Changes in phytoplankton and microzooplankton ... - Oxford Journals

JOURNAL OF PLANKTON RESEARCH

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Changes in phytoplankton and microzooplankton populations during grazing experiments at a Mediterranean coastal site M. MODIGH* AND G. FRANZE` LABORATORY OF ECOLOGY AND EVOLUTION OF PLANKTON, STAZIONE ZOOLOGICA ANTON DOHRN, NAPOLI, ITALY

*CORRESPONDING AUTHOR: [email protected] Received September 9, 2008; accepted in principle April 22, 2009; accepted for publication April 23, 2009; published online 17 May, 2009 Corresponding editor: John Dolan

Microzooplankton grazing on phytoplankton populations was investigated by means of eight dilution experiments in different seasons and trophic conditions at station Mare Chiara, a longterm study site in the Gulf of Naples, Tyrrhenian Sea. To check for changes in prey and predator populations, size-fractionated chlorophyll and HPLC analyses and microzooplankton counts were performed before and after the incubations. On average, 68% of daily phytoplankton production was consumed by the microzooplankton in spring and summer, and in winter, more than 100% of phytoplankton daily production was consumed. Nutrients were added in four of our experiments. Diatoms (as recorded by fucoxanthin concentrations) increased significantly in the nutrient-enriched experiments, and unexpectedly, also heterotrophic dinoflagellate growth was enhanced by nutrient additions. Remarkable changes in grazer abundance were observed in several experiments; a substantial difference was found in microzooplankton grazing pressure (g/k 100) as calculated by regression of apparent chlorophyll a growth against nominal dilution factor or against average predator concentrations, 72 + 19% and 60 + 23% of phytoplankton daily production, respectively. However, our observations suggest that if growth and mortality of grazers in the dilution series are not considered, microzooplankton grazing impact may be either underestimated, at low phytoplankton growth rates, or overestimated in more productive conditions.

I N T RO D U C T I O N Microzooplankton, mainly ciliates and heterotrophic dinoflagellates but also small metazoan such as copepod nauplii, is an important component of pelagic ecosystems (Calbet, 2008). Owing to the relatively high abundances, when compared with the larger zooplankton, and to generation times that are similar to those of their algal prey, the microzooplankton is generally able to control phytoplankton assemblages (Irigoien et al., 2005). A recent review of several hundreds of grazing experiments indicated on average 67% of phytoplankton production (PP) being consumed by microzooplankton (Calbet and Landry, 2004). The review of Calbet and

Landry (2004) was followed by a debate whether microzooplankton consuming two-thirds of PP may represent an overestimation of the amount of carbon channeled through the microbial grazers (Dolan, 2004; Dolan and McKeon, 2005; Landry and Calbet, 2005). Estimates of microzooplankton grazing on phytoplankton are commonly carried out by means of the dilution method (Landry and Hassett, 1982). A basic assumption of the dilution method is that phytoplankton growth is independent of phytoplankton concentration, whereas there is a linear relationship between dilution factor and predator abundance and grazing pressure. However, growth or mortality of protozoan grazers may

doi:10.1093/plankt/fbp035, available online at www.plankt.oxfordjournals.org # The Author 2009. Published by Oxford University Press. All rights reserved. For permissions, please email: [email protected]


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occur in the incubation bottles and should be accounted for to obtain a proper evaluation of microzooplankton grazing rates (Dolan et al., 2000). A long-term study of the pelagic ecosystem is being carried out at Station Mare Chiara (MC) in the Gulf of Naples, Tyrrhenian Sea (Scotto di Carlo et al., 1985; Ribera d’Alcala` et al., 2004). Information on the composition and annual variability of microzooplankton in the Gulf of Naples has been reported (Modigh, 2001; Modigh and Castaldo, 2002), but there are no data on microzooplankton grazing impact and, in general, there are only a few studies on microzooplankton grazing in the Mediterranean Sea (Dolan et al., 2000; Fonda-Umani and Beran, 2003; Latasa et al., 2005; Calbet et al., 2008). We investigated microzooplankton grazing in different seasons and contrasting trophic conditions at Stn MC by means of dilution experiments. To evaluate microzooplankton grazing impact on phytoplankton biomass concentrations and composition, sizefractionated chlorophyll a and HPLC analyses were performed. Growth or mortality of micrograzers during the incubations may severely alter grazing rate estimates, thus a number of microzooplankton samples collected before and after the incubations were analyzed.

