Seasonal Variability of the Red Tide-Forming Heterotrophic Dino

Plankton Benthos Res 8(1): 9–30, 2013 Plankton & Benthos Research © The Plankton Society of Japan

Seasonal variability of the red tide-forming heterotrophic dinoﬂagellate Noctiluca scintillans in the neritic area of Sagami Bay, Japan: its role in the nutrient-environment and aquatic ecosystem

1, 1 1 2,3 KOICHI ARA *, SACHIKO NAKAMURA , RYOTO TAKAHASHI , AKIHIRO SHIOMOTO 1 & JURO HIROMI

1 D epartment of Marine Science and Resources, College of Bioresource Sciences, Nihon University, Fujisawa, Kanagawa 252– 0880, Japan 2 N ational Research Institute of Fisheries Science, Fisheries Research Agency, Kanazawa-ku, Yokohama, Kanagawa 236– 8648, Japan 3 P resent Address: Department of Aquatic Bioscience, Faculty of Bioindustry, Tokyo University of Agriculture, Abashiri, Hokkaido 099–2493, Japan Received 19 June 2012; Accepted 14 January 2013

Abstract: The role of the heterotrophic dinoﬂagellate Noctiluca scintillans in affecting the nutrient-environment and aquatic ecosystem was investigated in the neritic area of Sagami Bay, Kanagawa, Japan, from January 2002 to De- cember 2006, based on abundance, intracellular nutrient content, excretion rate and response of phytoplankton (diatoms) to enrichment of nutrients extracted from N. scintillans cells. Seasonal variations in abundance and vertical distribution of N. scintillans were signiﬁcantly related to the physical structure of the water column, water tempera-

ture, chlorophyll a and primary productivity. Intracellular nutrient contents, except for Si(OH)4-Si, revealed clear seasonal fluctuations, which were significantly correlated to cell size variations. Thalassiosira rotula increased to higher + cell abundances at higher concentrations of nutrients, which were extracted from N. scintillans cells. NH4 -N and 3– + PO4 -P excretion rates were much higher during the first 1–3 h, and decreased rapidly with time. Daily NH4 -N and 3– PO4 -P supply by N. scintillans excretion was estimated to be on average 34.8% and 55.3%, especially 50.6–85.4% and 80.5–135.8% in April–July (monthly mean), of the daily N and P requirement for primary production, respectively. The large amounts of nutrients regenerated and released by N. scintillans excretion can increase the N and P concentrations in ambient seawater, especially in the upper layer in spring–summer, and consequently affect phytoplankton (diatom) abundance. These may exacerbate eutrophication in these waters by a mutually supportive relationship between phytoplankton and N. scintillans: bottom-up control (phytoplankton–N. scintillans) and nutrient supply by N. scintillans to phytoplankton through excretion.

Key words: excretion rate, intracellular nutrient contents, Noctiluca scintillans, nutrient pools and supplies, Sagami Bay

world (e.g. Kuroda 1990, Elbrächter & Qi 1998). Seasonal, Introduction year-on-year and/or spatial variations in abundance of Red tide outbreaks caused by the large heterotrophic di- Noctiluca have been extensively studied in temperate and noﬂagellate Noctiluca scintillans (Macartney) Ehrenberg subtropical estuarine and coastal waters (e.g. Kuroda & (Noctiluca hereafter), one of the most common red tide- Saga 1978, Uhlig & Sahling 1990, Huang & Qi 1997, Tada forming organisms, have been frequently observed in tem- et al. 2004, Miyaguchi et al. 2006). These studies found perate-to-tropical estuarine and coastal waters around the distinct seasonal variations with considerable peaks (i.e. blooms, red tide outbreaks) of Noctiluca abundance in * Corresponding author: Koichi Ara; E-mail, [email protected] spring–summer after the ﬁnal stage of spring phytoplank- 10 K. Ara et al.

Fig. 1. Map showing the sampling station in Sagami Bay with isobaths in meters. ton (diatom) blooms. quently to very low levels in spring–summer in temperate Noctiluca has extremely high liquid contents of ammo- waters, while nutrient supplies from the nutrient-rich + 3– nia (NH4-N) and phosphate (PO4 -P) within the cells deeper layer are obstructed by seasonal stratification. In (Okaichi & Nishio 1976, Montani et al. 1998, Pithakpol et these waters, Noctiluca may serve as a nutrient regenerator al. 2000a, b), which may contaminate the surface seawater due to its high contents of ammonia and phosphate. How- after secretion, exudation and lysis of Noctiluca cells. In ever, studies on Noctiluca as a nutrient regenerator and + 3– fact, abnormally high NH4-N and PO4 -P concentrations in supplier affecting aquatic ecosystems have been limited. the surface waters have been reported to be associated Sagami Bay, located on the southern coast of central with Noctiluca red tides and/or blooms (Schaumann et al. Honshu, the main island of Japan, is a semi-circular em- 1988, Elbrächter & Qi 1998, Montani et al. 1998, Pithakpol bayment facing the western North Pacific Ocean (Fig. 1), et al. 2000a, Al-Azri et al. 2007). Pithakpol et al. (2000a, and is geographically located in the transition zone where + 3– b) stated that NH4-N and PO4 -P pools within Noctiluca nutrient-rich estuarine water from neighboring Tokyo Bay, cells contributed high percentages to the water column one of the most eutrophic semi-enclosed embayments in + 3– NH4-N and PO4 -P pools, especially during the period at Japan, flows out to the Pacific Ocean. Although chemical which a Noctiluca red tide was formed in the upper waters oxygen demand (COD), dissolved inorganic nutrient and in spring. chlorophyll a (Chl-a) concentrations in Sagami Bay have It is well known that light and dissolved inorganic nutri- maintained much lower levels than in neighboring Tokyo ents are major limiting factors for phytoplankton growth. Bay (Saitou 1992, Ara & Hiromi 2008, Ara et al. 2011b), Phytoplankton species are limited to grow and increase in since the 1980s some signs of eutrophication (i.e. gradual the upper layer of the water column, within which dis- rise in COD and Chl-a concentration, decline in transpar- solved inorganic nutrient concentrations decrease fre- ency, red tide outbreaks) have occurred, partly in the Noctiluca scintillans in Sagami Bay 11 coastal waters of Sagami Bay (Yamada & Iwata 1992, Ya- gently vertical towing from 20 m depth to the surface, mada 1997, Machida et al. 1999, Yamada & Matsushita using a NORPAC net (45 cm in mouth diameter, 200 μm in 2005, Miyaguchi et al. 2006, Ara & Hiromi 2008, Ara et mesh opening size), and were carefully transferred into a al. 2011a). The neritic waters in the innermost open area of 20 L tank filled with the ambient surface seawater. For the Sagami Bay, especially the upper layer around Enoshima diatom culture experiment on April 20, 2004, live micro- Island, have higher phytoplankton standing crops (Chl-a) phytoplankton samples were collected by vertical tows and primary production than those in any other open area from 20 m depth to the surface, using a plankton net in the bay (Ara & Hiromi 2007, 2008, 2009, Ara et al. (30 cm in mouth diameter, 63 μm in mesh opening size), 2011a, b). The higher phytoplankton biomasses and pro- and transferred into 1 L bottles filled with ambient surface duction rates in these waters are induced by much higher seawater. These live samples were taken to the laboratory nutrient concentrations due to nutrient-rich run-off from within 1–2 h. Seawater for laboratory experiments was the Sakai and Hikiji Rivers (Saitou 1992, Yamada & Iwata taken at the sea surface (ca. 1 m depth) and filtered through 1992, Tanaka 1993, Yamada & Matsushita 2005, 2006, Ara a Whatman GF/F glass fiber filter (filtered seawater hereaf- & Hiromi 2007, 2008, 2009, Ara et al. 2011a, b). ter) prior to experiments. Studies on Noctiluca in the coastal waters of Sagami Physicochemical properties (i.e. water temperature, sa- Bay have dealt with aspects of red tides and/or red color- linity and density) and Chl-a concentration at this station ation phenomena, sometimes having originated in ex- were previously published by Ara & Hiromi (2007, 2008, panded from neighboring Tokyo Bay (Tanaka 1985, Ya- 2009), Ara et al. (2011a, b) and Okutsu et al. (2012), and mada 1997, Kamataki 2005), predominance in the micro- details of observation methods can be found in these pa- phytoplankton assemblages (Tatara & Kikuchi 2003, Baek pers. Prior to sample collection, water temperature and sa- et al. 2009), and seasonal and interannual variations in linity were recorded using a Memory STD (AST-1000/ abundance in relation to environmental factors (Miyaguchi P-64K, Alec Electronics, Japan), and density (σt) was cal- et al. 2006). However, in these waters as well as other wa- culated. Water samples taken at the seven depths, as de- ters of the world, there has been no attempt to investigate scribed above, were used for Chl-a and dissolved inorganic the availability of nutrients regenerated by Noctiluca for nutrient analyses. When a red tide of Noctiluca had formed phytoplankton growth or to evaluate the contribution of at the surface on April 19, 2006, after removing Noctiluca nutrient (N and P) supply by Noctiluca excretion to phyto- carefully using a 200 μm-mesh screen at the sea surface, plankton primary production. This study provides data on an additional water sample for dissolved inorganic nutri- (1) seasonal variations in abundance, cell size and intracel- ents was taken at the center of Noctiluca patches in the up- lular nutrient contents and concentration of Noctiluca in permost surface layer (0 to ca. 3 cm depth) using a plastic relation to abiotic/biotic properties and estimation of the bucket. For Chl-a analysis, 0.5 L of seawater aliquots were contribution of intracellular nutrient pools within Nocti- filtered through Whatman GF/F glass fiber filters (47 mm). luca cells to the total nutrient standing stocks, (2) response Chl-a concentration was determined with a fluorometer of phytoplankton (diatoms) to nutrients extracted from (TD-700, Turner Designs, USA) after extraction with 90% + 3– Noctiluca cells, and (3) NH4 -N and PO4 -P excretion acetone (Holm-Hansen et al. 1965, Parsons et al. 1984a). + rates of Noctiluca, estimating the contribution of NH4 -N Dissolved inorganic nutrient concentrations, i.e. ammo- 3– + – – and PO4 -P supply for primary production, over five years nium (NH4-N), nitrate+nitrite (NO3+NO2-N), phosphate 3– (2002–2006) in the neritic area of Sagami Bay. (DIP: PO4 -P) and silicate (DSi: Si(OH)4-Si), were determined using an Autoanalyzer (AACSIII, Bran+Luebbe, Germany) (Parsons et al. 1984a, Hansen & Koroleff 1999). Materials and Methods Primary productivity (PP) was determined by the in situ 13C tracer method (Hama et al. 1983), as described by Ara Field investigation & Hiromi (2007, 2009) and Ara et al. (2011b). Water sam- A series of field investigations were conducted mostly ples were taken from depths corresponding to 100, 50, 25, every two weeks (i.e. twice a month) from January 2002 to 10, 5 and 1% photon fluxes of that just above the sea sur- December 2006, at a station (Lat. 35o16′22.0′′N, Long. face using a Niskin bottle. Dissolved inorganic carbon 139o29′41.0′′E; local depth: ca. 55 m) located in the neritic (DIC) concentration in the seawater was determined using area, ca. 4.5 km off Enoshima Island, Fujisawa, of Sagami a Total Organic Carbon (TOC) Analyzer (TOC-VCPH, Shi- Bay (Fig. 1). On each sampling date, water samples for madzu, Japan). After removing large zooplankton with a Noctiluca enumeration were taken at depths of 0, 5, 10, 20, 200 μm-mesh, seawater samples were immediately trans- 30, 40 and 50 m, using duplicate Van Dorn bottles (10 ferred into clean 1 or 0.5 L polycarbonate bottles. After the 13 L×2). For Noctiluca enumeration, 20 L water samples addition of NaH CO3 (Cambridge Isotope Laboratories, were concentrated using a hand-net (63 μm mesh size), ca. 10% of the total inorganic carbon in ambient water), the transferred into 100 mL bottles and immediately preserved bottles were placed at the same depths at which the water in 1% glutaraldehyde (final concentration). For laboratory samples were taken, and were incubated in situ for 24 h. experiments, live Noctiluca samples were collected by The samples were then filtered through precombusted (at 12 K. Ara et al.

