Growth and grazing mortality rates of Prochlorococcus ...

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Growth and grazing mortality rates of Prochlorococcus, Synechococcus and eukaryotic picophytoplankton in a bay of the Uwa Sea, Japan MIHO HIROSE1†, TOSHIYA KATANO2,3 AND SHIN-ICHI NAKANO1* 1

3-5-7, MATSUYAMA 790-8566, EHIME, JAPAN, 2CENTER FOR MARINE ENVIRONMENTAL STUDIES, EHIME UNIVERSITY, 3 BUNKYO-CHO 3, MATSUYAMA 790-8577, JAPAN AND DEPARTMENT OF LIFE SCIENCE, HANYANG UNIVERSITY, SEOUL 133-791, SOUTH KOREA LAFWEDY, EHIME UNIVERSITY, TARUMI

†

PRESENT ADDRESS: NIPPON BECTON DICKINSON COMPANY, LTD, AKASAKA DS BUILDING,

5-26, AKASAKA 8-CHOME,

MINATOKU, TOKYO,

107, JAPAN

*CORRESPONDING AUTHOR: [email protected] Received July 4, 2007; accepted in principle November 5, 2007; accepted for publication November 29, 2007; published online December 4, 2007 Communicating editor: K.J. Flynn

Growth and grazing mortality rates of Synechococcus, Prochlorococcus and eukaryotic picophytoplankton were determined using dilution experiments in a bay of the Uwa Sea, Japan. Seasonal changes in the vertical distribution of each picophytoplankter were interpreted based on previous physico-chemical measurements and potential ecophysiological differences. Significant relationships between dilution and picophytoplankton growth rate were found only during the stratified period, suggesting that food linkage between the picophytoplankton and grazers was active during the stratified period. We found an almost 1:1 balance between growth and grazing mortality rates of Prochlorococcus and Synechococcus in the presence of both nanoflagellates and ciliates, suggesting that the abundance of the cyanobacteria is in a quasi-steady-state system with grazing losses compensated by growth. The relationship between growth and grazing mortality rate of eukaryotic picophytoplankton was unclear. We discuss the dynamics of each picophytoplankter abundance with special reference to the characteristics of the microbial food web in the bay, together with two physical events responsible for matter cycling within the bay.

I N T RO D U C T I O N It is well known that two cyanobacterial genera Synechococcus and Prochlorococcus and eukaryotic picophytoplankton are widely distributed as the principal components of phytoplankton biomass in the world’s oceans (Stockner and Antia, 1986; Partensky et al., 1999). The abundance of the picophytoplankters varies both horizontally and vertically (Hall and Vincent, 1990; Blanchot and Rodier, 1996; Vaquer et al., 1996; Cavender-Bares et al., 2001). Seasonal changes in horizontal and vertical abundances of cyanobacteria and picoeukaryotes are also found (Li, 1994; Katano et al., 2004, 2005, 2007). The ecology of picophytoplankters has mainly been studied in pelagic areas so far. In

contrast, others (Shimada et al., 1995; Crosbie and Furnas, 2001; Worden et al., 2004; Katano et al., 2004, 2005, 2007) have reported cell densities of the cyanobacteria and picoeukaryotes in coastal waters but we still have limited information about the ecology of picophytoplankters in coastal waters. Abundance of picophytoplankters is regulated by several environmental parameters such as light (Arumbrust et al., 1989; Vaquer et al., 1996; Jacquet et al., 2001), water temperature (Iriarte and Purdie, 1994; Agawin et al., 2000a, b), nutrient levels (Hall and Vincent, 1990; Li, 1994; Mann and Chisholm, 2000; Vaulot et al., 1996) and grazing (Landry et al., 1995; Reckermann and Veldhuis, 1997; Worden and Binder, 2003), and the variations in horizontal and vertical

doi:10.1093/plankt/fbm101, available online at www.plankt.oxfordjournals.org # The Author 2007. Published by Oxford University Press. All rights reserved. For permissions, please email: [email protected]


