This article was downloaded by: [Shikata, Tomoyuki] On: 28 November 2008 Access details: Access Details: [subscription number 906062392] Publisher Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

European Journal of Phycology Publication details, including instructions for authors and subscription information: http://www.informaworld.com/smpp/title~content=t713725516

Growth dynamics of Heterosigma akashiwo (Raphidophyceae) in Hakata Bay, Japan Tomoyuki Shikata a; Souta Yoshikawa a; Tadashi Matsubara a; Wataru Tanoue a; Yasuhiro Yamasaki a; Yohei Shimasaki a; Yukihiko Matsuyama b; Yuji Oshima a; Ian R. Jenkinson c; Tsuneo Honjo a a Laboratory of Marine Environmental Science, Division of Bioresource and Bioenvironmental Science, Kyushu University, Hakozaki, Fukuoka, Japan b National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency 2-17-5 Maruishi, Hatsukaichi, Hiroshima, Japan c Agence de Conseil et de Recherche Océanographiques, France Online Publication Date: 01 November 2008

To cite this Article Shikata, Tomoyuki, Yoshikawa, Souta, Matsubara, Tadashi, Tanoue, Wataru, Yamasaki, Yasuhiro, Shimasaki,

Yohei, Matsuyama, Yukihiko, Oshima, Yuji, Jenkinson, Ian R. and Honjo, Tsuneo(2008)'Growth dynamics of Heterosigma akashiwo (Raphidophyceae) in Hakata Bay, Japan',European Journal of Phycology,43:4,395 — 411 To link to this Article: DOI: 10.1080/09670260801979295 URL: http://dx.doi.org/10.1080/09670260801979295

PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.informaworld.com/terms-and-conditions-of-access.pdf This article may be used for research, teaching and private study purposes. Any substantial or systematic reproduction, re-distribution, re-selling, loan or sub-licensing, systematic supply or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.

Eur. J. Phycol., (2008), 43(4): 395–411

Growth dynamics of Heterosigma akashiwo (Raphidophyceae) in Hakata Bay, Japan

TOMOYUKI SHIKATA1, SOUTA YOSHIKAWA1, TADASHI MATSUBARA1, WATARU TANOUE1, YASUHIRO YAMASAKI1, YOHEI SHIMASAKI1, YUKIHIKO MATSUYAMA2, YUJI OSHIMA1, IAN R. JENKINSON3 AND TSUNEO HONJO1 1

Laboratory of Marine Environmental Science, Division of Bioresource and Bioenvironmental Science, Kyushu University, Hakozaki, Fukuoka, 812-8581, Japan 2 National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency 2-17-5 Maruishi, Hatsukaichi, Hiroshima, 739-0452, Japan 3 Agence de Conseil et de Recherche Oce´anographiques, Larvergne, 19320 La Roche Canillac, France

Downloaded By: [Shikata, Tomoyuki] At: 12:58 28 November 2008

(Received 22 March 2007; accepted 6 February 2008) In Hakata Bay, Japan, from April to June 2006, Heterosigma akashiwo dynamics were investigated by daily to weekly sampling in relation to environmental parameters. To test how well the water in the samples supported H. akashiwo growth, bioassays of growth of a cultured strain of H. akashiwo were also conducted on these samples. In Hakozaki, a semi-enclosed fishing port, H. akashiwo had bloomed at almost all stations, one week after DIN and DIP concentrations increased by the end of May. Thereafter, H. akashiwo declined and fluctuated at low densities. The H. akashiwo population almost completely disappeared associated with a rapid decrease in salinity at the end of June. In the bioassays, addition of phosphate and nitrate often promoted H. akashiwo growth during the investigation, but 1 week before the H. akashiwo bloom, the growth rates in seawater samples without addition of these nutrients became close to those with nutrients. Addition of vitamins and metals barely affected growth rates during the investigation period. Moreover, even when some phytoplankton species also bloomed densely, H. akashiwo grew in the seawater samples at rates similar to that when phytoplankton abundance was low, indicating absence of significant allelopathic control by other phytoplankton. The present study shows that H. akashiwo growth is limited by N- and P-sources in this bay. Key words: cell division, competition, diatoms, ecology, growth, harmful algal blooms, nutrients, phosphate uptake