400oC for 4 h) Whatman GF/F filters (47 mm). These fil- Table 1. The initial concentrations of dissolved inorganic nu- ters were dried at 60oC for 1–2 h, treated with HCl fumes trients in control and test media for the culture experiment on the for 3 h to remove inorganic carbon, completely dried at diatom Thalassiosira rotula. 60oC for 1–2 h and stored in a vacuum desiccator. The iso- Concentration (μM) topic concentration of 13C and 12C was determined using a Medium/ nutrients + －－ 3－ mass-spectrometer (ANCA-SL, Europe Scientific, UK). NH4 -N NO3 +NO2 -N PO4 -P Si(OH)4-Si The dark uptake was always corrected for primary productivity. Control 0.17 0.68 0.07 5.12 Medium 1 21.30 1.91 1.33 21.71 Sample treatments Medium 2 11.69 1.35 0.76 14.17 Medium 3 1.31 0.69 0.13 5.60 Noctiluca from 10–100% subsamples were counted under a microscope. Cell diameter was measured using an eyepiece micrometer for 100–300 cells per sample or all (culture experiment seawater hereafter). Filtered seawater cells when samples contained <100 cells. Cell volume was containing intracellular nutrients extracted from 1000 calculated from cell diameter assuming spherical configu- Noctiluca cells was prepared (NS medium hereafter), as ration. described above. Usually, 15 mL of culture experiment seawater was introduced into each 20 mL glass test tube. Intracellular nutrient contents Test media (i.e. Medium 1, 2 and 3) were made by adding 3 The methods for preparing and determining intracellular and 1.5 mL of NS medium and 1.5 mL of 10% NS medium nutrient contents of Noctiluca were based on Montani et al. (diluted with filtered seawater) to 15 mL of culture experi- (1998), an outline of which is briefly given as follows. ment seawater, respectively. The T. rotula cells were intro- After standing for 2–3 h, 200 buoyant Noctiluca cells were duced into each glass test tube containing culture experi- separated from mixed samples into separate petri dishes, ment seawater and test media. The initial cell abundance in using a wide-bore pipette. In each analysis, 3–5 replicates each test tube was adjusted to be 1.6–2.0×103 cells mL–1. were prepared. These cells were carefully washed with fil- The initial concentration of dissolved inorganic nutrients tered seawater several times by sieving through a 200 μm- in control and test media were determined, as described mesh screen to remove small algae and particles. Then, the above, and are presented in Table 1. For the control and cells with a small amount of filtered seawater (5–10 mL) each test medium, triplicates were incubated for 288 h were smashed up with a Teflon homogenizer, final volume under the same laboratory conditions as for monoculture. was adjusted to 50 mL using filtered seawater, and this The cell abundance of T. rotula in each test tube was mixture was filtered through Whatman GF/F filters (25 counted under a microscope at intervals of 6 h for the first mm). These samples were transferred into 50 mL clean 12 h, and every 12 h from 12 to 288 h. polypropylene bottles, and kept in a freezer (–40oC) until Excretion rate analysis. Dissolved inorganic nutrient concentrations were determined, as described above. Intracellular nutrient con- Excretion rate of Noctiluca was measured by a water- + – – tents were quantified by subtracting the NH4-N, NO3+NO2 bottle method (Ikeda 1974). After acclimating for 4–6 h in 3– -N, PO4 -P and Si(OH)4-Si concentrations measured in fil- an incubator adjusted to the in situ surface temperature at + – – o tered seawater from the respective NH4-N, NO3+NO2-N, capture (16.7–25.8 C), buoyant Noctiluca cells were sepa- 3– PO4 -P and Si(OH)4-Si concentrations measured in filtered rated from mixed samples into petri dishes using a wide- seawater containing extracted intracellular nutrients and bore pipette, and were carefully washed with filtered sea- by dividing the number of cells (i.e. 200) that had been ex- water several times by sieving through a 200 μm-mesh tracted in each analysis. screen. Usually, 400 Noctiluca cells were transferred into each 100 mL glass DO bottle filled with filtered seawater. Diatom response to enrichment with nutrients ex- In each experiment, at least three replicates and one con- tracted from Noctiluca cells trol (without Noctiluca) were prepared. The experiment Cell chains of the diatom Thalassiosira rotula Meunier, was run for 1–72 h in an incubator adjusted to the same one of the dominant species in the microphytoplankton as- temperatures as for acclimation under dark conditions. semblages at our study site (Ara et al. 2011a), were isolated Noctiluca was not fed during the experiment. After the in- from natural assemblages and mono-cultured in modified cubation, seawater was siphoned off through a 200 μm- Erd-Schreiber medium (Nakajima 1988) under laboratory mesh screen into 50 mL clean polypropylene bottles, and o + 3– conditions (i.e. temperature: 23.0±1.0ºC; white fluorescent kept in a freezer (–40 C) until analysis. NH4-N and PO4 -P –2 –1 + 3– light at photon flux density of 60 μmol m sec ; light : dark were determined, as described above. NH4-N and PO4 -P + photoperiod of 12L : 12D). Photon flux density was mea- excretion rates were quantified by subtracting NH4-N and 3– + sured using an underwater quantum sensor (LI-1925SA/ PO4 -P concentrations in the control bottles from the NH4- 3– LI-250, LI-COR, USA). Filtered seawater, previously auto- N and PO4 -P concentrations in the respective experimen- claved at 121oC for 20 min, was prepared for the controls tal bottles at the end of each incubation period, and by di- Noctiluca scintillans in Sagami Bay 13

Fig. 2. Seasonal variations in vertical proﬁles of water temperature, salinity and density (σt) in Sagami Bay from January 2002 to December 2006. Arrows denote sampling dates. viding by the number of cells (i.e. 400) in each experimen- Results tal bottle. Data analysis Temperature, salinity, density and dissolved inorganic nutrients The depths where 25, 50 and 75% of the Noctiluca population was distributed (25, 50 and 75%D, respectively) on Water column temperature, salinity and density (σt) var- each sampling date were calculated from the abundance at ied from 12.34 to 28.25oC, from 30.28 to 34.74 and from depths of 0, 5, 10, 20, 30, 40 and 50 m, according to Pen- 19.91 to 26.01, respectively, and showed similar seasonal nak (1943). Correlations between abiotic/biotic variables variations during all ﬁve years (Fig. 2). In autumn–spring

(water temperature, salinity, Chl-a and PP) and Noctiluca (November–April), temperature, salinity and density (σt) abundance in the water column (0–50 m depth) for all sam- were homogeneous throughout the water column. pling dates in 2002–2006 and in each season of 2002–2006 Thermo-, halo- and pycnoclines developed at 20–30 m (winter: December–February; spring: March–May; sum- depth in summer (June–September), while at the surface mer: June–September; autumn: October–November) and temperatures rose to 25–28oC and salinities declined to at the depth of 50%D were analyzed by Spearman rank 30–32. The difference between densities (σt) at the surface correlation. and lower layer, i.e. vertical stability of seawater in the water column (0–50 m depth), was higher in spring–autumn, especially during the summer stratiﬁed period, and lower in winter. + NH4-N concentration varied from below the detection 14 K. Ara et al.