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abundance of the picophytoplankters are attributable to the relative importance of those parameters at each location. Direct sinking and/or sinking mediated by zooplankton activity are another important loss process of picophytoplankters in the surface waters (Richardson and Jackson, 2007). Significant attention has been paid to mortality rates of Synechococcus and Prochlorococcus due to grazing. Protistan grazers have been considered as consumers of the cyanobacteria, and laboratory studies have often shown that cyanobacteira are ingested by flagellates (Caron et al., 1991; Dolan and Simek, 1998, 1999; Guillou et al., 2001) and ciliates (Caron et al., 1991; Christaki et al., 1999). Chroococcoid cyanobacteria have been observed in the food vacuoles of nanoplanktonic protists ( Johnson et al., 1982). Two methods have been mainly applied to determine grazing rates on the cyanobacteria in marine environments: the dilution (Landry and Hasset, 1982; Rivkin et al., 1999; Quevedo and Anadon, 2001; Worden et al., 2004) and fluorescently labeled surrogate (Sherr et al., 1991; Sanders et al., 2000; Karayanni et al., 2005). Using these methods, we already have the information about grazing on the cyanobacteria in the North Atlantic, including the Sargasso Sea (Kuipers and Witte, 2000; Sanders et al., 2000; Karayanni et al., 2005), the California Current (Worden and Binder, 2003), the Arabian Sea (Reckermann and Veldhuis, 1997), the central equatorial Pacific (Landry et al., 1995), the subarctic Pacific (Rivkin et al., 1999), the California Bight (Worden et al., 2004) and the Indian Ocean (Paterson et al., 2007). In Asian seas, only Chang et al. (Chang et al., 2003) is available for Synechococcus, and no studies of grazing on Prochlorococcus have so far been reported from Asian seas. Moreover, the information about grazing on eukaryotic picophytoplankton is limited relative to that on the cyanobacteria. To elucidate the scale of organic matter transfer between the picophytoplankters and their grazers, more data are required from other seas. The Uwa Sea is the coastal area of Shikoku Island where cultivation of the pearl oyster Pinctada martensii is the most productive in Japan. There are two major physical events in the Uwa Sea: kyucho and bottom intrusion. The former is an intrusion of warm, oligotrophic surface water from the south of the Bungo Channel to the west coast of Shikoku Island, which occurs mainly in summer (Takeoka and Yoshimura, 1988; Takeoka et al., 1992). High abundances of Prochlorococcus and Synechococcus were carried by advection with kyucho into the Uwa Sea (Katano et al., 2005, 2007). The bottom intrusion consists of deep, cold, nutrient-rich water that flows just over the continental shelf (Takeoka et al., 2000; Kaneda et al., 2002a, b), serving as the major nutrient input to the coastal areas

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(Koizumi and Kohno, 1994; Takeoka et al., 2000; Kaneda et al., 2002a, b). Blooms of diatoms (Koizumi and Kohno, 1994) and eukaryotic picophytoplankton (Katano et al., 2005) promoted by the nutrient inputs of the bottom intrusion have been observed in a bay of the Uwa Sea, while the bottom intrusion had negative effects on the abundance and growth of Prochlorococcus and Synechococcus (Katano et al., 2005). In the present study, to determine growth and grazing mortality rates of Synechococcus, Prochlorococcus and eukaryotic picophytoplankton in Uchiumi Bay, we conducted dilution experiments (Landry and Hasset, 1982). To identify which grazers are important consumers for the picophytoplankters, we used ,5 and ,200 mm seawater filtrates in the experiments.