Introduction Hakata Bay, Japan, fronts Fukuoka City, with 1.4 million inhabitants, and drainage from the city and factories flow there. In Hakata Bay, especially in semi-enclosed sections, phytoplankters, such as diatoms, dinoflagellates and raphidophytes, often bloom. Heterosigma akashiwo (Hada) Hada ex Hara et Chihara, which is a member of the Raphidophyceae, is one of the main phytoplankters causing blooms. From our investigation over 10 years, we know that H. akashiwo blooms occur regularly in the bay in early summer (May–June). H. akashiwo causes red tide blooms in subpolar to subtropical eutrophic coastal waters (Honjo, 1993; Smayda, 1998). The physiological and ecological features of this raphidophyte were reviewed by Honjo (1992, 1993) and Smayda (1998). Correspondence to: Tomoyuki Shikata. E-mail: shikata@agr. kyushu-u.ac.jp

Water temperature is closely linked with the bloom dynamics of H. akashiwo in the field, acting on both vegetative growth and cyst germination (Tomas, 1980; Imai & Itakura, 1999). A close relationship exists also between bloom frequency and eutrophication in some sea areas (Honjo, 1993), perhaps explained by its requirement for abundant nitrogen and phosphorus sources (Watanabe & Nakamura, 1984a). Its growth also has a high requirement for iron (Brand, 1991) and trace metals, including manganese (Takahashi & Fukazawa, 1982), and vitamin B12 (Nishijima & Hata, 1984). In nature, allelopathic substances (Honjo & Tabata, 1985; Yamasaki et al., 2007) from competing phytoplankters as well as pH (Watanabe & Nakamura, 1984b; Hosaka, 1992; Hansen, 2002) may also control the growth of H. akashiwo. Nonetheless, the relative importance of nutrients and allelopathy to blooming by H. akashiwo remains unknown.

ISSN 0967-0262 print/ISSN 1469-4433 online/08/040395–411 ß 2008 British Phycological Society DOI: 10.1080/09670260801979295

T. Shikata et al. Thus, we investigated the relationship between environmental factors and short-term dynamics of H. akashiwo in Hakata Bay from spring to early summer, based on daily to weekly sampling. Materials and methods

Downloaded By: [Shikata, Tomoyuki] At: 12:58 28 November 2008

Study site Hakata Bay is a small, shallow bay (20 km from east to west; 10 km from north to south; maximum depth: 23 m; Fig. 1). The tidal range is 2 m. In this bay, water temperature starts to increase in March, following air temperature, thermal stratification is established and remains until September or October. The south-west area of this bay is semi-enclosed behind a big control dyke. Because it was expected that semi-enclosure might strongly influence water quality and the dynamics of H. akashiwo, we conducted regular sampling at as many stations as possible. These sampling stations were the two fishing ports of Hakozaki (3 m depth) and Aratsu (4 m depth) and ‘bay’ stations H (10.5 m depth) and I (12.5 m depth) in the semienclosed section, as well as the fishing port of Meinohama and the bay stations A–G and J (depth, Stn A: 22 m, B: 14 m, C: 13.5 m, D: 7 m, E: 8 m, F: 7.5 m, G: 8 m and J: 4.5 m) outside the control dyke (Fig. 1).

