless there is a threshold, should be linearly related to grazing activity (Gaul et al., 1999). Although nitrogen in the form of nitrate was added in excess to incubation containers, regeneration of ammonium could have favored growth of small phytoplankton (more so in less diluted treatments than in dilute treatments). If so, the difference in growth rates among treatments would have to be greater than the difference in mortality due to grazing. It is interesting to note that, in 15 grazing experiments

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Table 2 Mean plankton densities (n = 2), apparent growth rates and regression statistics (n = 4) from the Link Port July 2012 dilution experiment.

Copepodites (individuals) Copepodites (μg C) Nauplii (individuals) Nauplii (μg C) Tintinnid ciliates Empty tintinnid loricas Aloricate ciliates Protoperidinium sp. Other dinoflagellates Diatoms (≥30 μm) Chaetoceros sp. Pennate diatoms (b30 μm) Centric diatoms (b30 μm) Other (b30 μm)

Initial density (L−1) — undiluted

Apparent growth rate (d−1) — 20%

Apparent growth rate (d−1) — undiluted

Slope

r2

p-Value

41 9 296 7 958 173 1245 610 418 47,617 65,194 28,951 247,668 65,308

– 0.71 – −0.12 −2.88⁎⁎ 1.47⁎⁎

– 0.11 – 0.01 −3.60⁎⁎ 1.69⁎⁎ −2.13⁎⁎ −1.18⁎⁎

– −0.76 – 0.16 −0.90 0.27 −3.07 2.70 0.35 0.08 −0.69 1.15 0.06 2.12

– 0.47 – 0.04 0.10 0.33 0.99 0.52 0.19 0.03 0.52 0.28 0.01 0.62

– 0.3142 – 0.7889 0.6846 0.4282 0.0029 0.2854 0.5660 0.8224 0.2804 0.4726 0.9227 0.2137

0.32 −3.33⁎⁎⁎ −0.98⁎⁎ −0.20 −2.62⁎⁎⁎ 0.17 −1.52 −1.86

−0.70 −0.13 −3.17⁎⁎⁎ 1.09 −1.46 −0.17

⁎⁎ Indicates significant (α = 0.05) difference in abundance or carbon content between initial and final treatments. ⁎⁎⁎ α = 0.01.

Table 3 Mean plankton densities (n = 2), apparent growth and regression statistics (n = 4) from the South Canal July 2012 dilution experiment.

Copepodites (individuals) Copepodites (μg C) Nauplii (individuals) Nauplii (μg C) Tintinnid ciliates Empty tintinnid loricas Aloricate ciliates Protoperidinium sp. Other dinoflagellates Diatoms (≥30 μm) Pennate diatoms (b30 μm) Centric diatoms (b30 μm) Other (b30 μm)

Initial density (L−1) — undiluted

Apparent growth rate (d−1) — 20%

Apparent growth rate (d−1) — undiluted

Slope

r2

p-Value

219 52 563 15 2788 365 455 142 88 50,437 250,431 550,268 17,360

– 0.61 – −0.02 −1.42⁎⁎ 2.06⁎⁎ −4.16⁎⁎⁎

– −0.24 – 0.05 −2.16⁎⁎⁎ 2.60⁎⁎⁎ −1.77⁎⁎⁎

−2.03 −1.77 −0.60 1.26 −1.98⁎⁎⁎ 1.55

−0.10 −2.14 0.43 1.91⁎⁎⁎ −1.08⁎⁎⁎

– −1.05 – 0.09 −0.92 0.68 2.99 2.41 −0.46 1.29 0.81 1.14 −1.83

– 0.58 – 0.02 0.76 0.79 0.95 0.76 0.01 0.45 0.31 0.95 0.14

– 0.2368 – 0.8486 0.1274 0.1117 0.0245 0.1278 0.8898 0.3315 0.4460 0.0278 0.6227

0.09

⁎⁎ Indicates significant (α = 0.05) difference in abundance or carbon content between initial and final treatments. ⁎⁎⁎ α = 0.01