METHODS The present study was conducted at the station Ub (33820 N, 1328280 E; depth, ca. 53 m) in Uchiumi Bay located in Iegushi, Uchiumi Village, Ehime Prefecture, Japan, and opens to Bungo Channel in the Uwa Sea (Hashimoto and Nakano, 2003; Ichinotuska et al., 2006). In the bay, there is extensive culture of pearl oysters, and fish culture is rare. Our sampling station was located in the center of the pearl oyster culture farm. Water samples were collected monthly using a 6 L Van-Dorn water sampler from nine depths between 0 and 50 m, at the station Ub (Hashimoto and Nakano, 2003; Nakano et al., 2004) from October 2000 to September 2001. Vertical distribution of water temperature and salinity were determined with a Chlorotech profiler (Arec Electronics Co., ACL-208-DK). To determine chlorophyll (Chl) concentration, a 100 mL water sample was filtered through a ,0.2 mm Nuclepore filters to retain seston. Each filter retaining seston was put into a glass test tube, and 6.5 mL of N, N-dimethylformamide (DMF) was added to extract chlorophyll. The amount of Chl thus extracted was determined by the fluorometric method (Moran and Porath, 1980). Cells of Prochlorococcus, Synechococcus and picoeukaryotes were enumerated with a flow cytometer FACS Vantage SE (Becton Dickinson) under 488-nm excitation (1W Argon laser, Coherent). The forward scatter, which is related to cell size, and side scatter, which is related to the internal structure (granularity) of the particle, were recorded; the two florescence signals referred to as “orange” (564–586 nm) and “red” (675–715 nm) that are related to the phycoerythrin and Chl contents of the cells, respectively. For analyses of fresh samples, 0.1 mm pore size filtered seawater was used as the sheath fluid.

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Cell parameters were normalized to 2 mm fluorescent beads (Polysciences). Data were collected as listomode files and then analyzed on a computer using Cell Quest (Becton Dickinson). The signatures of Prochlorococcus and Synechococcus were confirmed using isolated strains of Prochlorococcus SS120, SS51, SS52 and SS2 (CCMP), and Synechococcus OK08 and OK01 (Katano et al., 2001, 2005). Immediately after the collection of a water sample, another 100 mL portion was fixed with glutaraldehyde at a final concentration of 1% for enumeration of nanoflagellates. After filtering the fixed water sample through a black-stained 0.8 mm Nuclepore filter, cells of nanoflagellates were counted using an epifluorescence microscope under ultraviolet excitation by the primulin method (Caron, 1983). Owing to the weakness of autofluorescence from autotrophic nanoflagellates, we counted both autofluorescent and non-autofluorescent nanosized flagellates as nanoflagellates. At least 30 cells of nanoflagellates were enumerated for each sample. Data on vertical distribution of ciliates were available only from March to September 2001. For enumeration of ciliates, a 500 mL portion of the water sample was fixed with acid Lugol’s solution to a final concentration of 1%, and the ciliates were concentrated by sedimentation. Ciliate cells were enumerated in a haematocytometer at 200 or 400 magnification. Ciliate taxa were identified using the classification guides of Foissnner (Foissnner, 1994), Chihara and Murano (Chihara and Murano, 1997), Struder-Kypke et al. (Struder-Kypke et al., 2002) and Struder-Kypke and Montagnes (Struder-Kypke and Montagnes, 2002). To determine growth and grazing mortality rates of picophytoplankton, dilution experiments were conducted (Landry and Hasset, 1982). A water sample was collected from 10 m depth and used for the following size fractionations: 200 mm mesh plankton net for removing metazoan zooplankton, 5 mm Nuclepore filter for removing metazoan zooplankton and large protists and a Gelman Culture Capsule ( pore size 0.2 mm) for removing all planktonic organisms. We assumed that the ,200-mm filtrate contained nanoflagellates and microzooplankton such as ciliates acting as grazers of picophytoplankters, and that the ,5-mm filtrate contained only nanoflagellates. The ,200 or ,5-mm filtrates were diluted in the 0.2-mm filtrate at dilution levels of 1.0, 0.9, 0.8, 0.7, 0.5, 0.3, 0.2 and 0.05. Each dilution was poured into duplicate 1 L polycarbonate bottles washed by acid cleaning before use. To compensate for any decrease in nutrient availability, NH4Cl and NaH2PO4 were added to the bottles at concentrations of 30 mmol N L21 and 2 mmol P L21, respectively. However, there is a controversy over the effect of nutrient addition on picophytoplankton growth, positive or negative (Worden and