Sampling Frequencies and subjects of the present investigations are summarized in Table 1. A flow chart of the investigation is also shown in Fig. 2. In 2006, surface seawater was sampled at each sampling station from April to June 2006, the seasons when H. akashiwo had previously bloomed yearly in Hakata Bay. We went to fishing ports by bicycle or car, sampled seawater between 9:00 and 10:30 every morning and went back our laboratory within 1 h. Every week,

396 surface seawater samples were taken at the bay stations using Kyushu University research vessels Isoshigi and Wakasugi. When sampling at bay stations, we left port at 10:00 in the morning and returned to our laboratory within 4 h.

Measurement of environmental factors Surface water temperature, salinity, chlorophyll a (chl a) and pH were measured at the surface using a thermosalinity meter (model 85, YSI/Nanotech Inc., Ohio, USA), a conductivity-temperature-depth meter with a fluorescence probe (ASTD 687, ALEC ELECTRONICS Co. LTD., Kobe, Japan), and a pH meter (F-51, HORIBA TECHNO SERVICE Co. LTD., Kyoto, Japan), respectively. Because dissolved oxygen (DO) concentrations control sediment-water exchange and dynamics of nutrients, particularly phosphate (Pomeroy et al., 1965; Sannigrahi & Ingall, 2005), we measured DO concentration in the bottom layer (bottom-30 cm) using a DO meter (model 550A, YSI/ Nanotech Inc., Ohio, USA). The seawater samples were filtered through 0.22 mm syringe filters (MILLEX-GV, Millipore Corporation, MA, USA), and then frozen (at –80 C) for analysis of dissolved inorganic nitrogen (DIN) including nitrate, nitrite and ammonium, and dissolved inorganic phosphorus (DIP). Concentrations of DIN and DIP were subsequently determined with an autoanalyzer (TRAACS 800, Bran þ Luebbe Co., Hamburg, Germany) in accordance with the method of Strickland & Parsons (1968).

Counting phytoplankton Heterosigma akashiwo and other dominant species of phytoplankton were counted in 10 ml to 1 ml of each sample under a light microscope immediately after sampling. Seawater samples for counting phytoplankton

Fig. 1. Sampling stations, fishing ports of Hakozaki (1), Aratsu (2) and Meinohama (3) and bay stations (Stns A–J) in Hakata Bay (black circle). White and black stars indicate the locations of our laboratory and meteorological observatory, respectively.

Growth dynamics of Heterosigma

397 strain can actively proliferate at 20–25 C and grow without a lag-phase after salinity decreases from 30 to 10 psu. Seawater for culture medium to maintain the strain was collected from the Tsushima Warm Current around Oki Island (34 240 5800 N; 130 120 2000 E) and aged under laboratory conditions for more than 1 year. When the oceanic water was collected, only a few cells of phytoplankton occurred in 1 ml of the open seawater. The aged oceanic water was enriched with modified SWM-3 (Itoh & Imai, 1987) without any calcium pantothen, nicotinic acid, -aminobenzonic acid, inositol, folic acid or thymine addition (Yamasaki et al., 2007). Cultures were maintained in 100 ml flasks containing 50 ml of the modified SWM-3 medium with a salinity of 30 psu at 20 C under 530 mmol m2 s1 of cool-white fluorescent illumination on a 12:12 h light–dark cycle. Irradiance in the

were not fixed with chemicals after seawater sampling because the time from sampling to the start of counting was short,  4 h.

Precipitation measurement Precipitation was recorded by the Japan Meteorological Agency (http://www.data.kishou.go.jp/etrn/index.html) at the meteorological observatory (Fig. 1) in Fukuoka City.

Bioassay The bioassay protocol is summarized in the upper right of Fig. 2. An axenic clone, NIES-10, of H. akashiwo was used to bioassay seawater samples. From our preliminary experiments, the NIES-10

Downloaded By: [Shikata, Tomoyuki] At: 12:58 28 November 2008

Table 1. Summary of investigations in Hakata Bay. Sampling points

Hakozaki

Atatsu

Meinohama

Stns A–J

Investigation frequency Investigation items Cell density of Heterosigma akashiwo Salinity DIN and DIP Water temperature Chl a Species composition of phytoplankton pH Dissolved oxygen

Every day

Every day

Every day

Every week

þ þ þ þ þ þ þ þ

þ þ þ þ þ þ þ þ

þ þ þ þ þ þ þ 

þ þ þ þ

Note: þ: investigated; : not investigated.