(from five sampling dates) in the IRL, Putland and Sutton (2010) added ammonium rather than nitrate to experimental treatments and did not observe positive slopes. On the other hand, diatoms, which mostly exhibited positive slopes, are thought to prefer nitrate (Glibert, 2010), which was added in excess. A toxic contaminant in the filtrate could have also caused differences in phytoplankton instantaneous growth rates among dilution treatments and produced positive slopes, given that the toxin would be most concentrated in the more dilute samples (Landry et al., 1995). If that were the case, however, positive slopes would have been observed in all experiments. Toxicity from vacuum filtration, sample spinning, or plastic containers is also unlikely since the same methods have been used in previous experiments that produced only negative slopes (Dix et al., 2013; Phlips et al., 2002; Quinlan et al., 2009). Changes in individual grazer clearance rates among dilution treatments, which may arise from selective grazing, may have been a factor in producing positive slopes. For example, in a mesocosm experiment by Nejstgaard et al. (1997), a coccolithophore bloom was thought to be facilitated by microzooplankton grazing on other species. Also, grazer food selection may be different at different food levels (Teixeira and Figueiras, 2009). The success of small pennate diatoms, usually benthic in origin, in the July experiments points to the possibility that they were

resistant to planktonic grazing. Then again, planktonic grazers in the IRL may be adapted to benthic prey given the shallow, well-mixed environment. The most likely explanation for the frequent positive slopes observed in the IRL is differential changes in grazer abundance among dilution treatments over the incubation period caused by trophic cascades (demonstrated with models by Calbet and Saiz, 2013). In the July experiments, there was clear evidence that net growth rates of aloricate ciliates were not constant among the two dilution treatments examined. Given that marine ciliates are important omnivorous grazers (Sherr and Sherr, 2002), changes in their abundance over the course of an experiment can result in either positive or negative relative changes in CHL depending on the number of trophic levels present. The negative slope for aloricate ciliates at LP and the positive net growth rate in the 20% treatment (initial CHL approximately 0.6 μg L− 1) is contrary to the theory of dilution-induced mortality suggested by Dolan et al. (2000) who attributed positive slopes of tintinnid and oligotrich ciliates to starvation at high dilutions and predicted ciliate mortality when treatment CHL concentrations are around 1 μg L− 1. Protoperidinium spp., on the other hand, did show signs of dilution-induced mortality, which, to the authors' knowledge, has not been described previously.

Fig. 3. a. Regressions of CHL-based apparent growth rate versus relative dilution for Link Port experiments throughout 2012. Solid regression lines were statistically significant (see Table 1). b. Regressions of CHL-based apparent growth rate versus relative dilution for South Canal experiments throughout 2012. Solid regression lines were statistically significant (see Table 1).

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Fig. 4. Apparent growth rates of phytoplankton (based on chlorophyll a) and grazers (based on changes in abundance or carbon content) over the 24-hour incubation period in July experiments. Solid regression lines were statistically significant (see Tables 2 and 3).

While aloricate ciliates were the only grazer group examined that exhibited statistically significant differences in net growth rates among dilution treatments, actions of other grazers were likely important in shaping the positive CHL slopes. Negative tintinnid and dinoflagellate growth rates unassociated with dilution level (zero slopes) suggest that either the communities were at the end of their life cycle initially or they were affected by a natural source of mortality (e.g., pathogens and/or parasites; Coats and Bachvaroff, 2013; Mayali and Azam, 2004). Positive net gains in empty tintinnid loricas add to the evidence of tintinnid mortality at both dilution levels. In both LP and SC July experiments, carbon-based copepodite regression slopes were inversely proportional to CHL-based slopes (Fig. 4). Given that the net growth rates of most other grazers were negative, with the exception of aloricate ciliates in the LP 20% treatment, it is possible that responses of copepodites (mostly Oithona spp.) directly influenced the responses of phytoplankton. Oithona spp. prefer ciliates, however, and ingestion of diatoms is thought to be rare (Calbet and Saiz, 2005; Castellani et al., 2005; Nishibe et al., 2010; Zamora-Terol and Saiz, 2013). So, if changes in copepodites did affect changes in CHL, it was