Binder, 2003). The series of dilutions thus prepared were incubated at 10 m depth for 24 h on the pier of the pearl oyster culture farm where the water depth was ca. 40 m. Subsamples taken from the bottles before incubation were fixed with glutaraldehyde at a concentration of 0.1% and frozen using liquid nitrogen, and this was repeated at the end of the incubation period. The frozen samples were stored in a freezer at 2808C until they could be analyzed using a flow cytometer (Vaulot et al., 1989). The cell densities of Synechococcus, Prochlorococcus or eukaryotic picophytoplankton were determined at the beginning and at the end of the incubation using the flow cytometer as previously described, and the population growth rates of each picophytoplankter were calculated as follows:

m¼

ln N t ln N 0 t

where m is growth rate (day21), N0 and Nt are the cell densities of Prochlorococcus, Synechococcus or eukaryotic picophytoplankton at the beginning and at the end of incubation, respectively, and t is the incubation time (day). The linear regression of the observed net growth rates versus dilution was examined in order to estimate growth and grazing mortality rates of picophytoplankters. Statistical analyses were conducted using Kaleida Graph v.3.5 (Synergy Software).

R E S U LT S In Uchiumi Bay, thermal stratification usually develops between May and September (Katano et al., 2004, 2005; Nakano et al., 2004), and this was also the case in the present study (Fig. 1A). Thus, we can divide the study period into two: stratified (from May to September) and mixing (from October to March), although thermal stratification in May of the present study was relatively weak (Fig. 1A). High Chl a concentrations were detected in the surface mixing layer during the stratified period (Fig. 1B). Thus, clear vertical distributions of Chl a concentration were found during the stratified period, whereas the Chl a was almost uniformly distributed during the mixing period (Fig. 1B). Thus, the changing pattern of the vertical profile of Chl a was attributable to vertical mixing or changes in associated environmental parameters. Cell densities of Synechococcus varied from 1.2 103 (at 30 m depth in February 2001) to 4.6 105 cells mL21 (at 0 m in June 2001) during the study period,

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Fig. 1 Seasonal changes in the vertical distributions of (A) water temperature (8C) and (B) Chl a concentration (mg L21) at the station Ub in Uchiumi Bay.

and high densities were detected during the stratified period, especially from June to August (Fig. 2A). Cell densities of Prochlorococcus ranged between 1.5 102 and 1.8 104 cells mL21 (Fig. 2B). High abundance of Prochlorococcus was usually detected in the layer from 2 to 20 m depths (Fig. 2B), although relatively high abundance of Prochlorococcus was found not only during the stratified period but also during the mixing period (Fig. 2B). The cell densities of eukaryotic picophytoplankton ranged between 208 (at 40 m depth in September 2001) and 3.6 104 cells mL21 (at 10 m depth in March 2001) during the study period (Fig. 2c). The abundance of the picoeukaryotes was relatively high in the whole water column between March and May 2001. Nanoflagellate cell densities varied from 0.2 102 (at 50 m depth in August 2001) to 2.9 103 cells mL21 (at 20 m depth in March 2001) during the study period (Fig. 3A). High cell densities of nanoflagellates were detected between February and May, except in April, and the depth at which we found high nanoflagellate cell densities was seasonally variable (Fig. 3A). The density of ciliates was relatively high in April, and high cell densities were detected between the surface and 20 m: 15 000 cells L21 at 15 m depth

Fig. 2 Seasonal changes in the vertical distributions of cell densities of (A) Synechococcus (104 cells mL21), (B) Prochlorococcus (103 cells mL21) and (C) eukaryotic picophytoplankton (103 cells mL21) at the station Ub in Uchiumi Bay.