Sampling of surface seawater in three fishing ports and 10 bay stations

Seawater samples of three fishing ports and bay Stn A

Filtration Subculture at 8-day intervals

Counting phytoplankters

Filtration

Making media (addition of nutrients)

Cell inoculation

Starvation culture 1 week previously

Heterosigma Measurement of fluorescence

Abundances of H. akashiwo

Abundances of competitive phytoplankters

Calculation of the growth rate

Nutrients analysis

Measurements of environmental factors •Water temperature •Salinity at surface layer •pH •Chlorophyll a •Dissolved oxygen at bottom layer

Environmental factors

Fig. 2. Flow chart of investigations in this study.

Bioassay

þ þ þ (Stn A (Stn A (Stn A (Stn A 

only) only) only) only)

Downloaded By: [Shikata, Tomoyuki] At: 12:58 28 November 2008

T. Shikata et al.

398

incubator was measured with a Quantum Scalar Laboratory Irradiance Sensor (QSL-2101, Biospherical Instruments Inc., San Diego, USA). Every 7–8 days, 2 ml of culture was inoculated into fresh medium. A further 250 ml of the culture, which was cultured in modified SWM-3 for 7–8 days, was inoculated into 100 ml oceanic water in a 200 ml Erlenmeyer flask and was cultured under the same conditions of temperature and light as in the subculture also for 7–8 days (hereafter referred to as starvation culture). The oceanic water collected around Oki Island, filtered with Steri-cups (pore diameter of 0.22 mm; Millipore Corporation, MA, USA) and adjusted to pH 7.8 with tris-aminomethane and HCl. The aims of the starvation culture were to minimize the introduction of extra- and intra-cellular nutrients from the subculture, to acclimatize cultured cells to the low nutrient conditions and to keep physiological states of cells as constant as possible throughout the investigation periods. From mid-April to late-June 2006, seawater samples for bioassay experiments were collected from surface layers in the three fishing ports and Stn A. To protect seawater samples from contamination as much as possible, the sampling was conducted separately from other sampling for other investigations, using a disposable, high-quality paper cup. The frequencies of sampling in the three fishing ports and at Stn A were every 2 or 3 days and every week, respectively. The seawater samples were filtered using Steri-cups (pore diameter of 0.45 mm; Millipore Corporation, MA, USA), and to them were added components to make the different media shown in Table 2. Under clean-bench conditions, each medium

Table 2. Media for bioassay using seawater of (A) fishing ports and (B) Stn A. Abbreviation (A). Seawaters collected from three fishing ports þNone þNaH2PO42H2O þNaH2PO42H2OþNaNO3 þNaH2PO42H2OþFeEDTA þNaH2PO42H2OþP1-metal mixture solution þNaH2PO42H2OþP2-metal mixture solution þNaH2PO42H2OþNa2EDTA þNaH2PO42H2OþVitamin mixture solution þAll (B). Seawaters collected from Stn A þNone þNaH2PO4–2H2O þNaNO3 þNaH2PO4–2H2OþNaNO3 þNaH2PO4–2H2OþNaNO3þFeEDTA þNaH2PO4–2H2OþNaNO3þP1-metal mixture solution þNaH2PO4–2H2OþNaNO3þP2-metal mixture solution þNaH2PO4–2H2OþNaNO3þNa2EDTA þNaH2PO4–2H2OþNaNO3þVitamin mixture solution þAll