likely the result of a trophic cascade, as observed in Liu and Dagg (2003) and Zöllner et al. (2009). The very inclusion of small copepods in the incubations of the current study suggests that, in productive waters, or at least in this region of the IRL, it may be extremely difficult to restrict the dilution technique to the defined microzooplankton size class (b200 μm). Future grazing experiments inclusive of all size classes (i.e., omitting the step of filtering out mesozooplankton) may give more realistic results, but chlorophyll size fractionation and microscopic analysis of all size classes is recommended to assess trophic relationships and interpret model results. 5. Conclusions The grazing impact of microzooplankton in the south-central IRL estimated from this study was low (0–49% standing crop was grazed) on a global scale (Schmoker et al., 2013), but comparable to estimates from another well-flushed estuary in northeast Florida (Dix et al., 2013). It is clear from plankton community analysis of the July experiments that the assumption of a constant balance between grazer

Fig. 5. Phytoplankton apparent growth rates over the 24-hour incubation period in July experiments. Solid regression lines were statistically significant (see Tables 2 and 3).

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growth and mortality throughout dilution levels was not met. It is hypothesized that similar situations occurred in the experiments during the rest of the year, especially when positive slopes were observed. Given the apparent assumption violations, and limited data to support the trophic cascade hypothesis, it would be risky to make conclusions about the ecological implications of the results from this study. Because the dilution technique is designed to measure community grazing rates, interactions among distinct groups (ciliates, flagellates, and metazoans) are not directly apparent and are difficult to tease apart even when samples are analyzed microscopically. More detailed studies of trophic dynamics in estuarine plankton communities are critical to advance our understanding of ecosystem-level grazing impacts, especially in the IRL where there is a pressing need for such information given major algal blooms in recent years (Gobler et al., 2013; Phlips et al., 2014). If trophic cascades are in fact common in the IRL, it is possible that insufficient control by microzooplankton grazers contributes to bloom development of small phytoplankton species; however, the impact of microbial grazers on harmful algal species cannot be quantified until appropriate methods for resolving complex trophic interactions are identified. Acknowledgments Research was funded by the Florida Specialty License Plate Program's (Grant #HBO403) Save Our Seas plate. The authors are grateful for the hard work of the following volunteers, whose contributions to field work and experiments made this project possible: Debbie Langley, Stephanie Lear, Lisa Heise, Carol Hoeman, Debbie Dix, Harold Fisher, Haille Carter, Elizabeth Urban, Penn Prett, and Evelyn Kopke. The authors would also like to thank Drs. Bill Louda, Paul Hargraves, and Edward Phlips for their support throughout the project, including review of the manuscript.[SS] References Agis, M., Granda, A., Dolan, J.R., 2007. A cautionary note: examples of possible microbial community dynamics in dilution grazing experiments. J. Exp. Mar. Biol. Ecol. 341 (2), 176–183. Badylak, S., 2004. Spatial and temporal patterns of phytoplankton composition in subtropical coastal lagoon, the Indian River Lagoon, Florida, USA. J. Plankton Res. 26 (10), 1229–1247. Berninger, U.-G., Wickham, S.A., 2005. Response of the microbial food web to manipulation of nutrients and grazers in the oligotrophic Gulf of Aqaba and northern Red Sea. Mar. Biol. 147 (4), 1017–1032. Calbet, A., 2008. The trophic roles of microzooplankton in marine systems. ICES J. Mar. Sci. 65 (3), 325–331. Calbet, A., Landry, M.R., 2004. Phytoplankton growth, microzooplankton grazing, and carbon cycling in marine systems. Limnol. Oceanogr. 49 (1), 51–57. Calbet, A., Saiz, E., 2005. The ciliate–copepod link in marine ecosystems. Aquat. Microb. Ecol. 38 (2), 157–167. Calbet, A., Saiz, E., 2013. Effects of trophic cascades in dilution grazing experiments: from artificial saturated feeding responses to positive slopes. J. Plankton Res. 35 (6), 1183–1191. Calbet, A., Saiz, E., Almeda, R., Movilla, J.I., Alcaraz, M., 2011. Low microzooplankton grazing rates in the Arctic Ocean during a Phaeocystis pouchetii bloom (Summer 2007): fact or artifact of the dilution technique? J. Plankton Res. 33 (5), 687–701. Castellani, C., Irigoien, X., Harris, R.P., Lampitt, R.S., 2005. Feeding and egg production of Oithona similis in the North Atlantic. Mar. Ecol. Prog. Ser. 288, 173–182. Chen, B.Z., Liu, H.B., Landry, M.R., Chen, M., Sun, J., Shek, L., Chen, X.H., Harrison, P.J., 2009. Estuarine nutrient loading affects phytoplankton growth and microzooplankton grazing at two contrasting sites in Hong Kong coastal waters. Mar. Ecol. Prog. Ser. 379, 77–90. Chen, E., Gerber, J., 1990. Climate. In: Meyers, R., Ewel, J. (Eds.), Ecosystems of Florida. University of Central Florida Press, Orlando. Coats, D.W., Bachvaroff, T.R., 2013. Parasites of tintinnids. In: Dolan, J.R., Montagnes, D.J.S., Agatha, S., Coats, D.W., Stoecker, D.K. (Eds.), The Biology and Ecology of Tintinnid Ciliates: Models for Marine Plankton. John Wiley & Sons, United Kingdom, pp. 145–170. Dix, N., Phlips, E., Suscy, P., 2013. Factors controlling phytoplankton biomass in a subtropical coastal lagoon: relative scales of influence. Estuar. Coast. Shelf Sci. 36 (5), 981–996. Dolan, J.R., Gallegos, C.L., Moigis, A., 2000. Dilution effects on microzooplankton in dilution grazing experiments. Mar. Ecol. Prog. Ser. 200, 127–139. Eaton, A.D., Clesceri, L.S., Rice, W.E., Greenberg, A.E., 2005. Standard Methods for the Analysis of Water and Wastewater. 21 ed. American Public Health Association, Baltimore, Maryland.

