Selectivity and grazing impact of microzooplankton on phytoplankton in two subtropical semi-enclosed bays with different chlorophyll concentrations

Journal of Experimental Marine Biology and Ecology 390 (2010) 149–159

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Journal of Experimental Marine Biology and Ecology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j e m b e

Selectivity and grazing impact of microzooplankton on phytoplankton in two subtropical semi-enclosed bays with different chlorophyll concentrations Alle A.Y. Lie, C. Kim Wong ⁎ Department of Biology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong

a r t i c l e

i n f o

Article history: Received 6 August 2009 Received in revised form 9 September 2009 Accepted 4 May 2010 Keywords: Dilution method HPLC pigments Microzooplankton grazing Size fractionation

a b s t r a c t Microzooplankton grazing rates were compared between two sites (S1 and S2) in the coastal seas of eastern Hong Kong with similar physio-chemical parameters, but different chlorophyll concentrations. During the period from March 2007 to January 2008, six sets of dilution experiments, combined with high performance liquid chromatography and phytoplankton size fractionation (b 200 μm, b 20 μm and b 5 μm), were carried out to study the microzooplankton grazing rate on phytoplankton of different taxonomic groups and sizes. Although total chlorophyll a concentrations were much higher in S1 (4.98–18.42 μg l− 1) than in S2 (0.29– 1.68 μg l− 1), size composition of phytoplankton was relatively similar between the two sites. Measured as chlorophyll a, phytoplankton growth rates (− 0.84–1.91 d− 1 in S1; 0.03–2.85 d− 1 in S2) and microzooplankton grazing rates (0.00–2.26 d− 1 in S1; 0.00–1.49 d− 1 in S2) for all three size fractions were similar between the two bays. Phytoplankton growth rates and microzooplankton grazing rates measured as other pigments for phytoplankton of different size fractions did not show strong variations. Microzooplankton grazing impact, expressed as the ratio of microzooplankton grazing rate to phytoplankton growth rate, was generally higher in S1 than in S2, although the difference was not statistically significant. High microzooplankton grazing impact on alloxanthin (1.00–45.85) suggested strong selection toward cryptophytes. Our results provided no evidence for size selective grazing on phytoplankton by microzooplankton. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Tolo Harbour lies on the northeastern corner of Hong Kong and is well-known for its severe eutrophication and almost continuous algal blooms during the 1970s and 1980s (Lam and Ho, 1989; HKEPD, 2006). Tolo Harbour opens into Mirs Bay, a much bigger bay that is continuously flushed by water currents from the open ocean. Measures to reduce nutrient loading into Tolo Harbour were implemented in the 1990s (HKEPD, 2006). In recent years, the concentrations of nitrogen and phosphorus in Tolo Harbour have declined to levels comparable to those in Mirs Bay (HKEPD, 2006; HKEPD, 2007), but despite the decrease in nutrient concentrations, phytoplankton biomass and the number of recorded algal bloom occurrences are still much higher in Tolo Harbour than in Mirs Bay (HKEPD, 2006; Liu and Wong, 2006; HKEPD, 2007). While large stores of nutrients in bottom sediments may sustain algal growth, the roles of grazers may also impact the dynamics of these semi-enclosed bays, but such studies have not been conducted in this region. Microzooplankton, such as ciliates, flagellates and copepod nauplii, have long been considered as important grazers of phytoplankton

⁎ Corresponding author. Tel.: + 852 26096771; fax: +852 26035391. E-mail address: [email protected] (C.K. Wong). 0022-0981/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2010.05.001

(Pomeroy, 1974) because of their high abundance, ubiquitous distributions, fast growth rates (e.g. Jonsson, 1986; Bernard and Rassoulzadegan, 1990), and ability to ingest a large size range of food particles (e.g. Hansen et al., 1994; Peters, 1994). Calbet and Landry (2004) have shown that microzooplankton grazing can remove on average ∼60% of the phytoplankton primary production in coastal and estuarine systems, and impose a higher grazing impact on phytoplankton than mesozooplankton. Despite their feeding plasticity, microzooplankters are often considered to feed primarily on smaller phytoplankton (Burkill et al., 1995; Froneman and McQuaid, 1997; Palomares-Garcia et al., 2006; Calbet, 2008). Ciliates in particular feed optimally on prey that are ∼ 8–10× smaller than themselves, even though the size ratio between predator and prey can vary from 1:1 to 30:1 (Hansen et al., 1994). This is possibly due to an increase in handling time and a decrease in feeding efficiency when prey size is not optimal, leading to energetic costs (Peters, 1994). As important grazers of phytoplankton, microzooplankton can significantly affect the structure, composition and biomass of phytoplankton communities (Burkill et al., 1987; Strom and Welschmeyer, 1991; Suzuki et al., 2002; Irigoien et al., 2005; Calbet, 2008). Because Tolo Harbour and Mirs Bay have relatively similar nutrient concentrations and physio-chemical characteristics, microzooplankton grazing may potentially be an important factor that contributes to the differences in phytoplankton biomass between the two bays. The first