þnone þP þNP þNFe þPP1 þPP2 þPED þPVi þall þnone þP N þNP þNPFe þNPP1 þNPP2 þNPED þNPVi þall

was filter-sterilized with 0.22 mm syringe filters and 3 ml of it was dispensed into each of three polystyrene test tubes. We inoculated 1 ml of the starvation culture into 3 ml of each different medium, made from the oceanic water samples from the four stations of Hakata Bay (Table 2), and modified SWM-3 made from oceanic water around Oki Island as a positive bioassay in polystyrene test tubes, and cultured for 10 days under the same conditions of temperature and light as the subculture and the starvation culture. As a negative bioassay, 4 ml of the starvation culture were poured into three polystyrene test tubes with nothing and cultured alongside other cultures. After the start of incubation, in vivo fluorescence (Brand, 1981) was measured with a Turner Designs Model 10-00R fluorometer every 2 days. Growth rates were calculated from three consecutive data points in the incubation period using the method of Brand (1981) and then maximum growth rates during the incubation period were determined. As we needed to know the growth potential of oceanic water used for the starvation cultures, 10 different media were made from the open seawater, according to the recipe for the bioassay of water samples from Stn A (Table 2B) and culturing was conducted as described above.

Statistical analysis The data in the bioassay were tested for homogeneity of variances using Levene’s test. If the variances were homogeneous, a one-way ANOVA and the multiple comparison with Tukey’s HSD test or Dunnett’s pairwise multiple comparison t-test were employed to test differences among growth rates in treatments. All data that did not show homogeneous variances were log-transformed, and a Levene’s test was performed once again. When the variances were not homogeneous even after log-transformation, a Games–Howell post-hoc was used. All analyses were performed using the programme SPSS for Windows (SPSS, Chicago, IL, USA). A significance level of p < 0.05 or p < 0.01 was used in all tests.

Results Fluctuations of H. akashiwo in Hakata Bay Fluctuations of H. akashiwo in Hakata Bay are shown in Fig. 3. Cell density of H. akashiwo peaked at the end of May 2006 at most stations. The maximum cell density of H. akashiwo in the fishing ports, Hakozaki, Aratsu and Meinohama, was 1,200 cells ml1 on 28 May, 460 cells ml1 on 24 May and 240 cells ml1 on 21 May. At Stns B, F, G, H and J, the cell densities of H. akashiwo also reached >102 cells ml1 on 22 May 2006. Although the cell density of H. akashiwo also reached >102 cells ml1 at some sampling stations other than during the bloom period, they did not do so all on the same days.

Growth dynamics of Heterosigma

Between 15 April and 30 June, surface water temperatures in the fishing ports of Hakozaki, Aratsu and Meinohama, as well as at Stn A, increased progressively from 13–15 C on 15 April to reach 23–26 C on 24 June (Fig. 4). Surface salinities in the fishing ports of Hakozaki, Aratsu and Meinohama and at the bay stations ranged from 10 to 32.3 psu, from 15.5 to 32.2 psu and from 5.3 to 32.6 psu and from 8.7 to 34.1 psu, respectively (Fig. 4). The surface salinities at all sampling stations in Hakata Bay fluctuated concurrently and decreased on rainy days. Especially at near-shore stations, the salinities fluctuated widely. Near the end of June, a particularly marked decrease in surface salinities occurred after heavy rain, during and after which H. akashiwo disappeared completely. During the investigation periods, DO concentrations in bottom layers in the fishing ports of Hakozaki and Aratsu ranged from 0.10 to 14.3 mg l1 and from 0.07 to 11.4 mg l1, respectively (Fig. 4). Dissolved oxygen concentrations steadily decreased during the investigation period and remained 8.5 in the fishing ports of Hakozaki and Aratsu when phytoplankon bloomed from mid- to late June, pH hovered around 8.0. During the investigation period, surface chl a concentrations in the fishing ports of Hakozaki and Aratsu and at Stn A ranged from 0.8 to 76.2 mg l1, from 0.4 to 73.5 mg l1 and from 0.4 to 3.8 mg l1, respectively (Fig. 4). Peaks of chl a were observed in late April, late May, early June, and particularly in mid- and late June. The peaks in chl a were contributed by both diatoms and flagellates (Fig. 5). The dominant and sub-dominant taxa in the phytoplankton peaks were the raphidophyte H. akashiwo, the diatoms Chaetoceros sp., Skeletonema costatum, Rhizosolenia sp., and the dinoflagellates, Prorocentrum dentatum, Prorocentrum minimum, Prorocentrum triestinum. Concentrations of DIN and DIP in the three fishing ports and at the bay stations are shown in Figs 6 and 7, respectively. In Hakozaki,