METHOD Eight microzooplankton grazing experiments were carried out at Stn MC (40848.500 N, 14814.900 E; 80 m depth) in the Gulf of Naples between 2004 and 2008. Microzooplankton grazing rates and phytoplankton growth rates were determined using the dilution method of Landry and Hassett (1982). The water for the experiments was collected at 1 m depth with an automatic Carousel sampler of SeaBird Electronics, equipped with 12 L Niskin bottles and a CTD (SBE 911 plus). The day prior to the experiment,

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water was collected at Stn MC, filtered (GF/F Whatman glass fiber filter; 47 mm) and stored cool and in the dark until the next day. On the morning of the experiment, water was collected at Stn MC, siphoned from the Niskin bottles into carboys by submerged silicon tubing and transported to the laboratory. All experimental equipment was soaked in 10% HCl overnight and thoroughly rinsed with distilled water. Screening for mesozooplankton was performed by gently passing the water through a 200 mm mesh plankton net into a large 150 L tank. To ensure the homogeneity of subsamples dispensed into the incubation bottles, the tank was gently and continuously mixed by means of two long glass rods. The filtered seawater was mixed with decreasing volumes of whole water (ww) from the tank to obtain duplicate series of five or six dilutions (undiluted—100%, 75%, 50%, 25%, 5% or 100%, 80%, 60%, 40%, 20%, 5% ww). Nutrients were added in the four experiments carried out in 2007/ 2008 (Table I) both to the natural sample and to the filtered seawater (Guillard’s f/2 medium, 2 mM NO3 and 0.1 mM PO4 added per liter). Incubations were made in 13 L polycarbonate carboys in a walk-in thermostat chamber where in situ temperature and light:dark cycles were simulated according to seasonal conditions. Cool-white fluorescent lights provided 150 mmol photons m22 s21 in the February and December experiments, and 250 mmol photons m22 s21 in the other experiments. All incubations were carried out for 24 h and, to avoid settling, the incubation bottles were gently turned upside down every 2–3 h during daytime. Phytoplankton was generally dominated by small diatoms and phytoflagellates (Diana Sarno, Zoological Station of Naples, unpublished data), as has been reported for most of the year at Stn MC (Ribera d’Alcala` et al., 2004), thus sedimentation should have been minimal during the night. Samples for inorganic nutrient analyses were collected from the initial batch before dispensing the water

Table I: In situ conditions at the time of our experiments and TIN (NO3 þ NO2 þ NH4) concentrations at T24 Experiment

Nutrients added

T0

In situ TIN (mM)

T24 100% ww TIN (mM)

Chl (mg L21)

Cil (Cells L21)

Dino (Cells L21)

Np (ind. L21)

May 2004 June 2004 September 2004 March 2005 February 2007 July 2007 December 2007 February 2008

No No No No Yes Yes Yes Yes

18 25.5 24.5 14.2 15.2 25 17 14.5

3.62 1.10 0.41 12.31 1.26 0.44 1.71 2.46

2.79 0.98 n.d. 7.43 3.53 2.41 n.d. 3.41

1.92 0.71 0.21 9.39 0.48 0.61 0.45 4.96

13 110 2862 2223 42 631 3423 2754 2037 9908

2944 3980 n.d. n.d. 1332 980 592 3006

65 183 72 119 26 35 10 46

Cil, ciliates; Dino, heterotrophic dinoflagellates .20 mm; Np, nauplii; n.d., not determined.