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objective of this study is to compare the microzooplankton grazing rates between Tolo Harbour and Mirs Bay. Higher microzooplankton grazing rates in Mirs Bay than in Tolo Harbour would provide evidence for top-down control of phytoplankton biomass. Previous studies on the taxonomic composition of phytoplankton in Tolo Harbour and Mirs Bay have focused predominately on large and easily identifiable groups, such as diatoms and dinoflagellates (Lam and Ho, 1989; HKEPD, 2006; HKEPD, 2007), while little is known about the size composition of the phytoplankton community (Yung et al., 1997; Wong and Wong, 2004). Because areas of low chlorophyll biomass are often dominated by small-sized phytoplankton (Chisholm, 1992), the phytoplankton community in Mirs Bay may be dominated by smaller phytoplankton and may be more susceptible to microzooplankton grazers. Microzooplankton grazing rate may also vary among phytoplankton of different taxonomic groups due to selective grazing (Burkill et al., 1987; Strom and Welschmeyer, 1991; Suzuki et al., 2002). While previous studies on microzooplankton grazers have not revealed selectivity on any particular groups of phytoplankton, preference on phytoplankton groups with higher growth rates has been documented (e.g. Burkill et al., 1987; Strom and Welschmeyer, 1991; Gaul and Antia, 2001; Strom, 2002). Hence the second objective of this study is to measure microzooplankton grazing rates on phytoplankton of different size fractions and taxonomic groups. Phytoplankton growth rates and microzooplankton grazing rates were estimated using the dilution method (Landry and Hassett, 1982). High performance liquid chromatography (HPLC) (Burkill et al., 1987) and phytoplankton size fractionation (e.g. Strom et al., 2001; Safi et al., 2007) were combined with the dilution method to estimate the microzooplankton grazing rate on phytoplankton of different taxonomic groups and size fractions. The incorporation of HPLC with the dilution method allows the measurements of pigmentspecific microzooplankton grazing rates (Burkill et al., 1987). As some phytoplankton pigments can be used as chemotaxonomic markers of certain phytoplankton taxonomic groups (Jeffrey et al., 1997), this method provides a rapid and effective way to obtain information on

the interactions between microzooplankton grazers and phytoplankton. The phytoplankton community was divided into three different size fractions, b200 μm, b20 μm and b5 μm, to provide information on microzooplankton grazing on the overall phytoplankton, nanophytoplankton and picophytoplankton communities respectively. 2. Materials and methods Six sets of dilution experiments were conducted between March 2007 and January 2008 at S1 (22°26′N; 114°13′E) and S2 (22°27′N; 114°27′E), two fixed sites located in Tolo Harbour and Mirs Bay respectively (Fig. 1). Temperature, salinity and dissolved oxygen (DO) at the surface (0.5 m) were measured using a Hydrolab H20 water quality monitoring system (Hydrolab Corporation). Surface seawater for dilution experiments was collected with a 3 l plastic bucket and filtered through a 200 μm mesh to remove mesozooplankton. Water samples (250 ml) for nutrient analysis were filtered through Whatman GF/F glass-fiber filters (0.7 μm pore size, 47 mm diameter) to remove all particles, and frozen at − 20 °C until analysis. Concentration of ammonium, nitrite and nitrate, silica, and phosphate were analyzed with a SKALAR Continuous Flow Analyzer (SKALAR Analytical). Particle-free seawater (FSW) was prepared by filtering seawater through a Millipore 0.22 μm membrane filter. FSW was used to dilute unfiltered seawater (UFSW) into four different dilutions (25 UFSW:75 FSW, 50 UFSW:50 FSW, 75 UFSW:25 FSW and 100 UFSW:0 FSW). Nutrients were added to provide a final concentration of 20 μM NO3and 1 μM PO42– to prevent nutrient depletion during incubation. Incubations without nutrient addition (unenriched incubations) were also carried out using 100 UFSW:0 FSW to determine phytoplankton growth rates under ambient nutrient conditions. Dilution experiments were carried out in 1.2 l glass bottles with duplicates for all incubations. The bottles were incubated for 24 or 48 h at the surface (∼0.5 m) in an outdoor tank (∼4 m diameter; ∼ 2 m deep) containing natural seawater from Tolo Harbour (Fig. 1). Dilution experiments for S2 usually lasted 48 h to provide higher final pigment concentrations

Fig. 1. A map of Hong Kong: the two study sites are located in Tolo Harbour (S1) and Mirs Bay (S2). Dilution experiments were conducted at IS.