104

103

103

102

102

101

101

0

0

104 Aratsu

104

103

103

102

102

101

101

0

0

104

Meinohama

104

103

103

102

102

101

101

0

15 Apr 25 Apr 5 May 15 May 25 May 4 Jun 14 Jun 24 Jun

A B C D

E F G

H I J

0

15 Apr 25 Apr 5 May 15 May 25 May 4 Jun 14 Jun 24 Jun

Fig. 3. Variations in Heterosigma akashiwo abundance (cells ml1) in the three fishing ports (left) and at the ten bay stations (right).

T. Shikata et al.

400

Aratsu Hakozaki Meinohama *Symbols of salinity at offshore stations A B C D G H I J

Stn A E

F

30 Water temperature (°C) 20 10 100 Precipitation (mm) 50

Downloaded By: [Shikata, Tomoyuki] At: 12:58 28 November 2008

0 30 20 10

Salinities (psu) in fishing ports

0 35 25 15

Salinities (psu) at bay stations*

5 15 10 5

DO (mg l−1)

0 9

pH

8 7 100

Chl a (mg l−1)

10 1 0.1 15 Apr

25 Apr

5 May

15 May

25 May

4 Jun

14 Jun

24 Jun

Fig. 4. Variations of surface water temperature, precipitation, surface salinity, near-bottom dissolved oxygen, surface pH, surface chl a in fishing ports of Hakozaki, Aratsu and Meinohama and at the bay stations. Two-headed arrow and downpointing arrow indicate the period when the bay-wide bloom of Heterosigma akashiwo occurred and H. akashiwo population was rapidly declined, respectively.

Growth dynamics of Heterosigma 105 104

Cell density (cells ml−1)

105 Hakozaki (Diatoms)

104

103

103

102

102

101

101

0

0

105

105

104

Downloaded By: [Shikata, Tomoyuki] At: 12:58 28 November 2008

401

Aratsu (Diatoms)

104

103

103

102

102

101

101

0

0 105

105 104

Meinohama (Diatoms)

104

103

103

102

102

101

101

0

0

105 104

Hakozaki (Flagellates)

Aratsu (Flagellates)

Meinohama (Flagellates)

105 Stn A (Diatoms)

104

103

103

102

102

101

101

0 15 Apr 25 Apr

Stn A (Flagellates)

0 5 May 15 May 25 May 4 Jun

14 Jun 24 Jun

15 Apr 25 Apr 5 May 15 May 25 May 4 Jun 14 Jun 24 Jun

Fig. 5. Variations of dominant diatoms Skeletonema costatum (white circle), Rhizosolenia sp. (black circle), Chaetoceros sp. (white triangle) and Leptocylindrus danicus (black triangle) and flagellates Prorocentrum minimum (white circle), Prorocentrum triestinum (black circle), Prorocentrum dentatum (white triangle) in three fishing ports and at Stn A. Two-headed arrow and down-pointing arrow indicate the period when the bay-wide bloom of Heterosigma akashiwo occurred and H. akashiwo population rapidly declined, respectively.

Aratsu and Meinohama, DIN concentrations ranged from 0.1–303 mM, 10.5–226 mM and 6.0– 57 mM. Corresponding ranges of DIP were