+ －－ Fig. 3. Seasonal variations in vertical proﬁles of ammonium nitrogen (NH4-N), nitrate+nitrite (NO3 +NO2 -N), phosphate 3－ (PO4 -P) and silicate (Si(OH)4-Si) in Sagami Bay from January 2002 to December 2006. Arrows denote sampling dates.

– – limit (0.02 μM) to 5.56 μM, and there was no consistent the summer stratified period (June–September), NO3+NO2 3– trend in seasonal and vertical distributions (Fig. 3). The -N, PO4 -P and Si(OH)4-Si concentrations were very low – – 3– concentrations of NO3+NO2-N, PO4 -P and Si(OH)4-Si in the upper layer (0–20 m depth), whereas they main- showed similar seasonal variations during all five years. tained high levels below the stratified layer. When a red – – 3– NO3+NO2-N, PO4 -P and Si(OH)4-Si concentration ranged tide of Noctiluca was formed at the surface on April 19, from 0.15 to 13.60 μM, from below the detection limit 2006, dissolved inorganic nutrient concentrations, except + (0.02 μM) to 0.91 μM and from 0.22 to 32.75 μM, respec- for Si(OH)4-Si, were extremely high (NH4-N: 92.0 μM; – – – 3– tively. In late autumn–spring (November–March), NO3 NO3+NO2-N: 28.6 μM; PO4 -P: 7.9 μM; Si(OH)4-Si: – 3– +NO2-N, PO4 -P and Si(OH)4-Si concentrations were high 7.0 μM) at the center of Noctiluca patches in the uppermost and homogeneous throughout the water column. During layer (0–3 cm depth). Noctiluca scintillans in Sagami Bay 15

Fig. 4. Seasonal variations in vertical proﬁles of chlorophyll a concentration and primary productivity in Sagami Bay from January 2002 to December 2006. Arrows denote sampling dates.

Chl-a concentration and primary production the upper layer (0–10 m depth). Drastic increases in abun- Chl-a concentration varied from 0.03 to 20.36 μg L–1 dance at the surface were associated with increases in (Fig. 4). In November–January, Chl-a concentrations were water temperature and with decreases in salinity, and these low (<1 μg L–1) throughout the water column. Chl-a con- appeared during or just after the period of spring phyto- centrations were very high throughout the water column or plankton blooms. Higher abundances (>1.0×102 cells L–1) in the upper layer (0–20 m depth) during the period of were found in May–July 2002, April–May, July and Sep- spring phytoplankton blooms (February–May). The maxi- tember 2003, April and July 2004, April, August and Octo- mum Chl-a concentration during the spring phytoplankton ber 2005 and June 2006. The maximum abundance in blooms (February–May) in 2004 (20.4 μg L–1) was greater 2002 (1.8×103 cells L–1) was higher than that in other years than that in other years (5.3–5.9 μg L–1). Relatively high (2.1×102–9.7×102 cells L–1). Chl-a concentrations (3–5 μg L–1) remained in the upper The depth where 50% of the Noctiluca population was layer (0–10 m depth) during summer (June–September). distributed (50%D) varied from 0 to 40 m, with an overall The maximum Chl-a concentration during the autumn mean±SE of 6.7±0.7 m (Fig. 5). The depth of 50%D was phytoplankton blooms (October–November) in 2006 found frequently in the upper layer (0–10 m depth) during (14.9 μg L–1) was greater than in other years (0.9–3.4 μg most of the study period, especially at the surface-to-sub- L–1). surface (0–2 m depth) in spring–summer, whereas during The depth of the euphotic zone (i.e. the depth corre- late autumn–early spring when Noctiluca was scarce (<5 sponding to 1% of sea surface sunlight intensity) varied cells L–1), the depth of 50%D was found below 20 m depth. from 4.5 m to the bottom, with an overall mean±SE of In almost all cases, Noctiluca abundance showed signifi- 26.5±1.1 m. PP varied from ca. 0 to 1321.4 μg C L–1 d–1 cantly positive correlations with Chl-a and PP, and signifi- (Fig. 4). Higher rates (>300 μg C L–1 d–1) of PP were ob- cantly negative correlations with salinity (Table 2). Nocti- served in the upper layer (0–10 m depth) in April–October, luca abundance showed significantly positive correlations and the rates were low (<40 μg C L–1 d–1) throughout the with water temperature in spring and summer in the water water column in December–January. column and during all periods at the depth of 50%D, but significantly negative correlation in winter in the water Abundance column. Noctiluca abundance varied from 0 to 1.8×103 cells L–1, Cell diameter and volume with an overall mean±SE of 2.5×101±3 cells L–1 (Fig. 5). The abundance was high in spring–summer, especially in The mean (±SE) cell diameter and volume of Noctiluca 16 K. Ara et al.

Fig. 5. Seasonal variations in abundance of Noctiluca scintillans and the midpoint distribution depth (50%D) in Sagami Bay from January 2002 to December 2006. Arrows denote sampling dates. Filled circles and vertical lines in the bottom panel denote 50%D and the range of 25–75%D, respectively.

Table 2. Coefﬁcients of Spearman rank correlation between abiotic/biotic variables (water temperature, salinity, chlorophyll a and primary productivity) and the abundance of Noctiluca scintillans (cells L－1) in the water column (0–50 m depth, WC) and at the midpoint distribution depth (50%D) in Sagami Bay, from January 2002 to December 2006. T: water temperature (°C); S: salinity; Chl-a: chlorophyll a (μg L－1); PP: primary productivity (μg C L－1 d－1). Signiﬁcant correlation: *0.01

Abiotic/biotic variables Period/depth T Chl-a PP S (°C) (μg L－1) (μg C L－1 d－1)

All period (WC) 0.023 －0.148*** 0.437*** 0.364*** Winter (WC) －0.414*** 0.249*** 0.410*** 0.133 Spring (WC) 0.440*** －0.397*** 0.178** 0.397*** Summer (WC) 0.171** －0.463*** 0.443*** 0.512*** Autumn (WC) －0.029 －0.180* 0.245** 0.198* All period (50%D) 0.209* －0.328** 0.311** 0.444***

varied from 325±13 to 826±10 μm and from 1.83×107± and Chl-a. Temperature and Chl-a showed similar contri- 1.99×106 to 3.15×108±1.27×107 μm3, respectively (Fig. 6). butions to cell size variations (Table 3). Cell size (i.e. diameter and volume) of Noctiluca showed Intracellular nutrient content and concentration seasonal fluctuations: their cell sizes were generally larger in winter–early spring and smaller in summer. There were The mean (±SE) intracellular nutrient content and con- significantly negative correlations between abiotic/biotic centration of Noctiluca varied from 0.45±0.01 to 4.95± variables (i.e. temperature and Chl-a) and cell sizes (Fig. 0.39 nmol cell–1 and from 10.1±0.28 to 49.0±1.79 amol –3 + –1 7), while there was no statistically significant correlation μm for NH4-N, from 0.03±0.01 to 0.64±0.02 nmol cell –3 – between temperature and Chl-a (p>0.05). With the present and from 0.37±0.17 to 6.52±<0.01 amol μm for NO3 – –1 multiple regression model, 43% and 42% of variance in +NO2-N, from 0.07±<0.01 to 0.40±0.01 nmol cell and –3 3– cell diameter and volume were explained by temperature from 0.89±0.04 to 2.75±0.05 amol μm for PO4 -P, and Noctiluca scintillans in Sagami Bay 17

Fig. 6. Seasonal variations in cell diameter and volume of Noctiluca scintillans in Sagami Bay from January 2002 to Decem- ber 2006. Data are means±SE.

Fig. 7. Relationships between abiotic/biotic variables, i.e. water temperature (T) and chlorophyll a (Chl-a) concentration at the depth of 50%D, and cell sizes, i.e. cell diameter and volume (CD and CV, respectively), of Noctiluca scintillans in Sagami Bay from January 2002 to December 2006. Data are means±SE. For each relationship, logarithmic or exponential approximation was applied to obtain a higher correlation coefﬁcient in regression analysis. 18 K. Ara et al.

Table 3. Regression statistics for cell diameter and volume of Noctiluca scintillans (Y and ln Y, μm and μm3, respectively) with respect －1 to ambient water temperature (X1, °C) and chlorophyll a (Chl-a) (X2, μg L ). a, b and c are constants.