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into the incubation bottles and again from each bottle after the incubation, stored at 2208C and analyzed according to Hansen and Grasshof (1983) with a Technicon II autoanalyzer. Samples for chlorophyll a (Chl a) concentrations were collected from each experimental container before (T0) and after (T24) the incubation. In addition, chlorophyll size fractions (total and ,5 mm Chl a) were measured before and after the incubations in the undiluted and the most diluted (100% and 5% ww) samples. Samples were first passed through the 5 mm Nuclepore membranes and then collected on GF/F filters. Filters were stored in liquid nitrogen until fluorometric analysis according to Neveux and Panouse (1987). For determination of diagnostic phytoplankton pigments (HPLC), 2 – 3 L was collected at the beginning and the end of the experiment from the 100% and 5% ww experimental bottles, filtered onto a GF/F glass fiber filter (47 mm, Whatman) and stored in liquid nitrogen until analysis (Vidussi et al., 1996). In May and June 2004, HPLC analyses were also performed on the ,5 mm fraction in all carboys at T0 and T24; size fractionation was obtained as described for the Chl a analyses. The diagnostic pigments for which there were distinguishable peaks on the chromatograms were fucoxanthin (Fuco), hexanoyloxyfucoxanthin (Hex), 190 -butanoyloxyfucoxanthin (Bf ), alloxanthin (Allo), chlorophyll b (Chl b), peridinin (Per) and zeaxanthin (Zea). To check for photoacclimation of the microalgae, the concentrations of the photoprotective pigments diadinoxanthin (DD) and diatoxanthin (Dt) were determined (Brunet et al., 2008). Samples for microscope counts of microzooplankton were always taken from the two 100% ww bottles of each experiment at T0 and T24. Additional microzooplankton counts at T0 and T24 were performed on samples collected from 2 –4 bottles from the dilution series. The microzooplankton samples were fixed with acid Lugol’s solution (2% final concentration) and stored in the dark at 48C. Samples were concentrated by settling from 300 to 100 mL then an aliquot (2.5 – 50 mL) was transferred to a sedimentation chamber and scanned with an inverted microscope. At least 100 ciliates and 100 dinoflagellates were counted for each experimental container in one or more subsamples. However, this could not always be achieved due to very low abundances in the more diluted bottles (20% and 5% ww) and thus the calculated changes in grazer abundance in the diluted containers must be considered with caution. The microzooplankton was divided into groups (naked ciliates ,20 mm ESD, naked ciliates .20 mm ESD, tintinnids, Myrionecta rubra, heterotrophic

dinoflagellates, all dinoflagellates ,20 mm ESD and nauplii). For most of our analyses, all ciliates except M. rubra were pooled when not stated otherwise. All ciliates, large .20 mm heterotrophic dinoflagellates and nauplii were counted over the whole sedimentation chamber. Dinoflagellates were recognized as heterotrophic based on epifluorescence observations on some live samples and on the basis of taxonomy (Lessard and Swift, 1986). For the estimate of small ,20 mm dinoflagellate numbers, two or four diameters of the sedimentation chamber(s) were counted. Nauplii mortality was not observed to occur during incubations, and for a better estimate of nauplii abundances, all counts (3 – 13 counts) for any single experiment were pooled taking into account the dilution factor to obtain average nauplii concentration in each experiment (Table I). Biomass in terms of carbon was determined from biovolume and appropriate conversion factors for each group (0.19 pg mm23 for ciliates Putt and Stoecker, 1989; 0.14 pg mm23 for dinoflagellates Lessard, 1991; 0.045 pg mm23 for nauplii Gifford and Caron, 2000).

Data analysis Phytoplankton growth rate for each experimental bottle was calculated as m (day21) ¼ (1/t) ln(ChlT24/ChlT0) for t ¼ 24 h. Linear regression was used to plot the best-fit relationship between the phytoplankton growth and the dilution level. Phytoplankton growth (k) and grazing mortality (g) rates are defined as the y-intercept and the negative slope of this relationship, respectively (Landry and Hassett, 1982). The portion of PP consumed was calculated as the ratio (g/k) 100 (Calbet and Landry, 2004). On the basis of microscope counts at T0 and T24, microzooplankton growth rates were calculated as m (day21) ¼ (1/t) ln(Conc.T24/Conc.T0). Growth or mortality of predators, ciliates and heterotrophic dinoflagellates, may have resulted in higher or lower grazing pressure than what is assumed from the dilution factor. Phytoplankton growth and microzooplankton grazing rates were calculated by regression of apparent Chl a growth against average predator concentrations in the incubation carboys (GMPA). Mean ciliate and heterotrophic dinoflagellate abundance (Cil þ Dino Ab) during the 24 h incubation was calculated as (Cil þ Dino AbT0 Cil þ Dino AbT24)0.5 and thus converted to %T0 predator concentration (Dolan et al., 2000). In some experiments, microzooplankton counts were performed on one of the two dilution series and to allow for the comparison between grazing rates obtained from GMPA or the nominal dilution factors, phytoplankton growth and microzooplankton grazing rates are