9

Evans, G.T., Paranjape, M.A., 1992. Precision of estimates of phytoplankton growth and microzooplankton grazing when the functional response of grazers may be nonlinear. Mar. Ecol. Prog. Ser. 80, 285–290. Ferrier-Pagès, C., Rassoulzadegan, F., 1994. Seasonal impact of the microzooplankton on pico- and nanoplankton growth rates in the northwest Mediterranean Sea. Mar. Ecol. Prog. Ser. 108 (3), 283–294. First, M.R., Lavrentyev, P.J., Jochem, F.J., 2007. Patterns of microzooplankton growth in dilution experiments across a trophic gradient: implications for herbivory studies. Mar. Biol. 151 (5), 1929–1940. First, M.R., Miller III, H.L., Lavrentyev, P.J., Pinckney, J.L., Burd, A.B., 2009. Effects of microzooplankton growth and trophic interactions on herbivory in coastal and offshore environments. Aquat. Microb. Ecol. 54 (3), 255. Gallegos, C.L., 1989. Microzooplankton grazing on phytoplankton in the Rhode River, Maryland: nonlinear feeding kinetics. Mar. Ecol. Prog. Ser. 57, 23–33. Gaul, W., Antia, A.N., Koeve, W., 1999. Microzooplankton grazing and nitrogen supply of phytoplankton growth in the temperate and subtropical northeast Atlantic. Mar. Ecol. Prog. Ser. 189, 93–104. Gifford, D.J., 1993. Consumption of protozoa by copepods feeding on natural microplankton assemblages. In: Kemp, P.F., Sherr, B.F., Sherr, E.B., Cole, J.J. (Eds.), Handbook of Methods in Aquatic Microbial Ecology. Lewis Publishers, pp. 723–729. Glibert, P.M., 2010. Long-term changes in nutrient loading and stoichiometry and their relationships with changes in the food web and dominant pelagic fish species in the San Francisco Estuary, California. Rev. Fish. Sci. 18 (2), 211–232. Gobler, C.J., Koch, F., Kang, Y., Berry, D.L., Tang, Y.Z., Lasi, M., Walters, L., Hall, L., Miller, J.D., 2013. Expansion of harmful brown tides caused by the pelagophyte, Aureoumbra lagunensis DeYoe et Stockwell, to the US east coast. Harmful Algae 27, 29–41. Hargraves, P.E., Hanisak, M.D., 2011. The significance of chlorophyll size fractionation in the Indian River Lagoon, Florida. Fla. Sci. 74 (3), 151–167. Landry, M., Hassett, R., 1982. Estimating the grazing impact of micro-zooplankton. Mar. Biol. 67, 283–288. Landry, M., Constantinou, J., Kirshtein, J., 1995. Microzooplankton grazing in the central equatorial Pacific during February and August, 1992. Deep-Sea Res. II 42 (2–3), 657–671. Lawerence, C., Menden-Deuer, S., 2012. Drivers of protistan grazing pressure: seasonal signals of plankton community composition and environmental conditions. Mar. Ecol. Prog. Ser. 459, 39–52. Lessard, E.J., Murrell, M.C., 1998. Microzooplankton herbivory and phytoplankton growth in the northwestern Sargasso Sea. Aquat. Microb. Ecol. 16 (2), 173–188. Li, W.K., 1990. Particles in “particle-free” seawater: growth of ultraphytoplankton and implications for dilution experiments. Can. J. Fish. Aquat. Sci. 47 (7), 1258–1268. Liu, H., Dagg, M., 2003. Interactions between nutrients, phytoplankton growth, and microand mesozooplankton grazing in the plume of the Mississippi River. Mar. Ecol. Prog. Ser. 258, 31–42. Mahoney, R.K., Gibson, R.A., 1983. Phytoplankton ecology of the Indian River near Vero Beach, Florida. Fla. Sci. 46, 212–232. Mayali, X., Azam, F., 2004. Algicidal bacteria in the sea and their impact on algal blooms. J. Eukaryot. Microbiol. 