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for HPLC analysis. All apparatus used were washed with 10% HCl, then rinsed with Milli-Q H2O and finally rinsed with FSW prior to the experiments to remove any nutrients adhered on the surface. Seawater samples were filtered through meshes of different sizes to collect phytoplankton of different size fractions (b200 μm, b20 μm and b5 μm) before cells were eventually collected on Whatman GF/F glass-fiber filters (0.7 μm pore size, 47 mm diameter). The filters were blotted dry and stored at −80 °C until pigment extraction. Pigments were extracted in 90% HPLC grade acetone, sonicated for 30 min and stored in darkness at 4 °C for 24 h. Extracts were centrifuged and passed through a NALGENE syringe filter with PTFE membrane (0.2 μm pore size, 13 mm diameter) before injection into the HPLC machine (Hewlett Packard HP 1100 series). Pigments were separated using 80 HPLC grade methanol:20 0.5 M ammonium acetate as solvent A, 90 HPLC grade acetonitrile: 10 Milli-Q water as solvent B, and pure HPLC grade ethyl acetate as solvent C. Pigment concentrations in the 5– 20 μm and 20–200 μm size fractions were calculated by subtracting the b5 μm concentration from the b20 μm concentration and the b20 μm concentration from the b200 μm concentration, respectively. A 250 ml subsample of the b200 μm size fraction from each 100 UFSW:0 FSW incubation was preserved in Lugol's solution (2% final concentration) for identification and enumeration of phytoplankton and microzooplankton. All samples were stored in darkness at 4 °C until analysis. Samples for analysis were concentrated (5–10×) by sedimentation, transferred to a Sedgwick–Rafter counting chamber and counted under an inverted microscope. Pigment-specific phytoplankton growth rates under nutrient enrichment (μn) and microzooplankton grazing rates (g) were estimated by the methods described by Landry and Hassett (1982). In the cases of positive slopes and non-significant grazing rate estimates (p N 0.05), g was assumed to be 0 d− 1 and μn was obtained from the pigment-specific apparent growth rate of the enriched 100 UFSW:0 FSW incubations. The pigment-specific phytoplankton growth rates in ambient nutrient conditions (µ0) were calculated by adding g to the pigment-specific apparent growth rate of unenriched incubations. The percentage of the pigment-specific phytoplankton standing stock grazed (SS) by microzooplankton was calculated using the equation (Safi et al., 2007): −g


SS = 1−e

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Table 1 Physio-chemical parameters of surface seawater at S1 and S2 during the study period from March 2007 to January 2008. Site Date

Salinity NH4+ NO2-+NO3- PO42- SiO2 Temperature DO (μM) (μM) (μM) (μM) (°C) (mg l−1)

S1 15 Mar 07 3 May 07 27 Aug 07 20 Sep 07 8 Nov 07 11 Jan 08 S2 29 Mar 07 9 May 07 30 Aug 07 28 Sep 07 15 Nov 07 17 Jan 08

19.9 25.2 28.3 27.7 22.8 21.0 20.6 24.2 28.9 28.1 22.7 17.4

8.9 8.5 8.7 6.5 7.8 10.5 8.8 8.3 7.1 6.5 8.0 9.3

32.2 31.4 28.5 30.4 30.6 32.0 34.0 34.1 31.3 32.8 33.1 32.8

2.2 2.2 2.8 1.7 1.7 2.2 1.7 2.2 1.7 2.2 1.7 3.3

6.5 5.9 5.7 5.7 5.4 5.7 5.4 5.9 5.7 5.2 6.7 5.7

4.2 2.3 3.9 3.1 4.1 4.8 3.5 4.7 2.8 4.4 3.1 3.2

27.4 5.3 27.8 22.1 15.3 16.7 14.2 22.8 12.8 23.1 22.1 30.3

most of the study period. The largest size fraction (20–200 μm) never constituted more than 50% in the samples collected from S2, and was the dominant size fraction (N50%) only in the samples collected from S1 during the warmest months of August and September. Concentrations of accessory pigments were also much higher in S1 than in S2

× 100

The percentage of the pigment-specific phytoplankton production grazed (PG) was calculated using the equation (Safi et al., 2007): −g


PG = 100 × 1−e

= ð1–e

−μ0

Þ

3. Results 3.1. Field conditions and initial stock and composition Surface temperatures and DO were almost similar between the two sites (Table 1). Surface water temperature rarely fell below 20 °C, and was nearly 30 °C in the hottest months of August and September. Salinity was slightly lower in S1, and dropped to a minimum in August at both sites (Table 1). Surface water nutrient concentrations were comparable between the two sites (Table 1). Ammonium, nitrite and nitrate, silica, and phosphate exhibited similar variation patterns but did not show discernable seasonal and spatial trends. Total chlorophyll a concentrations in S1 were about 3–20× higher than in S2 (Fig. 2). Chlorophyll a concentrations at both sites tended to be higher during the colder months, except for a peak recorded in September at S2. Despite the large difference in total chlorophyll a concentrations, contribution by phytoplankton of different size fractions seemed to be quite similar between the two sites during

Fig. 2. Seasonal changes in chlorophyll a concentrations for phytoplankton in the 20– 200 μm, 5–20 μm and b 5 μm size fractions.