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presented separately for the two parallel series of experimental bottles. However, significant differences between the two parallel series were not found, t-test P . 0.5 for all experiments, except in May (t-test P ¼ 0.03).

R E S U LT S Initial experimental conditions Environmental parameters at Stn MC, at the time of our experiments, varied from mesotrophic conditions in winter and spring to more oligotrophic conditions in summer (Table I). In particular, TIN concentrations ranged between 0.41 and 12.31 mM and the contribution of NH4 to TIN varied with the seasons. Ammonia was 35% of TIN in winter (February and December), around 55% in spring (March and May), increased to 74% in June and was 88% in September and 90% in July. When compared with TIN, PO4 concentrations were much less variable ranging between 0.1 and 0.2 mM in situ in all seasons as well as at T24. Chl a concentrations ranged from 0.21 to 9.39 mg L21. The ,5 mm Chl a fraction accounted for a minimum of 18% of total Chl a concentration in May 2004 and up to 75% in December 2007. Coefficient of variation for replicate samples varied between 1 and 22 for total Chl a measurements and from 3 to 31 for the ,5 mm Chl a fraction. Abundance and biomass of ciliates ranged 2037 – 42 631 cells L21 and 1.5 – 53.5 mg C L21 and those of heterotrophic dinoflagellates ranged 592 – 3980 cells L21 and 0.7– 7.9 mg C L21, respectively. Nauplii reached up to 183 ind. L21, biomass ranged between 0.1 and 4.9 mg C L21 (Table I). Other microzooplankton, such as rotifers and sarcodines, were only occasionally encountered as single specimens and were not considered. Replicate counts (2 – 4 counts from the same experimental bottle) were performed on eight samples, coefficient of variation ranged from 3 to 20.

Fig. 1. Regression analyses of dilution experiments conducted at Stn MC (a) in winter in conditions of water column mixing and (b) in the period of water column stratification. The trendline fit to data pooled for all experiments of each panel.

between the changes in Chl a concentrations and dilution factor were found in all the three experiments (Fig. 1a, Table II). The microzooplankton consumed .100% of PP as microzooplankton grazing rates in winter always exceeded phytoplankton growth; no correlation was found between phytoplankton growth and grazing mortality rates (Fig. 2). In the February 2007 experiment, negative phytoplankton growth rates were the same in the 100%, 75% and 50% ww bottles, i.e. a non-linear relationship was observed between apparent Chl a growth rates and dilution factor. However, calculating microzooplankton grazing as the difference between chlorophyll growth rates in the absence of grazers (5% ww) and in the natural sample (100% ww) gave an estimate of g ¼ 0.64 + 0.07 day21, close to the value g ¼ 0.66 + 0.16 day21 obtained from the linear regression.

Results of grazing experiments

Stratified water column

Our experiments can be divided into two groups in relation to water column conditions.

Stable stratification of the water column is observed at Stn MC from April through October. The results of the experiments of May, June (no nutrient additions) and July (nutrients added) are shown in Fig. 1b and Table II. In addition, transient stratification of the water column was observed at the time of our experiment in March 2005 due to a slight warming (D18C) of the upper few meters of the water column. On average, 68% of PP was consumed by microzooplankton under stratified water column conditions. Grazing rates (range

Mixed water column Complete mixing of the water column occurs at Stn MC from November till March. In the three experiments carried out in winter (December 2007, February 2007 and February 2008), phytoplankton growth rates were low, ranging between 0.15 + 0.12 and 0.63 + 0.01 day21, and significant linear regressions (P , 0.05)