(Fig. 3B). The dominant taxa found in the present study were the oligotrichous genera Strombidium and Strobilidium, similar to those of Ichinotsuka et al. (Ichinotuska et al., 2006). Significant relationships between dilution and growth rate tended to be found during the stratified period (Table I). Ten cases were successful in the experiment using ,200 mm filtrates, and seven cases in the experiment using the ,5 mm filtrates (Table I). Grazing mortality rates of Prochlorococcus in ,200 mm filtrates showed positive responses to growth rates of Prochlorococcus in the filtrates (Fig. 4). This was also likely for Synechococcus, although there were only three cases for Synechococcus in

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Fig. 3 Seasonal changes in the vertical distributions of (A) nanoflagellates (cells mL21) and (B) ciliates (103 cells L21) at the station Ub in Uchiumi Bay.

the present study (Table I; Fig. 4). Pearson correlation analysis showed that the correlation between growth and grazing mortality rate of Prochlorococcus in the ,200 mm filtrates was statistically significant (n = 5, r = 0.950, P , 0.05) with a slope of 1.14 (Fig. 4). The correlation between growth and grazing mortality rate of Synechococcus in the ,200 mm filtrates had r = 0.947, but it was insignificant because of small dataset (n = 3, P . 0.05). We also compared the grazing mortality rate of picophytoplankters in ,5 mm filtrates with growth rates of picophytoplankters in the filtrates, but no clear trend was found.

DISCUSSION Seasonal dynamics of microbial abundance in Uchiumi Bay Previous studies conducted in Uchiumi Bay have reported that the abundance of Synechococcus becomes high between June and August (Katano et al., 2004, 2005; Nakano et al., 2004), which was also the case in the present study (Fig. 2A). In contrast, the seasonal

changing pattern of Prochlorococcus in the present study was different from that of previous studies (Katano et al., 2005). We found high Prochlorococcus cell densities in October, January and March; all of which are included in the mixing period (Figs 1A and 2B), whereas Katano et al. (Katano et al., 2005) demonstrated high cell densities of Prochlorococcus during the stratified period. Katano et al. (Katano et al., 2005) hypothesized that Prochlorococcus in Uchiumi Bay would be brought in by kyucho from the surface water of the Pacific Ocean (see Methods section). Water temperature during the mixing period is almost isothermal vertically, and water temperatures from December 2000 to March 2001 in Uchiumi Bay (Center for Marine Environmental Studies, 2006) were 1 – 28C higher than those in Katano et al. (Katano et al., 2005). This was probably due to the high frequency of kyucho occurrence. Hence, relatively high cell densities of Prochlorococcus detected during the mixing period in the present study were likely attributable to Prochlorococcus being frequently brought in when kyucho occurred. For the eukaryotic picophytoplankton, the pattern of seasonal changes in the present study was similar to that described by Katano et al. (Katano et al., 2005) who noted that the growth of the eukaryotes was enhanced by nutrients supplied by the bottom intrusion (see Methods section). During the stratified period of 2001, there were bottom intrusions between June and August, and the supplementation of nutrients accelerated the growth of diatoms and dinoflagellates (Nakano et al., 2004). Hence, it is likely that the high cell densities of eukaryotic picophytoplankton, noted during the stratified period in the present study, were due to nutrients supplied by the bottom intrusion, although neither the present study nor the Katano et al.’s study (Katano et al., 2005) does not have an appropriate explanation for high cell densities of the eukaryotes in April. In the present study, there were no clear seasonal trends in the vertical distribution of nanoflagellate densities (Fig. 3A). Clear trends might be found if we could have separately enumerated nanoflagellates as autotrophic and heterotrophic (see Methods section), although the seasonal changing pattern of nanoflagellate cell density is different from year to year in Uchiumi Bay (Nakano et al., 2004). Ciliate cell densities were high in April (Fig. 3B). In Uchiumi Bay, Chl a concentration or phytoplankton abundances are usually high between March and May (Hashimoto and Nakano, 2003; Nakano et al., 2004; Ichinotuska et al., 2006). Cell densities of ciliates have been shown to increase during this season (Ichinotuska et al., 2006), and this was also the case in the present study (Fig. 3B). The dominant taxa in the present study were the oligotrichous genera Strombidium and Strobilidium; both of