Y or ln Y=aX1+bX2+c Cell size r2 n p a b c

Cell diameter －15.6 －12.3 877 0.426 92 <0.0001 Cell volume －0.079 －0.081 20.0 0.416 92 <0.0001

+ －－ 3－ Fig. 8. Seasonal variations in intracellular nutrient (NH4-N, NO3 +NO2 -N, PO4 -P and Si(OH)4-Si) content and concentration of Noctiluca scintillans in Sagami Bay from January 2002 to December 2006. Data are means±SE. from 0.13±0.03 to 0.99±0.02 nmol cell–1 and from -P contents and concentrations exhibited seasonal ﬂuctua- –3 0.88±0.18 to 16.2±1.46 amol μm for Si(OH)4-Si, respec- tions: the intracellular nutrient contents were higher in + – – 3– tively (Fig. 8). Intracellular NH4-N, NO3+NO2-N and PO4 spring and lower in summer, which was similar to the Noctiluca scintillans in Sagami Bay 19

+ 3– trend seen in cell size variations, whereas the intracellular time and proportion (%) of NH4-N and PO4 -P excretion to + 3– nutrient concentrations were higher in summer and lower intracellular NH4-N and PO4 -P content. There was no sta- in spring, which translates to a mostly inverse relationship tistical difference (p>0.05) between these two regression to the intracellular nutrient contents. For intracellular equations of experiment time–proportion of nutrient excre- + Si(OH)4-Si content, there was no consistent trend in the tion to intracellular nutrient content relationships for NH4- 3– seasonal fluctuations during the study period. There were N and PO4 -P. significant correlations between cell volume and intracel- + – – 3– lular NH4-N, NO3+NO2-N and PO4 -P content and be- + 3– Discussion tween cell volume and intracellular NH4-N, PO4 -P and Si(OH) -Si concentration, respectively (Fig. 9). 4 Seasonal variations in abundance The intracellular nutrient concentrations quoted above + were 10,100–49,000 μM for NH4 -N, 374–6,520 μM for The present study found clear seasonal variations in – – 3– NO3+NO2-N, 891–2,790 μM for PO4 -P and 878–16,200 Noctiluca abundance: although the variations (i.e. exact μM for Si(OH)4-Si (Table 4). The ratio of intracellular nu- duration of occurrence, abundance, distribution) differed trient concentration to nutrient concentration in the sur- depending on the year, overall variation patterns were es- rounding seawater (i.e. the concentration ratio) at each sentially similar during all five years, and these were simi- sampling date and depth was 3.5×103–1.1×106-fold for lar to those reported for other temperate coastal waters + 1 4 – – NH4-N, 6.1×10 –1.4×10 -fold for NO3+NO2-N, 1.4× (e.g. Uhlig & Sahling 1990, Pithakpol et al. 2000a, b, Tada 3 5 3– 1 4 10 –2.6×10 -fold for PO4 -P, and 6.7×10 –3.2×10 -fold for et al. 2004, Miyaguchi et al. 2006). For Noctiluca, such Si(OH)4-Si. seasonal variations occur commonly in temperate seas, being explainable by local abiotic/biotic variables (i.e. Diatom response to enrichment with nutrients ex- physical structure of ambient seawater, water temperature, tracted from Noctiluca cells food availability) and the abiotic/biotic variable-dependent The cell abundance of Thalassiosira rotula in the con- growth of Noctiluca. The seasonal variations in vertical trol and enriched solutions (i.e. Medium 1, 2 and 3) showed distribution of Noctiluca were correlated significantly to similar values at 6–60 h, increasing to ca. 3×103 cells the physical structure of the water column: Noctiluca was mL–1 at 60 h (Fig. 10). The cell abundances in enriched so- distributed most densely in the upper layer (0–10 m depth) lutions increased rapidly at 60–84 h, whereas at that time in late spring–early autumn (April–October), and in even those in the control solution remained low. Thereafter, greater numbers during the summer stratified period until the end of experiment (72–288 h), the cell abundances (June–September), whereas during other periods of verti- in the test solutions remained at higher levels than in the cal mixing of waters (November–March), it was rare and control. The cell abundance in the control was largely con- dispersed throughout the water column. The depth where stant (ca. 3.5×103 cells mL–1) at 72–204 h and then de- 50% of the Noctiluca population was distributed (50%D) creased gradually. The maximum cell abundances in the exhibited a mostly inverse relationship to the vertical sta- control, Medium 1, 2 and 3 were found at 144, 276, 252 bility of the water column (Figs. 2, 5), and there was a sig- and 240 h, respectively. nificantly negative correlation between them (exponential approximation, r2=0.177, p<0.0001). This indicates that Excretion rate seasonal stratification, i.e. increasing vertical stability of + 3– The mean (±SE) NH4-N and PO4 -P excretion rate of the water column (0–50 m depth), was one of the principal Noctiluca varied from 2.4±0.9 to 242.6±7.4 pmol cell–1 factors causing accumulation of positively buoyant cells in h–1 and from 0.2±0.1 to 24.2±10.8 pmol cell–1 h–1, respec- the upper layer, due to the lower specific gravity of Nocti- + 3– tively (Fig. 11). Both NH4-N and PO4 -P excretion rates de- luca cells than that of seawater (Nozawa 1943). Aggrega- creased exponentially with time: the rates were higher at tion of Noctiluca in the surface water has been found in 1–12 h, especially at 1–3 h, and were almost constant at many other coastal waters (e.g. Kuroda & Saga 1978, 24–72 h. There were significant correlations between ex- Schaumann et al. 1988, Elbrächter & Qi 1998, Tada et al. + 3– periment time and NH4-N and PO4 -P excretion rates, re- 2004). In littoral waters, after ascent to the upper layer spectively. With the present multiple regression model, caused by seasonal stratification, the passive accumulation + 3– 91% and 88% of variance in NH4-N and PO4 -P excretion of Noctiluca cells due to wind and/or tidal currents has rates were explained by experiment time, ambient water been reported as an important mechanism for the forma- + 3– temperature and intracellular NH4-N and PO4 -P contents tion of Noctiluca blooms or aggregations (Le Fèvre & (Table 5). The proportion (%) of nutrient excretion to intra- Grall 1970, Huang & Qi 1997, Dela-Cruz et al. 2003, Mi- cellular nutrient content varied from 0.1 to 23.2% h–1 for yaguchi et al. 2006). + –1 3– NH4-N and from 0.1 to 22.5% h for PO4 -P. The propor- At our study site, Noctiluca abundance showed signifi- + 3– + tions of NH4-N and PO4 -P excretion to intracellular NH4- cantly positive correlations with water temperature, Chl-a 3– N and PO4 -P content decreased exponentially with time. and PP, and an inverse correlation to salinity: in almost all There were significant correlations between experiment seasons, these correlations were significant. Significantly 20 K. Ara et al.

Fig. 9. Relationships between cell volume (CV) and intracellular nutrient [NH+-N (ICT ), NO－+NO－-N (ICT ), 4 NH4 3 2 NO3+NO2 PO3－-P (ICT ) and Si(OH) -Si] content and between cell volume and intracellular nutrient [NH+-N (ICC ), NO－+NO－-N, 4 PO4 4 4 NH4 3 2 PO3–-P (ICC ) and Si(OH) -Si (ICC )] concentration in Sagami Bay from January 2002 to December 2006. Data are 4 PO4 4 Si(OH)4 means±SE. For each relationship, linear, logarithmic, exponential or power approximation was applied to obtain a higher correlation coefﬁcient in regression analysis. Noctiluca scintillans in Sagami Bay 21

+ －－ 3－ Table 4. The mean (±SE) intracellular nutrient (HN4-N, NO3 +NO2 -N, PO4 -P and Si(OH)4-Si) content and concentration within Noctiluca scintillans cells, and the ratio of intracellular nutrient concentration to nutrient concentration in the surrounding seawater (i.e. concentration ratio) at each sampling date and depth in Sagami Bay, from January 2002 to December 2006. Data in parentheses are overall mean±SE.

Intracellular content Intracellular concentration Concentration ratio (nmol cell－1) (μM) (-fold)

0.45±0.01–4.95±0.39 10,100–49,000 3.5×103–1.1×106 HN+-N 4 (2.16±0.15) (20,600±1,230) (1.5×104±4.2×103) 0.03±0.01–0.64±0.02 374–6,520 6.1×101–1.4×104 NO－+NO－-N 3 2 (0.27±0.03) (2,560±242) (9.2×102±1.3×102) 0.07±<0.01–0.40±0.01 891–2,790 1.4×103–2.6×105 PO3－-P 4 (0.19 ± 0.01) (1,720±72) (7.0×103±1.3×103) 0.13±0.03–0.99±0.02 878–16,200 6.7×101–3.2×104 Si(OH) -Si 4 (0.44±0.04) (5,040±604) (9.7×102±1.9×102)

Fig. 10. Temporal variation in cell abundance of Thalassiosira rotula under three different enrichment conditions with nutrients extracted from Noctiluca scintillans cells. positive correlations between Noctiluca abundance and than with water temperature. In the present study, it is ex- Chl-a have been reported in other coastal waters (e.g. pected that salinity would affect abundances minimally Schaumann et al. 1998, Painting et al. 1993, Dela-Cruz et because it varied within the range (30.3–34.7) of suitable al. 2002). Signiﬁcant correlations with Chl-a and PP sug- salinities for continual Noctiluca population growth (14– gest that Noctiluca and phytoplankton are in a grazer–prey 34, Lee & Hirayama 1992). In summer, especially in the relationship: the variations in abundance of Noctiluca are upper layer (0–10 m depth) at temperatures of 26–28oC, affected by Chl-a concentration (i.e. bottom-up control), even though Chl-a and PP remained relatively high, Nocti- while Chl-a concentration would not be affected mainly by luca abundances were very low (Figs. 2, 4, 5), as similarly Noctiluca abundance (i.e. top-down control), because in found in other waters (e.g. Uhlig & Sahling 1990, Huang & the latter case prey and grazer organism abundances nor- Qi 1997, Schoemann et al. 1998, Tada et al. 2004, Miya- mally would show an inverse relationship and there would guchi et al. 2006). Laboratory-culture experiments have be signiﬁcantly negative correlation between them (Huang shown that growth and production rates of Noctiluca are & Qi 1997, Isinibilir et al. 2008). In spring, Noctiluca temperature- and food-dependent: Noctiluca has enhanced abundance showed stronger correlations with water tem- growth rates at temperatures ranging from 5 to 21–23oC perature, salinity and PP than Chl-a, whereas in summer it and under higher prey (phytoplankton) concentrations, showed stronger correlations with salinity, Chl-a and PP whereas it could not grow at temperatures higher than 26– 22 K. Ara et al.