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Table II: Phytoplankton growth (k) and microzooplankton grazing (g) rates from the dilution experiments. Regression analyses using dilution factor (left) or GMPA (Geometric Mean Predator Abundance), (right) in the experimental bottles Dilution

GMPA

Experiment

Nutrients added

g

k

r2

g

k

r2

May 2004 A May 2004 B June 2004 September 2004 A September 2004 B March 2005 A March 2005 B February 2007 A February 2007 B July 2007 A July 2007 B December 2007 A December 2007 B February 2008 A February 2008 B

No No No No No No No Yes Yes Yes Yes Yes Yes Yes Yes

1.33 0.89 0.96 n.d. n.d. 0.15 0.17 0.55 0.77 1.20 1.53 0.83 1.10 1.05 0.69

1.62 1.09 1.04 21.22 20.97 0.40 0.40 0.31 0.44 1.85 1.96 0.06 0.23 0.64 0.62

0.86 0.92 0.94 0.96 0.78 0.66 0.58 0.85 0.73 0.99 0.96 0.83 0.89 0.90 0.56

1.06 n.d. 1.05 n.d. n.d. n.d. 0.16 0.61 0.69 0.75 1.08 n.d. n.d. n.d. n.d.



Fig. 2. Relationship between microzooplankton grazing rate and phytoplankton growth rate in the three winter experiments February 2007, December 2007 and February 2008 (closed symbols), and in the experiments conducted in May 2004, June 2004, March 2005 and July 2007 (open symbols, regression equation). Each data point is the mean between the two replicates. The dashed line represents a 1:1 relation between g and k.

0.17 + 0.01 to 1.37 + 0.23 day21) were linearly correlated with phytoplankton growth rates (range 0.40 + 0.01 to 1.91 + 0.08 day21) (Fig. 2). The grazing experiments carried out in oligotrophic conditions in September did not give the expected results. A small increase in Chl a concentrations was observed only in the undiluted carboy where nutrient turnover by means of grazing may have sustained the tiny increment. Phytoplankton mortality increased linearly with dilution factor and the regression analysis resulted in a positive slope (Fig. 3). This suggests an equilibrium between phytoplankton uptake and nutrient turnover by heterotrophic consumption, i.e. mainly regenerated production occurred in these low-nutrient conditions, and, in fact, TIN was almost exclusively ammonium (NH4 88% of TIN).

Fig. 3. Regression analysis of the dilution experiment of September 2004.

Grazing rates as related to abiotic and biotic parameters At low Chl a concentrations, Chl a ,1 mg L21, grazing rates varied between 0.47 and 1.53 day21 without any apparent relationship between these two parameters, whereas grazing rates decreased linearly at higher chlorophyll concentrations (Fig. 4a). Temperature showed a positive correlation with phytoplankton growth rates (r 2 ¼ 0.58, P , 0.05) and with microzooplankton grazing rates (r 2 ¼ 0.44, P , 0.05). No correlation was found between nauplii abundance and any of the abiotic or biotic parameters considered.

Changes in microzooplankton abundance and composition Microzooplankton counts conducted on samples collected from the carboys at T0 and T24 showed shifts in ciliate and heterotrophic dinoflagellate abundances.

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Fig. 4. Relationship between initial Chl a concentrations and microzooplankton grazing rates as estimated by dilution experiments (a) at Stn MC; (b) based on data from McManus et al. (1992), Landry et al. (1998), Lessard and Murrell (1998), Dolan et al. (2000), Latasa et al. (2005) and Calbet et al. (2008). Regression line (filled triangle) and equation in bold refer to Chl a .1 mg L21, data points (cross) and equation in italic refer to Chl a ,1 mg L21.

Table III: Growth rates of ciliates and heterotrophic dinoflagellates based on microscope counts on samples collected at T0 and T24 from the experimental bottles Experiment May 2004 Ciliates Dinoflagellates June 2004 Ciliates Dinoflagellates September 2004 Ciliates Dinoflagellates March 2005 Ciliates February 2007 Ciliates Dinoflagellates July 2007 Ciliates Dinoflagellates December 2007 Ciliates Dinoflagellates February 2008 Ciliates Dinoflagellates

100% ww

80% ww

60 –40% ww

20% ww

5% ww

20.11 0.62

0.11 0.12

n.d. n.d.