51 (2), 139–144. Modigh, M., Franzè, G., 2009. Changes in phytoplankton and microzooplankton populations during grazing experiments at a Mediterranean coastal site. J. Plankton Res. 31 (8), 853–864. Murrell, M.C., Stanley, R.S., Lores, E.M., DiDonato, G.T., Flemer, D.A., 2002. Linkage between microzooplankton grazing and phytoplankton growth in a Gulf of Mexico estuary. Estuaries 25 (1), 19–29. Nejstgaard, J., Gismervik, I., Solberg, P., 1997. Feeding and reproduction by Calanus finmarchicus, and microzooplankton grazing during mesocosm blooms of diatoms and the coccolithophore Emiliania huxleyi. Mar. Ecol. Prog. Ser. 147, 197–217. Nishibe, Y., Kobari, T., Ota, T., 2010. Feeding by the cyclopoid copepod Oithona similis on the microplankton assemblage in the Oyashio region during spring. Plankon Benthos Res. 5 (2), 74–78. Phlips, E., Badylak, S., Christman, M., Lasi, M., 2010. Climatic trends and temporal patterns of phytoplankton composition, abundance, and succession in the Indian River Lagoon, Florida, USA. Estuar. Coast. Shelf Sci. 33 (2), 498–512. Phlips, E., Badylak, S., Lasi, M., Chamberlain, R., Green, W., Hall, L., Hart, J., Lockwood, J., Miller, J., Morris, L., Steward, J., 2014. From red tides to green and brown tides: bloom dynamics in a restricted subtropical lagoon under shifting climatic conditions. Estuar. Coast. Shelf Sci. 1–19. Phlips, E.J., Badylak, S., Grosskopf, T., 2002. Factors affecting the abundance of phytoplankton in a restricted subtropical lagoon, the Indian River Lagoon, Florida, USA. Estuar. Coast. Shelf Sci. 55 (3), 385–402. Putland, J., Sutton, T., 2010. Microzooplankton grazing and productivity in the central and southern sector of the Indian River Lagoon, Florida. Fla. Sci. 73 (3), 236. Quinlan, E.L., Jett, C.H., Phlips, E.J., 2009. Microzooplankton grazing and the control of phytoplankton biomass in the Suwannee River estuary, USA. Hydrobiologia 632 (1), 127–137. Redden, A.M., Sanderson, B.G., Rissik, D., 2002. Extending the analysis of the dilution method to obtain the phytoplankton concentration at which microzooplankton grazing becomes saturated. Mar. Ecol. Prog. Ser. 226, 27–33. Redfield, A., Ketchum, B., Richards, F., 1963. The influence of organisms on the composition of sea-water. In: Hill, M. (Ed.), The Composition of Sea-water Comparative and Descriptive Oceanography. The Sea 2. Interscience Publishers, New York. Reynolds, C.S., 2006. Ecology of Phytoplankton. Cambridge University Press, Cambridge. Sabatini, M., Kiorboe, T., 1994. Egg production, growth and development of the cyclopoid copepod Oithona similis. J. Plankton Res. 16 (10), 1329–1351. Sartory, D.P., Grobbelaar, J.U., 1984. Extraction of chlorophyll a from freshwater phytoplankton for spectrophotometric analysis. Hydrobiologia 114 (3), 177–187.