+ 3－ Fig. 11. Relationships between elapsed experiment time (ET) and nutrient (NH4-N and PO4 -P) excretion rate of Noctiluca scintillans and between experiment time and proportion of nutrient excretion to the intracellular nutrient content (ER and %NH4 ER ). Data are means±SE. %PO4

+ 3－－1 －1 Table 5. Regression statistics for ammonia (NH4-N) and phosphate (PO4 -P) excretion rates (Y, pmol cell h ) with respect to + 3－－1 elapsed experiment time (X1, hours), water temperature (X2, °C) and intracellular NH4-N or PO4 -P content (X3, nmol cell ). a, b, c and d are constants.

ln Y=a ln X +bX +cX +d Excretion 1 2 3 r2 n p rate a b c d

Ammonia －0.92 0.11 1.25 0.67 0.913 24 <0.0001 excretion Phosphate －1.05 －0.12 －3.87 6.33 0.882 24 <0.0001 excretion

27oC (Lee & Hirayama 1992, Buskey 1995, Kiørboe & tion growth of Noctiluca in the upper layer in spring–au- Titelman 1998, Nakamura 1998a, b, Pithakpol et al. 2000a, tumn, except during the periods of temperatures higher Tada et al. 2004). At our study site, the abundance and bio- than ca. 26oC in summer, since Noctiluca has been known mass of planktonic organisms (i.e. pico-, nano-, micro- and to feed on a variety of prey items, including phytoplank- mesozooplankton) and the concentrations of particulate ton, bacteria, micro- and mesozooplankton, fish eggs and carbon and nitrogen (PC and PN, respectively) were higher detritus (e.g. Kirchner et al. 1996, Shanks & Walters 1996, in spring–autumn, especially in the upper layer, whereas Sautour et al. 2000, Fonda Umani et al. 2004). these were lower throughout the water column (0–50 m The present study found that Noctiluca abundance and depth) in winter (Ara & Hiromi 2007, 2009, Ara et al. Chl-a concentration followed similar seasonal variations as 2011a, b, Okutsu et al. 2012). These could enhance popula- outlined above. However, they showed drastic variations Noctiluca scintillans in Sagami Bay 23 within the scale of a few days or weeks, with increases in tus and growth of Noctiluca. During the period of lower Noctiluca abundance following increases in Chl-a concen- temperatures and Chl-a concentrations (winter–early trations with a time lag of a few weeks (Figs. 4, 5). This is spring), larger cells would be exposed to conditions of similar to the seasonal variation pattern where heterotroph lower food availability, have lower growth rates and sur- (i.e. zooplankton) biomass peaks appear after phytoplank- vive longer, as mentioned above, and thus they would ac- ton biomass (i.e. Chl-a) peaks in open seas (e.g. Parsons et cumulate lower nutrient concentrations but higher nutrient al. 1984b). In the upper layer (0–10 m depth), the time lag contents, whereas during the other periods of higher tem- in the fortnightly present time-series of Chl-a concentra- peratures and Chl-a concentrations (late spring–summer), tion and Noctiluca abundance (n, cells L–1) when converted smaller cells would have higher growth (cell division) rates to log (n+1), and examined by a cross-correlation analysis, and accumulate higher nutrient concentrations but lower was roughly estimated to be 39 days (r=0.294, p<0.0001). nutrient contents. Our preliminary laboratory-culture experiments on Noc- Seasonal variations in cell size tiluca found that under conditions of sufficient food (prey: The cell size of Noctiluca exhibited seasonal fluctua- raphidophycean flagellate Heterosigma akashiwo (Hada) tions, with larger sizes in winter–early spring and smaller Hada, initial prey abundance: 1.3×104 cells mL–1), intra- + – – 3– sizes in summer. The cell size was negatively correlated cellular NH4-N, NO3+NO2-N and PO4 -P contents in- with water temperature, as has been found in the Inland creased gradually until the 35th day, whereas intracellular

Sea of Japan (Pithakpol et al. 2000a). Laboratory-culture Si(OH)4-Si contents revealed neither increasing nor de- experiments on Noctiluca have shown that cell size increasing trends during the experiment. Under starvation + – – 3– creases with decreasing food availability (i.e. phytoplank- conditions, intracellular NH4-N, NO3+NO2-N, PO4 -P and ton cell abundance) and the cells swelled when the dura- Si(OH)4-Si contents stayed at similar levels at least until tion of the starved condition was prolonged. Furthermore, the 13th day, and then most of the Noctiluca cells died be- growth rates decreased with increasing cell size (Buskey fore reaching the 25th day (Ara unpublished data). These + 1995, Kiørboe & Titelman 1998), although the cell size of results indicate that Noctiluca regenerates NH4-N and 3– other heterotrophic dinoflagellates has been reported to in- PO4 -P by active feeding and accumulates these sub- – – crease with increasing food (phytoplankton) concentration stances within the cells, whereas NO2-N and NO3-N might (e.g. Protoperidinium hirobis (Abé) Balech, Jacobson & be nitrified (oxidized) by-product substances derived from + Anderson 1993). In the present study, cell size of Noctiluca NH4-N, due to the high acidity of the intracellular liquids decreased with increasing Chl-a concentration, as has been (pH 4–5, Okaichi & Nishio 1976). In addition, these data found in other coastal waters (Dela-Cruz et al. 2003). The indicate that Noctiluca contains silicate, derived from dia- seasonal variations in cell size imply that during the period tom frustules from its phytoplankton prey (diatoms), that of lower temperatures and Chl-a concentrations (winter– would be neither digested nor regenerated by Noctiluca, as early spring) Noctiluca would be exposed to lower food has been mentioned by Montani et al. (1998). availability, have lower growth rates and survive longer The proportion of intracellular nutrient contents in the than during periods of higher temperatures and Chl-a con- water column (0–50 m depth) and in the euphotic zone at centrations (late spring–summer), and this was consistent each sampling date accounted for 0–82.5% (mean: 22.7%) + with the variations in abundance. Thus, the cell size could and 0–91.7% (mean: 30.5%) for NH4-N, 0–40.4% (mean: – – reveal the nutritional state and information on the popula- 1.2%) and 0–71.1% (mean: 2.0%) for NO3+NO2-N, tion growth of Noctiluca at our study site. 0–74.1% (mean: 7.6%) and 0–89.0% (mean: 11.8%) for DIN (dissolved inorganic nitrogen: NH++NO–+NO–-N), and Intracellular nutrient content and concentration, and 4 3 2 0–81.4% (mean: 9.3%) and 0–91.8% (mean: 15.1%) for nutrient pools 3– PO4 -P of the total nutrient standing stocks (i.e. intracellu- The present study showed that Noctiluca had high intra- lar nutrient contents plus nutrient concentrations in the am- cellular nutrient contents and concentrations. The values bient seawater), respectively (Fig. 12). The intracellular nu- + 3– for intracellular NH4-N and PO4 -P contents obtained in trient contents comprised larger proportions during the pe- the present study were comparable to or slightly lower than riod of higher Noctiluca abundances in April–July, ac- + those obtained for the Inland Sea of Japan (NH4-N: 1.7– counting for an average in the euphotic zone and in the –1 3– –1 6.8 nmol cell , PO4 -P: 0.1–0.6 nmol cell , Montani et al. water column (0–50 m depth) of 49.2–63.7% and 33.9– + – – 1998, Pithakpol et al. 2000a, b). The intracellular nutrient 54.5% for NH4-N, 6.4–16.6% and 2.8–6.4% for NO3+NO2 contents, except for Si(OH)4-Si, revealed clear seasonal -N, 28.0–48.8% and 15.5–30.2% for DIN, 39.2–62.7% and 3– fluctuations that were positively correlated to cell size, as 16.7–35.5% for PO4 -P of the total nutrient standing stocks, has also been reported in the Inland Sea of Japan (Montani respectively. et al. 1998, Pithakpol et al. 2000a, b). The intracellular nu- Diatom response to enrichment with nutrients ex- trient concentrations were negatively correlated to cell tracted from Noctiluca cells size. This indicates that intracellular nutrient contents and concentrations are dependent mainly on the nutritional sta- Thalassiosira rotula increased to higher cell abundances 24 K. Ara et al.