21.65 21.22

20.78 0.02

20.09 0.05

0.22 20.56

1.64 21.16

n.d. n.d.

21.05 20.28

20.60 20.90

n.d. n.d.

n.d. n.d.

n.d. n.d.

n.d. n.d.

0.18

0.23

0.46

20.04

21.47

20.27 + 0.13 20.15 + 0.35

n.d. n.d.

0.69 + 0.21 1.09 + 0.19

n.d. n.d.

21.89 20.17 0.16 0.25

21.09 + 0.13 20.17 + 0.09

n.d. n.d.

21.15 + 1.14 20.56 + 0.25

n.d. n.d.

n.d. n.d.

20.03 + 0.10 1.31 + 0.01

n.d. n.d.

n.d. n.d.

n.d. n.d.

n.d. n.d.

n.d. n.d.

n.d. n.d.

n.d. n.d.

n.d. n.d.

Growth (or mortality) rates in the experimental bottles are reported in Table III. Nanociliates (,20 mm) contributed 39 + 14% to total ciliate abundance in the T0 as well as in the T24 samples in the less diluted containers. The few ciliates found in samples from 5% ww bottles at T24 were mainly nanociliates. An increase in tintinnid numbers was not observed in any of our experiments; however, the relative contribution of tintinnids to total ciliate numbers changed sometimes quite dramatically during the incubations due to high mortality recorded for the naked ciliates. Myrionecta rubra was found in low numbers in most samples.

Heterotrophic dinoflagellates .20 mm ESD were mainly Protoperidinium spp., large Gyrodinium (.90 mm in length) and some specimens of the Diplopsalis group. Ciliate numbers remained fairly stable in the undiluted sample (100% ww) except in September and December when we observed very high ciliate mortality (m ¼ 20.60 and 21.89 day21, respectively), and in July when an increase in ciliate abundance (m ¼ 0.69 + 0.21 day21) was recorded. A decrease in ciliate numbers was always recorded in the 20% and 5% ww samples. Heterotrophic dinoflagellate numbers increased in May and, in particular, in July in the 100% ww

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containers and remained relatively stable in the other experiments. In the 5% ww, in contrast to the significant mortality observed for the ciliates, dinoflagellate numbers remained fairly stable or increased (Table III). In July, a more than 2-fold increase in ciliate and heterotrophic dinoflagellate numbers and biomass was recorded in the 100% ww, and total protozoan biomass was 3.3 + 0.4 mg C L21 at T0 and reached 8.5 + 2.4 mg C L21 at T24. Growth rates of the large heterotrophic dinoflagellates were correlated with Chl a and Fuco growth rates in the nutrient amended experiments (P , 0.05), whereas no correlation was found in the experiments where no nutrients were added (Fig. 5). Ciliate growth rates were not correlated with the growth rates of any pigment neither in the nutrient amended nor in the non-amended experiments. Small (,20 mm) dinoflagellates increased in May and in the four nutrient amended experiments in the 100% ww (0.92 + 0.34 day21). In the nutrient amended experiments, but not in the experiments where no nutrients were added, small dinoflagellates were correlated with growth rates of Chl a, Fuco, Bf and Hex (P , 0.05; n ¼ 7). However, as no distinction was made between autotrophic and heterotrophic small dinoflagellates, it is not possible to discern between an increase in the heterotrophic dinoflagellate growth and stimulation of autotrophic dinoflagellate growth due to nutrient additions.

Fig. 6. Relationship between (a) grazing rates estimated using the dilution factors (gDil ) and the absolute difference between gDil and the grazing rate obtained using mean predator abundance (gGMPA); (b) relationship between phytoplankton growth rate (kDil ) and the difference between PP consumed by the microzooplankton as estimated using dilution factors or mean predator abundance [y-axis: ((gDil/kDil ) 2 (gGMPA/kGMPA)) 100].