10

N. Dix, M.D. Hanisak / Journal of Experimental Marine Biology and Ecology 465 (2015) 1–10

Schmoker, C., Hernández-León, S., Calbet, A., 2013. Microzooplankton grazing in the oceans: impacts, data variability, knowledge gaps and future directions. J. Plankton Res. 35 (4), 691–706. Sherr, E., Sherr, B., 2002. Significance of predation by protists in aquatic microbial food webs. Anton Leeuw. Int. J. G. 81 (1), 293–308. Smith, N., 2001. Seasonal-scale transport patterns in a multi-inlet coastal lagoon. Estuar. Coast. Shelf Sci. 52 (1), 15–28. Smith, N.P., 1993. Tidal and nontidal flushing of Florida's Indian River Lagoon. Estuaries 16 (4), 739–746. Strom, S.L., Macri, E.L., Olson, M.B., 2007. Microzooplankton grazing in the coastal Gulf of Alaska: variations in top-down control of phytoplankton. Limnol. Oceanogr. 52 (4), 1480. Teixeira, I.G., Figueiras, F.G., 2009. Feeding behaviour and non-linear responses in dilution experiments in a coastal upwelling system. Aquat. Microb. Ecol. 55, 53–63.

Youngbluth, M., Gibson, R., Blades, P., Meyer, D., Stephens, C., Mahoney, R., 1977. Plankton in the Indian River Lagoon. In: Young, D.K. (Ed.), Indian River Coastal Zone Study Third Annual Reort. Harbor Branch Consortium, pp. 40–60. Zamora-Terol, S., Saiz, E., 2013. Effects of food concentration on egg production and feeding rates of the cyclopoid copepod Oithona davisae. Limnol. Oceanogr. 58 (1), 376–387. Zhang, W., Xiao, T., Wang, R., 2001. Abundance and biomass of copepod nauplii and ciliates and herbivorous activity of microzooplankton in the East China Sea. Plankton Biol. Ecol. 48 (1), 28–34. Zöllner, E., Hoppe, H.-G., Sommer, U., Jürgens, K., 2009. Effect of zooplankton-mediated trophic cascades on marine microbial food web components (bacteria, nanoflagellates, ciliates). Limnol. Oceanogr. 54 (1), 262–275.