Fig. 12. Seasonal variations in the proportion (%) of intracellular nutrient contents of Noctiluca scintillans to total (poten- tial) nutrient standing stocks (i.e. intracellular nutrient contents of Noctiluca scintillans plus actual nutrient concentrations in seawater) in the water column (0–50 m depth) and in the euphotic zone.

Excretion rate and nutrient supplies when higher concentrations of nutrients extracted from + 3– Noctiluca cells were added. This indicates that nutrients NH4-N and PO4 -P excretion rates of Noctiluca varied regenerated by Noctiluca could be available for phyto- depending on elapsed experiment time, temperature and its plankton (diatom) growth and increase. Thus, Noctiluca cell size (volume), and were most strongly affected by time would play an effective role as a nutrient regenerator in (Table 5, Fig. 11). These rates were much higher during the aquatic ecosystems through it regenerating and releasing first 1–3 h and decreased rapidly with time. This seems to nutrients that could subsequently be utilized by phyto- be common in zooplankton with metabolic (i.e. respiration plankton. and excretion) rates being high in freshly-caught specimens and decreasing rapidly with time under laboratory conditions (Mayzaud 1976, Le Borgne 1979, Ikeda & Sk- Noctiluca scintillans in Sagami Bay 25 joldal 1980, Skjoldal et al. 1984, Fernández-Urruzola et al. high in the uppermost layer (0–3 cm depth), as also found 2011). Two possible explanations can be offered for the in the Inland Sea of Japan (Montani et al. 1998, Pithakpol phenomenon: (1) higher rates for freshly caught specimens et al. 2000a). In addition, relatively high nutrient concen- are due to the increased activity during sampling, and (2) trations associated with high Noctiluca abundances at the lower rates after prolonged laboratory maintenance of surface have been observed in other waters (Schaumann et specimens are due to laboratory effects, such as depletion al. 1988, Elbrächter & Qi 1998, Al-Azri et al. 2007). Ac- of the food source. The former scenario supposes that the cordingly, we addressed whether the concentrations of dis- + 3– lower rates in specimens maintained over long periods solved inorganic nutrients, especially NH4-N and PO4 -P, would better estimate the rates under natural conditions, in ambient seawater were influenced by Noctiluca. The + 3– while the latter supposes that the higher rates of freshly timing of intermittently high NH4-N, PO4 -P, Chl-a and/or caught specimens are closer to their natural values (Omori PP in the upper layer (0–10 m depth) followed high Nocti- + 3– + 3– & Ikeda 1984). Actually, NH4-N and PO4 -P excretion luca abundances and presumably high NH4-N and PO4 -P rates of Noctiluca were higher when Chl-a concentrations supply by Noctiluca excretion (Fig. 13). Thus, dense Nocti- in ambient seawater were higher during our study, espe- luca populations could cause increases in dissolved inor- cially early in the experiment (i.e. the first 1–6 h). This in- ganic nutrient concentrations in ambient seawater and con- dicates that Noctiluca excretion rates reflect food availabil- sequently in Chl-a and PP. In this case, the timing of both ity in the ambient seawater on each sampling date, and that the high Noctiluca abundances and Chl-a concentrations the rapid decreases in Noctiluca excretion rate with could be explained by a mutually supportive relationship elapsed experiment time were due to the effect of starva- (e.g. Harris 1959, Dugdale & Goering 1967, Corner & tion during the experiment. Our preliminary laboratory- Davis 1971) between phytoplankton and Noctiluca: bot- culture experiments on Noctiluca found that under starved tom-up control (i.e. phytoplankton–Noctiluca) and nutrient conditions the intracellular nutrient contents remained supply by Noctiluca excretion stimulating phytoplankton constant for several days, as mentioned above, which im- growth. On the other hand, in April 2004, in April 2003 plies that during this period Noctiluca accumulated nutri- and June 2006, and in April and August 2005, intermit- + 3– ents and decreased its excretion rates to very low levels. In tently high NH4-N, PO4 -P, Chl-a and/or PP could have + 3– addition, the proportion (%) of NH4-N and PO4 -P excre- been caused mainly by the intrusion of nutrient-rich waters + 3– tion rates to intracellular NH4-N and PO4 -P contents ex- into the bay, i.e. by the expansion of Tokyo Bay Water and hibited similar variations to each other (Fig. 11), indicating by the intrusion of Intermediate Oyashio Water and of the + that Noctiluca excreted liquids containing both NH4-N and bottom slope water (deep water) from the continental 3– PO4 -P that had been regenerated and accumulated within slope, respectively (Ara & Hiromi 2008, Ara et al. 2011b), the cells in the same proportions. Thus, it was assumed as well as nutrient supply by Noctiluca excretion also + 3– that the highest NH4-N and PO4 -P excretion rates, ob- being high. tained during the first 1 h (243 pmol N cell–1 h–1 and We evaluated the role of Noctiluca in the marine ecosys- –1 –1 + 3– 24 pmol P cell h ), would have been less influenced by tem by estimating the contribution of NH4-N and PO4 -P starvation and would therefore be closer to the actual ex- supply from excretion to the DIN and DIP requirement for cretion rate for Noctiluca in ambient seawater. These val- phytoplankton primary production at our study site. Daily + ues were applied to the following calculations. The NH4-N DIN and DIP requirement for phytoplankton was esti- 3– and PO4 -P excretion rates of Noctiluca were 7.5–10.3-fold mated from PP values obtained in the present study, as- and 7.9–15.3-fold higher than those of micro- and meso- suming a nutrient uptake molar C : N : P ratio of zooplankton (i.e. planktonic protists and metazoans) with 106 : 16 : 1 (i.e. Redfield ratio, Redfield et al. 1963, Brzez- the same body weights (188 ng C or 428 ng DW) at the inski 1985, Brzezinski & Nelson 1995). The depth-inte- same water temperature (24.5oC). These values were calcu- grated daily DIN and DIP requirement was calculated by lated using the regression equations of cell volume-cell the integral of daily DIN and DIP requirement in the eu- carbon weight relationship and assuming carbon weight to photic zone, i.e. from the sea-surface to the depth corre- + be 43.9% of dry weight for Noctiluca (Omori 1969, Tada et sponding to 1% of sea-surface sunlight intensity. NH4-N 3– –1 –1 al. 2000), and the regression equations of body weight-ex- and PO4 -P excretion rates (243 pmol N cell h and cretion rate relationships for mesozooplankton and plank- 24 pmol P cell–1 h–1) were converted to daily rates by mul- + tonic protists (Ikeda 1985, Dolan 1997). tiplying by 24 (hours). The contribution of daily NH4 -N 3– During spring–summer, seasonal stratification inter- and PO4 -P supply by Noctiluca excretion was estimated to fered with nutrient supply to the upper layer and kept dis- be 0–3,118% (mean: 34.8%) and 0–4,959% (mean: 55.3%) solved inorganic nutrients at low concentrations in the to the daily DIN and DIP requirement for primary produc- upper layer (0–20 m depth) due to nutrient uptake by phy- tion, respectively (Fig. 14). The estimated values of daily + 3– toplankton (Figs. 2–4, Ara & Hiromi 2008, Ara et al. NH4-N and PO4 -P supply by Noctiluca excretion were 2011a, b). However, when a Noctiluca red tide was formed higher during the period of higher Noctiluca abundances, at the surface on April 19, 2006, dissolved inorganic nutri- contributing on average 11.2–85.4% and 17.9–135.8% in ent concentrations, except for Si(OH)4-Si, were extremely March–October, especially 50.6–85.4% and 80.5–135.8% 26 K. Ara et al.

+ Fig. 13. Seasonal variations in abundance of Noctiluca scintillans, estimated N and P supplies by Noctiluca excretion, NH4-N 3－ and PO4 -P concentrations in ambient seawater, chlorophyll a and primary productivity in the upper layer (0–10 m depth) in Sagami Bay from January 2002 to December 2006. Data are means±SE. Hatched bar denotes the period of high Noctiluca + 3－ + 3－ abundances and high NH4-N and PO4 -P supply by Noctiluca excretion associated with intermittently high NH4-N, PO4 -P, Chl-a and/or primary productivity. Dark shading denotes the period of vertical mixing between surface and deeper waters. Noctiluca scintillans in Sagami Bay 27

+ 3－ Fig. 14. Seasonal variations in N and P requirement for phytoplankton primary production, NH4-N and PO4 -P supplied by + 3－ Noctiluca scintillans in the euphotic zone, and the ratio (%) of NH4-N and PO4 -P supplied by Noctiluca scintillans to the N and P requirements for primary production respectively, in Sagami Bay from January 2002 to December 2006.