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(Fig. 3). As expected, pigments that are associated with small phytoplankton, such as alloxanthin (cryptophytes), zeaxanthin (cyanobacteria), and chlorophyll b (green algae), tended to be most-prevalent in the b5 μm size fraction. Peridinin, a chemotaxonomic marker for dinoflagellates, was most abundant in the 20– 200 μm size fraction. Fucoxanthin, a marker for diatoms, was the most abundant accessory pigment at both sites throughout the entire study period. 3.2. Plankton community composition Phytoplankton densities were consistently higher in S1 than in S2. Averaged over the entire study period, phytoplankton densities were in the order of 105 cells ml− 1 in S1 and 103 cells ml− 1 in S2. Diatoms represented the most-dominant taxa in both S1 and S2, and the prevalence of small diatoms was confirmed by the presence of high proportions of fucoxanthin in the b5 μm size fraction (Fig. 3). The diatoms Pseudo-nitzchia, Chaetoceros, Leptocylindrus, and Skeletonema were common in both sites. Dinoflagellates common at both sites included Prorocentrum, Heterocapsa, Karenia, and Scrippsiella. Cryptophytes were also commonly found in both sites, but their densities (average: 297 ± 144 cells ml− 1 in S1; 56 ± 85 cells ml− 1 in S2) were

Fig. 3. The average concentrations of various pigments (excluding chlorophyll a) for phytoplankton in the 20–200 μm, 5–20 μm and b 5 μm size fractions.

extremely low compared to those of diatoms. Averaged over the entire study period, microzooplankton densities were higher in S1 (45 ± 24 individuals ml− 1) than in S2 (12 ± 9 individuals ml− 1). The most common microzooplankton grazers found were ciliates, but heterotrophic dinoflagellates, such as those from the genera Gyrodinium and Ceratium, were also common. Microscopic inspections of 100 UFSW:0 FSW incubations did not reveal any noticeable changes in microzooplankton community composition, but we cannot rule out the possibility of such changes (Agis et al., 2007), especially in treatments with long incubation time (e.g. 48 h). 3.3. Dilution experiment results Regression analyses for some dilution experiments produced positive slopes. For unknown reasons, positive slopes were encountered most frequently among dilution experiments carried out in January. Positive slopes also appeared to be common when the number of points available for regression analysis and the coefficient of determination (r2) were low. Pigment-specific phytoplankton growth rates under nutrient enrichment (μn) were usually positive due to nutrient enrichment (Fig. 4). Among all three size fractions, only 1 out of 36 μn for chlorophyll a was negative. For peridinin, 6 out of 28 measurable μn were negative. μn for all pigments were comparable between S1 and S2 and among the three size fractions. In November, however, μn for most pigments were lower in S1 than in S2. Alloxanthin was the only pigment that did not fit into this pattern. In general, μn for alloxanthin was less variable than those of other pigments. Alloxanthin also had slightly lower μn compared to the other pigments, especially in S1, where the maximum μn for alloxanthin was ∼3× lower than the maximum μn for other pigments. As expected, μ0, the pigment-specific phytoplankton growth rates in ambient nutrient conditions, tended to be lower than μn for all pigments (Fig. 5), especially for the S1 incubations. Negative μ0 was recorded for all pigments measured. Among all size fractions, 6 out of 36 μ0 for chlorophyll a were negative. For peridinin, 12 out of 29 measurable μ0 were negative. Negative μ0 occurred mostly when g was assumed to be 0 d− 1. Paired t-tests showed that the values of μ0 and μn for all pigments, including chlorophyll a, did not differ significantly between S1 and S2 (p N 0.05). For all pigments, the seasonal patterns exhibited by μ0 appeared to be similar to that observed for μn. The pigment-specific microzooplankton grazing rate (g) for most pigments did not differ between the two sites and among the three size fractions (Fig. 6). Indeed, g and μ for most pigments exhibited similar seasonal trends such as higher rates in August and lower rates in January. A noticeable exception included chlorophyll a in the b200 μm size fraction in S1. In addition, g for pigments, including chlorophyll a, fucoxanthin and 19'-hex-fucoxanthin, at S2 in November were not very high even when their μ values had reached peaks. For all pigments, including chlorophyll a, paired t-tests showed no significant difference in g between S1 and S2 (p N 0.05). g/μ0 for fucoxanthin, alloxanthin and chlorophyll a seemed higher in S1 than in S2 (Fig. 7), but the difference was not statistically significant (paired t-test, p N 0.05). Exceptionally high g/μ0 ratios were recorded in S1 for alloxanthin (45.8 and 44.6) and fucoxanthin (45.0) when μ0 were extremely low. g/μ0 ratios for alloxanthin were high at both sites, with all values ≥1, except in cases where no grazing was detected. Although g/μ0 were often lower for the b200 μm size fraction than the b20 μm and b5 μm size fractions, the trend was neither obvious nor consistent throughout the study period. For most pigments, g tended to correlate significantly with its corresponding μn and μ0 at both sites (Pearson's correlation, p b 0.05). Despite its comparatively larger sample size (n = 10), correlation between g and μ for alloxanthin was not as strong as those of other pigments. When g was plotted against μ0, the regression line approximated the equilibrium curve (g = μ0) better at S1 than at S2 (Fig. 8).