When calculating g and k using average predator abundance in the incubation bottles (GMPA) instead of the dilution factor, we found both lower and higher values for g, whereas only minor changes were found for k (Table II). The absolute difference between the grazing rates obtained from the regression of DChl a against dilution factor (gDil ) or against average predator abundance (gGMPA) increased exponentially with grazing rate (gDil ) (Fig. 6a). Moreover, the difference in PP consumed by the microzooplankton, (g/k) 100, whether estimated from dilution factor or from grazer abundance, increased linearly with increasing phytoplankton growth rate (Fig. 6b).

Changes in phytoplankton biomass and composition

Fig. 5. Correlation between apparent growth rates of heterotrophic dinoflagellate and of fucoxanthin: (a) in the nutrient-enriched experiments, (b) in the experiments where no nutrients were added.

Chlorophyll concentrations remained essentially stable in the 100% ww except in July (m ¼ 0.54 + 0.28 day21) and in December (20.75 + 0.05 day21). The contribution of the small ,5 mm fraction was the same at T0 and T24 in May (18% of total Chl a) and in June (53%). In the other experiments, minor reductions in the percentage contribution of the small Chl a fraction were recorded; the most relevant reductions occurred in July

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when Fuco was the most abundant pigment, and in June when we found Hex to be the most abundant pigment.

DISCUSSION

Fig. 7. Growth rates [ln(Pigment concentrationT0/Pigment concentrationT24)] in the dilution series in May. Each point is the mean value calculated from the two replicate bottles of the dilution series. For pigment abbreviations see Method section.

and February 2008 from 26% to 19% and from 20% to 13% at T0 and T24, respectively. Phytoplankton diagnostic pigment analyses on samples at T0 showed Fuco (typical of diatoms) as the most abundant pigment (Fuco/Chl a 0.35 – 0.86) except in September and December when Hex was the most abundant pigment. Major changes in the relative contribution of each diagnostic pigment did not occur in the 100% ww samples except in July and February 2008 when Fuco increased from 62% to 77% and from 86% to 95% of total pigments at T0 and T24, respectively. In the most diluted containers, 5% ww, the maximum total Chl a growth rates were observed in July (1.68 + 0.12 day21) and the minimum in December (0.11 + 0.03 day21). Changes in the relative contribution of single diagnostic pigments were observed in all experiments due to different pigmentspecific growth rates (Fig. 7). Chlorophyll growth rates, recorded in the 5% ww containers, were higher in the ,5 mm fraction when compared with total Chl a growth rates except in July and February 2008 (Table IV). Diagnostic pigments were determined also for the ,5 mm fraction in May,

The aim of our study was to estimate microzooplankton grazing at our long-term study site MC in the Gulf of Naples. Mesotrophic conditions generally characterize Stn MC (Ribera d’Alcala` et al., 2004) and to minimize manipulations of the system a first series of grazing experiments were performed without nutrient additions. However, nutrient depletion may occur in the surface layer at Stn MC after some months of seasonal stratification of the water column. In fact, our experiment conducted in September failed due to nutrient limitation in the dilution series. Thus, a second series of experiments were conducted with nutrient additions. Our study focused on changes in grazer populations during incubations as such changes may profoundly alter the outcome of the experiments.

Microzooplankton grazing at Stn MC Microzooplankton consumed most of phytoplankton daily production, between 40% and 92% of PP in stratified water column conditions and more than 100% of PP in winter when mixing of the water column occurred. Calbet and Landry (2004) reported an overall average of 67% of PP consumed by microzooplankton in the world oceans; however, consumption exceeding 100% has been frequently reported (Landry et al., 1998; Lessard and Murrell, 1998; Murrell and Hollibaugh, 1998; Bo¨ttjer and Morales, 2005; First et al., 2007). A basic assumption for the dilution method is that phytoplankton growth is independent of concentration and thus not nutrient limited in any of the dilution

Table IV: Phytoplankton growth rates for total chlorophyll (Chl tot) and for the ,5 mm size fraction in the undiluted (100% ww) and in the most diluted (5% ww) bottles. Mean + SD for replicate samples from the two series of experimental bottles 100% ww

5% ww

Experiment

Chl tot