+ 3– in April–July, to the daily DIN and DIP requirement for that of extremely high intracellular NH4-N and PO4 -P + 3– primary production, respectively. These mean and maxi- contents and high NH4-N and PO4 -P excretion rates. In mum percentages obtained in the present study were com- addition, nutrients regenerated and released by Noctiluca parable to, or relatively higher than, those calculated for could be available for phytoplankton growth and increase. micro- and mesozooplankton in other waters (e.g. Verity At our study site, Noctiluca can therefore be expected to 1985, Harrison 1992, Hernándes-León et al. 2008). play an important role as a nutrient regenerator and supplier due to the significant amount of NH+-N and PO3–-P The role of Noctiluca in the nutrient-environment and 4 4 regeneration, the pools within the cells, and its supply to aquatic ecosystems primary production. This would be especially true during One of the most characteristic aspects of Noctiluca was the period of higher Noctiluca abundances and lower nutri- 28 K. Ara et al. ent concentrations in the upper layer in late spring–sum- ami Bay, Japan. J Oceanogr 67: 87–111. mer. This would stimulate phytoplankton (diatom) abun- Baek SH, Shimode S, Kim H-C, Han M-S, Kikuchi T (2009) dance, biomass and production, and may exacerbate eutro- Strong bottom-up effects on phytoplankton community caused phication by the mutually supportive relationship between by a rainfall during spring and summer in Sagami Bay, Japan. phytoplankton and Noctiluca: bottom-up control (i.e. phy- J Mar Syst 75: 253–264. toplankton–Noctiluca) and nutrient supply by Noctiluca Brzezinski MA (1985) The Si : C : N ratio of marine diatoms: in- excretion to phytoplankton. Moreover, increasing eutrophi- terspecific variability and the effect of some environmental cation, especially Si deficiency relative to N and P, can re- variables. J Phycol 21: 347–357. sult in ecosystem-level changes by reducing the role of dia- Brzezinski MA, Nelson DM (1995) The annual silica cycle in the toms in aquatic ecosystems, e.g. frequent Noctiluca red Sargasso Sea near Bermuda. Deep-Sea Res I 42: 1215–1237. tides and/or other noxious phytoplankton (i.e. non-diatom) Buskey E (1995) Growth and bioluminescence of Noctiluca scintillans on varying algal diets. J Plankton Res 17: 29–40. blooms, which have been observed in other estuarine and Corner EDS, Davis AC (1971) Plankton as a factor in the nitro- coastal waters of Japan, e.g. Tokyo Bay (Nomura 1998, Yo- gen and phosphorus cycles in the sea. Adv Mar Biol 9: 101 – shida et al. 1998, Kamataki 2005, Yoshida & Ishimaru 204. 2008) and Osaka Bay and/or the Inland Sea of Japan (Ma- Dela-Cruz J, Ajani P, Lee R, Pritchard T, Suthers I (2002) Tem- nabe et al. 1994, Hori et al. 1998, Imai et al. 2006, Yoshi- poral abundance patterns of the red tide dinoflagellate Nocti- matsu 2008). luca scintillans along the southeast coast of Australia. Mar Ecol Prog Ser 236: 75–88. Acknowledgments Dela-Cruz J, Middleton JH, Suthers IM (2003) Population growth and transport of the red tide dinoflagellate, Noctiluca We thank Mr. Kazuharu Yuasa, captain/owner of the scintillans, in the coastal waters off Sydney Australia, using fishery boat “Genshun-maru”, for his continuous helpful cell diameter as a tracer. Limnol Oceanogr 48: 656–674. assistance in fieldwork. We thank Dr. Kuninao Tada, Dolan JR (1997) Phosphorus and ammonia excretion by plank- Kagawa University, for technical advice in measurement of tonic protists. Mar Geol 139: 109–122. intracellular nutrient contents and Dr. Yukio Yanagisawa, Dugdale RD, Goering JJ (1967) Uptake of new and regenerated Nihon University, for advice in statistical analysis. Thanks forms of nitrogen in primary production. Limnol Oceanogr 12: are also due to all members who participated in “Project 196–206. SHONAM” for helping with fieldwork and analyzing sam- Elbrächter M, Qi ZY (1998) Aspect of Noctiluca (Dinophyceae) ples. This research was financially supported in part by the population dynamics. In: Physiological Ecology of Harmful Ministry of Education, Culture, Sports Science and Tech- Algal Blooms (eds Anderson DM, Cembella AD, Hallegraeff nology of Japan through the “Open Research Center Proj- MG). NATO ASI Series, Vol. G. 41. Springer-Verlag, Berlin, ect” of Nihon University. pp. 315–335. Fernández-Urruzola I, Packard TT, Gómez M (2011) GDH activity and ammonium excretion in the marine mysid, Leptomysis References lingvura: effects of age and starvation. J Exp Mar Biol Ecol Al-Azri A, Al-Hashmi K, Goes J, Gomes H, Rushdi AI, Al- 409: 21–29. Habsi H, Al-Khusaibi S, Al-Kindi R, Al-Azri N (2007) Sea- Fonda Umani S, Beran A, Parlato S, Virgilio D, Zollet T, De sonality of the bloom-forming heterotrophic dinoflagellate Olazabal A, Lazzarini B, Cabrini M (2004) Noctiluca scintil- Noctiluca scintillans in the Gulf of Oman in relation to envi- lans Macartney in the Northern Adriatic Sea: long-term dy- ronmental conditions. Int J Oceans Oceanogr 2: 51–60. namics, relationships with temperature and eutrophication, Ara K, Hiromi J (2007) Temporal variability in primary and co- and role in the food web. J Plankton Res 26: 545–561. pepod production in Sagami Bay, Japan. J Plankton Res 29 Hama T, Miyazaki T, Ogawa Y, Iwakuma T, Takahashi M, Ot- (Suppl. 1): 185–196. suki A, Ichimura S (1983) Measurement of photosynthetic pro- Ara K, Hiromi J (2008) Temporal variability and characteriza- duction of a marine phytoplankton population using a stable 13 tion of physicochemical properties in the neritic area of Sag- C isotope. Mar Biol 73: 31–36. ami Bay, Japan. J Oceanogr 64: 195–210. Hansen HP, Koroleff F (1999) Determination of nutrients. In: Ara K, Hiromi J (2009) Seasonal variability in plankton food Methods of Seawater Analysis, 3rd Edition (eds Grasshoff K, web structure and trophodynamics in the neritic area of Sag- Kremling K, Ehrhardt M). Wiely-VCH, Weinheim, pp. 159– ami Bay, Japan. J Oceanogr 65: 757–779. 228. Ara K, Fukuyama S, Tashiro M, Hiromi J (2011a) Seasonal and Harris E (1959) The nitrogen cycle in Long Island Sound. Bull year-on-year variability in chlorophyll a and microphytoplank- Bingham Oceanogr Coll 17: 31–65. ton assemblages for 9 years (2001–2009) in the neritic area of Harrison WG (1992) Regeneration of nutrients. In: Primary Pro- Sagami Bay, Japan. Plankton Benthos Res 6: 158–174. ductivity and Biogeochemical Cycles in the Sea (eds Ara K, Yamaki K, Wada K, Fukuyama S, Okutsu T, Nagasaka S, Falkowski PG, Woodhead AD). Plenum Press, New York, pp. Shiomoto A, Hiromi J (2011b) Temporal variability in physico- 385–409. chemical properties, phytoplankton standing crop and primary Hernándes-León S, Fraga C, Ikeda T (2008) A global estimation production for 7 years (2002–2008) in the neritic area of Sag- of zooplankton ammonium excretion in the open ocean. J Noctiluca scintillans in Sagami Bay 29