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Table I: Results of the dilution experiments March

Size fraction (mm)

Picophytoplankter

Growth (day21)

Grazing (day21)

r

,200

S P E S P E S P E S P E S P E S P E S P E S P E S P E S P E S P E S P E S P E S P E

NS 1.16 NS NS NS NS NS Ns NS NS NS NS 0.25 1.34 0.92 NS 1.40 20.60 NS NS NS NS NS NS NS 2.15 NS NS 2.06 0.19 0.96 0.67 NS 0.82 1.32 20.15 1.39 1.23 0.70 NS NS NS

NS 1.22 NS NS NS NS NS NS NS NS NS NS 0.65 1.41 0.94 NS 1.89 0.87 NS NS NS NS NS NS NS 2.64 NS NS 1.57 0.45 0.95 0.99 NS 0.62 1.43 0.70 1.54 1.72 0.94 NS NS NS

– 0.758 – – – – – – – – – – 0.915 0.851 0.906 – 0.871 0.941 – – – – – – – 0.941 – – 0.781 0.738 0.956 0.749 – 0.953 0.744 0.780 0.942 0.892 0.954 – – –

,5

April

,200

,5

May

,200

,5

June

,200

,5

July

,200

,5

August

,200

,5

September

,200

,5

r = P , 0.05; S, Synechococcus; P, Prochlorococcus; E, eukaryotic picophytoplankton; NS, not significant.

which may ingest bacteria-sized prey (Ichinotuska et al., 2006). Hence, eukaryotic picophytoplankton, which actively proliferate between March and May, may support the high production of ciliates during that period, and hence ciliate abundance may be subject to bottom-up control.

Growth and grazing mortality rates of picophytoplankters Significant relationships between dilution and growth rate were found only during the stratified period (Table I). In the other period of the present study, the regression of net growth rate against dilution was found to be statistically insignificant (Table I). This could be

the result of non-linear grazing response (Worden and Binder, 2003) and/or very low grazing rates. The abundances of Synechococcus and Prochlorococcus are usually high during the stratified period (Katano et al., 2004, 2005; Nakano et al., 2004), and this was the case also in the present study. Thus, it is likely that food linkage between the picophytoplankton and grazers was active during the stratified period. The growth rates of picophytoplankters in the present study overlapped the upper estimates of previous studies (Table II). The growth rates of Prochlorococcus have generally been considered as around 0.69 (day21) because of their cell cycle which is tightly phased to the light – dark cycle with DNA replication during the afternoon, followed by binary fission after dark (Liu et al., 1997;

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Fig. 4 Relationship between growth and grazing mortality rates of Prochlorococcus in the ,200 mm (closed circles), ,5 mm (open circles) and Synechococcus in the ,200 mm (closed triangles) filtrates. The solid line shows a significant correlation by Pearson correlation analysis between growth and grazing mortality rate of Prochlorococcus in the ,200 mm filtrates (n = 5, r = 0.950, P , 0.05, slope = 1.14). The dotted line shows the 1:1 balance between growth and grazing rates.