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Fig. 4. Seasonal changes in pigment-specific phytoplankton growth rates under nutrient enrichment (μn) for phytoplankton in the b200 μm, b20 μm and b5 μm size fractions: dilution experiments were conducted in S1 and S2 between March 2007 and January 2008. Pigments measured included peridinin, fucoxanthin, 19'-hex-fucoxanthin, alloxanthin, zeaxanthin, chlorophyll b and chlorophyll a. Missing data appeared when pigment concentrations were too low to be detected by HPLC.

For various pigments, the percentages of standing stock grazed (SS) were 24.5–92.0% for the b200 μm size fraction, 30.7–97.6% for the b20 μm size fraction and 41.4–89.6% for the b5 μm size fraction in S1. In S2, the percentages were 20.4–77.2% for the b200 μm size fraction, 38.4–80.2% for the b20 μm size fraction and 47.9–80.6% for the b5 μm size fraction. SS measured in terms of chlorophyll a are summarized in Table 2, in which the average values for the b200 μm size fraction were 64.6% in S1 and 60.0% in S2. The percentage of production grazed (PG) was usually higher in S1 than in S2, but the difference was not statistically significant (Mann–Whitney rank sum test, p N 0.05). In S1, the values were 90.4–3508% for the b200 μm size fraction, − 559– 797% for the b20 μm size fraction and 90.1–3574% for the b5 μm size fraction. In S2, the percentages were 56.6–283% for the b200 μm size fraction, 49.7–167% for the b20 μm size fraction and 66.4–150% for the b5 μm size fraction. At both S1 and S2, the maximum values came from alloxanthin. Three negative PG values were produced in the b20 μm size fraction in S1 when μ0 was negative. For chlorophyll a in the b200 μm size fraction, PG averaged 163% in S1 and 81.4% in S2 (Table 2). Temperature, salinity and DO correlated significantly with several of the μn for the b20 μm and b200 μm size fractions (Pearson's correlation, p b 0.05). In contrast, μ0 did not correlate with temperature, salinity and DO (Pearson's correlation, p N 0.05). The importance of nitrite and nitrate concentrations on μ0 was demonstrated by the

presence of significant correlation (Pearson's correlation, p b 0.05) between these nutrients and the μ0 of several pigments from all size fractions. Temperature, along with salinity and DO also correlated significantly with the g of some pigments from various size fractions (Pearson's correlation, p b 0.05). 4. Discussion 4.1. Comparison between the sites The study was conducted at two sites with different chlorophyll a concentrations. Physio-chemical parameters including temperature, DO, salinity and nutrients were quite similar between the two sites. According to Chisholm (1992), waters with low chlorophyll levels tend to be dominated by small phytoplankton. However, the size compositions of phytoplankton in the two sites were similar. In S1, the site with much higher chlorophyll a concentrations, the phytoplankton community was dominated by the largest size fraction (20– 200 μm) only during August and September. Since Chisholm's rule may be more applicable to open ocean systems where more data are available (e.g. Le Bouteiller et al., 1992; Blanchot and Rodier, 1996; Marañón et al., 2000), it can be argued that nutrient concentrations in S2, the site with lower chlorophyll a concentrations, may be high enough to support a phytoplankton community with a relatively high

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Fig. 5. Seasonal changes in pigment-specific phytoplankton growth rates in ambient nutrient conditions (μ0) for phytoplankton in the b200 μm, b20 μm and b5 μm size fractions: dilution experiments were conducted in S1 and S2 between March 2007 and January 2008. Pigments measured included peridinin, fucoxanthin, 19'-hex-fucoxanthin, alloxanthin, zeaxanthin, chlorophyll b and chlorophyll a. Missing data appeared when pigment concentrations were too low to be detected by HPLC.

proportion of large phytoplankton. Nitrite and nitrate concentrations at both S1 and S2 were N5 μM (Table 1). Indeed, according to the three-dimensional growth rate model of Parsons and Takahashi (1973), 2.2 μM of nitrogen will be enough for the large phytoplankton Dithylum brightwellii to grow faster than the smaller phytoplankton Coccolithus huxleyi under high light intensity. Alternatively, since the density of copepods was higher in Tolo Harbour than in Mirs Bay (Wong unpublished data), selective grazing by copepods may reduce the abundance of large-sized phytoplankton in S1. Paracalanus and Oithona, the most abundant mesozooplankton grazers in Tolo Harbour (Wong et al., 1993), have been shown to be able to feed on or even prefer food particles N20 μm (Paffenhöfer, 1984; Tsuda and Nemoto, 1988; Castellani et al., 2005). Unfortunately, little information is available on the grazing impact of these copepods on the phytoplankton in Tolo Harbour. We had speculated that the difference in phytoplankton biomass between the two bays, Tolo Harbour (S1) and Mirs Bay (S2) was due to microzooplankton grazing preferences toward smaller-sized phytoplankton, leading to a lower grazing rate (g) or grazing impact (g/μ0) in systems dominated by large phytoplankton, and consequently allowing the formation of higher phytoplankton biomass. This was not supported by our results. During August and September, when proportion of chlorophyll a in the 20–200 μm size fraction was high in S1, chlorophyll a concentrations there were low, and g and g/μ0 were also not evidently lower when compared to values recorded in

other months at both S1 and S2. Our results also provided no evidence of size selectivity in microzooplankton grazing. Therefore, the large difference in phytoplankton biomass between Tolo Harbour and Mirs Bay could not have been due to differences in microzooplankton grazing rates or grazing patterns. S1 was located in Tolo Harbour, a landlocked bay with poor tidal flushing (Lam and Ho, 1989). Long water residence time, poor vertical mixing and lack of horizontal transport may allow phytoplankton biomass to increase rapidly without being diluted by water masses of low phytoplankton biomass.