Plankton Res 30: 577–585. Lee JK, Hirayama K (1992) Effects of salinity, food level and Holm-Hansen O, Lorenzen CJ, Holmes RW, Strickland JDH temperature on the population growth of Noctiluca scintillans (1965) Fluorometric determination of chlorophyll. J Cons (Macartney). Bull Fac Fish Nagasaki Univ 71: 163–168. Perm Int Explor Mer 30: 3–15. Machida M, Fujitomi M, Hasegawa K, Kudo T, Kai M, Ko- Hori Y, Miyahara K, Nagai S, Tsujino K, Nakajima M, Yama- bayashi T, Uede T (1999) Red tide of Ceratium furca along the moto K, Yoshida Y, Araki N, Sakai Y (1998) Relationships be- Pacific coast of central Japan in 1997. Nippon Suisan Gakkai- tween the dominant phytoplankton and DIN : DIP ratios in shi 65: 755–756. (in Japanese) Osaka Bay and Harima-nada. Nippon Suisan Gakkaishi 64: Manabe T, Tanda M, Hori Y, Nagai S, Nakamura Y (1994) 243–248. (in Japanese with English abstract) Changes in eutrophication and phytoplankton in Harima- Huang C, Qi Y (1997) The abundance cycle and influence factors nada–results of environmental monitoring for 20 years. Bull on red tide phenomena of Noctiluca scintillans (Dinophyceae) Coast Oceanogr 31: 169–181. (in Japanese with English ab- in Dapeng Bay, the South China Sea. J Plankton Res 19: 303– stract) 318. Mayzaud P (1976) Respiration and nitrogen excretion of zoo- Ikeda T (1974) Nutritional ecology of marine zooplankton. Mem plankton. IV. The influence of starvation on the metabolism Fac Fish Hokkaido Univ 22: 1–97. and the biochemical composition of some species. Mar Biol 37: Ikeda T (1985) Metabolic rates of epipelagic marine zooplankton 47–58. as a function of body mass and temperature. Mar Biol 85: Miyaguchi H, Fujiki T, Kikuchi T, Kuwahara VS, Toda T (2006) 1–11. Relationship between the bloom of Noctiluca scintillans and Ikeda T, Skjoldal HR (1980) The effect of laboratory conditions environmental factors in the coastal waters of Sagami Bay. J on the extrapolation of experimental measurements to the Plankton Res 28: 313–324. ecology of marine zooplankton. VI. Changes in physiological Montani S, Pithakpol S, Tada K (1998) Nutrient regeneration in activities and biochemical components of Acetes sibogae aus- coastal seas by Noctiluca scintillans, a red tide-causing dino- tralis and Acartia australis after capture. Mar Biol 58: 285– flagellate. J Mar Biotechnol 6: 224–228. 293. Nakajima T (1988) Effects of deep sea water on the growth of a Imai I, Yamaguchi M, Hori Y (2006) Eutrophication and occur- marine diatom species Skeletonema costatum. Bull Plankton rences of harmful algal blooms in the Seto Inland Sea, Japan. Soc Jpn 35: 45–55. (in Japanese with English abstract) Plankton Benthos Res 1: 71–84. Nakamura Y (1998a) Growth and grazing of a large heterotro- Isinibilir M, Kideys AE, Tarkan AN, Yilmaz IN (2008) Annual phic dinoflagellate, Noctiluca scintillans, in laboratory cul- cycle of zooplankton abundance and species composition in tures. J Plankton Res 20: 1171–1720. Izmit Bay (the northeastern Marmara Sea). Estur Coast Shelf Nakamura Y (1998b) Biomass, feeding and production of Nocti- Sci 78: 739–747. luca scintillans in the Seto Inland Sea, Japan. J Plankton Res Jacobson DM, Anderson DM (1993) Growth and grazing rates of 20: 2213–2222. Protoperidinium hirobis Abè, a thecate heterotrophic dinofla- Nomura H (1998) Changes in red tide events and phytoplankton gellate. J Plankton Res 15: 723–736. community composition in Tokyo Bay from the historical Kamataki H (2005) Occurrence of red tides in Tokyo Bay and plankton records in a period between 1907 and 1997. Ocean- Sagami Bay from 1975 to 2003. Bull Kanagawa Pref Fish Res ogr Jpn 7: 159–178. (in Japanese with English abstract) Inst 10: 49–53. (in Japanese) Nozawa K (1943) Specific gravity and its adaptation to environ- Kiørboe T, Titelman J (1998) Feeding, prey selection and prey mental sea water in Noctiluca. Zool Soc Jpn 55: 305–314. (in encounter mechanisms in the heterotrophic dinoflagellate Noc- Japanese) tiluca scintillans. J Plankton Res 20: 1615–1636. Okaichi T, Nishio S (1976) Identification of ammonia as the toxic Kirchner M, Sahling G, Uhlig G, Gunkel W, Klings KW (1996) principle of red tide of Noctiluca miliaris. Bull Plankton Soc Does the red tide-forming dinoflagellate Noctiluca scintillans Jpn 23: 75–80. (in Japanese with English abstract) feed on bacteria? Sarsia 81: 45–55. Okutsu T, Ara K, Hiromi J (2012) Microbial food web structure Kuroda K (1990) Noctiluca scintillans (Macartney) Ehrenberg. of the plankton ecosystem in the neritic area of Sagami Bay, In: Red Tide Organisms in Japan̶An Illustrated Taxonomic Japan: seasonal variability in chlorophyll a <20 μm, bacteria, Guide̶(eds Fukuyo Y, Takano H, Chihara M, Matsuoka K). heterotrophic nanoflagellates and microzooplankton. Bull Uchida Rokakuho, Tokyo, pp. 78–79. (in Japanese with Eng- Plankton Soc Jpn 59: 1–19. (in Japanese with English abstract) lish notes) Omori M (1969) Weight and chemical composition of some im- Kuroda K, Saga S (1978) The distribution and ecology of Nocti- portant oceanic zooplankton in the North Pacific Ocean. Mar luca scintillans in Osaka Bay. Bull Japan Soc Fish Oceanogr Biol 3: 4–10. 32: 56–67. (in Japanese) Omori M, Ikeda, T (1984) Methods in Marine Zooplankton Ecol- Le Borgne RP (1979) Influence of duration of incubation on zoo- ogy. A Wiley-Interscience Publication, New York, 332 pp. plankton respiration and excretion results. J Exp Mar Biol Ecol Painting SJ, Lucas MI, Peterson WT, Brown PC, Hutchings L, 37: 127–137. Mitchell-Innes BA (1993) Dynamics of bacterioplankton, phy- Le Fèvre J, Grall JR (1970) On the relationships of Noctiluca toplankton and mesozooplankton communities during the de- swarming off the western coast of Brittany with hydrological velopment of an upwelling plume in the southern Benguela. features and plankton characteristics of the environment. J Mar Ecol Prog Ser 100: 35–53. Exp Mar Biol Ecol 4: 287–306. Parsons TR, Maita Y, Lalli CM (1984a) A Manual of Chemical 30 K. Ara et al.

and Biological Methods for Seawater Analysis. Pergamon trogen content of Noctiluca scintillans in the Seto Inland Sea Press, Oxford, 173 pp. of Japan. J Plankton Res 22: 1203–1211. Parsons TR, Takahashi M, Hargrave B (1984b) Biological Tanaka K (1993) A condition of water pollution in Sagami Bay Oceanographic Processes, 3rd Edition. Pergamon Press, Ox- and inflowing rivers. Bull Japan Soc Fish Oceanogr 57: 249– ford, 329 pp. 255. (in Japanese) Pennak RW (1943) An effective method of diagramming diurnal Tanaka N (1985) IV Biology. In Coastal Oceanography Research movements of zooplankton organisms. Ecology 24: 405–407. Committee. In: Oceanography in Japanese Islands (ed the Pithakpol S, Tada K, Montani S (2000a) Nutrient regeneration Oceanographic Society of Japan). Tokai University Press, during Noctiluca scintillans red tide in Harima Nada, the Seto Tokyo, pp. 417–427. (in Japanese) Inland Sea, Japan. In: Proceedings of the 15th Ocean Engi- Tatara E, Kikuchi T (2003) Seasonal change of standing stocks neering Symposium (ed the Society of Naval Architects of of phytoplankton and some ecological parameters in the Japan). pp. 127–134. coastal waters of Sagami Bay. Actinia 15: 15–24. (in Japanese Pithakpol S, Tada K, Montani S (2000b) Ammonium and phos- with English abstract) phate pools of Noctiluca scintillans and their supplies to the Uhlig G, Sahling G (1990) Long-term studies on Noctiluca scin- water column in Harima Nada, the Seto Inland Sea, Japan. La tillans in the German Bight population dynamics and red tide mer 37: 153–162. phenomena 1968–1988. Neth J Sea Res 25: 101–112. Redfield AC, Ketchum BH, Richards FA (1963) The influence of Verity PG (1985) Grazing, respiration, excretion, and growth organisms on the composition of seawater. In: The Sea, Vol. 2 rates of tintinnids. Limnol Oceanogr 30: 1268–1282. (ed Hill MN). John Wiley, New York, pp. 26–77. Yamada Y (1997) The strange color of sea occurred in Tokyo Saitou K (1992) Year to year variations in water quality in the Bay and Sagami Bay, May 1996. Bull Kanagawa Pref Fish Res coastal areas in Sagami Bay. Bull Jpn Soc Fish Oceanogr 56: Inst 2: 65–75. (in Japanese with English abstract) 328–334. (in Japanese) Yamada Y, Iwata S (1992) Recent trend of marine environment in Sautour B, Artigas LF, Delmas D, Herbland A, Laborde P (2000) Sagami Bay. Bull Japan Soc Fish Oceanogr 56: 323–327. (in Grazing impact of micro- and mesozooplankton during a Japanese) spring situation in coastal waters off the Gironde estuary. J Yamada Y, Matsushita S (2005) Environmental properties in the Plankton Res 22: 531–552. coastal area of Sagami Bay. Bull Japan Soc Fish Oceanogr 69: Schaumann K, Gerders D, Hesse K-J (1988) Hydrographic and 208–212. (in Japanese) biological characteristics of a Noctiluca scintillans red tide in Yamada Y, Matsushita S (2006) Estimation of pollution loads of the German Bight, 1984. Meeresforsch 32: 77–91. nitrogen, phosphorus and COD to Sagami Bay by using water Schoemann V, de Baar HJW, de Jong JTM, Lancelot C (1998) quality data of the influent rivers. Bull Kanagawa Pref Fish Effects of phytoplankton blooms on the cycling of manganese Tech Center 1: 43–49. (in Japanese) and iron in coastal waters. Limnol Oceanogr 43: 1427–1441. Yoshida K, Ishimaru T (2008) Long term change of the phyto- Shanks A, Walters K (1996) Feeding by a heterotrophic dinofla- plankton community in Tokyo Bay. Bull Plankton Soc Japan gellate in marine snow. Limnol Oceanogr 41: 177–181. 55: 45–47. (in Japanese with English abstract) Skjoldal HR, Båmstedt U, Klinken J, Laing A (1984) Changes Yoshida Y, Mishima Y, Sato M (1998) Relationships between the with time after capture in the metabolic activity of the carniv- dominant phytoplankton and DIN : DIP ratios in the inner part orous copepod Euchaeta norvegica Boeck. J Exp Mar Biol of Tokyo Bay. Nippon Suisan Gakkaishi 64: 259–263. (in Japa- Ecol 83: 195–210. nese with English abstract) Tada K, Pithakpol S, Montani S (2004) Seasonal variation in the Yoshimatsu S (2008) Long-term variation in phytoplankton in abundance of Noctiluca scintillans in the Seto Inland Sea, southern part of Harimanada. Bull Plankton Soc Jpn 55: 41– Japan. Plankton Biol Ecol 51: 7–14. 44. (in Japanese with English abstract) Tada K, Pithakpol S, Yano R, Montani S (2000) Carbon and ni-