Vaulot et al., 1995). Higher growth rates of Synechococcus have so far only been reported in nutrient-enriched Mediterranean mesocosms (.3 day21, Agawin et al., 2000b) and of Prochlorococcus in the Arabian Sea (.2 day21, Reckermann and Veldhuis, 1997). Moore et al. (Moore et al., 1995) also reported high growth rates

of Synechococcus and Prochlorococcus in laboratory systems. Agawin et al. (Agawin et al. 2000b) have noted that growth rates of Mediterranean Synechococcus were accelerated by the supply of nutrients, but they were suppressed under higher concentrations of dissolved inorganic nitrogen (.8 mM). Worden and Binder (Worden and Binder, 2003), on the other hand, noted that nitrogen and phosphorus amendments had little effect on growth rates of Synechococcus and Prochlorococcus. Although we believe that our estimates of growth rates of the picophytoplankters in the present study were valid, further studies of the relationship between picophytoplankter growth and nutrient supply are needed. Katano et al. (Katano et al., 2005) demonstrated numerous negative growth rates of Prochlorococcus in Uchiumi Bay and concluded that the environmental conditions there are unfavorable to the growth of Prochlorococcus. However, these authors also noted that there were several cases where cyanobacteria could proliferate when oceanic (Kuroshio) water dominated in the bay. Thus, it is possible that we were fortunate to have positive growth rates of Prochlorococcus since we had frequent occurrence of kyucho in the present study as mentioned earlier. Grazing rates of picophytoplankters estimated in the present study also fall into the upper range of those reported in previous studies (Table II), although we

Table II: Studies of growth and grazing rates determined by the dilution experiments Phytoplankton

Location

Growth rate (day21)

Grazing rate (day21)

Source

Synechococcus

Central Equatrial Pacific East China Sea Eastern Indian Ocean Gulf of Aden North-east Atlantic Sargasso Sea Somali Basin Southern California Bight Uchiumi Bay Central Equatrial Pacific Eastern Indian Ocean Gulf of Aden North-east Atlantic Sargasso Sea Somali Basin Southern California Bight Uchiumi Bay Central Equatrial Pacific Eastern Indian Ocean Gulf of Aden North-east Atlantic Somali Basin Southern California Bight Uchiumi Bay

0.47 + 0.16 0.42 + 0.22 0.33 + 0.44 0.18a 0.40 + 0.11 0.54 + 0.11 0.51 + 0.34 0.66 + 0.13 0.87 + 0.58 0.27 + 0.23 20.45 + 0.99 2.20a 0.35 + 0.10 0.49 + 0.13 0.60a 0.33 + 0.14 1.31 + 0.54 0.81 + 0.18 0.00 + 0.25 0.31a 0.32 + 0.12 0.49 + 0.10 0.99 + 0.28 0.81 + 0.16

0.76 + 0.20 0.21 + 0.14 0.21 + 0.19 0.06a 0.33 + 0.10 0.33+0.11 0.50 + 0.24 0.25 + 0.10 1.05 + 0.45 0.73 + 0.21 0.16 + 0.26 0.82a 0.26 + 0.07 0.41 + 0.18 0.59a 0.36 + 0.06 1.60 + 0.64 0.62 + 0.17 0.08 + 0.10 1.78a 0.22 + 0.08 0.48 + 0.27 0.45 + 0.33 0.94 + 0.00

Landry et al. (1995) Chang et al. (2003) Paterson et al. (2007) Reckermann and Veldhuis (1997) Quevedo and Anadon (2001) Worden and Binder (2003) Reckermann and Veldhuis (1997) Worden et al. (2004) This study Landry et al. (1995) Paterson et al. (2007) Reckermann and Veldhuis (1997) Quevedo and Anadon (2001) Worden and Binder (2003) Reckermann and Veldhuis (1997) Worden et al. (2004) This study Landry et al. (1995) Paterson et al. (2007) Reckermann and Veldhuis (1997) Quevedo and Anadon (2001) Reckermann and Veldhuis (1997) Worden et al. (2004) This study

Prochlorococcus

Eukaryotic picophytoplankton

All values are Mean + SD. For Reckermann and Veldhuis (Reckermann and Veldhuis, 1997) and the present study, the data of ,200 mm fraction were used. a Standard deviation was not calculated due to n = 1.