4.2. Phytoplankton growth rates and microzooplankton grazing rates Many investigators have reported negative phytoplankton growth rates in dilution experiments (e.g. Strom and Strom, 1996; James and Hall, 1998; Kim et al., 2007). Since µ0 was assumed to be the sum of the apparent phytoplankton growth and g, negative values for µ0 appeared most often when g was 0 d− 1. Correlation between μ0 and nitrite and nitrate concentrations suggested that local phytoplankton growth was limited by nitrogen, specifically nitrate (Lee and Arega, 1999; Arega and Lee, 2000). Peridinin, a marker of dinoflagellates, produced some negative μn, especially at S1. But dinoflagellates may be heterotrophic or mixotrophic instead of photoautotrophic, especially in warm tropical waters (Jeffrey and Vesk, 1997) and thus may not benefit much from the addition of nutrients.

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Fig. 6. Seasonal changes in pigment-specific microzooplankton grazing rates (g) for phytoplankton in the b200 μm, b 20 μm and b 5 μm size fractions: dilution experiments were conducted in S1 and S2 between March 2007 and January 2008. Pigments available for analysis included peridinin, fucoxanthin, 19'-hex-fucoxanthin, alloxanthin, chlorophyll b and chlorophyll a. Missing data appeared when pigment concentrations were too low to be detected by HPLC.

Our g of 0.00–2.26 d− 1 for chlorophyll a fits into the reported range of 0.00–3.86 d− 1 for dilution experiments carried out in subtropical or coastal waters (e.g. Strom and Strom, 1996; Landry et al., 1998; Ruiz et al., 1998; Landry and Calbet, 2004; Kim et al., 2007; Palomares-Garcia et al., 2006). Our lowest μ0 of −0.84–2.85 d− 1, however, was lower than the reported range of − 0.63–3.41 d− 1. For August, our g of 2.26 d− 1 in S1 and 1.48 d− 1 in S2 for chlorophyll a in the b200 μm size fraction were higher than the 0.71 d− 1 and 0.56 d− 1 reported by Sun et al. (2003) from dilution experiments conducted in waters in the west and southeastern parts of Hong Kong in August 2000. Chen et al. (2009) conducted dilution experiments at sites used by Sun et al. (2003) during the period from February 2007 to February 2008. Their ranges of 0.02–2.94d− 1 for μn and 0–1.88 d− 1 for g for chlorophyll a in the b200 μm size fraction were comparable to μn of 0.31–2.87 d− 1 and g of 0–1.57 d− 1 obtained in this study. On the other hand, our μ0 of −0.34–2.85 d− 1 for chlorophyll a in the b200 μm fraction was apparently larger than that presented by Chen et al. (2009). Temperature is often regarded as an important factor affecting microzooplankton grazing, and often correlated with grazing rates (Peters, 1994; Caron et al., 2000; Strom et al., 2001; Obayashi and Tanoue, 2002). The absence of strong correlation between g and physio-chemical parameters such as temperature and salinity in our results suggested, as explained by Caron et al. (2000), that g at any particular condition may also be influenced by other factors such as

prey and grazer abundance. For example, ciliates have a wide range of sensory capabilities, and may be able to respond to different stimuli and environmental conditions (Fenchel and Jonsson, 1988). Physiochemical parameters also had little effect on the level of microzooplankton grazing control in our study, as indicated by the lack of correlation between many of the physio-chemical parameters and the g/μ0 ratios of various size fractions and pigments.

4.3. Microzooplankton selectivity Previous dilution experiments have produced conflicting results on microzooplankton grazing on different phytoplankton size fractions. While Froneman and McQuaid (1997) and Zhang et al. (2005) reported slightly higher grazing rates on smaller phytoplankton size fractions, Strom et al. (2001) and Safi et al. (2007) did not find such pattern. Our own results also did not show any substantial evidence for size selective grazing by microzooplankton. The g for the b20 μm and b5 μm size fractions were often higher than that for the b200 μm size fraction, but the pattern was not consistent. Froneman and Perissinotto (1996a,b) studied size selective grazing by microzooplankton in dilution experiments by comparing the percentage change in different size fractions, and found that nanophytoplankton and picophytoplankton usually had negative percentage changes. However, even when our own data were analyzed in this way (data