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already have even higher grazing rates (.2 day21) of picophytoplankters in previous studies (Reckermann and Veldhuis, 1997; Agawin et al., 2000b). Worden and Binder (Worden and Binder, 2003) found an increase in grazing mortality of Synechococcus and Prochlorococcus after nutrient addition and discussed the possibility that the increase might be due to changes in the food quality of the cyanobacteria available as food for grazers. Hence, high grazing mortality rates in the present study were feasible, since we added nutrients into our experimental bottles. There is a controversy over the importance of Synechococcus or Prochlorococcus as food for protists. Growth rate (Caron et al., 1991) and growth yields (Caron et al., 1991; Guillou et al., 2001) of flagellates fed on Synechococcus (Caron et al., 1991; Guillou et al., 2001) or Prochlorococcus (Guillou et al., 2001) were low, relative to those of flagellates fed on heterotrophic bacteria (Caron et al., 1991; Nakano, 1994). This is also the case for ciliates (Verity and Villareal, 1986; Caron et al., 1991). Christaki et al. (Christaki et al., 1999) reported that Synechococcus was better prey for ciliates relative to Prochlorococcus, but we can see that growth rates and growth yields of Strombidium and Uronema fed on Synechococcus or Prochlorococcus in Christaki et al. (Christaki et al., 1999) were low relative to those of ciliates fed on heterotrophic bacteria by Caron et al. (Caron et al., 1991). Thus, we still cannot determine important grazers of the cyanobacteria (Dolan et al., 2005) and identify a major loss process of the cyanobacteria, although there are some cases where the growth of some ciliates was supported by field populations of Synechococcus in the NW Mediterranean (Perez et al., 1996) and a eutrophic lake (Simek et al., 1995, 1996). In the present study, the almost 1:1 balance between growth and grazing rates of Prochlorococcus in the ,200 mm filtrates (Fig. 4) suggests that the cyanobacterium is in a quasi-steady-state system with grazing losses compensated by growth. This appears also the case for Synechococcus, although the relationship was insignificant (Fig. 4). Previous studies have also demonstrated a high correlation between phytoplankton growth and microzooplankton grazing rates (Burkill et al., 1987; Verity, 1986). Grazers in the ,200 mm filtrate were not only flagellates but also microzooplankton such as ciliates, and it is likely that both nanoflagellates and ciliates are important grazers of Prochlorococcus and Synechococcus in Uchiumi Bay, at least during the stratified period (Table I). Eukaryotic picophytoplankton usually dominate during spring when the abundances of Synechococcus and Prochlorococcus are relatively low (Katano et al., 2005), and this was also the case in the present study. So, the food linkage between eukaryotic picophytoplankton and

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grazers may be active during spring, although the present study failed to demonstrate that for some reason. We need to collect more information about ecology of eukaryotic picophytoplankton in marine environments.

AC K N OW L E D G E M E N T S We thank K. Hyodo, T. Hirose and the staff of Ainan Institute of Oceanic and Fishery Center and the students of Ehime University for their help in field monitoring. Thanks are also due to Prof. Suzuki and other members of the Aquatic Biology and Ecology Laboratory of CMES, Ehime University, for their advice, discussions and encouragement throughout the study. We thank Dr. M.J. Morris for correction of our English and constructive comments on the manuscript.

FUNDING The present study was partly supported by the Grant-in-Aid for Scientific Research No. 16201004, JSPS, by the Center of Excellence (COE) Program at the “Global Center of Excellence for Interdisciplinary Studies on Environmental Chemistry”, and by the Research Fund of coastal environment in Uchiumi Bay, Ainan Town, Ehime Prefecture.

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