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Fig. 7. Seasonal changes in the ratios of pigment-specific microzooplankton grazing rates (g) to the pigment-specific phytoplankton growth rates in ambient nutrient conditions (μ0) for phytoplankton in the b200 μm, b20 μm and b5 μm size fractions: dilution experiments were conducted in S1 and S2 between March 2007 and January 2008. Pigments available for analysis included peridinin, fucoxanthin, 19'-hex-fucoxanthin, alloxanthin, chlorophyll b and chlorophyll a. Missing data appeared when pigment concentrations were too low to be detected by HPLC.

not shown), there were no consistent patterns in percentage changes among different size fractions. Microzooplankton exhibited strong preference toward cryptophytes. g/μ0 values for alloxanthin were consistently ≥1. Cryptophytes have also been found to be strongly preferred by the marine cladoceran Penilia avirostris in Tolo Harbour (Wong et al., 2006). Cryptophytes are used by dinoflagellates as a source of kleptochloroplasts (Eriksen et al., 2002). The ciliate Mesodinium rubrum, once considered exclusively photosynthetic, has also been found to feed on cryptophytes (Gustafson et al., 2000; Yih et al., 2004). To our knowledge however, there is no previous dilution experiment reporting microzooplankton preference on cryptophytes specifically. Some investigators reported that microzooplankton grazers prefer fast-growing phytoplankton groups, regardless of their abundance (Burkill et al., 1987; Strom and Welschmeyer, 1991; Gaul and Antia, 2001; Strom, 2002). In our study, this pattern is supported by the positive correlation between μ0 and g which can be interpreted as an indication of the ability of microzooplankton to respond to changes in phytoplankton growth rates. Such behavioural capability in microzooplankton may provide tight coupling between phytoplankton growth and microzooplankton grazing (e.g. Strom and Welschmeyer,

1991; Kim et al., 2007; Safi et al., 2007). As the regression curve produced by g/μ0 fitted an equilibrium curve better in S1 than in S2 (Fig. 8), microzooplankton grazing responses to changes to phytoplankton growth may be stronger in S1 than in S2. 4.4. Food web dynamics Safi et al. (2007) suggested that if microzooplankton grazed selectively on small phytoplankton, large phytoplankton would be left to sink, transferring the carbon and nutrient contents they contain to bottom waters. Although the phytoplankton communities at our study sites were often dominated by cells b5 μm, microzooplankton exhibited no size selectivity. Nutrients should therefore mainly be recycled, especially when the microzooplankters were the major consumers of phytoplankton. Since microzooplankton fecal pellets are either unconsolidated or slow-sinking (Buck and Newton, 1995), nutrient regeneration from these fecal pellets may occur mostly in the euphotic zone (Sieburth et al., 1978). The increasing evidence of mesozooplankton feeding preferentially on microzooplankton due to their optimal size, nutritional values and swimming behavior (e.g. Gasparini et al., 2000; Henjes

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Fig. 8. The relationship between pigment-specific microzooplankton grazing rate (g) and pigment-specific phytoplankton growth rate in ambient nutrient conditions (μ0) in S1 and S2: black: b 200 μm, gray: b20 μm, and white: b5 μm. Solid lines: regression lines. Dotted line: equilibrium curve (g = μ0). Data for various pigments and phytoplankton size fractions were lumped to obtain the regression lines. Points with g assumed to be 0 d− 1 were excluded from the regression analysis. p b 0.01 for both regressions.

et al., 2007; Calbet, 2008) adds to the importance of the role played by microzooplankton in transferring energy to higher trophic levels. Since the abundance of mesozooplankton is higher in Tolo Harbour than in Mirs Bay (Wong unpublished data) and microzooplankton grazing impacts based on g/μ0 values obtained in this study were also higher in S1 than in S2, microzooplankton may play a heavier role in transferring energy to higher trophic levels in S1 than in S2. Measured in chlorophyll a and in cases where microzooplankton grazing was detectable, only 1 out of 12 g/μ0 values was b1 in S1, whereas 9 out of 13 g/μ0 values obtained in S2 were b1. Since the

values of g/μ0 were mostly N1 at S1, one might expect phytoplankton densities to be low in S1 due to higher grazing pressures. However, this was not the case. In fact, much higher phytoplankton biomass in S1 than in S2 implied that the in-situ grazing pressure in S1 may be reduced, most probably by the removal of microzooplankton by mesozooplankton. Dilution experiments are performed conventionally with the exclusion of mesozooplankton to study grazing by grazers b200 μm. (e.g. Landry and Hassett, 1982; Burkill et al., 1987; Landry, 1993). However, since mesozooplankton can affect the microbial food web

Table 2 Summary of the percentage of standing stock grazed (SS) and production grazed (PG) for chlorophyll a in six sets of dilution experiments conducted during the period from March 2007 to January 2008 in S1 and S2. Site

S1

S2

a b

Size fraction

Mar 07 SS (%)

PG (%)

May 07 SS (%)

PG (%)

Aug 07 SS (%)

PG (%)

Sep 07 SS (%)

PG (%)

Nov 07 SS (%)

PG (%)

SS (%)

PG (%)

b200 μm b 20 μm b5 μm b200 μm b 20 μm b5 μm

79.2 68.0 66.3 –a 54.2 47.9

97.6 111.2 102.7 –a 100.2 108.4

60.8 56.0 62.6 45.5 48.6 –a

120.1 319.2 135.1 58.9 68.2 –a

–a 88.0 89.6 76.4 63.7 77.4

–a 121.9 105.1 94.0 92.9 93.9

76.5 68.6 –a 47.3 52.9 73.8

298.1 210.3 –a 98.1 155.4 115.7

52.3 43.8 –a 70.4 71.4 70.6

198.0 − 501.2b –a 74.7 77.7 77.8

54.4 –a –a –a –a –a

100.8 –a –a –a –a –a

Microzooplankton grazing was undetectable. Negative value was due to a negative estimation of phytoplankton growth rate in ambient nutrient conditions (μ0).

Jan 08

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by selective feeding on microzooplankton, the exclusion of mesozooplankton does not accurately evaluate the balance between growth and grazing mortality of phytoplankton (Strom and Welschmeyer, 1991). It would therefore be interesting to design dilution experiments that would allow the measurement of microzooplankton grazing impacts on phytoplankton in the presence and absence of mesozooplankton. 5. Conclusion This was the first study of microzooplankton–phytoplankton interactions in the subtropical coastal waters of northeastern Hong Kong. The results revealed that despite the large differences in chlorophyll a concentrations, the size composition of the phytoplankton communities in the two study sites were relatively similar. Phytoplankton growth rates, microzooplankton grazing rates and grazing impact did not differ significantly between the two sites. Microzooplankton did not display size selectivity toward phytoplankton, but showed evidence of preference towards cryptophytes. Acknowledgements This paper is based on a thesis submitted by A.A.Y. Lie in partial fulfillment of the requirements for the MPhil degree in the Department of Biology, The Chinese University of Hong Kong. The work was funded by the Hong Kong RGC Research Grant-Direct Allocation to C.K. Wong (Project Number 476909). The authors would like to thank P. Tse, K.C. Cheung and Y.H. Yung for their technical assistance in the field. Suggestions and comments given by two anonymous reviewers are greatly appreciated. [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, 176–183. Arega, F., Lee, J.H.W., 2000. Long-term circulation and eutrophication model for Tolo Harbour, Hong Kong. Water Qual. Ecosyst. Model. 1, 169–192. Bernard, C., Rassoulzadegan, F., 1990. Bacteria or microflagellates as a major food source for marine ciliates: possible implications for the microzooplankton. Mar. Ecol. Prog. Ser. 64, 147–155. Blanchot, J., Rodier, M., 1996. Picophytoplankton abundance and biomass in the western tropical Pacific Ocean during the 1992 El Niño year: results from flow cytometry. Deep Sea Res. Part I 43, 877–895. Buck, K.R., Newton, J., 1995. Fecal pellet flux in Dabob Bay during a diatom bloom: contribution of microzooplankton. Limnol. Oceanogr. 40, 306–315. Burkill, P.H., Mantoura, R.F.C., Llewellyn, C.A., Owens, N.J.P., 1987. Microzooplankton grazing and selectivity of phytoplankton in coastal waters. Mar. Biol. 93, 581–590. Burkill, P.H., Edwards, E.S., Sleigh, M.A., 1995. Microzooplankton and their role in controlling phytoplankton growth in the marginal ice-zone of the Bellingshausen Sea. Deep Sea Res. Part II 42, 1277–1290. Calbet, A., 2008. The trophic roles of microzooplankton in marine systems. ICES J. Mar. Sci. 65, 325–331. Calbet, A., Landry, M.R., 2004. Phytoplankton growth, microzooplankton grazing, and carbon cycling in marine systems. Limnol. Oceanogr. 49, 51–57. Caron, D.A., Dennett, M.R., Lonsdale, D.J., Moran, D.M., Shalapyonok, L., 2000. Microzooplankton herbivory in the Ross Sea, Antarctica. Deep Sea Res. Part II 47, 3249–3272. 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. Chisholm, S.W., 1992. Phytoplankton size. In: Falkowski, P.G., Woodhead, A.D. (Eds.), Primary Productivity and Biogeochemical Cycles in the Sea. Plenum Press, New York, pp. 213–237. 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. Eriksen, N.T., Hayes, K.C., Lewitus, A.J., 2002. Growth responses of the mixotrophic dinoflagellates, Cryptoperidiniopsis sp. and Pfiesteria piscicida, to light under preysaturated conditions. Harmful Algae 1, 191–203. Fenchel, T., Jonsson, P.R., 1988. The functional biology of Strombidium sulcatum, a marine oligotrich ciliate (Ciliophora, Oligotrichina). Mar. Ecol. Prog. Ser. 48, 1–15. Froneman, P.W., McQuaid, C.D., 1997. Preliminary investigation of the ecological role of microzooplankton in the Kariega Estuary, South Africa. Estuar. Coast. Shelf Sci. 45, 689–695.

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